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Comparison of pathological and biochemical studies of transplanted morris hepatomas
COMPARISON OF PATHOLOGICAL AND BIOCHEMICAL STUDIES OF TRANSPLANTED MORRIS HEPATOMAS LYNNARD SLAUGHTER, HAROLD P. MORRIS and WAYNE E. CRISS Howard University,College of Medicine, School of Human Ecology.Cancer Center Washington, D.C. 20059
INTRODUCTION
Over the past three decades, investigators have utilized a series of transplantable rat hepatocellular carcinomas which were developed in our laboratory (1-6). During these years a large body of literature concerning the biochemistry of this spectrum of hepatocellular carcinomas has accumulated. The Morris hepatoma system has thus provided an ideal model to study the relationships that exist between the biochemistry, the morphology, and behavior patterns of cancer cells as elucidated by G. Weber and S. Weinhouse and their colleagues (7-12). A principal concern of our laboratory has been to assure the constant stability and uniformity of these tumors which would allow for the ease of reproducing biologically un-biased experiments and the confidence that the data derived by numerous investigators using these tumors would be a valid representation of each tumor cell line during several transplant generations. Over the past five years, 10 consecutive generations of each of 57 hepatoma cell lines have been carefully monitored with respect to their salient morphologic features, including the per cent of viable tumor cells, other tissue elements, and the degrees of differentiation of each tumor cell line (Table I). Also, the malignant potential of these tumors was monitored for the presence or absence of metastatic lesions, particularly throughout the respiratory system. In the present report we have presented a critical evaluation of five of these hepatoma lines with regard to their composite morphological features. in addition, we have reviewed four biochemical research programs which have made extensive use of these five tumor lines.
RESULTS
Tumor Morphology Transplantable tumors: 16 and 9618A. Morphologically these tumors are highly differentiated and resemble normal hepatocytes in which 65% of the malignant cells are viable. T u m o r 9618A tissue (4) growth pattern (Figs. I and 19
20
LYNNARD SLAUGHTER, et al. TABLE I. MORPHOLOGIC AND BIOLOGIC CHARACTERISTICS: SELECTED T R A N S P L A N T A B L E H E P A T O C E L L U L A R CARCINOMAS
Tumor number Generations
Average of 10
Growth rate (months) Chromosome number
16
9618A
44
7800
3924A
3-12
4--13
17-26
10 NA
12 42
5 NA
2 42
I 69-74
+ + + + m o + 80 h
+ + r + m o + 67 h
+ + + + + m o + 60 w
+ + + + m o + 50 w
+ + + r + e f + 35 u
+ +
+ +
+ +
+ +
+ -
-
(-)
+
+
-
+
(-)
-
(-)
+
110--119 365-374
Epithelial cell morphologic patterns
Nodules Cords Trabecular Compact sheet Cysts Pleomorphism Anaplasia Mitosis Necrosis c// of viable cells Degree of differentiation Mesen~.h.vmal.leature.~
Vascular channels Fibrosis. Pulmonarl" metatases Pneumonia
+ = present; - = absent: r = rare; m -- minimal: e = extreme; f = frequent; o = occasional; h = highly differentiated; w = well differentiated: u = undifferentiated; (-) = variable and minimal; NA = not available. 2) c o n s i s t s o f m u l t i p l e c o m p a c t s o l i d v a r i a b l e s i z e d n o d u l e s o f t u m o r c e l l s t h a t are either c o m p l e t e l y or partially s u r r o u n d e d by b r o a d and n a r r o w straps of m a t u r e f i b r o u s t i s s u e . T r a n s p l a n t a b l e t u m o r 16 t i s s u e g r o w t h p a t t e r n c o n s i s t s of broad compact
t r a b e c u l a e . In c o n t r a s t w i t h t u m o r 9 6 1 8 A , t u m o r
16
o c c a s i o n a l l y has m i t o t i c f i g u r e s a n d n a r r o w e l o n g a t e d s t r a p s o f f i b r o u s t i s s u e between some trabeculae. e i t h e r t u m o r 16 o r 9 6 1 8 A . 7800, t h e f o r m e r t u m o r s monitis, peribronchiolitis
P u l m o n a r y m e t a s t a s e s r a r e l y o c c u r in r a t s b e a r i n g i n c o n t r a s t w i t h a n i m a l s b e a r i n g t u m o r s 44 (4) a n d have extensive nonsuppurative interstitial pneuand perivasculitis.
T r a n s p l a n t a b l e t u m o r s : 4 4 a n d 7800. T h e s e t u m o r s a r e m o r p h o l o g i c a l l y ( F i g s . 3,-4) c h a r a c t e r i z e d as well d i f f e r e n t i a t e d h e p a t o c e i l u l a r c a r c i n o m a s in w h i c h 5 0 % o f t h e m a l i g n a n t cells a r e v i a b l e . T i s s u e g r o w t h p a t t e r n s v a r y f r o m n a r r o w t o b r o a d c o r d s a n d / o r b r o a d e l o n g a t e d t r a b e c u l a e o f cells t h a t resemble normal hepatocytes. Mitotic figures and fibrosis are occasionally o b s e r v e d t h r o u g h o u t the tissue. Rats bearing these t r a n s p l a n t a b l e t u m o r s
FIG. I. Transplantable
FIG. 2. Transplantable
heptocellular
hepatocellular
carcinoma
carcinoma
16, generation
9618A.
generation
12. 400X
IO. 400X.
FIG.
FIG.
3. I’ransplantable
4. Transplantable
hepatocellular
hepatoeellular
carcinoma
carcinoma
44. generation
7800, generation
24. 400X.
118. 400X
FIG.
5. Transplantable
hepatocellular
carcinoma
3924A.
generation
372. 400X.
PATHOLOGY
AND
BIOCHEMISTRY
21
OF HEPATOMAS
regularly have multiple disseminated viable tumor cellular metastases throughout all lung lobes. The lungs of animals bearing tumor 44 usually do not have evidence of pneumonia whereas animals bearing 7800 occasionally have focal areas of nonsuppurative pneumonitis. Transplantable tumor: 3924A. This highly undifferentiated (Fig. 5) carcinoma is usually composed of less than 30% viable tumor cells (5, 6). Sixty-five to 70% of this tumor is composed of degenerating and necrotic epithelial cells. The tissue growth pattern consists of loose or compact sheets of cells that do not resemble hepatocytes. Numerous mitotic figures and occasional narrow straps of fibrous tissue exist throughout the tumor. Rats bearing this tumor do not have pulmonary metastases. The lungs of these animals usually have extensive nonsuppurative pneumonitis, peribronchiolitis and perivasculitis. Survey of Several Current Research Programs Hormone requiring tumors. Several human and experimental tumors are hormone dependent or hormone responsive. Such terminology implies that these tumors require certain hormones for growth and/or functioning. In the late 1960s S. Weinhouse and W. E. Criss (unpublished data) noted that certain Morris hepatomas required insulin and cortisol for either optimal growth and/or expression of certain hormone responsive enzyme systems. Recently, S. Mishkin and his colleagues (13, 14) have observed that the growth of Morris hepatomas 44 and 7800 is dramatically slowed in hypothyroid rats. In the latter studies, Buffalo rats carrying Morris hepatomas 44 or 7800 were made hypothyroid with propylthiouracil or ll31. The hypothyroid state inhibited both tumor growth and lung metastases. and increased survival time of the rats bearing the tumors. Exogenously added thyroxine reversed all of these effects (Table 2). Therefore, it is quite possible
TABLE2.
INHIBlTlONOFGROWTHOFHEPATOMA44INHYPOTHYROlDRATS(l4) Decreased growth (o/c of controls)
Treatment
66 87 75
PTU 1”’ Surgical TX Tumor-bearing P. Value PTU + T, P. value
controls
Xi PT
(ll2.l)f9.9
285.1 f 25.5 0.00 I 296.1 f 17.7 NS
PTU = 6-propylthiouracil(O.I9c in Purina Chow): TX = surgical thyroidectomy: (8 ug/ kg body weight); NS = not significant. SGPT = Serum glutamic pyruvate transaminase, E.C. 2.6.1.2.
T, = thyroxin
22
LYNNARD
SLAUGHTER.
cr al.
that several of the Morris hepatomas are hormone requiring not necessarily require the same hormonal combination.
tumors,
but may
Cholesterol feedback inhibition system. Feeding of cholesterol to most laboratory animals (and humans) produces a marked decrease in the rate at which the livers of such animals synthesize cholesterol. Feedback inhibition of cholesterologenesis is one of the most sensitive regulatory systems in higher animals (15). This feedback regulatory control is missing in Morris hepatomas (16-19). The control is located at the conversion site of R-hydroxy-g-methylglutaric acid to mevalonic acid which occurs in the membrane portion of liver cell’s microsomes. Hydroxymethylglutaryl-CoA reductase (NADPH) (EC I. I. 1.34) is therefore the logical specific enzymatic system for the molecular lesion of this control point in the hepatomas. There are several possible reasons which might contribute to a molecular lesion at this point: (a) faulty active site on the enzyme; (b) faulty regulatory site on the enzyme; (c)inability of the enzyme to undergo allosteric shifts; (d) a regulatory signal from some other source not reaching the enzyme and (e) alteration in an inhibitor (signal) to the enzyme. It is anticipated that further studies by M. D. Siperstein and his colleagues using Morris hepatomas will provide for a more complete understanding of the feedback control involved in cholesterol synthesis. System of cyclic nucleorides. Cyclic AMP and cyclic GMP are intracellular “second messenger” systems which respond to extracellular stimuli and, in turn, regulate a variety of cellular functions in all mammalian tissues. Most of the major components of this cyclic nucleotide regulatory system have been examined in the Morris hepatomas (8, 9, 20-24). Whereas the cyclic AMP sub-system is highly responsive to hormones (glucagon and epinephrine) in normal liver tissues, in hepatomas the cyclic AMP responsiveness is almost completely lost. The latter is due to loss of membrane receptors for these hormones, decreased basal adenylate cyclase activity (EC 4.6. I. I). decreased responsiveness of adenylate cyclase to these hormones, and increased cyclic AMP specific phosphodiesterase (EC 3. I .4.17). In direct contrast, the cyclic GMP system is highly active. Increased levels of membrane guanylate cyclase (EC 4.6. I .2) and decreased levels of cyclic GMP specific phosphodiesterases (EC 3.1.4.17) have resulted in greatly elevated levels of cyclic GMP. In addition, increased levels of the calcium ion and the cytoplasmic form of calmodulin are found. Cyclic AMP and cyclic GMP responsive protein kinases (EC 2.7.1.37) have been observed. Further, other protein kinases which are independent of the cyclic nucleotides but respond to various growth regulators such as the polyamines have been identified. Additional studies are needed to determine if these trends are typical of all rapidly growing tissues, or whether they are characteristic only to the Morris hepatomas.
PATHOLOGY
AND BIOCHEMISTRY
OF HEPATOMAS
23
Combination experimental tumor therap.v. W. 0. Looney and his colleagues have been performing a series of studies using single and combined (radiation and chemical) therapy on several of the Morris hepatomas (25-28). They have determined cell doubling times, cell loss factors, growth fractions, and cell cycle intervals (G,, S, Gr. M) for hepatomas with IO-fold or more differences in growth rates (26). Using single chemotherapeutic agents (such as 5-fluorouracil, adriamycin and cyclophosphamide) tumor responses can be classified as complete regression, definite local regression, pseudo regression, and slowdown. Similar types of tumor responses are observed when single doses of radiation (375-2250R) are given to the tumors. Collectively, tumor tissue diversity (in terms of cell growth fraction, cell loss factor, cell doubling time, relationship of interstitial to vascular connective tissue, and parenchymal versus connective cell densities) plays a major role in the tumor’s response to either or both types of therapy. Utilization of combined modality therapy on hepatomas with various growth rates has also been useful. It has become apparent that radiation and/ or cell cycle specific chemotherapeutic agents (e.g., 5-fluorouracil) are capable of not only killing tumor cells but also of synchronizing many of the remaining viable cells in the tumor. For example, increased synchrony is achieved at 12 hr post-radiation and 24 hr post 5-fluorouracil treatment of Morris hepatoma 3924A. Further studies of the effects of 5-fluorouracil and radiation of this tumor model now indicate that whereas neither therapeutic agent will control tumor growth when used alone, in proper combination (dosage levels and treatment intervals) the two modes of therapy will indeed control tumor growth in Morris hepatoma 3924A. These results imply that therapeutic strategies for combined tumor therapy should be based upon: (a) recovery of lost and critical organs of the host from previous treatment; and (b) administration of second subsequent treatments at times of maximum tumor susceptibility (maximum rate of tumor cell proliferation) (27).
SUMMARY
Ten consecutive generations of each of 57 hepatoma cell lines, 5000 transplantable tumors, have been monitored to define their salient morphologic features. Also the lungs of rats bearing these tumors were evaluated for evidence of transplantable tumor cellular metastases. These evaluations have led us to conclude the following. (a) The pattern of tumor cell growth is uniform and consistent from generation to generation. (b) Tumor cell pleomorphism is extremely variable. (c) All tumors are well vascularited. (d) Mitoses are variable between generations. (e) Varying amounts of necrosis are always present in all tumor cell lines. (f) The degree of differentiation is uniform in a given tumor line. (g) All well differentiated transplantable tumors metastasize to normal lungs but not to inflamed lungs. (h)
24
LYNNARD
SLAUGHTER,
ef of.
Undifferentiated or poorly differentiated transplantable tumor cells rarely metastasize to the lungs. Four major program areas are reviewed. (a) Several of the Morris hepatomas may be hormone dependent or hormone responsive with regard to glucagon, insulin, cortisol or thyroxine. (b) The feedback regulatory control in cholesterol synthesis, which is missing in every Morris hepatoma, is probably located at the hydroxymethylglutaryl-CoA reductase site ofaction. (c) The intracellular “second messenger” system in the hepatomas shows lost responsiveness of the cyclic AMP sub-system and increased reponsiveness of the cyclic GMP sub-system. (d) The major factors in determining a tumor tissue’s response to radiation and/or chemotherapy are cell doubling times, cell loss factors, cellular growth fractions, and cell cycle intervals.
REFERENCES
I.
2.
3.
4.
5.
6.
7. 8.
9
IO.
I I. 12. 13.
1.. J. SLAUGHTER and H. P. MORRIS.Themorphologyoflungtissuefrom ratscarrying transplantable Morris hepatomas. p. I in Seven/h Bi-Annual .Y,t,mposium: Morris Heparoma Symposium (H. P. MORRIS and W. E. CRISS. eds.) (1979). 1.. J. SLAUGHTER and H. P. MORRIS. The morphologic and biologic behavior of the Morris hepatoma, pp. 16-21 in Proc. Annual Meeting American Assoc. of Lahoraror,v Animal Science ( 1979). Histopathology of transplantable hepatic carcinomas induced by M. D. REUBER. chemical carcinogens, pp. 43-54 in Gann Monograph I: Biologic and Biochemical Evaluation of Ma1ignanc.v in Experimental Heparomas (0. HAYAISHI and H. P. MORRIS. eds.). The Japanese Foundation for Cancer Res. and The Japanese Cancer Assoc. ( 1966). P. C. NOWELL. H. P. MORRIS and V. R. POTTER. Chromosomes of minimum deviation hepatomas and some other transplantable rat tumors. Cancer Res. 27,1565-1579 ( 1967). H. P. MORRIS and L. J. SLAUGHTER. Development. growth rate of malignancy and chromosome pattern of Morris transplantable hepatoma.Journa/ Toxicol. Environ. Heal/h 5. 263-282 (1979). H. P. MORRIS and L. J. SLAUGHTER. Historical development of transplantable hepatomas. pp. l-19 in Morris Hepalomas: Mechanisms qf Regubrion (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). G. WEBER. Ordered and specific pattern of gene expression in neoplasia. AJ~wws in Enz,vme Regulation I I, 79-I I5 ( 1973). G. WEBER. The molecular correlation concept of neoplasia and the cyclic AMP system. pp. 57- 102 in The Role qf Colic Nucleorides in Carcinogenesis (J. SCHULTZ and H. G. GRATZNER. eds.). Academic Press, New York (1973). J. F. CLARK. H. P. MORRIS and G. WEBER. Cyclic adenosine 3’. 5’ monophosphate phosphodiesterase activity in normal, differentiating. regenerating. and neoplastic liver. Cancer Res. 33, 356-36 I ( 1973). G. WEBER. E. OLAH. M. S. LUI and D. TZENG, Biochemical programs and enzymepattern-targeted chemotherapy in cancer cells. Adrwmes in Enzt.me Regulurion 17, l-21 ( 1979). S. WEINHOUSE. J. B. SHATTOS. W. E. CRISS. F. A. FARINAand H. P. MORRIS. Isozymes in relation to differentiation in transplantable rat hepatomas. pp. I-19. GANN. Vd 13. Japanese Cancer Assoc., Univ. Tokyo Press. Tokyo. Japan. (1973). W. E. CRISS. A review of isozymes. Cuncer Res. 31. 1523-1526 (1971). S. MISHKIN. H. P. MORRIS. M. A. YALOVSKY and P. V. N. MURTHY. Increased survival of rats bearing Morris hepatoma 7800 after induction of hypothyroidism. Comer Re.s. 39. 2371-2375 (1979).
PATHOLOGY 14.
15. 16. 17.
18. 19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
S. MISHKIN. Hypothyroidism
AND
BIOCHEMISTRY
OF HEPATOMAS
25
H. P. MORRIS, R. POLLACK. M. YALOVSKY and S. MISHKIK. inhibits the primary and metastatic growth of Morris hepatoma #44 and
prolongs survival: effect of administering I”’ at different stages of tumor development. Proc. Sot. Am. Assoc. Study of Liver Diseases (1980). M. D. SIPERSTEIN and M. J. GUEST, Studies on the site of the feedback control of cholesterol synthesis, J. C/in. Invesrig. 39, 642-646 (1960). M. D. SIPERSTEIN. V. M. GAGAN and H. P. MORRIS, Further studies on the deletion of cholesterol feedback system in hepatomas. Cancer Res. 26, 7-I I ( 1966). M. D. SIPERSTEIN. A. M. HYDE and H. P. MORRIS. Loss of control of hydroxyglutaryl coenzyme A reductasc in hepatomas. Proc. Nat/. Acad. .Gi.. USA 68. 315-317 (1971). L. A. BRICKER. H. P. MORRIS and M. D. SIPERSTEIN. Loss of the cholesterol feedback system in the intact hepatoma bearing rat. J. C/in. Intwrig. 51,206-215 (1972). M. H. WII.EY and M. D. SIPERSl-EIN. Control of cholesterol synthesis in normal and malignant cells. pp. 343-350 in Conlrol Mec+~ani.sm.cin Cancer (W. E. CRISS. I. ON0 and J. R. SABINE. eds.). Raven Press. New York (1976). W. E. CRISS and F. MURAD. Cyclic nucleotide metabolism in solid tumors. pp. 429-451 in Clinical Aspwrs of Colic Nuckorides (L. VOI.ICER. ed.). Spectrum Publishing. Kew York (1977). R. A. HICKIE. Regulation of cyclic AMP and cyclic GMP in Morris hepatoma. pp. 451-488 in Morris Heparomas: Mechanisms of Regulation (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). W. E. CRISS. P. MUGANDA. A. SAHAI and H. P. MORRIS. Cyclic nucleotide metabolism in solid tumor tissues, pp. 489-516 in Morris Hepa/oma.t: Mec~hanisnu o/ Regularion (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). W. E. CRISS. M. YAMAMOTO. Y. TAKAI. Y. NISHIZUKA and H. P. MORRIS. Requirement of polycation for enzymatic activity of a new protein kinase-substrate complex from Morris hepatoma 3924A. Cancer Res. 38. 3532-3539 (1978). K. UENISHI. W. E. CRISS and S. KAKIUCHI. Calcium activatable phosphodiesterase activity and calcium dependent modulator protein in transplantable hepatoma tissues. Biochem. J. 87,601~608 ( 1980). W. B. LOONEY. J. S. TREFIL, H. A. HOPKINS. C. J. KOVACS. E. R. RITENORand J.G. SCHAFFNER. Solid tumor models for the assessment of different treatment modalities: therapeutic strategy for sequential chemotherapy with radiotherapy. Proc,. Nor/. Acad. Si.. USA 74, 1983-1987 (1977). W. B. LOONEY. H. A. HOPKINS and J. S. TREFII.. Solid tumor models for the assessment of different treatment modalities: VI. Perturbations in the kineticsoftumorand host organ cellular therapy. pp. 677-7$7 in Morri.v Hepawmas: Mechankms of Rrgularwn (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). W. B. LOONEY. E. R. RITENOUR and H. A. HOPKISS. Changes in growth rate ofan experimental solid tumor following increasing doses of cyclophosphamide. Cancer Res. 40. 2179-2183 (1980). J. V. MOORE. H. A. HOPKINS and W. B. LOONEY. Dynamic histology of a rat hepatoma and response 10 5-fluorouracil. Cell 7is.,uc~ Kin&.\ 13. Q-63 ( 1980).
INTRODUCTION
Over the past three decades, investigators have utilized a series of transplantable rat hepatocellular carcinomas which were developed in our laboratory (1-6). During these years a large body of literature concerning the biochemistry of this spectrum of hepatocellular carcinomas has accumulated. The Morris hepatoma system has thus provided an ideal model to study the relationships that exist between the biochemistry, the morphology, and behavior patterns of cancer cells as elucidated by G. Weber and S. Weinhouse and their colleagues (7-12). A principal concern of our laboratory has been to assure the constant stability and uniformity of these tumors which would allow for the ease of reproducing biologically un-biased experiments and the confidence that the data derived by numerous investigators using these tumors would be a valid representation of each tumor cell line during several transplant generations. Over the past five years, 10 consecutive generations of each of 57 hepatoma cell lines have been carefully monitored with respect to their salient morphologic features, including the per cent of viable tumor cells, other tissue elements, and the degrees of differentiation of each tumor cell line (Table I). Also, the malignant potential of these tumors was monitored for the presence or absence of metastatic lesions, particularly throughout the respiratory system. In the present report we have presented a critical evaluation of five of these hepatoma lines with regard to their composite morphological features. in addition, we have reviewed four biochemical research programs which have made extensive use of these five tumor lines.
RESULTS
Tumor Morphology Transplantable tumors: 16 and 9618A. Morphologically these tumors are highly differentiated and resemble normal hepatocytes in which 65% of the malignant cells are viable. T u m o r 9618A tissue (4) growth pattern (Figs. I and 19
20
LYNNARD SLAUGHTER, et al. TABLE I. MORPHOLOGIC AND BIOLOGIC CHARACTERISTICS: SELECTED T R A N S P L A N T A B L E H E P A T O C E L L U L A R CARCINOMAS
Tumor number Generations
Average of 10
Growth rate (months) Chromosome number
16
9618A
44
7800
3924A
3-12
4--13
17-26
10 NA
12 42
5 NA
2 42
I 69-74
+ + + + m o + 80 h
+ + r + m o + 67 h
+ + + + + m o + 60 w
+ + + + m o + 50 w
+ + + r + e f + 35 u
+ +
+ +
+ +
+ +
+ -
-
(-)
+
+
-
+
(-)
-
(-)
+
110--119 365-374
Epithelial cell morphologic patterns
Nodules Cords Trabecular Compact sheet Cysts Pleomorphism Anaplasia Mitosis Necrosis c// of viable cells Degree of differentiation Mesen~.h.vmal.leature.~
Vascular channels Fibrosis. Pulmonarl" metatases Pneumonia
+ = present; - = absent: r = rare; m -- minimal: e = extreme; f = frequent; o = occasional; h = highly differentiated; w = well differentiated: u = undifferentiated; (-) = variable and minimal; NA = not available. 2) c o n s i s t s o f m u l t i p l e c o m p a c t s o l i d v a r i a b l e s i z e d n o d u l e s o f t u m o r c e l l s t h a t are either c o m p l e t e l y or partially s u r r o u n d e d by b r o a d and n a r r o w straps of m a t u r e f i b r o u s t i s s u e . T r a n s p l a n t a b l e t u m o r 16 t i s s u e g r o w t h p a t t e r n c o n s i s t s of broad compact
t r a b e c u l a e . In c o n t r a s t w i t h t u m o r 9 6 1 8 A , t u m o r
16
o c c a s i o n a l l y has m i t o t i c f i g u r e s a n d n a r r o w e l o n g a t e d s t r a p s o f f i b r o u s t i s s u e between some trabeculae. e i t h e r t u m o r 16 o r 9 6 1 8 A . 7800, t h e f o r m e r t u m o r s monitis, peribronchiolitis
P u l m o n a r y m e t a s t a s e s r a r e l y o c c u r in r a t s b e a r i n g i n c o n t r a s t w i t h a n i m a l s b e a r i n g t u m o r s 44 (4) a n d have extensive nonsuppurative interstitial pneuand perivasculitis.
T r a n s p l a n t a b l e t u m o r s : 4 4 a n d 7800. T h e s e t u m o r s a r e m o r p h o l o g i c a l l y ( F i g s . 3,-4) c h a r a c t e r i z e d as well d i f f e r e n t i a t e d h e p a t o c e i l u l a r c a r c i n o m a s in w h i c h 5 0 % o f t h e m a l i g n a n t cells a r e v i a b l e . T i s s u e g r o w t h p a t t e r n s v a r y f r o m n a r r o w t o b r o a d c o r d s a n d / o r b r o a d e l o n g a t e d t r a b e c u l a e o f cells t h a t resemble normal hepatocytes. Mitotic figures and fibrosis are occasionally o b s e r v e d t h r o u g h o u t the tissue. Rats bearing these t r a n s p l a n t a b l e t u m o r s
FIG. I. Transplantable
FIG. 2. Transplantable
heptocellular
hepatocellular
carcinoma
carcinoma
16, generation
9618A.
generation
12. 400X
IO. 400X.
FIG.
FIG.
3. I’ransplantable
4. Transplantable
hepatocellular
hepatoeellular
carcinoma
carcinoma
44. generation
7800, generation
24. 400X.
118. 400X
FIG.
5. Transplantable
hepatocellular
carcinoma
3924A.
generation
372. 400X.
PATHOLOGY
AND
BIOCHEMISTRY
21
OF HEPATOMAS
regularly have multiple disseminated viable tumor cellular metastases throughout all lung lobes. The lungs of animals bearing tumor 44 usually do not have evidence of pneumonia whereas animals bearing 7800 occasionally have focal areas of nonsuppurative pneumonitis. Transplantable tumor: 3924A. This highly undifferentiated (Fig. 5) carcinoma is usually composed of less than 30% viable tumor cells (5, 6). Sixty-five to 70% of this tumor is composed of degenerating and necrotic epithelial cells. The tissue growth pattern consists of loose or compact sheets of cells that do not resemble hepatocytes. Numerous mitotic figures and occasional narrow straps of fibrous tissue exist throughout the tumor. Rats bearing this tumor do not have pulmonary metastases. The lungs of these animals usually have extensive nonsuppurative pneumonitis, peribronchiolitis and perivasculitis. Survey of Several Current Research Programs Hormone requiring tumors. Several human and experimental tumors are hormone dependent or hormone responsive. Such terminology implies that these tumors require certain hormones for growth and/or functioning. In the late 1960s S. Weinhouse and W. E. Criss (unpublished data) noted that certain Morris hepatomas required insulin and cortisol for either optimal growth and/or expression of certain hormone responsive enzyme systems. Recently, S. Mishkin and his colleagues (13, 14) have observed that the growth of Morris hepatomas 44 and 7800 is dramatically slowed in hypothyroid rats. In the latter studies, Buffalo rats carrying Morris hepatomas 44 or 7800 were made hypothyroid with propylthiouracil or ll31. The hypothyroid state inhibited both tumor growth and lung metastases. and increased survival time of the rats bearing the tumors. Exogenously added thyroxine reversed all of these effects (Table 2). Therefore, it is quite possible
TABLE2.
INHIBlTlONOFGROWTHOFHEPATOMA44INHYPOTHYROlDRATS(l4) Decreased growth (o/c of controls)
Treatment
66 87 75
PTU 1”’ Surgical TX Tumor-bearing P. Value PTU + T, P. value
controls
Xi PT
(ll2.l)f9.9
285.1 f 25.5 0.00 I 296.1 f 17.7 NS
PTU = 6-propylthiouracil(O.I9c in Purina Chow): TX = surgical thyroidectomy: (8 ug/ kg body weight); NS = not significant. SGPT = Serum glutamic pyruvate transaminase, E.C. 2.6.1.2.
T, = thyroxin
22
LYNNARD
SLAUGHTER.
cr al.
that several of the Morris hepatomas are hormone requiring not necessarily require the same hormonal combination.
tumors,
but may
Cholesterol feedback inhibition system. Feeding of cholesterol to most laboratory animals (and humans) produces a marked decrease in the rate at which the livers of such animals synthesize cholesterol. Feedback inhibition of cholesterologenesis is one of the most sensitive regulatory systems in higher animals (15). This feedback regulatory control is missing in Morris hepatomas (16-19). The control is located at the conversion site of R-hydroxy-g-methylglutaric acid to mevalonic acid which occurs in the membrane portion of liver cell’s microsomes. Hydroxymethylglutaryl-CoA reductase (NADPH) (EC I. I. 1.34) is therefore the logical specific enzymatic system for the molecular lesion of this control point in the hepatomas. There are several possible reasons which might contribute to a molecular lesion at this point: (a) faulty active site on the enzyme; (b) faulty regulatory site on the enzyme; (c)inability of the enzyme to undergo allosteric shifts; (d) a regulatory signal from some other source not reaching the enzyme and (e) alteration in an inhibitor (signal) to the enzyme. It is anticipated that further studies by M. D. Siperstein and his colleagues using Morris hepatomas will provide for a more complete understanding of the feedback control involved in cholesterol synthesis. System of cyclic nucleorides. Cyclic AMP and cyclic GMP are intracellular “second messenger” systems which respond to extracellular stimuli and, in turn, regulate a variety of cellular functions in all mammalian tissues. Most of the major components of this cyclic nucleotide regulatory system have been examined in the Morris hepatomas (8, 9, 20-24). Whereas the cyclic AMP sub-system is highly responsive to hormones (glucagon and epinephrine) in normal liver tissues, in hepatomas the cyclic AMP responsiveness is almost completely lost. The latter is due to loss of membrane receptors for these hormones, decreased basal adenylate cyclase activity (EC 4.6. I. I). decreased responsiveness of adenylate cyclase to these hormones, and increased cyclic AMP specific phosphodiesterase (EC 3. I .4.17). In direct contrast, the cyclic GMP system is highly active. Increased levels of membrane guanylate cyclase (EC 4.6. I .2) and decreased levels of cyclic GMP specific phosphodiesterases (EC 3.1.4.17) have resulted in greatly elevated levels of cyclic GMP. In addition, increased levels of the calcium ion and the cytoplasmic form of calmodulin are found. Cyclic AMP and cyclic GMP responsive protein kinases (EC 2.7.1.37) have been observed. Further, other protein kinases which are independent of the cyclic nucleotides but respond to various growth regulators such as the polyamines have been identified. Additional studies are needed to determine if these trends are typical of all rapidly growing tissues, or whether they are characteristic only to the Morris hepatomas.
PATHOLOGY
AND BIOCHEMISTRY
OF HEPATOMAS
23
Combination experimental tumor therap.v. W. 0. Looney and his colleagues have been performing a series of studies using single and combined (radiation and chemical) therapy on several of the Morris hepatomas (25-28). They have determined cell doubling times, cell loss factors, growth fractions, and cell cycle intervals (G,, S, Gr. M) for hepatomas with IO-fold or more differences in growth rates (26). Using single chemotherapeutic agents (such as 5-fluorouracil, adriamycin and cyclophosphamide) tumor responses can be classified as complete regression, definite local regression, pseudo regression, and slowdown. Similar types of tumor responses are observed when single doses of radiation (375-2250R) are given to the tumors. Collectively, tumor tissue diversity (in terms of cell growth fraction, cell loss factor, cell doubling time, relationship of interstitial to vascular connective tissue, and parenchymal versus connective cell densities) plays a major role in the tumor’s response to either or both types of therapy. Utilization of combined modality therapy on hepatomas with various growth rates has also been useful. It has become apparent that radiation and/ or cell cycle specific chemotherapeutic agents (e.g., 5-fluorouracil) are capable of not only killing tumor cells but also of synchronizing many of the remaining viable cells in the tumor. For example, increased synchrony is achieved at 12 hr post-radiation and 24 hr post 5-fluorouracil treatment of Morris hepatoma 3924A. Further studies of the effects of 5-fluorouracil and radiation of this tumor model now indicate that whereas neither therapeutic agent will control tumor growth when used alone, in proper combination (dosage levels and treatment intervals) the two modes of therapy will indeed control tumor growth in Morris hepatoma 3924A. These results imply that therapeutic strategies for combined tumor therapy should be based upon: (a) recovery of lost and critical organs of the host from previous treatment; and (b) administration of second subsequent treatments at times of maximum tumor susceptibility (maximum rate of tumor cell proliferation) (27).
SUMMARY
Ten consecutive generations of each of 57 hepatoma cell lines, 5000 transplantable tumors, have been monitored to define their salient morphologic features. Also the lungs of rats bearing these tumors were evaluated for evidence of transplantable tumor cellular metastases. These evaluations have led us to conclude the following. (a) The pattern of tumor cell growth is uniform and consistent from generation to generation. (b) Tumor cell pleomorphism is extremely variable. (c) All tumors are well vascularited. (d) Mitoses are variable between generations. (e) Varying amounts of necrosis are always present in all tumor cell lines. (f) The degree of differentiation is uniform in a given tumor line. (g) All well differentiated transplantable tumors metastasize to normal lungs but not to inflamed lungs. (h)
24
LYNNARD
SLAUGHTER,
ef of.
Undifferentiated or poorly differentiated transplantable tumor cells rarely metastasize to the lungs. Four major program areas are reviewed. (a) Several of the Morris hepatomas may be hormone dependent or hormone responsive with regard to glucagon, insulin, cortisol or thyroxine. (b) The feedback regulatory control in cholesterol synthesis, which is missing in every Morris hepatoma, is probably located at the hydroxymethylglutaryl-CoA reductase site ofaction. (c) The intracellular “second messenger” system in the hepatomas shows lost responsiveness of the cyclic AMP sub-system and increased reponsiveness of the cyclic GMP sub-system. (d) The major factors in determining a tumor tissue’s response to radiation and/or chemotherapy are cell doubling times, cell loss factors, cellular growth fractions, and cell cycle intervals.
REFERENCES
I.
2.
3.
4.
5.
6.
7. 8.
9
IO.
I I. 12. 13.
1.. J. SLAUGHTER and H. P. MORRIS.Themorphologyoflungtissuefrom ratscarrying transplantable Morris hepatomas. p. I in Seven/h Bi-Annual .Y,t,mposium: Morris Heparoma Symposium (H. P. MORRIS and W. E. CRISS. eds.) (1979). 1.. J. SLAUGHTER and H. P. MORRIS. The morphologic and biologic behavior of the Morris hepatoma, pp. 16-21 in Proc. Annual Meeting American Assoc. of Lahoraror,v Animal Science ( 1979). Histopathology of transplantable hepatic carcinomas induced by M. D. REUBER. chemical carcinogens, pp. 43-54 in Gann Monograph I: Biologic and Biochemical Evaluation of Ma1ignanc.v in Experimental Heparomas (0. HAYAISHI and H. P. MORRIS. eds.). The Japanese Foundation for Cancer Res. and The Japanese Cancer Assoc. ( 1966). P. C. NOWELL. H. P. MORRIS and V. R. POTTER. Chromosomes of minimum deviation hepatomas and some other transplantable rat tumors. Cancer Res. 27,1565-1579 ( 1967). H. P. MORRIS and L. J. SLAUGHTER. Development. growth rate of malignancy and chromosome pattern of Morris transplantable hepatoma.Journa/ Toxicol. Environ. Heal/h 5. 263-282 (1979). H. P. MORRIS and L. J. SLAUGHTER. Historical development of transplantable hepatomas. pp. l-19 in Morris Hepalomas: Mechanisms qf Regubrion (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). G. WEBER. Ordered and specific pattern of gene expression in neoplasia. AJ~wws in Enz,vme Regulation I I, 79-I I5 ( 1973). G. WEBER. The molecular correlation concept of neoplasia and the cyclic AMP system. pp. 57- 102 in The Role qf Colic Nucleorides in Carcinogenesis (J. SCHULTZ and H. G. GRATZNER. eds.). Academic Press, New York (1973). J. F. CLARK. H. P. MORRIS and G. WEBER. Cyclic adenosine 3’. 5’ monophosphate phosphodiesterase activity in normal, differentiating. regenerating. and neoplastic liver. Cancer Res. 33, 356-36 I ( 1973). G. WEBER. E. OLAH. M. S. LUI and D. TZENG, Biochemical programs and enzymepattern-targeted chemotherapy in cancer cells. Adrwmes in Enzt.me Regulurion 17, l-21 ( 1979). S. WEINHOUSE. J. B. SHATTOS. W. E. CRISS. F. A. FARINAand H. P. MORRIS. Isozymes in relation to differentiation in transplantable rat hepatomas. pp. I-19. GANN. Vd 13. Japanese Cancer Assoc., Univ. Tokyo Press. Tokyo. Japan. (1973). W. E. CRISS. A review of isozymes. Cuncer Res. 31. 1523-1526 (1971). S. MISHKIN. H. P. MORRIS. M. A. YALOVSKY and P. V. N. MURTHY. Increased survival of rats bearing Morris hepatoma 7800 after induction of hypothyroidism. Comer Re.s. 39. 2371-2375 (1979).
PATHOLOGY 14.
15. 16. 17.
18. 19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
S. MISHKIN. Hypothyroidism
AND
BIOCHEMISTRY
OF HEPATOMAS
25
H. P. MORRIS, R. POLLACK. M. YALOVSKY and S. MISHKIK. inhibits the primary and metastatic growth of Morris hepatoma #44 and
prolongs survival: effect of administering I”’ at different stages of tumor development. Proc. Sot. Am. Assoc. Study of Liver Diseases (1980). M. D. SIPERSTEIN and M. J. GUEST, Studies on the site of the feedback control of cholesterol synthesis, J. C/in. Invesrig. 39, 642-646 (1960). M. D. SIPERSTEIN. V. M. GAGAN and H. P. MORRIS, Further studies on the deletion of cholesterol feedback system in hepatomas. Cancer Res. 26, 7-I I ( 1966). M. D. SIPERSTEIN. A. M. HYDE and H. P. MORRIS. Loss of control of hydroxyglutaryl coenzyme A reductasc in hepatomas. Proc. Nat/. Acad. .Gi.. USA 68. 315-317 (1971). L. A. BRICKER. H. P. MORRIS and M. D. SIPERSTEIN. Loss of the cholesterol feedback system in the intact hepatoma bearing rat. J. C/in. Intwrig. 51,206-215 (1972). M. H. WII.EY and M. D. SIPERSl-EIN. Control of cholesterol synthesis in normal and malignant cells. pp. 343-350 in Conlrol Mec+~ani.sm.cin Cancer (W. E. CRISS. I. ON0 and J. R. SABINE. eds.). Raven Press. New York (1976). W. E. CRISS and F. MURAD. Cyclic nucleotide metabolism in solid tumors. pp. 429-451 in Clinical Aspwrs of Colic Nuckorides (L. VOI.ICER. ed.). Spectrum Publishing. Kew York (1977). R. A. HICKIE. Regulation of cyclic AMP and cyclic GMP in Morris hepatoma. pp. 451-488 in Morris Heparomas: Mechanisms of Regulation (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). W. E. CRISS. P. MUGANDA. A. SAHAI and H. P. MORRIS. Cyclic nucleotide metabolism in solid tumor tissues, pp. 489-516 in Morris Hepa/oma.t: Mec~hanisnu o/ Regularion (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). W. E. CRISS. M. YAMAMOTO. Y. TAKAI. Y. NISHIZUKA and H. P. MORRIS. Requirement of polycation for enzymatic activity of a new protein kinase-substrate complex from Morris hepatoma 3924A. Cancer Res. 38. 3532-3539 (1978). K. UENISHI. W. E. CRISS and S. KAKIUCHI. Calcium activatable phosphodiesterase activity and calcium dependent modulator protein in transplantable hepatoma tissues. Biochem. J. 87,601~608 ( 1980). W. B. LOONEY. J. S. TREFIL, H. A. HOPKINS. C. J. KOVACS. E. R. RITENORand J.G. SCHAFFNER. Solid tumor models for the assessment of different treatment modalities: therapeutic strategy for sequential chemotherapy with radiotherapy. Proc,. Nor/. Acad. Si.. USA 74, 1983-1987 (1977). W. B. LOONEY. H. A. HOPKINS and J. S. TREFII.. Solid tumor models for the assessment of different treatment modalities: VI. Perturbations in the kineticsoftumorand host organ cellular therapy. pp. 677-7$7 in Morri.v Hepawmas: Mechankms of Rrgularwn (H. P. MORRIS and W. E. CRISS. eds.). Plenum Press. New York (1978). W. B. LOONEY. E. R. RITENOUR and H. A. HOPKISS. Changes in growth rate ofan experimental solid tumor following increasing doses of cyclophosphamide. Cancer Res. 40. 2179-2183 (1980). J. V. MOORE. H. A. HOPKINS and W. B. LOONEY. Dynamic histology of a rat hepatoma and response 10 5-fluorouracil. Cell 7is.,uc~ Kin&.\ 13. Q-63 ( 1980).