Studies On The Development, Biochemistry, And Biology Of Experimental Hepatomas

STUDIES ON THE DEVELOPMENT. BIOCHEMISTRY. AND BIOLOGY OF EXPERIMENTAL HEPATOMAS Harold P. Morris Laboratory of Biochemistry. NCI. NIH. PHS. DHEW. Betherda. Maryland

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I . Introduction I1. Some Basic Concepts of Cell Biochemistry I11. Development of Transplant.able Hepatomas I V . Ultrastructure and Cytochemical Studies . . . . . . . V. Carbohydrate Enzyme Activities and Metabolic Pathways A. Energy Pathways . . . . . . . . . . . . B. Glucose and Fructose Metabolism . . . . . . . . C. Enzyme Activities, Metabolic Pathways, and Growth Rate of Hepatomas . . . . . . . . . . . . . . VI . Protein and Amino Acid Metabolism . . . . . . . . A . Enzymes of Amino Acid Metabolism. . . . . . . . B. Amino Acid Incorporation and Oxidation . . . . . . . C. Lysine Metabolism . . . . . . . . . . . D . Hepatoma Proteins . . . . . . . . . . . VII . Ot.her Inducible Enzymes . . . . . . . . . . . A . Tryptophan Pyrrolase . . . . . . . . . B . Ethionine Fatty Hcpatomas . . . . . . . . . C. L-hspartic Amino 'I'rrmsferase . . . . . . . . . VIII . Niicleic Acid LZetabolism . . . . . . . . . . . A . Deoxyribonucleic 12(*idXuclcotidyl Transferase . . . . . 13. Ribonuclease Activity . . . . . . . . . . . C. Ribonriclease in Subcellular 1~'ractions . . . . . . . 1) . Other Microsomal Fraction Enzymes . . . . . . . I X . Metabolism of Purines and Pyrimidines and Cholesterol Synthesis by Hepatomas . . . . . . . . . . . . . . A . Metabolism of Purines . . . . . . . . . . . B. Metabolism of Pyrimidines . . . . . . . . . . C. Feedback Inhibition in Hepatomas . . . . . . . . D . Deletion of the Cholesterol Negat.ive Feedback Regulation in Liver Tumors . . . . . . . . . . . . . . E. Feedback Deletion . . . . . . . . . . . X . Other Activity Studies . . . . . . . . . . . A. Catalase Activity . . . . . . . . . . . . . . . . . . . . . B. Electron Spin Resonance . C. Tissue Culture . . . . . . . . . . . . D . Polyribosomes . . . . . . . . . . . . . . . . X I . Oxidative Phosphorylation and Phosphatide Synthesis A . Oxidative Phosphorylation and Adenosine Triphosphatase Activity of Mitochondria . . . . . . . . . . . . B. Phosphatide Synthesis . . . . . . . . . . .

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228 228 230 235 241 241 242 249 250 250 252 254 254 256 256 259 261 264 264 265 266 267 269 269 272 274 276 278 279 279 282 284 285 286 286 287

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HAROLD P. MORRIS

XII. Alterations of Dietary and Hormonal Regimens in Regulation of Enzyme Activity . . . . . . . . . . . . . . . XIII. Some Other Possible Mechanism(s) of Carcinogenesis a t the R‘Iolecnlar Level . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

287 291 296

I. Introduction

I t is the purpose of this chapter to assemble and review the literature that is primarily concerned with biological and biochemical characteristics of a spectrum of transplantable hepatomas developed in the author’s and other laboratories. For the most part, the literature published since 1961 will be covered, as this date coincides with the cut-off date of the literature included by the author in a previous review (Morris, 1963). This spectrum of transplantable hepatomas of different growth rate has provided the materials useful in describing the underlying molecular basis of neoplastic behavior. These tumors have provided the means to study biological and biochemical parameters associated with different rates of growth, and in those hepatomas having few or “minimal deviations” (cf. Potter, 1961) from normal liver it has been possible to study control mechanisms of both normal and tumor tissue. The fewer the alterations a tumor tissue has undergone in the development of neoplasia the fewer the parameters necessary to examine to determine those changes essential for the conversion of the normal cell to a cancer cell. It is believed the many contributions to new knowledge that have been made by the host of investigators who have studied one or another aspect of these newer hepatomas under a variety of conditions has helped to reveal many of the biological and biochemical differences between cells of normal liver and hepatomas. The reader should also consult other recent reviews including those of Weber (l96l), Potter (1962), Reid (1962), Farber (1963), Morris (1963), Potter (1963, 1964a,b). II. Some Basic Concepts of Cell Biochemistry

The organism is constantly faced with adaptations. The role of enzymes is crucial in maintaining homeostasis in both acute and chronic adaptations. Enzymes possess a variety of controlling mechanisms which maintain a harmonious association with their environment and under normal physiological situations maintain an extraordinary constancy of composition despite a continuous turnover of the numerous components of the system. The complexity of the system is well illustrated by Fig. 1, from Weber (1963b). At the base is the operon which is thought to contain two or three genes for each specific protein (enzyme). These are designated as regulator, operator, and structural genes. Thus, in Fig. 1 when the regulator gene is allowed to activate the

DEVELOPMEKT, BIOCHEMISTRY, BIOLOGY O F H E P A T O M A S

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FIG.1. Factors involved in enzyme activity and synthesis. The points are indimtetl at which actiriomyrin 11, priromycin, and ethionine attack the mechanisms of enzymca synthesis (From Weber, 1963b).

operat,or gene the latter inst,ructs t,he structural gene t o manufacture a specific enzyme protein. The DNA (deoxyribonucleic acid) of the structural gene t,ransmits genetic information to mRNA (messenger ribonucleic acid). If t,he mRiYA is cont'inuously present at the active site of the st>ructural gene DNA, it may be released on a n impulse from the operator gene. If in the presence of a balanced amino acid pool, high-energy phosphate donors, and soluble RNA (sRNA) amino acid complexes, the mRNA and the (t,ransfer) tRNA acting on available ribosomal sites will be able to synt,hesize t'he enzyme protein. The maintenance of specific enzyme populations is dependent on the opt'imum coricentration of the various comporient~s of bhis complex machinery including the regulating and coordinating influence of substrates, coenzymes, products, repressors, activators, inhibitors, hormones, etc., acting at various attacking points of the system (see Fig. 1). Thus, act,inomyciii D blocks the transfer of genet,ic information from DNA to RNA; sRNA is inhibited by puromycin. Ethionine is thought to lower ATP and unbalance the amino acid pool.

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HAROLD P. MORRIS

This complex constituting the enzyme-forming system may produce an inactive or latent enzyme or a proenzyme. The conversion of some inactive enzymes to active enzymes apparently occurs only under specified physiological situations when a suitable impulse converts them irreversibly to carry out a specific function (Weber, 1963b). Other enzymes, such as phosphorylase of the glycogen cycle, are produced in an inactive form so that they will be available for use when needed. They may be rendered inactive when they have completed their function but can again be reactivated if the necessity arises. Epinephrine, e.g., indirectly promotes the conversion of inactive or latent phosphorylase to the active enzyme by accelerating the conversion of ATP (adenosine triphosphate) to the true reactivator, cyclic-3’,5’-adenylic acid (Rall and Sutherland, 1960). Although the operator of the repressor system in mammalian organisms is not yet certain, it is included in Fig. 1 (Weber, 196313) as probably existing. It is believed (Pardee and Wilson, 1963) the enzyme molecules are made of interdependent parts, and the interaction of the parts determine the activity of the enzyme. Specific regulatory sites determine the interaction. The enzyme aldolase is one good example of this concept. It is a protein composed of three polypeptide chains held together in a compact form by noncovalent interactions (Schachman, 1964), and the cooperative folding of three of these chains is apparently required to make one active site for this enzyme (Schachman, 1963). It can be seen that the normal living cell has many mechanisms a t its disposal. Changes in its environment can quickly shift its enzymatic make-up. Many changes are likely to occur in the course of development and the induction of cancer. The subsequent sections of this chapter will review for the most part the enzymatic responses and differences of normal liver and newer transplantable hepatomas in a n effort to distinguish how the two types of cells respond to various environmental changes for the purpose of elucidating the complex mechanism(s) of neoplastic growth. 111. Development of Transplantable Hepatomas

A series of transplantable hepatomas have been developed in the author’s and other laboratories during the last decade. A summary of the carcinogen used, induction period, sex, strain, treatment period, histological type, and metastatic potential of these primary hepatomas are listed in Table I. It can be seen at once that the same, as well as a variety of, chemicals and treatments yield hepatomas with variable properties. Nine transplantab helepatomas have been developed by feeding the slowly acting carcinogen N-2-fluorenylphthalamic acid (FPA) in two separate experiments. Five of the nine hepatomas developed in male rats.

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Liver metastases to the lungs were found in all the males (Table I) but in only one female. The carcinogen was administered for 10 months and animals were sacrificed from 0.8 to 8.0 months later. A much longer period of exposure to FPA was used than Reuber (1961) used in the developing of H35 hepatoma, but a longer time was taken without FdiAA (N-2fluorenyldiacetamide) than for FPA before sacrificing the host and transplanting the tumor (Table I). No poorly differentiated hepatoma was produced with FPA but poorly differentiated transplanted hepatomas have been induced with FdiAA (Table I), although according to Miyaji (1965) some of the FPA primary hepatomas had some areas of less welldifferentiated hepatoma cells. Hepatoma 5123TC (tissue culture) after return to the animal retained the trabecular arrangement noted prior to tissue culture, but the cells and nuclei, according to Miyaji (1965), were much smaller. Bile secretion was noted by Miyaji (1965) in Hepatomas 5123,7793, and 731GB, tumors arising in female rats, and in 7794B and 7800, tumors developing in male rats. In general, Miyaji (1965) found stainable glycogen decreased from transplant generation to transplant generation. Two tumors, 7787 and 7794B, both slowly growing (Table 11), were found to have considerable stainable glycogen in the early generations. Some areas of 7794B hepatoma mere relatively free of glycogen while other areas showed deep glycogen staining. Hepatoma 7787, on the other hand, showed in early generations rather uniformly deep glycogen staining Miyaji (1965) (cf. Section V,B,l). Seven of the eight primary tumors, listed in Table I, and induced by FPA were of the “minimal deviation” type. Although the histological description is similar for these hepatomas they have different biochemical and enzymatic characteristics, as described in other sections of this report. Two transplantable tumors induced in the same liver in three instances where the conditions for tumor induction were most nearly identical possess different biological and biochemical characteristics, and also differences in rate of growth. Using the length of time between transfers as a criterion of growth rate, so that all tumor lines could be compared, the several hepatomas have been arranged in order of decreasing growth rate, as shown in Table 11. These data show a 25-fold difference in rate of growth between the slowest and fastest growing tumors. The three fastest growing-Hepatomas 3683,3924A, and 7288C-are not considered t o be tumors of the “minimal deviation” type (Potter, 1961). Hepatoma 7288C has an intermediate growth rate. The tumors can be transplanted in the strain of origin with takes usually ranging from 90 to 100%. The tumors grow progressively and eventually kill the host; mestastases to the lungs have been noted in many, but not all, of the primary or the transplantable

INDVCTION OF

Tumor, sex?and strain

5123, F, B“ 7777, F, B 7787, F, B 7793, F, B 7794 A, M, B 7794 B 7795“ M, B 7797’ M, B 7800 M, €3 H 35 M, AxCg 3683j M,AxC 3924 AiF, AxC 7288 B, M, B 7288 Cm 7316 A0 F, B 7316 E0

Drug

FPAb FPAb FPAb FPAb FPAb

FPAb FPAb PPAb

FPAb

FdiAAh FdiAAh FdiAAh FAA(F6)’ FAS (Fa)

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T.lULE I TR.INSPLINT.ABLE RAT IIEPATOMAS

Reference Morris et al. (1960) Morris and Wagner llorris and Wagner Morris and Wagner hforris and Wagner Morris and Wagner Morris and Wagner Morris and Wagner Morris and Wagner Reuber (1961) Morris and Wagner Morris and Wagner Morris and Wagner

(1965) (1965) (1965) (1965) (1965) (1965) (1965) (1964, 1965) (1964, 1965) (1964, 1965) (1965)

Morris and Wagner (1964, 1965) hlorris and Wagner (1965)

B = Buffalo-strain (inbred); F = female; M = male. FPA = N-2-fluorenylphthalamic acid. c C = continuous ingestion of the carcinogen. T = trabecular carcinoma, well-differentiated (Miyaji, 1965). e Some sublines during transplantation showed a few mucinsecreting cells, and a few cells that resemble cholangio cells (Miyaji, 1965). 1 Cholangiocarcinoma with bony metaplasia. AxC 9935, also ACI 9935 (inbred). h FdiAh = N-2-fluorenyldiacetamide. a

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Time without dnig (months)

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Histological type

Td Td

Td Td Td

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Td Td TP Tpk

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Presence of metastases

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Four 4-week periods of continuous feeding; each period separated by l week w/o carcinogen. I Rapidly growing-not “minimal deviation” type. FdiA.4 ingested continuously for 2 weeks, then alternately at weekly intervals for 14 weeks. Tp = trabecular carcinoma, poorly differentiated. FAA (F6) = N,N,2,7-fluorenylenebis-2,2,2-trifluoroacetamide. Intermediate growth rate, not “minimal deviation” type. TLW = trabecular carcinoma, less well-differentiated. 2,4,6-Trimethylaniline. O

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DEVELOPMEXT, BIOCHEMISTRY, BIOLOGY OF HEPATOMAS

TIMEBETWEENTRLNSFERS

TABLE I1 TR.\NSPL.\NTABLE R.4T LIVERTUMORS“

OF SEVER.\L

Period covered

3683 3Y24‘i 7288Ub H35TC9 H35TCIC 5123TC 7288C H 35 512313 5123C 5123.A 512311 5123e 7800 7794A 7316‘4 7795 7793 7316B 7787 77941<

Generation

Months

Average per generation (months)

221-276 13-178 4-17 1-3 3-18 4-25 2-25 10-28 14-42 17-42 17-40 17-38 1-17 1-12 1-10 1-11 1-8 1-7 1-8

25.5 25.8 8.7 1.4 16.0 28.1 34.1 20.3 46.9 19.0 47.4 46.1 48.0 34.5 33.6 37.8 33.4 29.1 38.3 29.4 26.7

0.5 0.6 0.7 0.7d 1. Id 1.3d 1.5 1.7 1.9 2.0 2.1 2.2 3.0 3.1 3.7 3.8 4.8 4.9 5.5 9.8 13.4

1-4

1-3

233

Range per generation (months) 0.3-0.9 0.4-1.0 0.4-1.2 0.6-0.8 0.6-1.6 0.8-2.0 0.8-2.4 1.2-2.6 1.3-3.5 1 .2-3.4 1.3-3.5 1.4-3.0 1.6-3.8 1.6-8.5 3.1-4.1 2.5-5.5 2.7-11.3 4.1-6.2 2.8-7.8 5.9-13.6 9.9-17.2

From Morris and IVngner (1965). Discontinued. c TC = tissue ridtiire. d After retiirii t o i n 13ir.opropagation. 5123 siiblines A, 13, C, and U were arbitrarily established from 5123 after the 16th serial transplant genrratioii. y

b

tumor lines (cf. Table I). The tumors seem to be remarkably stable from transplant generation to traiisplant generation, although the very slowly growing tumors seem to be growing more rapidly with transplantation. Attempts to grow some of the minimal deviation tumor lines in F, hybrid rats of two inbred strains (one being the strain of origin) have not shown any significant differelice in enzyme characteristics (cf. Roth et al., 1964), but the tumors grew in a very low percentage of the F, hybrids, precluding further efforts to grow the “minimal deviation” tumors in hybrid rats, and no attempts have been made to adapt these tumor lines to other inbred rat strains. The primary tumors selected for transplanting t o obtain tumors of the “minimal deviation” type mere from tumor nodules usually having an

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HAROLD P. MORRIS

appearance quite similar to host liver. Tumors of a more rapid rate of growth, such as 3683, 3924A, and 7288C, were less like liver in appearance and after many transplant generations have changed little in appearance, whereas the minimal deviation tumors have, grossly, retained in subsequent transplant generations much of their liverlike appearance. Rose bengal dye injected intravenously 30 minutes prior to sacrificing the primary tumor-bearing rat by decapitation (Reuber, 1965) results in more dye uptake by approximately one-third of well-differen tiated tumor nodules resembling liver, indicating the retention of some degree of function, while less well-differentiated tumor nodules were never colored by the dye. Although this procedure detects poorly differentiated from welldifferentiated primary hepatomas, the reliability of this dye uptake procedure t o distinguish primary hepatomas most like liver biochemically and enzymatically from hepatomas less like liver has not been fully established, but merits further study. Three hepatoma sublines, H35TC1, H35TC2, and 5123TC, were carried in tissue culture at the McArdle Laboratory (cf. Section X,C) for varying periods and then the in vitro hepatoma cells were returned to in vivo culture. These three sublines have been maintained in vivo for a few to many generations. Their growth rates have been accelerated somewhat. It will be noted in Table I1 that the growth rate in vivo of all three lines is somewhat more rapid than the growth rate of Hepatoma 7288C, a tumor considered to have an intermediate growth rate. If some enzymes or other biochemical characteristics of these tumors, after cultivation in vitro and return t o the animal, have deviated from the hepatoma line of origin biochemically or enzymatically then similar assays on the new sublines should be able to detect the changes and might afford one means of detecting how a “minimal deviation” hepatoma could be altered and possibly help explain how progression could occur in hepatomas. The glucokinase assays of H35TC1, and 5123TC, when compared t o those of H35 and 5123 (several sublines), (Weinhouse et al., 1963; Sharma et al., 1964) indicate lower glucokinase values for these two tissue culture sublines after return to in vivo growth than was present before in vitro growth. Are these lowered glucokinase activity values significant? The following of further generations of these tumors for glucokinase as well as for other biochemical parameters correlated with growth rate is indicated. Using the strong, rapidly acting carcinogen FdiAA, and induction techniques similiar to that used to induce transplantable hepatoma H35 (Reuber, 1961), Reuber (1965) has made further studies on the development of hepatomas. He found that change of parenchymal cells from hyperplastic to neoplastic took place gradually over a long period of time, By histological criteria, Reuber (1965) noted that areas of hyperplasia passed

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through the stages of nodules of hyperplasia, nodules with atypical cells, and small hepatomas before becoming well-developed carcinomas. Most of the carcinomas mere well differentiated while a few were poorly or highly differentiated. Of the earliest hyperplastic lesions a few progressed to hepatomas; some disappeared; while most underwent degenerative cytoplasmic changes. The nodules of hyperplasia present a t the time the FdiAA was withdrawn either remained as nodules or developed into hepatocellular carcinomas. The duration of ingestion of the carcinogen was important in the development of the hyperplastic lesions. Administration of the carcinogenic diet containing 0.025% FdiAA for four 4-week periods, with the diet free of carcinogen for 1 week between each period, resulted in the most typical development of the hyperplastic lesions. It was more difficult, according to Reuber (1965), to distinguish histologically between hyperplastic nodules and well-differentiated hepatomas during shorter or longer periods of exposure t o FdiAA, because the well-formed cords in the hepatomas made it difficult t o distinguish them from hyperplastic nodules. After 16 weeks of FdiAA ingestion, sacrifice of the animals 11 months later usually demonstrated that all the nodules in any one liver had become welldeveloped hepatomas. The problems of differentiating histologically according to Reuber between hyperplasia and carcinoma became minimal a t this time. These studies by Reuber (1965) in the evolution of hepatomas in rats ingesting the active carcinogen FdiAA can be used as a model for further studies t o aid in the development of hepatomas that are transplantable, but which must be studied biochemically in order to distinguish their biochemical differences. The failure of minimal deviation hepatomas to grow in other rat strains or in autologous hosts suggests that the immunological competence of the host may be of considerable influence in establishing transplantable rat hepatomas. Measures designed to decrease or circumvent the immunological competence of the host are now being studied in our laboratory in an effort t o develop transplantable tumors even more like liver than have been obtained thus far. It is believed that the fewer the enzymatic or biochemical deviations from normal liver that occur in a transplantable hepatoma, the more probable will be the chances of detecting key events in the development of liver neoplasia. IV. Ultrastructure and Cytochemical Studies

One parameter which could establish morphological differences in this spectrum of hepatomas is their ultrastructure which Esser arid Novikoff (1962) studied by electron microscopy and cytochemical staining in

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FIG.2. Morris 5123 hepatoma. The area adjarent to the nucleus (Nj contains relatively long lengths of Golgi membranes (C) with typical parallel orientation. A fenestra within the membiane is seen a t P.Dilated cisternae (Clj, containing homogeneous material of moderate electron opacity, are seen a t both ends. Material of similar appearance is present within small, isolated vesicles (L) in the vicinity of the Golgi membranes. Above and to the right of the Golgi apparatus rough surfaced endoplasmic reticulum (RERj may be seen. To the left and above, smooth-surfaced endoplasmic reticulum (SER) may be seen. A fenestration (FER) in the smooth-surfaced endoplasmic reticulum similar to that in the Colgi membrane is visible. Apparent continuity between rough- and smooth-surfaced endoplasmic reticulum is indicated a t CO. The zone indicated by lines a t T marks an area of possible transformation of elements of the smooth-surfaced

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Hepatoma H35 (Reuber, 1961) and Hepatoma 5123 (Morris et al., 1960). Although the similarity of 5123 to normal liver had been emphasized by Morris et al. (1960) by light microscopy and by Noviltoff (1960) by electron microscopy, the latter studies mere performed before the present methods were available for staining Golgi apparatus and endopIaemic reticulum in frozen sections. The two hepatomas were found to be remarliably similar but not identical. Bile canaliculae are present in both and both show nucleoside phosphatase activities. The 5123 canaliruli show numerous large dilations or acini (Novikoff, 1960) not present in Hepatoma H35 (Esser aiid Novikoff, 1962). Occasiorially these dilations in 5123 are filled with material giving positive reactions for lipid, mucopolysaccharide, and adenosineomonophosphatase activity. Small- t o medium-sized acid phosphatase-rich lysosomes are abundant over a wide area of pericarialicular cytoplasm but none reach the size of the spheres containing bile pigment in Hepatoma H35 (Esser and Novilioff, 1962). The Golgi apparatus is large and reticular and generally lies close to the iiucleus, and extensions toward the bile canaliculus are less common than in cells of Hepatoma H35. Both hepatomas show nucleoside diphosphatase activity in the eiidoplasmic reticulum. In frozen sections of 5123 Hepatoma, incubated with uridine, guanosine, inosiiie diphosphate or thiamine pyrophosphate as substrate, the endoplasmic reticulum appears as irregular strands in the cytoplasm (Esser and Noviltoff, 1962). Comparisons of the ultrastructure of these two hepatomas show that the mitorhondria, endoplasmic reticulum, and canalicular microvilli are essentially the same. The close proximity of the endoplasmic reticulum t o the mitochondria is also similar as well as the presence of unattached particles, which Esser and Novikofi (1962) believe to he ribonucleoprotein (RNP) particles. Both tumors contain an abundance of smooth surfaced endoplasmic reticulum especially in the Golgi areas. Isolated sacs and vesicles frequently observed in H35 Hepatoma are relatively rare in Hepatoma 5123. In many areas the ordered arrays of Golgi membranes (Fig. 2) can easily be distinguished from the loose, irregular arrangement of the reticulum while in other areas it was difficult for Esser aiid Novikoff (1962) to decide whether or not the membranes were part of the reticulum or belong t o the Golgi apparatus. Esser aiid Novikoff (1962) report that endoplasmir reticulum into Golgi membranes. Unmarked arrows indicate additional smooth-surfaced vesicles that may be transforming into Golgi membranes. hfitorhondria (hf) are also shown; note proximity of endoplasmic retirlilum to mitochondria. Apparently unattached particles (probably RNP particles) may be seen. A portion of the smooth-siirfaced endoplasmir reticulum (S) lies close to the plasma membrane (P). A microbody (MB) with internal niicleoid is also seen. Metharrylate-embedded; potassium permanganate staining ( X 33,000). (From Esser and Novikoff. 1962).

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HAROLD P. MORRIS

FIG.3. Part of a cell of Hepatoma 36%. Microvilli are present on the free surface of the cell, but the organization of the cytoplasm bears little resemblance to that of a hepatic cell. A few strands of organized ergastoplasm (ER) are present but many ribosomes lie free in the cytoplasmic matrix. Mitochondria (M) are relatively small and some possess longitudinal cristae (arrow). Multivesicular bodies (MV) are present and some smooth endoplasmic reticulum. A short tight junction (TJ) is present near the periphery a t the area of contact between the two cells (approximately X 26,000). (From Dalton, 1964.)

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FIG.4. Part of the cytoplasm of a cell of Hepatoma 7794B. Profiles of tubular endoplasmic reticulum (EN) are present in various parts of the figure. Electron-dense material is present within one of thesc profiles (arrow). Three microbodies (MB) in close association with endoplasmic reticulum are evident. In one of these the dense core exhibites striations with a spacing of approximately 300 A. The mitochondria (M) present are very similar to those of normal hepatic cells (approximately X 53,000). (From Dalton, 1964.)

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HAROLD P. MORRIS

FIG.5. Part of a cell of Hepatoma 7787. A fairly large amount of organized ergastoplasm (ER) is present with relatively few ribosomes lying free in the cytoplasmic matrix. At the left of the figure tubular endoplasmic reticulum is in continuity with the membranes of the ergastoplasm (ER) (arrow). Several of the mitochondria (M) exhibit the scarcity of cristae characteristic of many of the mitochondria of the cells of this hepatoma (approximately X 35,000). (From Dalton, 1964.)

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they were unable to identify smooth-surfaced vesicles when frozen tissues were incubated for enzyme activities because the ribosomes were generally not visible in the areas where reaction product formed in the membranes. Dalton (1964) also examined a later generation of Hepatoma 5123, subline B, and noted numerous small microbodies and a Golgi complex, giving evidence of secretory activity and suggesting that the cells of this tumor have retained some of the functions of normal liver hepatic cells. Although Hepatomas H35 and 5123 contain bile canaliculi they do not join in the organized fashion typical of liver, and, therefore, no drainage system into bile ducts exists. Esser and Novikoff (1962) distinguish tumor cells secreting bile as having a green color and showing spheres of varying size which they call secretory granules or vacuoles and also because the secretory granules stain for bilirubin. Dalton made ultrastructure studies of five hepatomas of different growth rate, Tumors 3683,7288C, 5123B, 7787, and 7794B. Using the same techniques for all five, Dalton (1964) was able to demonstrate a n inverse correlation between growth rate of these hepatomas and the degree of ultrastructural differentiation of the constituent cells. The presence in Hepatoma 3683, the most rapidly growing tumor, of little ergastoplasm, a relatively simple Golgi complex, and the absence of microbodies are all features consonant with differentiation. Dalton (1964) suggested from morphological evidence (Fig. 3) that 3683 hepatoma could have been derived from either bile duct cells or liver parenchymal cells. Hruban et al. (1964) noted the absence of microbodies in Novikoff and 3683 hepatomas but found microbodies in HC, LC (Rechcigl and Sidransky, 1962), 5123, and H35 hepatomas. In the cells of two very slowly growing hepatomas, 7787 and 7794B (Morris and Wagner, 1965), Dalton (1964) noted the presence of organized ergastoplasm, Golgi complexes, with evidence of secretory activity, and microbodies as large as or larger than those of normal hepatic cells (Fig. 4) (Dalton, 1964). I n case of 7787 (Fig. 5) (Dalton, 1964) found morphological evidence for glycogen storage and glycogenolysis (cf. Miyaji, 1965). I t is suggested that morphologically these two tumors, 7794B and 7787, have fewer deviations from normal liver than any others so far observed.

V.

Carbohydrate Enzyme Activities and Metabolic Pathways

A. ENERGY PATHWAYS One controversial aspect of cancer concerns the energy metabolism of tumors that was proposed by Warburg (1931, 1956) as the first essential event in the alteration of normal cells to become tumor cells. Now, however, transplantable liver tumors are available that do not possess those prop-

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HAROLD P. MORRIS

erties described by Warburg (1931, 1956) as characteristic of the energy metabolism of the malignant cell. In studies of the energy metabolism of Hepatomas 5123, 7800, and H35 (Reuber, 1961), Aisenberg and Morris (1961, 1963) report that all three hepatomas lacked significant aerobic or anaerobic glycolysis as measured either chemically or manometrically. The three neoplasms possessed a moderate respiratory rate which showed 4-to 5-fold stimulation on the addition of succinate and all three lacked a Crabtree effect i.e., there was no inhibition of respiration on the addition of glucose. The quotients for respiration and glycolysis closely resembled those of normal liver. The relationship between energy met.abolism to protein and DNA (deoxyribonucleic acid) synthesis for each of these three hepatomas indicated that they resembled normal liver in being unable to sustain either protein or DNA synthesis anaerobically, even in the presence of glucose. Aerobically, modest rates of incorporation of valine into protein and thymidine into DNA were achieved (Aisenberg and Morris, 1963). Glucose did not stimulate protein synthesis but did cause slight stimulation of DNA synthesis aerobically. Because these three hepatomas and several other slowly growing hepatomas were found by Weinhouse et al. (1963), Lin et al. (1962) and Elwood et al. (1963) to be low in glucokinase; these investigators also found that the addition of yeast hexokinase resulted in rapid uptake of glucose and lactate formation; the site of glycolysis impairment for these hepatomas was thought to be a t the glucokinase step. However, the very slowly growing, transplantable Hepatoma 7787 in an early transplant generation retained the hepatic pattern of glucose-ATP phosphotransferases (Sharma et al., 1964) (cf. Section V,B,l). The Warburg (1956) hypothesis that a universal correlation existed between aerobic glycolysis and malignancy does not hold for these slowly growing, welldifferentiated hepatomas. Despite these exceptions, anaerobic and aerobic glycolyses remain prominent biochemical features of the malignant cel I, although many more exceptions will no doubt be uncovered in the future. METABOLISM B. GLUCOSEAND FRUCTOSE 1. Glucose-ATP Phosphotransferases

The low aerobic glycolysis of Hepatoma 5123 in slices, as described by Weber et al. (1961b) and Aisenberg and Morris (1961, 1963), was confirmed by Weinhouse et al. (1963) using the whole liver homogenate system of Wenner et al. (1953). It was further established (Weinhouse et al., 1963; Lin et al., 1962; Elwood et al., 1963) that the site of glycolysis impairment for Hepatoma 5123 was at the glucokinase step (Figs. 6 and 7). In the absence of added yeast hexokinase, glucose uptake and lactic

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY OF HEPATOMAS

20

Time, mins.

40

243

60

FIG.6. Glucose uptake and lactate production (in pmoles) with and without added yeast hexokinase in whole homogenates of Hepatoma 51238; glucose uptake with ( A ) and without (A)hexokinase; lactate production with (0)and without ( 0 )hexokinase; 0 = lactate prodriction from glucose-6-phosphate; = lactate production from fructose 1,6-diphosphate. (From Elwood et al., 1963.)

al

20

Time, mins.

40

60

FIG.7. Effect of yeast hexokinase on glucose-U-Cl4 oxidation and on oxygen uptake (in pmoles) (cf. Fig. 6). 0 = respiration wit,hout, and = with hexokinase; A = C1402output without and A = with hexokinase. (From Elwood et al., 1963.)

244

HAROLD P. MORRIS

acid production proceeded at a very slow pace. It was noted that the addition of crystalline hexokinase from yeast resulted in a rapid uptake of glucose and formation of lactate which was increased from 5- to 20-fold. This is illustrated in a typical example in Figs. 5 and 6 for Hepatoma 5123. The data indicate that so long as hexokinase is present phosphorylation proceeded, the glycolytic system was activated, and there was a sustained production of lactate and ATP. The substrates glucose-G-P or fructose 1,6-DP, when added without hexokinase, resulted in almost as rapid production of lactate as when only glucose and hexokinase were added. One consistent finding was that no significant change in oxygen uptake by the stimulating effect of hexokinase on glycolysis occurred (Fig. 7). All five sublines of 5123 hepatoma showed a consistent and similar response t o the addition of yeast hexokinase, and invariably showed a striking increase of lactate production, a variable and often striking increase in glucose oxidation, and a n insignificant change in oxygen uptake. The glucokinase activity of a series of transplantable hepatomas has also been studied by Elwood et al. (1963). Of 17 transplantable hepatoma lines examined by these investigators, 9 were very low in glucokinase activity, varying from 1/10 to 1/5 that of normal liver. These nine hepatomas were all slowly growing tumors. The eight hepatomas that had high levels of glucokinase were more rapidly growing. Shonk et al. (1965) confirmed the above observations with respect to glucokinase. I n normal rat liver, increase in glucose concentration or addition of yeast hexokinase, the glucose uptake, lactate production, and glucose oxidation t o COJ were shown by Di Pietro and Weinhouse (1960) to increase. The low glycolytic activity of 5123 hepatomas, however, according to Elwood et al. (1963) appears to be due to the low activity of the enzyme and not to a low affinity for the substrates, since large increases in glucose concentration did not appreciably influence utilization. The low affinity of glucokinase in normal rat liver with a K , of 0.01-0.02 M (Di Pietro et al., 1962; Di Pietro and Weinhouse, 1960) ensures a considerable glucose uptake at high glucose concentrations. The very slowly growing, highly differentiated Hepatoma 7787 (Morris and Wagner, 1965), which has produced significant amounts of glycogen for at least three transfer generations, appears t o differ from the other low glucokinase hepatomas (Elwood et al., 1963). While 7787 has a low hexokinase and a moderate to high glucokinase (Sharma et al., 1964), it has a higher K , than the other slowly growing transplantable hepatomas. I n studies by Sharma et al. (1964) in the third and fourth transplant generations of Hepatoma 7787 a tendency toward higher hexokinase and lower glucokinase was noted in the fourth generation. If this hepatoma in subsequent transfers were to continue this

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245

trend it would represent a partial deletion of an important phosphotransferase (Sharma et al., 1964) and could be a molecular counterpart of the phenomenon of tumor progression (Weinhouse, 1960). Vinuela et al. (1963) and Sols et al. (1964) also describe a glucokinase enzyme in normal rat liver capable of phosphorylating glucose and mannose and a hexokinase which phosphorylates glucose to its 6-phosphate derivative. The glucokinase was found by Vinuela et al. (1963) to be quantitatively adequate for the first step in the synthesis of glycogen from glucose in liver. The manifestation of very low glucokinase activities of some hepatomas merits additional studies over several generations, beginning with primary tumors, and the use of several different liver carcinogens. Sharma et al. (1964) have made a start in this area by investigating glucoseATP transferases in “preneoplastic” liver of azo dye-fed rats. In the “preneoplastic” liver there was a progressive rise in the low K , hexoltinase to levels 5- to &fold higher than normal, and a &fold lowering of glucokinase, the high K , enzyme, during an ll-week period of 3’-DAB (3’-Methyldimethylaminoazobenzene) ingestion. Sharma et al. (1964) found cholangiocarcinomas induced by 3’-DAB to have no glucokinase but high hexokinase activities, whereas some of the hepatocarcinomas were moderate in both glucokinase and hexokinase activities. Burk et al. (1965) and Woods et al. (1965) reject some of the preceding results because they find that if an adequate intracellular DPN/DPNH2 ratio is maintained in the suspending media a consistent increase is obtained in the initial rates of anaerobic and aerobic glycolysis in rat hepatomas of different growth rate (cf. Table 11;Morris and Wagner, 1965) over that obtained from the host liver or the liver of tumor free animals. The DPN/DPNH, ratio was maintained in their experiments by the addition of suitable oxidants such as DPPI;, TPN, pyruvate, or prior exposure to 02.They report further that the total glycolysis determined manometrically has a largely endogenous glycolytic component and a smaller glucolytic component that is inhibited by 2-deoxyglucose or steroid. Furthermore, Woods et al. (1965) observe a positive correlation between growth rate and glycolysis which in the rapidly growing Hepatomas 3683 and H35TC2 had a Qt:G of approximately 12, the intermediate growth rate Hepatoma 7288C about 6, the slowly growing 5123 and 7794B hepatomas about 4-1, and adult liver 1-0 CmmC02/mg. dry wt./hour. Diethylstilbestrol a t a level of 50 p.p.m. inhibited glucolysis in the rapidly growing hepatomas 20-307,, in the slowly growing hepatomas 50-60Y0, and in adult liver 7Q-100~0, which they interpret as a dominant biochemical difference between a normal metabolism and a malignant hepatoma, and which, according t o Woods et al. (1965), involves a critical increase in ability to phosphorylate glucose. The divergent results obtained by Burk et al. (1965)

246

HAROLD P. MORRIS

and Woods et al. (1965) by manometric techniques contrasted with those found by Aisenberg and Morris (1961, 1963) and require further studies to clarify the fundamental alterations which have occurred in hepatomas with different growth rate. It is confidentially predicted that much new insight into malignant change(s) will be clarified by future work in this area, especially if the metabolites and/or enzymes involved can be precisely identified. 2. Phosphoglucomutase

Phosphoglucomutase, the enzyme responsible for channeling glucose-6-P into the glycogenic pathway, was found to be greatly decreased by Weber et al. (1964) in several transplantable liver tumors possessing different growth rates. Prior incubation in a magnesium-imadazole mixture (Najjar, 1962) showed that a quantitatively similar amount of activation occurred in control livers and in the hepatomas (Fig. 8); this observation, therefore,

FIG.8. Effect of activation on phosphoglucomutase in hepatomas. 7800, slowly growing; 3924A and 3683, rapidly growing. (From Weber et d.,1964.)

ruled out the possibility that low activity in the hepatomas was due to a lack of activation (Weber et al., 1964). In further studies of the kinetic properties of phosphoglucomutase and its affinity to glucose-l-P showed that the K , of the slowly growing Hepatoma 7800 was similar to that of normal liver. In contrast the K , values of two rapidly growing hepatomas, 3683 and 3924A, were higher than the K , of control liver of the ACI/N strain rat. Higher substrate concentrations were required in the rapidly growing tumors to achieve half-saturation of the enzyme. These observations of Weber et al. (1964) indicate only a quantitative difference in the

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

247

synthesis of enzyme protein in the slowly growing hepatoma. In contrast, the iiicreased K , values for the two rapidly growing tumors indicate a decreased affinity for the substrate which likely could mean a n alteration in the enzyme protein. Shonk et al. (1965) also found little phosphoglucomutase in rapidly growing hepatomas. These observations appear to support the view that in this case a progressive lesion is present in hepatomas affecting an early step in the synthesis of the protein. Further experiments will be required to determine if the template production mechanism of this enzyme involves one or more genes. 3. ddalic Dehydrogenase and Malic Enzyme in Hepatomas of Diferent Growth Rate These enzymes contribute to gluconeogenesis through the Krebs cycle. Weber et al. (1964) reported that malic dehydrogenase was decreased 15 to 3oy0 in the slowly growing Tumors 7800 and 5123D, approximately 3Oy0 in medium growth rate Hepatoma 7288C, and by 7Oy0 in rapidly growing Hepatomas 3924A and 3683. Shonk et al. (1965) also found relatively little malic dehydrogenase in either the mitochondria1 or cytoplasmic compartments of the rapidly growing hepatomas. The malic enzyme, on the other hand, in the spectrum of tumors studied by Weber et al. (1964) showed no correlation with hepatoma growth rate. 4. Phosphoenolpyruvate Carboxykinase This enzyme represents a n early step in the processes of gluconeogeneses (Wagle et al., 1963b). In the production of glucose from pyruvate according to Krebs (1954) in normal liver there are three thermodynamic barriers and the pathway of carbohydrate synthesis is made thermodynamically feasible by circumventing these energy barriers a t (1) the reversal of glucose-6-P formation; (2) the reversal of formation of fructose-l,6-diphosphate from fructose-Bphosphate and ATP; (3) the transfer of phosphate from phosphopyruvate to ADP. In cases 1 and 2 this is carried out by insertion in the pathway of the enzymes glucose-6-phosphatase and fructose diphosphatase, while for step 3 a cycle is inserted consisting of pyruvate carboxylase and phosphoenolpyruvate carboxykiriase (Krebs et al., 1964; Utter et al., 1964). Weber et al. (1964) found phosphoenolpyruvate carboxykinase activity in slowly growing tumors (5123D and 7800) to be in the same range or moderately decreased as compared to control values. The enzyme activity in rapidly growing hepatomas (3683 and 3924A), however, had decreased to 670 or less of the activity found in normal control rat livers. There are, therefore, progressive defects in phosphoenolpyruvate carboxykinase ( Weber et al., 1964), glucose-6-phosphatase, and fructose

248

HAROLD P. MORRIS

diphosphatase (Weber et al., 1961b; Weber and Morris, 1963; Weber, 1963b), which account for the progressive failure of gluconeogenesis in this spectrum of hepatomas of increasing growth rate and a t the molecular level constitute a triad of enzymatic lesions a t key steps of gluconeogenesis. 5. Fructose Metabolism The fructose metabolic pathways in transplantable liver tumors of different growth rate have been studied by Ashmore et al. (1963). A definite metabolic alteration in fructose metabolism was found for liver tumors of rapid growth rate, whereas the fructose metabolism of slowly growing hepatomas was similar to that of normal liver. This alteration was noted by a decreased fructose utilization through fructokinase to a preponderance of the hexokinase route of metabolism in the rapidly growing hepatomas. The carbohydrate metabolic features of these rapidly growing tumors, therefore, resemble those of muscle. Shonk et al. (1965) also found the levels of phosphofructokinase generally higher in the rapidly growing hepatomas although there were some exceptions. The phosphofructokinase activity in Hepatoma 7793, i.e., was higher than that found in normal liver and higher than in some rapidly growing hepatomas (3683 and 3924A) but not as high as the activity found in Noviltoff and Dunning hepatomas. Fructose diphosphatase, an enzyme required for gluconeogenesis, was found by Shonk et al. (1965) usually to be decreased in rat hepatomas. This decrease was greater in the rapidly growing hepatomas. 6. The Glycolytic Pathway The activities of the enzymes of the glycolytic pathway including intermediates and end products of anaerobic glycolysis have been studied by Ciaccio and Keller (1962) both in homogenates and intact tissue in situ for several rat hepatomas of different growth rates. The rapidly growing Hepatomas 3683 and 3924A as well as the slower growing 5123 hepatoma had appreciable levels glycerophosphate dehydrogenase and form under aerobic conditions a-glycerophosphate and lactate, but some of the sublines of 5123 hepatomas form less lactate and much more a-glycerophosphate than would be expected from the ratios of the respective dehydrogenase activities. The levels of glycerol phosphate dehydrogenase were also found to be appreciable in many hepatomas studied by Shonk et al. (1965) although still considerably lower than those observed in normal rat liver. Shonk et al. (1965) observed the ratio of lactate dehydrogenase to glycerol phosphate dehydrogenase to be increased in all hepatomas but the extent of the increase was greater in the rapidly growing than in the slowly growing hepatomas. It was thought (Ciaccio and Keller, 1962) that during the anaerobic metabolism of fructose-1,6-diphosphate,supplementation with

249

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

pyruvic acid, pyruvate-kinase, or high levels of ADP would channel the metabolic end products toward lactate. Furthermore, some of the alterations in 5123 hepatomas appeared to be in the metabolic sequence from 1,3diphosphoglyceric acid to pyruvate.

C. ENZYME ACTIVITIES, METABOLIC PATHWAYS, AND GROWTH RATE OF HEPATOMAS Several biochemical parameters have been determined for a spectrum of hepatomas possessing different growth rates by Weber and Morris (1963), TABLE I11 METABOLIC FACTORS A N D ENZYMES RATESOF HEPAIWM.W-~

CORRELATION

O F IN TERM EDI ARY

Correlated wit,h increasing growth rate

Not correlated with growth rate

WITH

GROWTH

Low or high in all hepatomas High

Increased Lactate production

Lacticdehydrogenase

C-l/C-6 oxidation of glucose Fructose into glycogen through hexokinase reaction Incorporation of the amino acids alanine, aspartate, glycine, serine, isoleucine, and valine into protein K , of phosphoglucomutase Pyruvate kinase Phosphofructokinase Glucokinase

Phosphohexoisomerase 6-Phosphogluconatedehydrogenase Malic enzyme Activation of phosphoglucomutase, maximum glucose phosphorylation, oxidation of alanine, aspartate, plyrine, serine, isoleucine, and valine Cellularity

Glucose 6-P-dehydrogenasec

Low Phosphoglucomutase Glycogen Fructose uptake Fructose to CO2 Fatty acid production

Decreased Glucose-6-phosphatase, glycerol dehydrogenase Fructodiphosphate Phosphoenolpyruvate carboxykinase Malic dehydrogenase Pyruvate to glucose Fructose to glycogen through fructokinase reaction Homogenate nitrogen content Supernatant nitrogen content ~~

The results are expressed as definite trends that fit into one of the three classes according to the increasing growth rate of the hepatomas. * From data of Wr-ber et a2. (1964) and Shonk et al. (1965). c Hepatoma 5123D was an exception where glucose 6-P-dehydrogenase was in the normal range. a

250

HAROLD P. MORRIS

Sweeney et al. (1963), Wagle et al. (1963b), Weber et al. (1964), and Shonk et al. (1965). As a result of these extensive studies the tumors have been grouped into three classes according to their growth rate. Growth rate as used here represents the time between transplantations (cf. Table 11). A summary of the results of these enzyme studies is presented in Table 111. These include (1) parameters correlated with growth rate either higher or lower; (2) those not correlated with growth rate; and (3) those low or high in all hepatomas so far studied. Many more hepatomas need to be examined before very broad conclusions can be made. The summary, however, provides a n opportunity to examine the phenomenon of liver neoplasia in the rat in terms of metabolic lesions differing in extent, a condition that may be comparable to the primary disease in man whose symptoms also show variable degrees of intensity of the neoplastic process (Weber et al., 1964, Boxer, G. E., 1964). VI. Protein and Amino Acid Metabolism

A. ENZYMES OF AMINOACID METABOLISM Assay values for several enzymes concerned in amino acid metabolism (Pitot et al., 1963) in a series of recently developed transplantable hepatomas (Morris and Wagner, 1965) are given in Table IV. It is apparent from these data that many rat hepatomas possess a number of enzymes of amino acid synthesis and degradation which are among normal tissues found almost exclusively in the rat liver. The levels of these enzymes vary greatly from hepatoma to hepatoma; when compared to normal liver some of the enzymes are several-fold higher, e.g., threonine dehydrase in Hepatomas 5123 and 7793. Proline oxidase is much lower in the hepatomas examined than in host or normal livers, but more than a 4-fold difference was found between the five tumors examined. Proline-5-carboxylate reductase was higher in Hepatoma 5123, lower in Hepatoma 7316A, and about the same level as host liver in the three other tumors assayed. Tyrosine a-ketoglutarate transaminase was equivalent to or as much as lo-fold higher in the tumor than in the host liver, while histidase was much lower in the hepatoma than in the liver; no consistent difference was found in histidase between tumors in male or female hosts. Although a hepatoma with enzymes concerned in amino acid metabolism represents a second primary site for amino acid synthesis and degradation, compared to liver this site in the tumor is unresponsive to stimuli, such as variations in dietary protein, which are effective stimuli in normal liver. If competition exists for host nitrogen between the host liver and the tumor, as has been suggested by Mider (1951) and LePage et al. (1952), it appears not to result from the demand of a rapidly growing cell population,

TABLE IV ENZYMES OF AMINOACID Tryptophan pyrrolaseb

Threonine dehydrasec

Serine dehydrased

MET.4BOLISMa

Proline oxidased

Proline-5carboxylate reductasee

Tyrosine 01ketoglutarate transaminasef

8<

Histidaseo

3!0

-zEm hj

Tumor H 35 7800 7793 7794A 7795 7777 5123 5123H2 7316~2 Normal liver

Host liver

Tumor

Host liver

Tumor

31 2 20 32

-

0.4 0.3 1.4 0.2 0.3 -

2.1

0.15

14 4

0 29 295 2 44 5 500 656 32

2.5

-

39

-

2.4 2.2 2.7 2.5 2.6

3 51

Pitot et al. (1963). b pmoles kyniirenine/honr/g. tissue. c pmoles a-keto acid/hoiir/g. tissue. d pmoles Oz/hoiir/g. tissue. pmoles DPNH2/hour/g. tissue. f pmoles p-hydroxphenylpyruvate/hour/g. 0 pmoles urocanate/hour/g. tissue.

Host liver Turnor

17 1.5 7 -

45

-

Host, liver Tumor

Host liver Tumor

174 177 177

150 140 138

I1 14 35

Host liver Tumor

Host liver

168 158 124

30 39 29

334 212 275

7.6 -

51 234 77

18.9 17.9 9.1 17.9

-

24 76.8

165 180

9 51

150 163

567 57

27 104 77

206

-

181

-

-

-

-

Tumor 2.5

-

3.7 3.3 1.3 1.5

---

0 . .

5 cj $

8

F

Y

0 kJ

z

tissue. t3

3

252

HAROLD

P.

MORRIS

but may be a consequence of the abnormal levels and stability of the enzymes of the tumor cells. This idea seems to be supported by the lowered plasma threonine in rats bearing hepatomas which have a very high threonine dehydrase activity. Hepatomas 7793 and 5123, the two hepatomas possessing very high levels of threonine dehydrase, were induced in female rats, but Hepatoma 7777 with a low threonine dehydrase level was also induced in a female. Hepatomas 7794A, 7795, and 7800 were induced in male rats; two of these are higher and one is lower in threonine dehydrase than host liver. All six tumors were induced by the same chemical, 2-FPA. Although Hepatomas 7800 and 7795 had about the same threonine dehydrase activity as normal liver, the activity of the enzyme in the host liver was greatly depressed. It is quite evident from the data of Table I V that in hepatomas several enzymes concerned in amino acid metabolism have patterns that are different in one or another aspect from normal liver enzyme patterns and show no indication of being in agreement with the idea of biochemical uniformity of tumors, as suggested by Greenstein (1956).

B. AMINOACID INCORPORATION AND OXIDATION Another aspect of protein metabolism has been explored by Wagle et al. (1963a) in normal liver, regenerating liver, and four hepatomas. The transplantable rat liver tumors possessed different rates of growth. These experiments measured the protein incorporation using six amino acids labeled with C14. Some of the data expressed in Table V as the percentage of incorporation compared t o the respective control show that regenerating liver and the TABLE V AMINOACID INCORPORATION INTO PROTEIN IN NORMAL AND NEOPLASTIC LIVER"-* Amino acid Tissue control

Alanine (100)

Aspartate (100)

Regenerating liver Increasing growth rate H 35 7285C 3924A 3683

16%

154O

135"

12gC

125"

126~

107 155c 494c 4530

95 151c 250" 21SC

97 123~ 291~ 215"

80 127" 383c 174.:

92 130 300~ 3Olc

85 1400 34F 302~

a

b

Glycine Serine Isoleucine Valine (100) (100) (100) (100)

From Wagle et al. (1963a). Expressed as per cent of respective normal control. P = <0.05 of respective normal control.

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

253

faster growing Hepatomas 7288C, 39248, and 3683 incorporated into protein significantly more of each of the six amino acids studied than was incorporated by the respective control tissue. Although regenerating liver, which has a growth rate approximately equal to the fastest growing hepatoma, incorporated significantly more of these amino acids into protein than normal liver it was much less than the incorporation shown by the rapidly growing Hepatomas 39248 and 3683. The highest incorporation rate was for alanine and this may indicate a preferential use of this amino acid (Wagle et al., 1963). The well-differentiated, slowly growing H35 hepatoma had incorporation rates within the normal range. One explanation for inability to show even a slight increase in incorporation rate could be the presence of a few necrotic and hemorrhagic areas in Hepatoma H35. It was suggested that the increased utilization of alanine and aspartate, which are gluconeogenic amino acids, may be connected with the striking decrease or absence of gluconeogeriic enzymes (Weber, 1963c; Weber et al., 1964; Wcher and Morris, 1962, 1963) and pyruvate to glucose production in these hepatomas (Sweeney et al., 1963; Weber et al., 1961b; Wagle et al., 1963b) . Tlie difl'erences between incorporation and oxidation of amino acids gives a measure of net change in the hepatoma. The two most rapidly growing hepatomas, 3924A and 3683, showed decreased oxidation of alanine and aspartate, no change in the oxidation of glycine and serine, but a marked increase in oxidation of isoleucine and valine (Wagle et al., 1963a). One explanation for the increased oxidation of these two amino acids was the possibility of their entering into fatty acid formation (Wagle et al., 1963a). These studies on amino acid incorporation and oxidation indicate a rough correlation with growth rate of this spectrum of liver tumors, but additional studies must be done to give a clearer picture of how these observations are related to tumor growth. Ciaranfi and Fonnesu (1962) found L-glyceraldehyde depressed the incorporation of amino acids into protein of the Yoshida ascites hepatoma, a rapidly growing neoplasm, and the D-isomer was less effective than the L-isomer (Guidotti et al., 1964) in inhibiting the incorporation of DL-leu~ine-1-C'~ into the protein of Yoshida AH 130 ascites hepatoma cells and in normal liver cells. The specific mechanism by which glyceraldehyde inhibited incorporation was unexplained, although it was noted that under anaerobic conditions L-glyceraldehyde markedly inhibited glycolysis as well as amino acid incorporation into protein ; but D-glyceraldehyde and 3-methylglyceraldehyde inhibited incorporation more effectively than glycolysis. Some of the inhibition of incorporation of amino acids into proteins was counteracted by glucose under aerobic conditions. Guidotti et al. (1964)

254

HAROLD P. MORRIS

suggested that the glyceraldehyde inhibition of glycolysis and amino acid incorporation into protein were accomplished by two different mechanisms. Since the Yoshida AH 130 ascites hepatoma is a rapidly growing hepatoma, c,omparisons of the possible inhibitory effects of glyceraldehyde in hepatomas of slower growth rate and in those that do not incorporate amino acids into proteins under anaerobic conditions (cf. Aisenberg and Morris, 1963) should yield useful information on the mechanism of this inhibition.

C. LYSINEMETABOLISM The intermediary metabolites of another amino acid, L-1y~ine-U-C~~ have been studied in normal liver, regenerating liver, Hepatoma 5123C, and in Wallter 256 carcinoma by Davis and Morris (1963). They noted that pipecolic acid accumulated to a greater extent in these two tumors of quite different origin than was found in normal liver or in regenerating liver. I n addition to pipecolic acid, as determined by radioactivity eluted from chromatographic columns, Hepatoma 5123C was found to be quantitatively higher than normal or regenerating liver for glutamate, aspartate, glutarate, and lysine, lower than regenerating liver for a-aminoadipate, but no different from either liver or regenerating liver for succinate. Davis and Morris (1963) noted that in one of the five unidentified peaks on their column, a much higher radioactivity occurred in normal liver than in regenerating liver. The determination of pipecolic acid in other hepatomas of different growth rate would be desirable to see if pepicolic acid accumulation is characteristic of minimal deviation hepatomas or to see how this metabolite varies in the different minimal deviation transplantable hepatomas. D. HEPATOMA PROTEINS Slices of normal rat liver incubated in vitro in the presence of ethylenediaminetetraacetic acid (EDTA) or citrate, agents which are thought to remove calcium, lose more of their protein into the incubation medium than do slices perfused with Ringer’s solution alone, according to Kalant and Miyata (1963). These effects do not result from breakage of cell membranes but have been taken as evidence of the role of calcium in the maintenance of normal selective permeability (Leeson and Kalant, 1961). EDTA or citrate should not affect, the permeability of malignant cells the same way as normal cells if calcium does not play a similar role in the malignant cell (Kalant et al., 1964). Apparently this effect of EDTA results from its chelation of calcium, as noted by Kaufmann and Wertheimer (1957). Kalant et al. (1964) suggest that EDTA may hasten the solubilization of an unidentified ‘lbasic protein 4” from an aggregated form in which it is held

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

255

intracellularly by a divalent cation. These investigators found little if any effect of EDTA on protein leakage from primary DAB-induced (dimethylaminoazobenzene) rat liver tumors, nor did the presence of EDTA in the medium alter the electrophoretic pattern of the proteins which leaked from the tumor slices. Kalant et al. (1964) distinguished a clear difference between DAB-induced primary hepatoma cells and normal liver cells with respect t o the compliment of intracellular proteins. Extending the EDTA effect to transplantable Hepatoma 5123TC Murray et al. (1964) reported a 51% increase in protein leakage in presence of EDTA in the normal Buffalo-strain rat liver and a 33% leakage in 5123TC hepatoma slices, indicating that with respect to protein leakage in the presence of EDTA the 5123TC hepatoma was more like normal liver than were the primary hepatomas induced by DAB. Furthermore, from starch gel electrophoresis of the sera of Hepatoma 5123TC tumor-bearing rats, Murray et al. (1964) showed (1) a decrease in the a-globulin zone, (2) an abnormal zone at the albumin front, (3) decrease in choline esterase and aromatic esterase, and (4) a n increase in esterase migrating with albumin. The soluble cytoplasmic protein from Hepatoma 5123TC, according to Kalant et al. (1964), showed (1) slight reduction in basic proteins which were markedly reduced or absent in primary DBA hepatomas, (2) a consistent slight retardation in tumor albumin, (3) a striking decrease in fast-moving esterases, and (4) relatively specific electrophoretic changes in tumor and serum proteins. These interesting findings merit further studies t o determine the relation of these basic proteins t o the slow-moving h-protein fraction of Sorof et al. (1958) and Sorof et al. (1963). Sorof et al. (1065) examined Hepatomas 5123C, 7793, 7787 for h proteins and their ability to form hz protein-conjugates of the fluorenyl carcinogen. The object was to determine if inability to form hz fluorenyl proteins could be associated with any key alteration in the induction of hepatomas closely resembling normal liver. These hepatomas had amounts of h proteins equivalent to or only slightly less than that found for host liver. The 2-FAA carcinogen metabolites were bound principally to proteins of the A component after column electrophoresis, the A fluorenylproteins have mobilities close to that of serum albumin. Sorof et al. (1965) observed several species of fluorenyl proteins and of the h protein classes present. Hepatoma 7787 was most like host liver, 5123 had less h and 7793 still less. All three hepatomas lack the ability to form more than trace amounts of h2 fluorenyl proteins. Further study will be required to determine whether this inability t o bind fluorenyl proteins is a secondary deletion or if the loss may have been involved in the original chemical induction of these hepatomas.

256

HAROLD P. MORRIS

VII. Other Inducible Enzymes

A. TRYPTOPHAN PYRROLASE Induction of tryptophan pyrrolase (TP) activity of livers of intact tumor-bearing rats and 14 “minimal deviation” transplantable hepatomas was reported by Dyer et al. (1964), using homogenates and the kinetic method of Greengard and Feigelson (1961) with and without the intraperitoneal administration of L-tryptophan as the inducer. The livers of rats bearing several different lines of transplantable hepatomas always showed greater tryptophan pyrrolase after induction, although there were variations among the individual animals in the absolute amounts of activity both before and after substrate induction. The livers of tumor-bearing rats showed lower absolute amounts and greater variations in tryptophan pyrrolase activity than did the livers of tumor-free control animals (Dyer et al., 1964). The possibility that insufficient inducer, L-tryptophan, reached the hepatoma was apparently ruled out by inducing the enzyme in Hepatoma H35 (Reuber, 1961) growing in the “tissue-isolated” kidney (Gullino and Grantham, 1961) and introducing L-tryptophan directly into the kidney artery. No greater activity was found in either the tumor or the host liver by this procedure than occurred following intraperitoneal administration of L-tryptophan. Pitot and Morris (1961) also showed that adequate inducer was available to the tumor so that the failure of response of the hepatoma t o L-tryptophan was not due to a lack of portal blood supply to the hepatoma (Cho et al., 1964). Dyer et al. (1964) separated the 14 hepatomas into three groups which responded differently to induction of tryptophan pyrrolase activity in the intact host. Figure 9, group 1, included those hepatomas with slight activity, in which there was no progressive increase with increasing time of incubation, and in which an erratic response often occurred in the tumor after L-tryptophan administration to the host. It was questionable whether this group of hepatomas, comprising half the entire group, possessed any tryptophan pyrrolase activity. Group 2 included a few hepatomas containing small amounts of activity that increased during incubation, and was significantly greater after L-tryptophan administration to the host. Group 3 included hepatomas with more activity than those of group 2; after induction the activity was as much as host liver (cf. Fig. 9). There were no recognizable morphologically significant differences in the tumors since all consisted mostly of liver (parenchymal) cells, usually without cholangiomatous areas (Miyaji, 1965). The absence or near absence of tryptophan pyrrolase in the tumor cells, however, does not interfere with the in vivo

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FIG.9. Average values for tryptophan pyrrolase (TP). Activities a t 0 time were substracted from activities a t 1 hour before averaging. Both sexes of tumor-bearing rats were used unless sex is specified. (From Dyer et al., 1964) (cf. Morris el al., 1964.)

growth of the tumors. Individual tumor lines were consistent in their behavior regarding tryptophan pyrrolase activity, a n observation that supports the view that the activity of this enzyme was inherent in individual hepatoma cells within any given tumor line. The absence or presence of activity in the tumor, therefore, appeared to be due to some intrinsic defect within the neoplastic cell. Pitot and Morris (1961) and Cho et al. (1964) also concluded that in intact hosts a few hepatomas were capable of tryptophan pyrrolase induction. Cho et al. (1964) and Dyer et al. (1964) were in agreement that in the majority of hepatomas examined the enzyme was at very low levels; according to Cho et al. (1964), most were not significantly responsive to any of the controls (hormonal or substrate) that are known to influence the synthesis of this enzyme (Feigelson and Greengard, 1962). Cho et al. (1964) also found that in adrenalectomized hosts the induction of tryptophan pyrrolase by L-tryptophan was lost in several hepatomas

258

HAROLD P. MORRIS

but not in the host liver. The administration of cortisone to the adrenalectomized host, however, increased in several hepatomas the responsiveness of their pyrrolase-forming system to corticosteroid hormones (Fig. 10). ADRX

10 0

00 cn

60

= 2

4.0

c

5?

e

20

a

n

0.4 02 Control

+ Try

t Try

+ 0.3 mg. CORT

FIG.10. Effect of cortisone on tryptophan pyrrolase (TP) induction in host liver and in Hepatoma H35 in adrenalectomized rats. Tryptophan pyrrolase units = pmoles kynurenine/hour/g. tissue; cortisone dosage given daily for 7 days after adrenalectomy. Each bar represents a single animal. Try = tryptophan; cort = cortisone acetate. (From Cho et aZ., 1964.)

The failure of substrate induction as noted by Cho et al. (1964) in adrenalectomized hosts bearing Hepatomas H35 and 7793, tumors possessing significant activity that responded in the intact host to substrate induction, is in contrast t o substrate induction in the liver of nontumor-bearing adrenalectomized rats as noted by Lee and Baltz (1962) and suggests that these hepatornas are completely dependent on the presence of corticosteroids for substrate induction of tryptophan pyrrolase activity. Cho and Pitot (1963) and Cho et al. (1964) tested the possibility that the structure of the enzyme in the hepatoma was different from that of the host liver by partially purifying the enzyme in liver and in Hepatoma 7793. No significant difference in the partially purified enzyme between liver and hepatoma enzyme was noted, either in certain of the kinetic constants or in the electrophoretic mobilities. Partially purified tryptophan pyrrolase from Hepatoma H35 also gave similar K,,, values to that of the liver enzyme. Experiments of Cho et al. (1964) seemed to exclude the possibility that

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these hepatomas are under exclusive hormonal control by showing that in the intact rat L-tryptophan did elicit the enzyme in Hepatoma H35, when the amino acids methionine or phenylalanine failed t o do so, although the latter two amino acids do induce the enzyme tyrosine transaminase in the intact rat, probably indirectly by adrenal stimulation (Kenney and Flora, 1961). Still another possibility was proposed by Cho et al. (1964) to explain pure substrate induction of TP in adrenalectomized hosts but not in the hepatomas (Fig. 10) by suggesting that the hepatomas have lost the capacity to maintain a stable RNA template for tryptophan pyrrolase synthesis. According to this idea the specific substrate induction of the enzyme in the hepatoma could be restored only if there is a renewal of RNA template by corticosteroid stimulation. Nemeth (1962) also suggested that tryptophan pyrrolase synthesis from amino acids or from preexisting protein required the simultaneous synthesis of mRNA. The inability of L-tryptophan t o induce activity in Hepatoma H35 in the adrenalectomized host, as illustrated above, gives support to this suggestion. Additional tests for synthesis of other enzymes in minimal deviation hepatomas could conceivably be used to test the stability of an RNA template, to give a more precise understanding of the essential characteristic(s) involved in the conversion of one normal cell type to its malignant counterpart (Cho et al., 1964).

B. ETHIONINE FATTY HEPATOMAS The dependence of adrenal cortical hormone for induction of tryptophan pyrrolase in hepatomas as described by Cho et al. (1964) raised the question of hormonal influence on other biochemical constituents in hepatomas. One of these has been studied by Miyaji (cited in Morris et al., 1964), who has investigated the lipid content of host liver and several transplantable hepatomas growing in female rats after intraperitoneal injections of ethionine. It will be noted from the data in Table VI that all host livers responded t o ethionine injection by a 2- to &fold increase in ether-extractable lipid, whereas only two of the five hepatomas listed responded. Hepatomas H35 and 731GA had a 2-fold increase in lipid following ethionine injections. This response was proportionately somewhat less than the response of the host liver t o ethionine injections. Female rats bearing Hepatoma 7316B injected with ethionine (not included in the data in Table VI) did not show an increase in lipid in the hepatoma following ethionine injections; yet this hepatoma arose in the same liver under the same treatment that induced Hepatoma 7316A. Hepatoma 7793 showed the greatest response in the intact animal to tryptophan pyrrolase induction but was unresponsive to increase in lipid

EFFECT O F ETHIONINEI N J E C T I O N S

ON THE

TABLE V I ETFIER-EXTRACTABLE LIPID CONTENT

OF

LIVER A N D TUMOR^

Liver

Tumor

Tumor lines

Generation used

Control lipid (%I

Ethionine-injected lipid (%I

Control lipid (5%)

E thionine-in jected lipid (%I

Ethionine-induced hepatomab H 35" 5123Dd 7316Ae 7793d

17 11 33 7 5

7.8 f 1 . 9 (3) 5 . 0 f 0 . 4 (3) 7 . 9 0 . 4 (3) 6 . 8 f 0 . 3 (3) 8 . 3 f 1 . 0 (3)

26.7 zf: 0 . 3 (3) 16.5 f 1 . 9 (4) 18.9 f 1 . 8 (3) 16.3 1 . 2 (3) 18.4 f 1 . 4 (4)

4 . 3 f 0 . 1 (3) 2 . 5 f 0 . 1 (3) 2 . 2 i-0 . 5 (3)

3 . 4 k 0 . 2 (3) 5 . 5 2 0 . 1 (4) 2 . 0 f 0 . 4 (3)

2.5 k 0.3 (3) 4 . 6 0 . 1 (3)

4 . 6 f 0 . 2 (3) 4 . 1 f 0 . 4 (4)

From iMiyaji (cited Morris et al., 1964). Ethionine-induced hepatoma (Sidransky, 1962). c FdiAA-induced hepatoma (see Table I). d 2FPAinduced hepatoma (see Table I). 2,4,6TMA-induced hepatoma (see Table I). Figures in parentheses indicate number of rats. a

*

*

+

F r c1

?

5 ti

s

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following ethionine injections. The only two hepatomas, of many examined, that were responsive to increased ether-extractable lipid after ethionine injections in Miyaji’s experiments were reported by Dyer et al. (1964) t o have small amounts of tryptophan pyrrolase; small amounts of activity could be induced by tryptophan, although Cho et al. (1964) have shown that the induction of tryptophan pyrrolase activity in hepatomas is dependent on the intact adrenal or the presence of adrenal cortical hormone. The effect of ethionine-induced fatty livers in fasted female rats (Farber et al., 1950; Jensen et al., 1951; Farber and Segoloff, 1955) may arise from the effect of pituitary hormones, the influence of ACTH on fatty acid mobilization, or the effect of growth hormone on protein metabolism. Future studies of this interesting phenomenon might well include studies of the role of these hormones on the development of fatty hepatomas after ethioriine injections in some and the lack of response of other hepatomas. Why do some hepatomas develop increased lipid after ethionine injections? Why do all hepatomas continue to synthesize cholesterol on a high dietary cholesterol (cf. Section IX,D)? What significance do these differences in response have to do (if anything) with development of neoplasia? These queries remain for future studies to elucidate.

C. L-ASPARTIC AMINOTRANSFERASE L-Aspartic amino transferase may exert an important alternate pathway control on the flow of aspartate into nucleic acid metabolism via aspartic carbamyl transferase to carbamyl aspartate (cf. Fig. 12, frame I). Two forms of L-aspartic amino transferase are present in liver, one primarily in the mitochondria and the other in the supernatant fraction [Moore and Lee (1960), Boyd (1961), Hook (1962), Hook and Vestling (1962), Devlin and Boxer (1963), Morino et al. (1963), Sheid and Roth (1965)]. Further studies by Boyd (1961), Hook (1962), and Hook and Vestling (1962) showed that by starch electrophoresis two enzymatic peaks of aspartic aminotransferase were present in normal rat liver. One of these, the cationic, corresponded to the mitochondria1 fraction, and the other, the anionic, t o the supernatant fraction. Dyer et al. (1960) found that whole homogenates of Hepatoma 5123 had an activity level approximately 2.5 times the level of normal liver. The question arose as t o whether this increase of activity in Hepatoma 5123 was due to one or the other of these two isozymes. If both were present in hepatomas did they behave similarly to normal liver isozymes in the electrophoretic field, and was there a relative quantitative difference in the isozymic peaks compared to normal liver? Five hepatomas with different growth rate have been studied by Otani and Morris (1964, 1965), namely, 3683, rapidly growing; 7288C with an

262

HAROLD P. MORRIS

intermediate growth rate; and slowly growing 5123A and B sublines, and 7316B. The specific activities (transaminase units per milligram of protein nitrogen per minute) of the aqueous extracts of normal liver, host liver, and the five hepatomas were determined. The data in Table VII indicate TABLE VII ASPARTIC AMINOTRANSFERASE ACTIVITIES OF NORMAL LIVER,HOSTLIVER,A N D SOME TRANSPLANTABLE HEPATOMA TISSUE" Specific activity Normal liver 21,530 16,260 20,790

Normal Buffalo-strain liver (M) Normal Buffalo-strain liver (F) Normal ACI/N-strain liver (F)

51238 (M) 7316 (M) 7288C (M) 7288C (F) 5123B (M) 3683 (F) 5

Hepatoma

Host liver

21,020 35,380 11,510 12,570 32,430 3,900

21,340 18,870 16,560 18,360 15,420 20,080

From Otani and Morris (1965). = male, F = female.

M

roughly that specific activity, except for 5123B, is inversely related to growth rate in this series of hepatomas. Normal liver, host liver, and all five hepatomas had two isozymic peaks. The relative distribution of the isozymes in normal and host liver was similar but was unlike the tumors. The ratio of soluble to mitochondrial isozyme was always larger in the neoplastic tissues than in normal or host liver; this is illustrated in Table VIII. Sheid and Roth (1965) have also noted about 70% of the total aspartic amino transferase activity in the soluble fraction of Hepatoma 5123, whereas about 2/3 of the total activity of normal liver was in the mitochondrial fraction. Devlin and Boxer (1963) have determined the aspartic amino transferase activity in the mitochondrial fraction and a cytoplasmic fraction of a series of 11 transplantable rat hepatomas. When compared to the activity of normal liver they found a very wide variation; some tumors, namely, 7288C and 7794A, were very similar to normal liver in their distribution of activity. Other hepatomas such as 5123 and 7793 had severalfold more activity in the cytoplasmic fraction than normal liver. The aspartic amino transferase activity in hepatomas so far examined, like

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TABLE VIII RATIOOF SOLUBLE TO h'hTOCHONDRIAL ASP.4RTIC AMINOTRANSFER.4SE h C T I V I T I E 5 I N CERTAINHEPATOMAS ARRANGEDIN ORDEROF INCREASING GROWTHRATE^ -

a

Tissue

Soluble/mitochondrial

Normal liver Host liver Hepatoma 7316B Hepatoma 5123'4 Hepatoma 51233 Hepatoma 7288C Hepatoma 3683

0.35 0.25 1.00 3.34 1.13 0.49 1.63

Prom Otani and Morris (1965).

many other parameters studied by others, has not revealed any two identical hepatomas. Further studies of the aspartic amino transferase of the subcellular fractions of rat liver and hepatomas by Sheid and Roth (1965) showed that the activity of Hepatomas H35 and 7787 resembled normal liver, whereas in other hepatomas the supernatant fraction increased and the mitochondrial fraction activity decreased with decreasing rate of growth of the hepatoma. The decreasing aspartic amino transferase activity in the mitochondria may be due in part to the decreasing number of mitochondria in the more rapidly growing tumors, although in electrophoretic studies Sheid and Roth (1965) showed that the increased enzyme activity in the supernatant fraction was not from leakage of mitochondria enzymes but was due t o increase in activity of the enzyme normally present in the supernatant fraction. I n other hepatomas the supernatant fraction aspartic amino transferase activity of normal male rats was increased some 300% following 6 days of subcutaneous administration of cortisone (Sheid and Roth 1965) but there was only IGOj, increase in activity in the mitochondria1 fraction. About half as much induction occurred in female as in male rats. L-Aspartate and ACTH also induced aspartic amino transferase activity but not thyroxine, growth hormone, estrogen, or testosterone in any subcellular fraction. Actinomycin D prevented induction of aspartic amino transferase activity by aspartate but had no effect on cortisone induction of activity. Puromycin, however, abolished induction of aspartic amino transferase activity by aspartate as well as most of the cortisone activity. Sheid and Roth (1965) were unable t o induce activity by cortisone or aspartate in the Novikoff, Dunning, 5123, or embryonic liver but both agents were effective in inducing activitjy in regenerating liver. These studies of Sheid and Roth (1965) again show a striking inability of a number of hepatomas to respond to

264

HAROLD P. MORRIS

induction of aspartic amino transferase activity by agents effective in normal or regenerating liver. VIII. Nucleic Acid Metabolism

A. DEOXYRIBONUCLEIC ACIDNUCLEOTIDYL TRANSFERASE In order to study DNA synthesis in malignant mammalian cells Matsavinos and Munson (1965) have partially purified DNA nucleotydyl transferase (E.C. 2,7.7.7) isolated from Hepatoma 5123TC (a subline obtained from 5123C that was grown for several months in tissue culture at McArdle Memorial Laboratory and then returned to the rat strain of origin (cf. Section X,C; Pitot et al., 1964). Its growth rate is noticeably more rapid than the other 5123 sublines (cf. Table 11). Some of the general properties of this enzyme indicate that maximal incorporation of [H3]dTTPinto DNA was attained in the presence of Mg++, an exogenous source of DNA, and the deoxyriboriucleoside5' triphosphates of adenine, cytosine, and guanine. Ca++ or Mn* were ineffective in replacing Mg++. Native double-stranded DNAs are more active primers than heat denatured or single-stranded DNAs while tobacco mosaic virus RNA is practically ineffective as primer. The double-stranded DNA primer was more active than single-stranded primer. The reaction is dependent on DNA because pancreatic DNase, but not pancreatic RNase, inhibits the reaction. Maximum rate of incorporation of [H3]dTTPwas dependent on the presence of the other three triphosphates-dATP, dCTP, and dGTP. Omission of dATP decreased the amount of incorporation by a factor of 7, whereas omission of all three triphosphates reduced incorporation by a factor of 20 suggesting that all four triphosphates participated in the reaction. No stimulation to incorporation occurred by substituting the complimentary triphosphates by the mono- or diphosphates. The basic requirements for the incorporation of deoxyribonucleotidesinto DNA by the 5123TC hepatoma enzyme is essentially similar to DNA nucleotidyl transferase described by Bollum (1960) for mammalian tissue and Bessman et al. (1958) for bacteria. It will be of interest to compare the DNA nucleotidyl transferases isolated from hepatomas of different growth rate to see if the basic requirements for the incorporation of deoxynucleotides into DNA in several hepatomas differ from normal mammalian tissues and from each other. This comparison would seem especially important in connection with the studies of Wheeler et aE. (1964), who report that the quantities of radioactive nucleotides are greatest for the rapidly growing tumors and progressively less for the slowly growing tumors; further, anabolic activity and catabolic activity should be evaluated simultaneously because Wheeler et al. (1964) have found that the quantities of radioactive catabolic products

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were lowest for the rapidly growing tumors and progressively greater for the slowly growing tumors. It is fully recognized, however, that the extracts of minces and sonically disrupted tissues used by Wheeler et al. (1964) contain many more enzyme systems than does the relatively pure system studied by Matsavinos and Munson (1965). Therefore, there seems to be little basis for comparison of the results of the two series of investigations at this time.

B. RIBONUCLEASE ACTIVITY The availability of hepatomas of different growth rate offered a means to attempt further clarification of the complexities of the RNase system in mammalian tissues, especially liver and hepatoma, and the relationship of the enzyme activity to cell proliferation and RNA metabolism, which is not well understood (Roth, 1963b). With improvement in assessment of enzyme and inhibitor activity Roth et al. (1964) resurveyed the RNase of normal rat liver and several transplantable rat hepatomas of different growth rate (Morris and Wagner, 1965) including the rapidly growing Novikoff and Dunning hepatomas and the slowly growing Reuber hepatoma H35 (Reuber, 1961). TABLE IX RIBONUCLEASE INHIBITOR AND LATENTRIBONUCLEASE ACTIVITY AS PER CENT CHANGE FROM NORMAL LIVER'

Novikoff Host liver Dunning (9. c . ) I. P. Host liver 7288C I. P. (8. c.) Host liver H 35 (9. c. and I. P.) Host liver 5123C 8.c. Host liver 5123D (s. c. and I. P.) Host liver 7800 (8. c.) Host liver

RNase inhibitor artivity

Latent6 RNase activity

-34 - 14 48 +240 f29 +241 +123 - 14 - 66 - 19 -69 -5 -51 -4 -80 -26

+43 39 170 47 13 32 -31 +I1 144 $146

+

+ + + + +

+

0

+68 +38 +54

From Roth et al. (1964). Inactive RNase. Normal liver for: Novikoff = Holtzman; Dunning = Fimher strain; H35 = ACI/N; strain; 7288C, 5123D, and 7800 = Buffalo-strain, and 5123C = LBF, hybrid, L = Lewis, B = Buffalo. b

266

HAROLD P. MORRIS

The change from normal liver of RNase inhibitor activity for several transplantable hepatomas, arranged according to decreasing growth rate as shown in Table IX (Roth et al., 1964), indicates decreased activity compared with normal liver except for Dunning and 7288C hepatomas where the RNase inhibitor activity was much elevated. The largest increase in inhibitor activity of these two hepatomas was found in tumors growing intraperitoneally. No correlations could be discerned between RNase inhibitor activity and growth rate of the hepatomas. Table IX also gives the change in latent or inactive RNase in the same group of hepatomas. There appears t o be an increase or no change in this latent RNase activity over that of normal liver in all the hepatomas except H35 where there was a decrease. I n the Dunning and 5123C hepatomas there was a considerable increase, although no clear explanation for these differences is available. It was suggested by Roth et al. (1964) that the RNase inhibitor may be related to the turnover of some important RNA component such as messenger, transfer, or ribosomal RNA.

C. RIBONUCLEASE IN SUBCELLULAR FRACTIONS 1. Mitochondria1 Fraction

The mitochondrial distribution of acid and alkaline RNase and the specific activity of these enzymes in the subcellular fractions of transplanted hepatomas as described by Roth et al. (1964) show that the percentage of acid and alkaline RNase activity of the mitochondrial fraction is greatly lowered. Since the specific activity of these enzymes in the mitochondrial fraction was based on N, Roth et al. (1964) found in those tumors in which the mitochondrial N was not greatly changed the specific RNase was significantly less than controls; where mitochondrial N was decreased to very low values the specific enzyme activity was usually considerably elevated over control values. 2. Nuclear Fraction The percentage of acid and alkaline RNase in the nuclear fraction was either unchanged or elevated. The percentage of N recovered in this fraction was considerably increased (Roth et al., 1964) probably because of an increased amount of unbroken or partially broken cells in the tumor nuclear fractions.

3. Microsomal Fraction A large precentage increase as well as a large increase in the specific enzyme activity of these RNase enzymes was found by Roth et al. (1964) in the microsomal fraction. The studies of Webb et al. (1964) show a signifi-

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267

cantly higher proportion of the lighter polyribosomes in hepatomas and a lower proportion of the heavier polyribosomes (cf. Section X,D). One hepatoma (7794A) studied by Webb et al. (1964) was least like liver in having very few of the heavy polyribosomes, but according to Webb et al. (1964) this was not the result of a higher nuclease activity. Further studies to determine whether there is a correlation between RNase activity and the capacity of the ribosomes to incorporate amino acids into protein would be valuable. 4. Supernatant Fraction Acid RNase is strikingly increased compared to liver in the supernatant fraction of all the hepatomas examined by Roth et al. (1964) except Noviltoff hepatoma, which was only slightly increased. Such observations are in agreement with reports by Reid (1962) and Roth (1963b). The increase was usually accompanied by an increase in N in the rapidly growing tumors but little change was noted in the N of slowly growing tumors (Roth et al. 1964). One explanation suggested by Roth et al. (1964) for this increase was a possible transfer of particulate-bound enzyme to the soluble portion of the cell. It appears, contrary to the suggestion of Daoust and Amano (1962), that the cancer cell does indeed contain RNase, and one of the problems is to learn more about these RNase’s and their function in both cancer and normal cells. D. OTHERMICROSOMAL FRACTION ENZYMES Even though the ultrastructural studies of Dalton (1964), and Esser and Noviltoff (1962) of the “minimal deviation” Hepatomas 5123 and H35 indicated that these hepatomas had retained many of the features characteristic of normal hepatic cells, it was important to study some of the biodiemical and enzymatic parameters in the microsomal fraction of normal, regenerating, and embryonal rat liver. Such studies have been carried out by Sugimura et al. (1965) on six hepatomas: two rapidly growing Yoshida ascites hepatomas, and four slowly growing Morris hepatomas. The microsomal fraction consists mainly of ribosomes and endoplasmic reticulum. Those reticulum structures which are not associated with ribosomes are called smooth-surfaced reticulum (Sieltevitz, 1963). Some ribosomes are free while others appear to be attached to the reticulum and comprise the rough-surfaced reticulum. Various firmly bound microsomal enzymes are present on the reticulum of the hepatic cells including hydrolytic, hydroxylating, elect,ron transporting enzymes, and specific hemoproteins such as cytochrome B6 (Strittmatter and Velick, 1956) and carbon monoxide-binding pigment, P-450 (Omura and Sato, 1962, 1963). The existence of these enzymes and hemoproteins are thought to be one of the

268

HAROLD P. MORRIS

characteristics of the differentiated hepatic cell, and Ernster el al. (1962) believe that the tissue-specific antigen is present in the microsomal fraction. The various biochemical, enzymatic, and protein measurements made by Sugimura et al. (1965) have been calculated as per cent using normal liver as 100, and are illustrated in Fig. 11. In microsomal protein the four

-

Normal liver I Regenerating liver2 Embryonal liver - 3 AH-371 4 AH-1305 7793 6 77948 7 7795 E 7316A 9

-

250

Microsornal protein

RNA

DNA

1250

200

200

I50

I50

100

100

50

50

0

E

2

150

L

0

E

u

t

I

Sulfalase

Hydroxylalian

~

250

.zoo

-

150

100

50 0 NADH-Cylo b5 NADH-Cylo c NADPH-Cyto c Reduclase I Reduclase Reductase

Cylochrorne b5

100

I00

50

50

0

0

FIG.11. Microsomal fraction biochemical and enzymatic assays for normal, regenerating, and embryonal liver; rapidly growing Hepatomas AH 371 and AH 130; and slowly growing Hepatomas 7793,77948,7795, and 73168 expressed as per cent of the respective values for normal liver. (From Sugimura et al., 1965.)

“minimal deviation’’ hepatomas were more like normal liver than they were like embryonal liver or the ascites hepatomas. I n RNA the ascites hepatoma AH 371 and three of the four minimal deviation hepatomas were more like regenerating liver than they were like either normal or embryonal liver. The AH 130 RNA was most like embryonal liver-both were much higher than normal liver. Hepatoma 7793 microsomal fraction RNA was similar to normal liver. In DNA all the hepatomas were more like normal liver than they were like either regenerating or embryonal liver except two-7316A) which was quite similar to embryonal liver, and 7795, which was halfway between normal and embryonal liver. I n phospholipid all the

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY OF HEPATOMAS

269

hepatomas were decreased from normal liver, but the ascites hepatomas were lower than the minimal deviation hepatomas and were more nearly like embryonal liver. The glucose-6-phosphatase in the microsomal fraction of Hepatoma 7793 was like normal liver; 7316A was slightly less than normal ; Hepatomas 7794A and 7795 were approximately 1/3 of normal; and the ascites hepatomas were about 1/20 of normal. Embryonal liver had half the value obtained for normal liver. The ATPase of regenerating and embryonal liver was slightly depressed from normal while all hepatomas were much higher than normal liver (125 to 264%) with Hepatoma 7793 the highest. I n the other assays embryonal liver was decreased to 1/3 or less of normal; the two ascites hepatomas were decreased more than the embryonal liver. In the microsomal fraction the sulfatase activity of 7793 was about normal and 7316A was higher than normal liver; but in cytochrome BE, 7316A was similar to normal liver. In all the other assays, except as noted above, the minimal deviation hepatomas were decreased from normal but in most instances they were decreased less than embryonal liver. From the comparisons presented in Fig. 11 one could conclude that in most of the assays made by Sugimura et al. (1965) the ascites tumors approached embryonal liver and were much depressed when compared to normal liver. None of the minimal deviation hepatomas were identical to each other but in most of the parameters examined they were more like normal liver than embryonal liver. All rapidly growing and slowly growing hepatomas had elevated ATPase activity in the microsomal fraction, from 1 1/4 to 2 1/2 times normal liver. While no explanation was found thus far for high ATPase values in the microsomal fraction of hepatomas, Sugimura et al. (1965) did suggest that these higher activity values could in some way be related t o a changed character of the microsomal membrane. An investigation of the influence of hormonal or other inhibitors of protein synthesis upon the microsomal-fraction measurements studied by Sugimura et al. (1965) should give important leads to some of the control mechanisms a t this level of the cell machinery. IX. Metabolism of Purines and Pyrimidines and Cholesterol Synthesis by Hepatomas

A. METABOLISM OF PURINES

It has been suggested that the loss of growth control associated with cancer might be related to loss of capacity to degrade nucleotides or nucleotide derivatives (Bennett et al., 1960). One approach to a study of this problem is that of Wheeler and Alexander (1961, 1961a) and Wheeler et al.

270

HAROLD P. MORRIS

(1962), who used suitably C14-labeled substrates and after suitable incubation with minces and sonic-disrupted hepatomas prepared paper chromatograms and radioautograms from alcoholic extracts and later assayed the radioactive areas. The extent of anabolism and catabolism in vitro of purines and purine ribonucleotides by rat tissues and of several hepatomas were determined (Wheeler and Alexander, 1961, 1961a,b), Wheeler et al. (1962). Hepatoma 5123 was more active catabolically than any of the host tissues examined including liver. Hepatoma H35 (Reuber, 1961), was about half as active catabolically as the host liver, while Novikoff hepatoma was much lessactive catabolically than the host liver. It was concluded (Wheeler et al., 1962) that a decrease in purine catabolism was not a requisite for uncontrolled neoplastic growth in these three hepatomas. A further series of hepatomas of different growth rates were studied by Wheeler et al. (1964) both in vitro and in vivo. The data obtained yielded information thought t o be pertinent to growth control mechanisms. In the in vitro experiments of Wheeler et al. (1964) the distribution of CI4 in anabolic and catabolic products of the hepatoma extracts after incubation with ~idenine-8-C'~ or hypoxanthine-8-C14 are presented in Table X (Wheeler et al., 1964). The tumors are arranged roughly in order of decreasing growth rate (cf. Table 11) as determined by frequency of transplantation (Morris and Wagner, 1965). Considerably more anabolism and considerably less catabolism with both substrates occurred in the minces of the rapidly growing hepatomas than in the minces of the slowly growing tumors or the host liver. 1. Catabolism of Xanthine

The xanthine oxidase activity of host liver was increased in rats bearing Novikoff and Morris 3683 hepatomas (Wu and Bauer, 1962), but no increase was noted in the livers of rats bearing Hepatoma 5123; from which Wu and Bauer concluded that tumor growth need not cause a change in the activity of xanthine oxidase in host liver. The xanthine oxidase activity of Hepatoma 5123 was similar to that found in the host liver but was barely measurable in 3683,39248, or Novikoff hepatomas (Wu and Bauer, 1962). A further study of catabolic peptidases by Wu and Bauer (1963) disclosed that among other tumors four hepatomas-Novikoff, 3683, 3924A (all rapidly growing neoplasms) and the slowly growing 5123 hepatomacontained high activities of five peptidases. However, the activities of L-prolylglycine, glycylglycine, and glycyl-L-leucine peptidases were similar to those of normal liver in these hepatomas. The L-leucylglycine peptidase activity was lower than that of normal liver, but the tripeptidase hydrolyzing glycylglycylglycine was higher. The data support the idea that many peptidases of liver tissue are retained when that tissue undergoes malignant

l?

0

TABLE X w DISTRIBUTION OF CI4AMONQ ANABOLIC PRODUCTS AND CATABOLIC PRODUCTS IN EXTRACTS O F HEPATOMAS AND LIVER AFTER INCUBATION

5

-3

WITH ADEN1NE-8-Cl4 OR HYPOX4NTH1NE-8-C1*

Total radioactivity

8

(yo)

d

Novikoff

3683

728%

5123TC

H35TC

51238

H35

Liver

Bz CI

Precursor: adenine-8-CI4 Anabolic products6 Catabolic productsC Precursor: hypoxanthine-8-C" Anabolic products* Catabolic productsc

8647

80 12

76 17

32 61

26 64

2 62

m

51 33

70 24

15 84

6 93

From m-heeler et al. (1964). Anabolic products include adenosine, AMP, ADP, ATP, NAD, inosine, and 0 Catabolic products include xanthine, xanthosine, uric acid, and allantoin.

-

1 97

40 30

39 42

61 28

0 99

2 96

2 95

-2 P 0

2 0

r

a

IMP.

B

s

+d

0

z

%

272

HAROLD P. MORRIS

transformation. Thus, Wu and Bauer (1963) observed as much variation among different tumor tissues in the activity of the peptidases examined as those found in normal liver and a convergence to relative uniformity as postulated by Greenstein (1956) was lacking.

B. METABOLISM OF PYRIMIDINES Some of the many enzymes of pyrimidine metabolism have been listed in five groups or frames and the reactions are numbered (Fig. 12). The Frame I: Ca9,bamyl phosohate alternatives

NH,

+

co2

2a Carbamyl aspartate

/

+ aspartat f

ATP-

1

Carbamyl phosphate

+ O*

2b Citrulline

-

Frame 11: U M P synthesis Carbamyl aspartate -Dihydroorotate 3

4

5

Frame 111: U M P alternatives

+ glutamine + ATP

7b

Ur

8b

UMP

9a CTP

=

++UTP

p* UMP

g

Orotic acid -0MP

U-

Qb

DHU -BUP-BAL 1Ob

llb

+ Cq

Frame IV: T M P synthesis dCDP-

CTP+-

CDP

-P 14

+ CH d C M P zd U M P d T M P 15 16

Frame V: T M P alternatives -t A

17a Y T T P

18a= DNA

TMP 17b

= DHT-BUIB-BAIB 20b

21b

+

Cot

FIG.12. Enzymes in pyrimidine metaboliEm expressed in five frames of reference. Abbreviations ATP, CTP, UTP, TTP; OMP = orotidylic acid; UR = uridine, U = uracil; DHU = dihydrouracil; BUP = p-ureidoproprionic acid; BAL = 8-alanine; Tdr = thymidine; T = thymine; DHT = dihydrothymine; BUIB = 8-ureidoisobutyric acid; BAIB = p-aminoisobutyric acid. (From Ono et al., 1963.)

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

273

activities of some of them have been studied in liver and several hepatomas by Ono et al. (1963). Several alternative pathways were included in the list because the ratio of anabolic to catabolic pathways for a given substrate can be calculated for the liver and tumors. The reactions of pathways found primarily in liver serve as “markers” because such reactions are present in much smaller amounts or absent in other tissues. In Fig. 12, frame I, is listed carbamyl phosphate synthetase (enzyme I), a n enzyme providing a mechanism for disposing of free NH, and converting it to a product, carbamyl phosphate, a product which can take the anabolic pathway, enzyme 2a, to form pyrimidine or the catabolic pathway to form urea via aspartic transcarbamylase (enzyme 2b). Aspartic transcarbamylase is widely distributed while carbamyl phosphate synthetase (enzyme 1) and ornithine transcarbamylase (enzyme 2a) are “liver markers.” Those enzymes listed in frame I1 are not limited to liver, but the catabolic enzyme, uracil reductase (9b, frame 111),is a useful liver “marker”; although kidney contains one tenth as much activity as liver, other tissues are essentially inactive. Wu and Bauer (1962) found a decrease of uracil reductase activity in the liver of rats bearing Hepatoma 5123 and 3924A and no change in rats bearing Novikoff and 3683 hepatomas; from which they concluded that tumor growth need not cause a change in activity of this enzyme in host liver. Thymidylate synthetase (enzyme 16, frame IV) is absent or very low in adult liver, but is present in regenerating liver after 18 hours (Maley and Maley, 1960). The dCMP deaminase (enzyme 15) is found in Noviltoff hepatoma (Maley and Maley, 1959), in liver with proliferating bile duct epithelium, and in regenerating liver (Maley and Maley, 1960). The catabolic enzyme, thymine reductase (enzyme 19b frame V), like uracil reductase is a liver “marker” enzyme which has the same distribution as uracil reductase (Potter et al., 1960). Enzymes that appear to be mandatory requirements in all DNA-synthesizing cells and no use in other cells are thymidylic kinase (enzyme 17a) (Bollum and Potter, 1959), and DNA polymerase (enzyme 18a) (Maley and Maley, 1960). Ono et al. (1963) found thymine reductase in Hepatoma 5123B to have values 70y0 of control values, while Hepatomas 7800, 7316A, and H35 had values ranging from 18 to 32% of normal, and h’ovikoff and Dunning hepatomas were 3% or less of normal liver. Emmelot et al. (1961) have reported for some mouse and rat hepatomas values ranging from o-52y0 of normal. It is clear, therefore, that neither the presence nor the absence of thymine reductase activity is a constant property of hepatomas. Examining the data of Ono et al. (1963) from several hepatomas for ornithine transcarbamylase, aspartate transcarbamylase, and carbamyl phosphate synthetase, it was apparent that no absolute correlation of

274

HAROLD P. MORRIS

qualitative or quantitative enzyme activities and growth rate or malignancy could be discerned. Jones et al. (1961) also found ornithine transcarbamylase to be absent in Hepatoma 3924A but increased more than 500-fold in Hepatoma 5123. From studies of Reid (1962), Reid and Morris (1963), and Bresnick (1962a), it was found that Hepatomas 5123A, C, and D, 7316A, 7800, and H35 possess aspartate transcarbamylase activities equivalent to that observed in adult normal liver. Partially purified aspartate transcarbamylase had essentially identical properties in liver and hepatomas including pH optimum, K,, and inhibition constants for a number of nucleotides that might be involved in feedback inhibition of this enzyme (Bresnick, 1962a). It may bc concluded from the investigations of Ono et al. (1963) that no single hepatoma thus far studied is a superior model for comparison with liver. Rather, these studies point to the need to check many other “minimal deviation”-type hepatomas for any change believed to he significant in the others. If tumor progression takes place from a slowly growing, minimal deviation hepatoma which results in a rapidly growing, multiple deviationtype tumor, such progression could well involve different sets of alterations in two or more progressions to attain an equally rapid growth rate in the multiple altered hepatoma. What the obligatory steps in the malignant process are remains unclear from these and other studies. It seems reasonable to assume that as one possibility the malignant change could occur as a break in the feedback mechanism that controls cell division. Among the pyrimidine-metabolizing enzymes the most probable candidates would seem to be enzymes that are normally low in adult parenchymal cells and which increase when cell division begins (Ono et al., 1963). These enzymes might include any or all of those listed in Fig. 12, frame IV, and 17a, 17b, and 18a in frame V (cf. Section XIII).

C. FEEDBACK INHIBITION IN HEPATOMAS The regulation of enzyme activity by products of the reaction has been termed end product or feedback inhibition. Of studies involving feedback inhibition, thymidine kinase possesses many of the attributes of a ratedetermining enzyme for DNA synthesis. Enzymatic activity is enhanced in regenerating liver (Bollum and Potter, 1959) and in livers of rats on a high protein diet after 4-12 days on a protein-free diet. Moreover, a greater effect of such a dietary regimen on enzymatic activity was found in H35 hepatoma (Gebert and Potter, 1964). The activity of this enzyme is inhibited in chick embryo by its distal product TTP (Maley and Maley, 1962) in Novikoff hepatoma (Ives et al., 1963), in regenerating liver (Breit-

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

276

man, 1963), and in human leucocytes (Bresnick and Karjala, 1964). In the latter report equal inhibition was noted by both TTP and dCTP. These observations lead to additional studies of the ability of these nucleotides to regulate thymidine kinase in liver, embryonic liver, regenerating liver, and in several hepatomas of different growth rates. Some of these results are presented in Table XI (Bresnick et al., 1964). The thymidine T.4BLE XI INHIBITION OF TAYMIDINE KINASEBY HEPATOMAS A N D LIVERS

Source of enzyme

TTPb

dCTPb

5.6 f 1.4 1.3 f 0.4 2.0 f 0.4 1.1 1.2

24.3 k 1 . 3 2.7 0.2 1.4 f 0.3 1.2 2.4

Intermediate growth rate 2.1 f 0.2 1.0 f 0.2

0.9 k 0 . 1

Rapidly growing 24.3 f 1 . 5 2.3 f 0.6 4 . 2 f 0.7 1.3 3.0

Slowly growing 1.7 5.5 3 . 2 rt: 0 . 6 2.3 4.0 3.6 4.6 4.0

H 35

5123A, B, and D 7800 77948 73168 7795 7793 7787

Adult Embryonic Regenera t h g a

b

None

bg.1

Novikoff ascites Dunning LC18 McCoy MDAB 3683 39248 7288C

Additions

Proteins

80 50 65

Liver 5.9 202 45

1.2 2.7 2.1 k 0.5 1.0 2.1 3.2 2.1 2.3 2.0 28.0 11.0

0.9 2.1 1.5 f 0.4 1.5 2.0 2.5 1.9 1.5 2.7 200.0 39.0

Revised from Bresnick et al. (1964). mpmoles TMP/mg. protein.

kinase was partially purified from each of the respective tissues. Both TTP and dCTP inhibited enzymatic activity of normal liver by 40 to 50y0and most host livers exhibit,ed patterns similar to normal liver. d C T P had little inhibitory effect on the partially purified enzyme from either embryonic liver or regenerating liver, but the inhibition by TTP amounted to 70-80%.

276

HAROLD P. MORRIS

In the hepatomas the partially purified thymidine kinase from Novikoff and Dunning LC 18 tumors was markedly sensitive to TTP but resistant to inhibition by dCTP. The McCoy MDAB tumor enzyme, however, was sensitive to both nucleotides. It appears doubtful if the rapidly growing 3683 hepatoma enzyme is inhibited by either nucleotide (Table XI). The thymidine liinase preparations from all the other hepatomas were inhibited by both TTP and dCTP, with the possible exception of TTP in Hepatoma 7795. Preliminary results with H3-dCTP indicated that little dCTP is either deaminated or dephosphorylated in any of the tumor preparat,ions. This seems to indicate that the lack of sensitivity of the enzyme preparation from either Novikoff or Dunning hepatomas was not due to a more active catabolism of dCTP than that of the other hepatoma extracts. Other studies by Maley and Maley (1963) noted that deoxycytidylate deaminase purified from chick embryo was effectively regulated by the end products associated with its metabolic pathway, dCTP and dTTP. The inhibition due to dGMP or dTTP could be completely reversed by dCTP (Maley and Maley, 1964), but they reported that deoxycytidylate deaminase from Novikoff and regenerating liver was not as effectively regulated by dCTP and dTTP as that from chick or rat embryo. Bukovsky and Roth (1964) studied feedback inhibition by TTP in a series of transplantable hepatomas of different growth rate in the presence of M g + +(T-TMP). Little evidence of T M P control with or without Mg++ was found in any of the hepatomas (Novikoff, McCoy DAB, and Morris 5123D) but in the presence of Mg++ 3 times as much TTP was required to obtain inhibition. It was found that the T M P phosphatase activity was greatly increased in Hepatoma 5123C by Mg++ but the rapidly growing Novikoff and McCoy DAB hepatomas showed little breakdown of TMP. D. DELETIONOF THE CHOLESTEROL NEGATIVEFEEDBACK REGULATION IN LIVERTUMORS The rate a t which the liver of rats can synthesize cholesterol can be greatly or almost totally depressed by the feeding of a diet containing cholesterol (Gould, 1951; Tomkins et al., 1954; Langdon and Bloch, 1953; Frantz et al., 1954; Siperstein and Fagan, 1964a,b). Siperstein and Guest (1960) have shown that the negative feedback reaction controlling cholesterol synthesis is located a t an early step in cholesterol biosynthesis, namely, a t the conversion of 0-hydroxy-0-methylglutaryl CoA to mevalonic acid; it is illustrated in abbreviated form by Fig. 13. The cholesterol diet suppressed the synthesis of mevalonic acid by over 200-fold and provided the first direct evidence that a depression of that extent was adequate to account for the inhibition of cholesterol synthesis (Siperstein and Fagan, 1962, 1964a,b). The mevalonate synthesis takes

277

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

1 Aceto- @ 0-HydroxyAcetyl Qacetyl ---+ P-methylCyA CoA glu$rate

Mevalonate -Squalene

-Cholesterol I

I

t

CO2

t Acetoacetic acid

I

\

\

I

! I

\

\.--- --___-

'

*

/

/

/

/

I

I

, 0

i

P-Hydroxythyric acid

FIG.13. Site of feedback control of cholesterogenesis. (From Siperstein and Fagan? 1964a.)

place primarily in the microsomal fraction of liver, although some also occurs in the soluble fraction of liver according to Siperstein and Fagan (1964a,b). Furthermore, these investigators showed that the activity of the microsomal fraction in cholesterol biosynthesis in normal livers is markedly depressed by dietary cholesterol while the soluble fraction activity is relatively unchanged and, therefore, probably not involved in feedback control. Siperstein and Fagan (1964a,b) concluded th a t cholesterol synthesis is restricted to microsome membranes (Table XII). This remarkTABLE XI1 ACID SYNTHESIS IN MICROSOMAL MEMBRANES ISOLATED FROM MEVALONIC RAT LIVER" Preparation

Mevalonate synthesized (yo) (added acetate-2-CI4)

Intact microsomes Microsomal membranes

0.50 1.45

From Siperstein and Fagan (1964b).

able example of a feedback mechanism in higher animals that inhibits a single reaction suggests the close similarity t o previously well-characterized synthetic feedback systems in bacteria where the end product specifically inhibits the first or an early step in the reaction sequence (Adelberg and Umbarger, 1953; Umbarger, 1956; Yates and Pardee, 1956). The possibility that a negative-feedback control mechanism might represent a characteristic of rapid cellular proliferation led Siperstein and Fagan (1964a,b) to examine regenerating rat and mouse liver; they found a normal cholesterol feedback system similar to that found in the normal liver of both species. The feedback control in cholesterol synthesis by exogenous cholesterol of hepatomas in three species-mouse, rat, and

278

HAROLD P. MORRIS

man-was also examined by Siperstein and Fagan (1964a,b) and found to be absent. One primary human hepatoma, the mouse Hepatoma BW7756, and Morris hepatoma 5123TC were all capable of very active cholesterol synthesis and completely lacked the feedback mechanism which normally regulates cholesterol synthesis when depressed by dietary cholesterol. Siperstein and Fagan (1964a) were able to show with C'*-labeled dietary cholesterol that this failure of feedback control was not due to inability of the end product to penetrate the tumor cell. A series of tcn additional rat hepatomas of different growth rate have now been examined by Siperstein et al. (1965) including the following Morris tumors: 3683, 39248 (both rapidly growing), and 7787, a very slowly growing tumor. All of these tumors display this failure of feedback control in cholesterol synthesis but to a variable extent. Thus, slowly growing Hepatoma 7800 has 20- to 40-fold greater capacity to synthesize cholesterol than does the rapidly growing Hepatoma 3683, and Hepatoma 7787 had a n even greater capacity to synthesize cholesterol. This defect in feedback regulation could be a factor in cellular malignancy and may possibly also be inversely related to growth rate. If, as seems likely from these studies, the absence of adequate feedback regulation may play a role in carcinogenesis, it would be the first direct experimental support for Potter's (1957,1958,1963) theory that a deranged negative fccdback control may bc one of the mechanisms involved in carcinogenesis, although the role of cholesterol synthesis is not now apparent (cf. Section XIII). The success of the studies by Siperstein gives hope of finding other negative feedback systems in higher animals which may not only correspond to those already known to exist in microorganisms but could result in a clearer understanding of some of the key events leading to carcinogenesis. The absence of a negative feedback of cholesterol synthesis in the many hepatomas studied by Siperstein and Fagan (1964a,b), Siperstein et al. (1965), appears to be a good example of the nth product, as described by Potter (1963) in a long chain of steps in cholesterol synthesis that are in the feedback loop (Fig. 13). The end product (in this instance, cholesterol) is hardly recognizable as the substance mevaloriate which in the normal liver is blocked by the high cholesterol diet.

E. FEEDBACK DELETION It has been proposed by Potter (1964a) that if in a series of L'minimal deviation" hepatomas one hepatoma line was found to lack a given enzyme and another was found to contain that enzyme, one would have to conclude that the enzyme was not essential to the conversion of a normal liver cell

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY OF HEPATOMAS

279

to a neoplastic cell although the deletion or absence of that enzyme might affect growth rate or other charactcristics of the tumor. On the other hand, other alternatives to this proposal of Potter (1964a) seem reasonable. (1) Partial or incomplete deletion in negative feedback control could occur. Although the relation of the deletion of negative fccdback control to liver neoplasia is unknown, Siperstein et al. (1965) has observed in different hepatomas a variable capacity for the biosynthesis of cholesterol when the tumor-bearing host was on a high cholesterol diet. (2) Theoretically, the deletion of two or more feedback control pathways simultaneously could be essential in the development of neoplasia. (3) Deletion of negative feedback control may be only one of several mechanisms iiivolved in the conversion of normal cclls to neoplastic cells (cf. Section XIII). An important approach to a better understanding of the mechanism(s) of carcinogenesis would be the development and biochemical characterization of hepatomas with fewer and fewer deviations from their cells of origin. Thus, it may be possible in the future to determine which changes are necessary for autonomous growth, for invasion, for metastasis, and many other factors which play a role in cancer development, such as hormones, wound healing, irritation, etc. (cf. Potter, 1964b). X. O t h e r Activity Studies

A. CATALASE ACTIVITY

It seemed important to reappraise the catalase activity of several newly developed hepatomas in light of Greenstein’s (1954) view that tumor tissue contained in geiieral no, or ncgligible amounts of, catalase. This view is now rather untenable in view of the development of two lines of transplantable hepatomas with greatly diff erent catalase activities (Rechcigl and Sidransky, 19G2). Therefore, a number of transplantable rat hepatomas have been assayed for catalase activhy by Rechcigl (Morris et al., 1964). The high catalase line (HC) has catalase activities higher than that found in normal liver (Fig. 14). The low line (LC) had values about 1/10 those for normal liver of approximately 180 to 190 units/g. fresh liver. Both were established in the first transplant generation. All three rapidly growing hepatomas had essentially no catalase activity (Fig. 14), but considerable catalase activity was found in each of several minimal deviation hepatomas (Fig. 14). From the preliminary values now available there seems to be a tendency for the catalase activity of Hepatoma 7794B [the slowest growing tumor in the group (cf. Table II)]t o be increasing. The HC and LC lines of Rechcigl and Sidransky (1962) retained their

280

HAROLD P. MORRIS

)AYS 10 15 16 27 31-99 31-94 58 58 167 4-79 261 69 I60 114 300 385 I32 I40 42 60 146 262 I49 42

-

TUMOR LlNFS NOVIKOFF 3683 3924 A 5123 TC 5123 A 5123 B 5123 C 5123 D 5123 D 5123 AVG. H-35 7787 7793 (P) 7793 (2) 7793 (5) 7794 (A) 7794 8 (1) 7794 B (2) 7794 8 (3) 7795 7800 HC LC 7316 A (2) 7316 B ( I ) 7316 B(2) 7288C 0

1

1

40

1

1

1

1

1

1

1

1

1

1

1

1

80 120 160 200 240 280 CATALASE ACTIVITY UNITS /GM. WET WEIGHT

1

1

320

1

31 0

FIG.14. Catalase activity in a series of transplantable hepatomas. Days represents the days of growth of the tumor assayed; P = primary; the numbers in parentheses refer to the transplant generation of the tumor assayed; single measurements = line only; a bar a t end of line represents range of values; 5123 avg. = all 5123 sublines with its standard error. [From Rechcigl (cf. Morris et al., 1964).]

high and low catalase activity values through several transplant generations. The activity of several other enzymes, however, in these two hepatoma lines did not differ significantly from normal liver (Rechcigl and Sidransky, 1962). The host liver of the high catalase line as well as Hepatoma 5123 during the growth of the tumor showed depressed catalase activity (Fig. 15) (Rechcigl et al., 1962). It is not certain whether this effect is due entirely to the size of the tumor or partly due to high catalase activity of the tumor. The HC line is somewhat more rapidly growing than the LC line. Other slowly growing hepatomas such as 7787, 7793, and 7795 (Fig. 14) seem to have lower catalase values than the faster though still slowly growing 5123 lines; however, 7800 with a growth rate somewhat more rapid than 7795 has considerably higher catalase activity. The catalase activities of the twenty transplantable hepatomas varied

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY OF HEPATOMAS

1200

L-

28 1

Normal liver

6 Tumor weight, g m .

FIG. 15. Tissue catalase activity of Buffalo-strain female rats bearing Hepatoma 5123; T.B. = liver of tumor-bearing host. (From Rechcigl et al., 1962.)

from essentially nil to values higher than found in normal liver. Catalase activity may be dependent on the presence of more or less well-organized ergastoplasm, although the site of catalase activity within the cell is not known. Higashi and Peters (1963a,b), however, obtained good agreement by both the enzymatic and immunochemical procedures for the distribution of catalase in the various cell fractions of normal rat liver. Most of the catalase was found in the mitochondria and supernatant fractions. The catalase of microsomes was associated with the granular rather than with the agranular fraction and was found in the granular fraction sedimenting between 40 and 4575 sucrose. Higashi and Peters (1963a) isolated cell fractions of rat liver at various times after injection of leucine-CI4 and determined the incorporation of C14 in these cell fractions. Catalase was synthesized by the ribosomes according to Higashi and Peters (1963a) and is first detectable in the granular zone of the reticulum. In from 10 to 60 minutes it either becomes soluble and finds its way to the mitochondria or it may be transferred directly from the reticulum membrane to the mitochondria, although Higashi and Peters (196313) were unable to ascertain the mode of transfer. It is well known, however, that tumor-bearing animals suffer from hypoproteinemia. The effect of a hepatoma on liver catalase may be mediated through a deficiency in one or more amino acids required for catalase

282

HAROLD P. MORRIS

synthesis, (Rechcigl, 1963). Because catalase activity of Hepatoma 5123 was unaffected by a protein-free diet (Rechcigl et al., 1962), whereas normal liver catalase activity is decreased, the tumor may have a preferential claim on available metabolic units needed for catalase synthesis. There is yet no explanation for the variable catalase activity found in the series of hepatomas thus far examined, but studies of the synthesis of this enzyme in the various cell fractions of hepatomas by the techniques of Higashi and Peters (1963a) could well give valuable insight into the alterations in the liver cell which give rise to various levels of catalase in different hepatoma lines. B. ELECTRON SPIN RESONANCE Electron spin resonance (ESR) spectroscopy (Nebert, and Mason, 1963, 1964) characterizes the kinds and concentrations of substances containing unpaired electrons such as exist in paramagnetic states of transition elements and in free radicals. Nebert and Mason (1964) have applied this technique to Hepatoma 5123B. On the microsomal fractions diff erenres in ESR signal intensities were statistically evaluated. The microsomal Fe, (heme) was significantly decreased in host liver and diminished more than 3-fold in Hepatoma 5123B as compared t o control normal rat livers, a finding that is consistent with similar measurements on mouse Hepatoma BW 7756. The ESR of microsomal manganese-protein was not significantly different in normal and host liver, but the manganous signal was decreased 2 1/2 times in the hepatoma. No difference in free-radical content was found in normal or host liver or hepatoma. Neither did spectrophotometric spectra a t liquid nitrogen temperatures or acetanilide hydroxylase show any significant differences between normal liver, host liver, and Hepatoma 5123B.

C. TISSUECULTURE One approach to a study of the molecular basis of biochemical defects occurring in neoplasms is through the use of tissue culture. Two rapidly growing hepatomas, Noviltoff and Dunning LC 18, and two slowly growing hepatomas, Morris 5123C and Reuber H35 (Gentry et al., 1962; Morse et al., 1964) have been cultivated in vitro for extended periods (Pitot et al., 1964) without, in the rapidly growing tumors, any appreciable alteration of their malignant potential, their enzymology or morphology. The 5123 tumor cells insofar as they have been examined for enzymatic activity after reinoculation into suitable hosts, were indistinguishable from the original tumor. The karyotypes of Morris 5123 and the in vitro line 5123H2 have been studied by Pitot et al. (1964). The former possessed the normal diploid chromosomal compliment, while the latter, after reimplantation into ani-

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

283

mals, had one or two more than the normal diploid number of chromosomes. Although continued in vitro cultivation of 5123H2 resulted in fibroblast contamination which gave rise to sarcomas, a n early explant of 5123H2 from tissue culture to the Buffalo-strain rat has been maintained in the author’s laboratory through more than 25 serial transfers. Morphologically the 5123TC cells, according to Miyaji (1965), were much smaller after return t o the animal but retained the trabecular arrangement. No certain distinguishable biochemical differences from the Morris 5123 hepatoma line of origin have been clearly identified. There was a slight but consistent increase in growth rate of the tissue culture line when it was first returned to the animal. The reinoculation into ACI/N strain rats by tissue cultures of H35H4 after 5 subcultures (Pitot et al., 1964) also resulted in some increased growth over that of the H35 hepatoma (H35TC, subline) (see Table 11). The H35 hepatoma culture (H35H4) after growth in the animal was again explanted to T flasks in vitro (Pitot et al., 1964). Very slowly growing epithelial colonies were found on the fibroblastic monolayer. All attempts at single cell cloning were unsuccessful; however, after three serial colony clonings a cell strain was obtained free of fibroblast contamination (Pitot et al., 1964) and consistently gave after in vitro cultivation for more than 1 year typical Reuber H35-type tumors in vivo. The growth rate of this tumor line (H35TCz) from very preliminary observations seems to be slightly more rapid than the first H35 tissue culture line (cf. Table 11). The morphology of the second tissue culture line in vivo and the H35 is almost identical (Pitot et al., 1964). The second tissue culture line, however, had a chromosomal mode of about 46 compared to 42 to 43 for the H35 hepatoma. The assay for a number of enzymes in H35 in vivo and in vitro are presented in Table XI11 (Pitot et al., 1964). Several enzymes present in liver and H35 hepatoma in vivo were absent from the H35H4 IIE in vitro, namely tryptophan pyrrolase, glucose-6phosphate dehydrogenase, and proline oxidase. Threonine dehydrase was present at a very low level. However, the hepatic “marker” enzymeshistidase, ornithine transaminase, tyrosine transaminase, thymine reductase, and glucokinase-were present in the cultured cells (Pitot et al., 1964). The absence of glucose-6-P-dehydrogenase, an important enzyme in the intermediary metabolism of carbohydrate, is of interest because this enzyme is strongly affected by adrenal hormones (Potter and Ono, 1961). The Novikoff hepatoma cells in vitro were shown to reproduce more cells in 3 days than the H35 hepatoma cells in vitro produced in 14 days, which corresponds roughly to the growth rate of the two hepatomas in vivo (Pitot et al., 1964). The inducibility of both tryptophan pyrrolase and threonine dehydrase in hepatoma in vivo is at a low level. Pitot et al. (1964) unsuccess-

284

HAROLD P. MORRIS

TABLE XI11 SOMELIVERENZYMES in Vivo, I N REUBER HEPATOMA H35 in Vivo, CULTURELINE H35H4 I I E in Vitroaab

AND

TISSUE

Enzyme

Liver

H35

H35H4 I I E

Gluc,ose-6-phosphate dehydrogenase Tryptophan pyrrolase Histadase Threonine dehydrase Proline oxidase Ornithine transaminase Tyrosine a-ketoglutarate transaminase Hexokinase Glucokinase Thymidine reductase

100-300 2-4 25-75 20-80 120-280 75-110 75-150 15-30 30-56 1.6-2.8

50-100 0.3-0.6 2.3 0-70 9-15 380-520 200-600 10-30 13-20 0 . 14c

0 0 0-3 0-20 0 320-400 80-200

18 19 0.01c

Values expressed as pmoles product/g. wet wt./hour. (1964). c Approximate values.

* From Pitot et al. Q

fully attempted to induce tryptophan pyrrolase with a-methyl tryptophan as the inducer i n uitro. Furthermore, even the maintenance of H35H4 IIE for 24 hours without glucose, a repressor of threonine dehydrase, did not affect the level of the enzyme. I n adrenalectomized hosts cortisone induces tyrosine transaminase in the host liver and in hepatoma H35 (Pitot, 1963). The addition of cortisone 24 hours before the assay to the tissue cultures of H35 hepatoma resulted in a significant induction of tyrosine transaminase (Pitot et al., 1964). TABLE XIV TYROSINE TRANSAMINASE IN LIVER, H35 HEPATOMA A N D H35H4 IIE HEPATOMA CELLSIN TISSUECULTURE",^ Liver Control in vivo Plus cortisone in vivo ADRX in vivo Control in vivo Control in vitro Plus cortisone in vitro a

15-50 220-300 20-55 120-450 -

-

H35 200-600 300 50-80 130-300 -

H35H4 I I E 80-200 400-680

Values expressed as rmoles product/g. wet wt./hour. From Pitot et al. (1964).

Maximum induction occurred 6 hours after addition of the hormone to the culture medium and the enzyme level remained high for a t least andther 18 hours (Pitot et al., 1964), probably because the hormone is not rapidly

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metabolized or removed in vitro. This enzyme, on the other hand, after induction in vivo rapidly returns to normal in Hepatoma H35, indicating that the kinetic characteristics of the induction of this enzyme in vitro may differ from that in vivo, possibly because the cortisone is more rapidly metabolized in vivo (Pitot et al., 1964). A number of defects are present in the control of enzyme synthesis in H35 hepatoma when compared to liver. Even after the elimination of all tumor-host interactions by the culture of Hepatoma H35 in vitro the basic abnormalities of defective control of this hepatoma still existed.

D. POLYRIBOSOMES Aggregates of ribosomes (Warner et al., 1963; Wettstein et al., 1963) rather than single ribosomes are currently believed to be the functional units of protein synthesis in mammalian and other organisms. The machinery for the assembly of the polypeptide chains may now be viewed as functional units consisting of several ribosomes held in a linear array by a single strand of mRNA (cf. Section 11). No11 et al. (1963), Goodman and Rich (1963), and others give evidence that the growing peptide chain remains attached to the individual ribosomes while the mRNA containing the codoiis moves relative to the ribosomes. The cytoplasmic polyribosome pattern of normal rat liver and several transplantable hepatomas has been studied by Webb et al. (1964). The hepatomas used were Novikoff, a rapidly growing, poorly differentiated tumor having multiple deviations from normal liver, and several welldifferentiated hepatomas (Miyaji, 1965) including tumors of different growth rate (cf. Table 11) varying as much as 10-fold in months between transfers. The well-diff erentiated hepatomas possess a “marker” enzyme pattern which qualitatively indicates their similarity to normal liver. The degree of confidence in comparing hepatomas with liver is greatly increased by using these well-differentiated hepatomas. Webb et al. (1964) approached the question of whether or not these well-diff erentiated hepatomas possess a common defect in their enzyme-forming systems by a study of the characteristics of the polyribosomes, an integral part of the enzyme-forming system. These authors, using the method of Wettstein et al. (1963), purified C ribosomes from normal liver, host liver, and several hepatomas. The C-ribosome patterns of all the various hepatomas studied differ markedly from that for normal liver. The C-ribosome pattern of the hepatomas was characterized by a smaller proportion of the heavier polyribosomes and a significantly higher proportion of the monomers and dimers, with the dimer peak always higher than the monomer peak. These high monomer and dimer peaks are not normally present in the pattern from normal rat liver which Webb et al. (1964) interpreted as an abnormality that might be

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HAROLD P. MORRIS

expected t o result (1) on basis of kinetic considerations, (2) from increased output of ribosomes, (3) decreased output of mRNA, or (4) a marked decrease in the stability of a fraction of mRNA. Although the hepatomas examined by Webb et al. (1964) had wide variations in growth rate and probably indirectly in synthesis of polypeptides, the data do not clearly distinguish any common alteration of the polyribosome pattern that may explain differences between the normal and neoplastic liver. Hepatoma 51238 was moat like normal liver insofar as its compliment of heavy polyribosomes recovered after protein deletion and 77948 was least like normal liver. Hepatoma 7794A appears to have a significantly lower concentration of many of the catabolic enzymes of amino acid metabolism (Pitot et al., 1963) and has a considerably slower growth rate than Hepatoma 5123A. I n further studies on the polyribosome pattern of some “minimal deviation” hepatomas Webb et al. (1965) found the fraction of bound polyribosomes in the postmitochondrial supernatant t o be approximately zero in immature liver and Novikoff Hepatoma, 20% or less in Hepatoma 7793 and 7794A, 40% in Hepatoma 7787 and 7800, and 60-70y0 in normal and regenerating adult liver. A proportion of bound ribosomes thus appears to be correlated with the degree of differentiation of the hepatomas and inversely with their growth rate. If, as suggested from recent evidence (Hess and Lugg, 1963), deoxycholate acts by displacing an equilibrium reaction between the ribosomes and the membrane, then the data of Webb et al. (1964) suggest that about 50% of the polyribosomes in normal liver are tightly bound, whereas there is complete recovery of polyribosomes from hepatoma cells without prior treatment with deoxycholate. Most of the polyribosomes appear t o be free in the hepatoma cells or else they are so loosely bound that they are released during homogenization and centrifugation. Pitot and Peraino (1964) have suggested that there is a faulty interaction between polyribosomes and the endoplasmic reticulum which might be the cause of changes in the stability of the mRNA in those hepatomas with the fewest deviations from normal liver. XI. Oxidative Phosphorylation and Phosphatide Synthesis

A. OXIDATIVEPHOSPHORYLATION AND ADENOSINETRIPHOSPHATASE ACTIVITYOF MITOCHONDRIA Isolated mitochondria from 5123, 3924A, 3683, and ethionine-induced hepatomas catalyzed coupled oxidative phosphorylation, as noted by Devlin and Pruss (1962), whereas no significant phosphorylation occurred in Novikoff and Dunning hepatomas except in the presence of bovine serum albumin (BSA). 2,CDinitrophenol uncoupled phosphorylation of all the tumors. Hepatomas 5123, 3924A, 3683, and ethionine-induced hepa-

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tomas had a n adenosine diphosphate (ADP)-dependent respiration but when the ADP-concentration approached zero only 5123, 3924A, and ethionine-induced hepatomas had a decreased rate of respiration. An ADPdependent respiration and a decreased rate of respiration when ADP concentration approached zero occurred with Novikoff and Dunning hepatomas by the addition of BSA. A low latent ATPase was present in untreated mitochondria from all hepatomas. ATPase activity was increased by Mg* in all the hepatomas except 5123 and ethionine-induced. In the absence of BSA, dinitrophenol stimulated the ATPase of the 5123 and ethionine-induced hepatomas but none of the other hepatomas. BSA elicited a 2,4-dinitrophenol-activatedATPase from the mitochondria of Novikoff and Dunning hepatomas according to Devlin and Pruss (1962), but had no significant effect on the various ATPase activities of the other tumors. Considering the dinitrophenolsensitive phosphorylating activity of the mitochondria of 39248 and 3863, the absence of a dinitrophenol-activated ATPase was surprising.

SYNTHESIS B. PHOSPHATIDE The enzyme system for methylating phosphatidyl aminoethanol to phosphatidyl choline in mouse ascites tumors and in rat hepatomas has been studied by Figard and Greenberg (1962). This enzyme system methylates cephalin to lecithin. Figard and Greenberg (1962) found this methylating-enzyme system largely depleted in mouse hepatoma 134, and in Dunning and Novikoff rat hepatomas. The activity of Morris hepatoma 5123D was considerably higher than Dunning and Novikoff hepatomas, although only 20y0 that of the normal liver, notwithstanding the lack of the enzyme system for forming lecithin from phosphatidyl aminoethanol in these hepatomas. Figard and Greenberg’s (1962) analytical studies of the phosphatides present did not show any striking abnormalities, although the total amount of phosphatides present in the tumors was much less than in normal liver. I n some of the hepatomas there was some lowering of the proportion of lecithin and a slight increase in cephalin. There was no drastic deviations in the fatty acid composition of tumors from that found in normal tissues. Since a wide variation in activity of several enzymes in different hepatomas has been observed it might be valuable to study the enzymes involved in the synthesis of lecithin from free choline for several other minimal deviation hepatomas, and to determine the extent of variation among such tumors in the enzymes involved in phosphatide synthesis. XII. Alterations of Dietary and Hormonal Regimens in Regulation of Enzyme Activity

The great diversity of the minimal deviation hepatomas under conditions of adaptation to sudden changes in the diet of the host, e.g., to a high

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(91%) protein diet was demonstrated by studies of the activity of threonine dehydrase (Bottomley et al., 1963a; Potter, 1964a,b) (Fig. 16). High levels

4 Low

+OW

H-35

One week Time on 91% protein diet

FIG. 16. Threonine dehydrase activity in several minimal deviation hepatomas after the host animal had been on a high protein diet for 1week. None of the hepatomas show a response similar to that obtained for normal liver. Each hepatoma line shows a different response. (Data from Bottomley et al. (1963a) (cf. Potter, 1964a,b.)

of the enzyme can be induced in normal liver by a high protein diet or high doses of cortisone for 7 days. It is noted that hepatoma threonine dehydrase values initially may be higher, lower, or about the same as the control liver but the activities of this enzyme in several hepatomas 1 week after the host was placed on the high protein diet showed a wide diversity of response (Fig. 16). However, it does not appear, from these experiments, that the activity of threonine dehydrase could be essential to the malignant process. Extending these effects of dietary alterations on the activity of threonine dehydrase (Bottomley el al., 1963b) showed that a reciprocal relationship existed in normal liver between threonine dehydrase and glucose 6-P-dehydrogenase in that the activity of both enzymes through

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various dietary alterations could not be induced to high levels at the same time as illustrated in Fig. 17. Further studies on transplantable minimal

3000 L

0)

._ 0

1

5

I

2000

a z

‘I Z

a t-

5

1000 e

0

+

AA

+

O

0

0 0

@A

pM a-Ketobutyrate / hr/g. liver

FIG.17. Threonine dehydrase activity (as a-ketobutyrate) and glucose 6-phosphate dehydrogenase (as T P N reduction) in normal rat liver under maximum conditions of induction. Each point represents both enzymes in a given sample. The data show that when the activity of one enzyme is high the activity of the other is low. (From Bottomley et al., 1963b) (cf. Potter, 1964a).

deviation hepatomas revealed a wide range of values for these two enzymes with some tumors having relatively high levels of threonine dehydrase and low levels of glucose 6-P-dehydrogenase, and some having high glucose 6-P-dehydrogenase and low levels of threonine dehydrogenase (Fig. 18). These differences between tumors and livers could not be ascribed to circulatory differences. Neither do these tumors conform to the “convergence” theory of Greenstein (1954). High levels of glucose 6-P-dehydrogenase can be accomplished in livers of normal fasted rats by refeeding a diet containing glucose or fructose and adequate protein. A high protein diet or high doses of cortisone for 7 days induce high levels of threonine dehydrase in livers of rats but in none of these conditions does the normal liver simultaneously have high levels of both enzymes. Bottomley et al. (1963b) were unable to select by these adaptation experiments any one of the ten transplanted hepatoma livers as most re-

290

HAROLD P. MORRIS

3000

A A

@

$&

I

I

El

1000 2000 3000 p M CI -Kelobutyrate /hr/g. tumor

40 10

FIG. 18. Threonine dehydrase activity and glucose-6-phosphate dehydrogenase activity in a spectrum of minimal deviation hepatomas on a standard diet (cf. Fig. 17). The activity of one enzyme is high in some hepatomas and the activity of the other enzyme is low, whereas in other hepatomas the reverse is true. The data indicate that the control mechanism is similar to that in normal liver (cf. Fig. 17) but that the internal milieu of the various hepatomas is not the same. (Data from Bottomley et aZ., 196313) (cf. Potter, 1964a.)

sembling normal liver. The ten hepatomas present a spectrum of levels of threonirie dehydrase and glucose 6-P-dehydrogenase activities ranging from very low t o relatively high levels; when the host receives a normal diet the levels of these two enzymes in the hepatomas stay within the range obtained by variabIe dietary and hormonal regimens of the liver of tumor-free animals. It is suggested from these observations that in the induction of rat hepatoma, cells are produced whose enzyme content may vary markedly from the parent cell under similar conditions of dietary and hormonal regimens but which do not have levels of threonine dehydrogenase and glusose 6-P-dehydrogenase outside those attainable in normal rat liver. It appears that the internal milieu of the hepatoma cells as well as their adaptive mechanisms are altered compared t o normal cells. Thus, the hepatoma cell has an altered blood supply (absence of portal circulation) altered excretory pathway (absence of biliary system), and altered enzyme systems for eliminating specific substances. All these factors seriously affect the interpretation of the results of studies dealing with enzyme levels. If the alteration of the internal milieu occurs, it implies

DEVELOPMENT, BIOCHEMISTRY, BIOLOGY O F HEPATOMAS

29 1

that as yet unknown changes in structure of qualitative enzyme patterns have occurred in the neoplastic cell. These dietary studies were extended by Potter (1964~)t o groups of normal and Hepatoma 5123TC-bearing rats maintained for 2 weeks on 0, 12, 30, 60, and 90% casein diets with glucose as the carbohydrate. The animals were allowed food only during 12 hours of darkness. KOfood was available during 12 hours of light. Labeling was accomplished by H3thymidine 1 hour before sacrifice a t each of 6, 12, 18, and 24 hour periods. Samples were fixed for autoradiographic identification of DNA. In addition, DNA was extracted from homogenized samples for counting and determinations were made on the homogenates for TdRkinase, ornithilie transaminase, serine dehydrase, and glycogen. Hepatoma 5123TC exhibited a cyclic alteration in DNA labeling, according to Potter (1964c), which was in general similar t o that found in normal or host liver but a t a much higher level. These unexpected findings showed complex systematic patterns not reflecting the variations found in normal and host liver and in certain instances in opposite phase. Additional studies must be made for a series of hepatomas to confirm the uniformity or diversity of minimal deviation hepatomas which may be related t o oscillatory metabolic functions before a general interpretation of the present observations can be made. Some additional suggestions as to mechanism(s) possibly involved in carcinogenesis are discussed in the final section. XIII. Some Other Possible Mechanism (s) of Carcinogenesis at the Molecular Level

The transplantable malignant neoplasms of the liver of rats have many different biological and biochemical properties, as have heen described in the preceding sections. No two of these neoplasms so far studied in considerable detail are identical even though in some instances they have been induced under identical conditions in the same liver. These irreversible changes which have been induced in the liver cell form a spectrum of liver neoplasms with fewer biochemical and biological deviations than were previously available, but the “minimal” deviation tumor has not yet been obtained. The present tumors afford the nearest approach to normal liver cells, and should be helpful in determining some initial key events or series of eveiits at the molecular level, whether they be single or multiple in nature, which will distinguish the neoplastic from the normal cell. It should be pointed out also that hepatomas are not unique in having many different biological and biochemical properties, for the urinary proteins in a series of transplantable plasma-cell mouse neoplasms studied by M. Potter et al. (1964) were structurally different in each of more than twenty tumor lines. Each type was heritable in that the same protein could

292

HAROLD P. MORRIS

be isolated repeatedly from different transfer generations. According t o Potter et al. (1964), the protein synthesized by each tumor reflects the sustained activity of the genes that control its synthesis and can be regarded as a heritable marker for that tumor. The type of protein formed by the tumor appears t o be established in the primary neoplasm and the ability to form each type is transmitted to the daughter cells during mitosis and provides an interesting system of neoplasia useful for the study of protein formation under genic direction, possibly through enzymatic or metabolic pathways. These observations of M. Potter et al. (1964) not only demonstrate the great variability among a spectrum of neoplasms induced by a single carcinogen but should provide information relating to both the biochemical nature of the neoplastic cell and the molecular basis of antibody specificity. These plasma-cell neoplasms with their extensive array of heritable markers offers another experimental neoplastic system quite comparable in diversity to the transplantable hepatomas. Both the plasmacell neoplasms and the transplantable minimal deviation hepatomas further illustrate the necessity to study neoplasia at the molecular level because it appears that the usual histological characterizations are insufficient to demonstrate the biochemical differences now known to occur in a spectrum of tumors arising from the same organ or tissue (cf. Section IV). Furthermore, the determination of tumor incidence, or the influence of a variety of environmental factors thereon (important as such studies may be), probably will not disclose the basic molecular mechanism(s) involved in carcinogenesis; they do not reveal the diversity of properties which the writer believes exists at the molecular level in tumors. Probably a variety of mechanisms are involved in the induction of cancer. Some additional possibilities are briefly described below. 1. A repair mechanism in a mutant strain of Escherichia Cali K12 which has lost the ability to reactivate UV-irradiated T I phage without light has been studied by Boyce and Howard-Flanders (1964). They have found this repair mechanism t o be controlled by three genes. A defect in any one will prevent the repair in DNA. Their observations indicate that the repair is effected by the binding together of two thymine molecules covalently linked t o form a thymine dimer (Wacker, 1963). Boyce and HowardFlanders were of the opinion that the thymine dimers, possibly as oligonucleotides, may be excised by specific nucleases from one-stranded DNA followed by insertion enzymatically of nucleotides in the excised portion with base-pairing complimentary to the opposite strand that had remained intact. The phosphodiester structure becomes rejoined, thus effecting the repair of the twin-stranded molecule. Boyce and Howard-Flanders (1964) suggest that the enzymatic removal of the damage followed by reconstruction of the DNA from the base sequence of the complimentary strand

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could, if it applies to mammalian organisms, be a n important mechanism in carcinogenesis or in the preservation of DNA. If the repair could occur by error from a noncomplimentary strand an abnormal or altered DNA structure might arise which, if it permitted cell survival, might eventually give rise to self-duplicating cells independent of host control. 2. Another possible mechanism for the regulation of gene action is that proposed by Allfrey (19G4) through certain atomic groups such as the acetyl group attached to histones. The histones are bound tightly to the genetic material of the cell and may inhibit chromosomal function by limiting RNA synthesis in differentiated cells. The removal of histones from the nucleus results in increased RNA synthesis (Allfrey et al., 1963; Allfrey and Mirsky, 1963). This histone suppression of RNA synthesis may be due in large part to complex formation between the added histones and the DNA needed to serve as a template in the polymerase reaction. The inhibition of RNA polymerases by histones has been observed in mammalian systems (Allfrey and Mirsky, 1963). Acetylation of the histones, however, no longer effectively inhibited RNA synthesis by the DNA-dependent RNA polymerase of calf thymus nuclei despite the ability of these altered histones to hind DNA. Allfrey (1964) is of the opinion that more unknown subtle mechanisms may exist which alter histone and chromosome structure to permit both inhibition and reactivation of RNA production a t different DNA loci along the chromosome. If the alterations leading to neoplasia occur by alteration of histone and chromosome structure it, appears reasonable t o believe that many different DNA loci along the chromosome may be attacked by chemical carcinogens or their metabolites, leading to a large variety of biochemically different neoplasms. 3. Such a view appears to be supported in other studies by Magee and Farber (1962), who noted that alliylating carcinogens alkylate transfer RNA much more than they do DNA and that the pattern of such alkylation is aberrant. Thus, Magee and Farber (1062) observed that sites of RNA which are normally free of methyl groups are alkylated; for example, 7-methylguanine, a normally rarely methylated base that occurs in abundance in transfer RNA of rats exposed to carcinogenic methylating agents. Such compounds may be carcinogens by causing aberrant or excessive methylations of DNA or RNA. 4. Srinivasan and Borek (1964) have found enzymes in microorganisms which activate transmethylation at the polymer level of the previously formed transfer RNA. The bases are methylated after the formation of the polymeric nucleic acids and are called by Srinivasan and Borek RNA methylases. According t o Gold and Hurwitz (1964a,b), in E . coli strain W there are no less than six enzymes involved in the introduction of methyl groups in various positions of transfer RNA and four other enzymes which

294

HAROLD P. MORRIS

activate both transfer RNA and ribosomal RNA; a separate enzyme fraction is specific for DNA in these organisms. These methylases are ubiquitously distributed. Every tissue examined contained the enzymes (Srinivasan and Borek, 1964), but they are not the same for the various bases in all species. Some possibilities which DNA might follow in the process of uncoiling and replication are ( a ) the damage might be repaired by insertion of a guanine molecule in place of methylguanine, ( b ) another base might be incorporated introducing an error in coding, (c) the gap in the DNA chain may be cleaved as a result of a process of /%elimination of a phosphate ester bond (Svrinivasan and Borek, 1964). Methylating enzymes foreign to the host cell could yield aberrant methylation of transfer RNA with anomalies both in the concentration and in the distribution of the methyl groups. If changes occur in patterns and levels of methylating enzymes the “minimal deviation” hepatomas which can be compared directly with normal liver from which they originated, especially if they possess all the liver ‘Lmarker’’enzymes, may be a useful neoplasm in providing experimental material for tests of such mechanisms of oncogenesis, because it is believed they represent examples of the earliest stages of cancer cells now available in transplantable form, and because they provide sufficient normal and neoplastic material from which t o compare the biochemical and biological differences whereby one can distinguish the key differences. 5 . A distinctive pattern of deoxynucleotide metabolism by every tumor cell type has been postulated by Roth (1963a) which would determine its rate of proliferation. The tumor pattern would differ in one or more ways from the normal adult pattern. Roth noted that patterns of deoxynucleotide metabolism differ strikingly in different hepatomas. According to Roth some hepatomas have a favorable balance with respect to some anabolic reactions (e.g., Novikoff hepatoma). I n other hepatomas (e.g., Dunning) this does not seem to be the case. Yet both tumors proliferate rapidly, leading one to postulate the existence of alternative anabolic pathways or greatly decreased catabolic degradation of deoxynucleotides. Roth (1963a) postulated that the initial steps leading t o a neoplastic cell induced by a chemical carcinogen were probably random. In such cells the alterations may effectively disrupt metabolic processes. Some cells would be unable t o divide and many may die. I n other cases the nature of the alterations may, for survival, force a change in the metabolic pattern of the cell. Some of these changes may actually favor cell proliferation in comparison t o normal adult cells. If these altered cells continued their growth they would eventually kill their host. This explanation seems feasible for rapidly growing hepatomas, but is not sufficient t o explain the alterations occurring in many of the slower growing hepatomas. The author has noted in his laboratory one transplantable hepatoma that weighed

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approximately 2 g. 22 months after inoculation into the new host, a rate of growth slower than that which occurs in the growth of the normal liver in a young growing rat. In the next transfer generation this hepatoma produced large tumors in 8 months. If several inactivation gene changes occurred, however, the amount of dCMP deaminase synthesis, for example, could be greatly affected. Substitution of one nucleotide in a triplet could lead to a triplet that codes for a different amino acid. If the final protein (enzyme) has one or more substitutions then there could occur (a) a completely inactive protein enzyme, (b) lower protein enzyme activity, (c) higher protein enzyme activity. Transplantable hepatomas as described herein are now available with many enzyme activities deleted, some deleted or depressed, others higher than normal, while many tumor enzyme activities appear much like the normal liver. Enzymes may have substrate binding sites, repressor sites, and feedback inhibitor sites (Pardee and Wilson, 1963). Changes in physicochemical properties, K,, etc., of the enzymes (cf. Table 111; Weber et al., 1964) may affect their reaction rates and the hormonal control may be deleted or repressed (Cho et al., 1964). Enzymes such as dCMP deaminase may be different in some rat hepatomas from the dCMP deaminase in normal rat liver. An almost unlimited number of alterations of the normal liver could lead to neoplastic livers and a vast amount of work remains to be done a t the molecular level for an understanding of the events leading to neoplastic growth. 6. A tentative model for chemical hepatocarcinogenesis has been proposed by Pitot and Peraino (1964) which includes an irreversible alteration in the structure of the endoplasmic reticulum. This alteration could result in changes in the rates of synthesis of protein enzymes and a partial failure in the ability of the synthesizing machinery to respond normally to exogenous regulators such as amino acids, hormones, glucose, etc. The changes must not cause cell death but must permit net protein synthesis and autonomous growth independent of critical factors that control growth of adjacent normal cells. Pitot and Peraino (1964) propose that these structural-functional membrane defects be transmitted to daughter cells via some non-DNA inheritance. Further, as growth and replication rates increase the genome may be secondarily altered in a way that would favor the perpetuation of the defects. Pitot and Peraino (1964) suggest that a rational approach to reverse the carcinogenic process would be a reversal or conversion of the malignant cell back to its normal counterpart. The model system proposed by Pitot and Peraino (1964) presents an entirely new approach to the procedures that might be useful in the control of the neoplastic disease. To have any practical value, however, it would have to be effective by reversing all of the cells in any given tumor. Such a concept seems unlikely from our present degree of oncogenesis sophistication. It appears more probable

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HAROLD P. MORRIS

that a n unrepairable defect (or defects) that becomes altered during repair in the genome occurs during the action of the chemical carcinogen or a metabolite thereof in hepatocarcinogenesis, which then secondarily could alter irreversibly the structure of the endoplasmic reticulum as proposed by Pitot and Peraino (1964) [cf. Roth (1963a), Srinivasan and Borek (1964), Magee and Farber (1962), and Allfrey (1964), Potter (1964b)l. It is believed that there must be heritable control mechanisms that determine how or when an enzyme-forming system (enzyme-protein) will function. It is remarkable that such a complex a machine as outlined in Fig. 1 (Weber, 1963b) should be so difficult to alter. Speaking now of the liver a single impulse furnished by a chemical carcinogen usually does not result in even one very well-differentiated hepatoma. Repeated doses of the carcinogen usually appear necessary. This leads one to the belief (a) that it is extremely difficult to alter irreversibly the normal homeostasis of the cell and (b) that a series of probably irreversible alterations must take place before that cell arises which no longer is under the full control of the organisms control mechanisms-one that no longer can make all the adaptations required to stay within the control mechanisms of the organism and, therefore, has become a neoplastic cell. We must explore many more tumors with fewer and fewer changes from the normal. We must also use the presently known methods of attack available to the molecular biologist, and apply newer and newer procedures as they become available or are developed. The above suggestions are not all inclusive or all exclusive. It is likely that not one but many mechanisms involving the activity of the genes may be operating during the induction of neoplasia. It is undoubtedly a many faceted problem and should be attacked from many angles. It is also believed that success in elucidating some of the mechanism(s) of carcinogenesis is more likely to come from multiple working hypotheses because they distribute the work as well as divide the affections of investigators who may become unwittingly biased in the support of a single hypothesis. A group of hypotheses encompass the causation of cancer from all sides and, therefore, the total outcome from many approaches should be full and profitable in their future accomplishments. ACKNOWLEDGMENTS Valuable criticisms of the first draft of this review by Dr. Helen M. Dyer and Dr. Michael Potter are gratefully acknowledged.

REFERENCES Adelberg, E. A., and Umbarger, H. E. 1953. J. Bid. Chem. 205,

Aisenberg, A. C., and Morris, H. P. 1961. Nature 191, 1314-1316. Aisenberg, A. C.,and Morris, H. P. 1963. Cancer Res. 23, 5&568.

475-482.

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