Carcinoplacental isoenzyme antigens

CARCINOPLACENTAL ISOENZYME ANTIGENS W. H. FISHMAN Cancer Research Center, Tufts University School of Medicine, New England Medical Center Hospitals, Boston, Massachusetts INTRODUCTION What is a Car¢inoplacental Isoenzyme Antigen ? This term was invoked to describe the identical antigenicity of placental alkaline phosphatase (E.C. 3.1.3.1) isoenzyme and the Regan isoenzyme in neoplastic tissue and to emphasize the absence of placental isoenzyme from fetal tissues and serum. The Regan isoenzyme is a placental type of alkaline phosphatase first identified in a metastatic squamous cancer of the lung in a patient, Mr. Peter Regan (1, 2). On the other hand, carcinofetal or oncofetal (3) antigens would refer to proteins which occur in tumors and in the fetus but are missing from the placenta. Both carcinoplacental and carcinofetal antigens would then belong to the general category of carcinoembryonic antigens. This term has already been employed by Gold (4) to refer to a gastrointestinal tract antigen in the fetus which appears in tumors of the gastrointestinal tract in adults. The following scheme may be helpful: carcinoembryonic antigens

I I

I

carcinofetal

carcinoplacental

Gold antigen ~-Fetoprotein

Regan isoenzyme Nagao isoenzyme Regan variantq Warnock

Non-carcinoembryonic antigens such as those attributed to virus or tissue transplantation phenotypes would form a separate category. Fetal forms of several enzymes have also been recognized but these have been studied most extensively in experimental tumors. Reviews on this subject have recently appeared (5-7). 293

294

w.H. FISHMAN

The purpose of this paper is to consider the phenomenon of the existence of a carcinoplacental antigen in human tumors which is at the same time a recognized isoenzyme. In addition to an account of the phenomenon, new evidence will be presented which relates to the identification of placental phenotypes of alkaline phosphatase in tumors and to the ultrastructural locations so far revealed for tumor alkaline phosphatases. The outlines of families of Regan and non-Regan isoenzyme phenotypes can be seen. Finally, an effort will be made to place this phenomenon into its place in the modern concepts of the nature of the cancer cell. REGAN ISOENZYME

In 1967 a 30-year-old white male had X-ray evidence of a right upper lobe infiltrate and after the biopsy of a large supraclavicular node was done, the diagnosis of metastatic bronchogenic squamous cell carcinoma was made. The brain metastases required dexamethazone and X-ray therapy. Beginning in July the patient exhibited a progressive rise in serum alkaline phosphatase which was not accompanied by any change in SGOT, SGPT and bilirubin levels. Several bone surveys were negative for bone metastases. Two months later he expired and the autopsy findings revealed a large tumor mass arising from the main stem bronchus of the right lung with extensive involvement of the mediastinal, periaortic and supraclavicular lymph nodes and both adrenal glands. Multiple tumor nodules were found in the spleen, kidneys and brain. At that time, we were able to distinguish the alkaline phosphatases prepared from liver, bone, intestine and placenta by a combination of enzyme tests involving L-phenylalanine inhibition, heat-inactivation, starch gel electrophoresis and neuraminidase treatment (8-12). It was then established that approximately 50 ~o of the elevated serum alkaline phosphatase was heatstable and L-phenylalanine-sensitive. These properties were most pronounced in extracts of tissues totally replaced by tumor which also exhibited the strongest alkaline phosphatase activity. The behavior of the heat-stable, L-phenylalanine-sensitive moiety on starch gel electrophoresis before and after neuraminidase treatment was indistinguishable from placental alkaline phosphatase. Intense cytoplasmic alkaline phosphatase activity, often associated with large granules, was observed in tumor cells of thin sections processed for the Kaplow enzyme-staining reaction for alkaline phosphatase. This staining reaction was completely abolished by the presence in the medium of L-phenylalanine but not by o-phenylalanine. Subsequent studies have centered on the enzyme kinetics of placental (13) alkaline phosphatase, L-phenylalanine inhibition (14, 9, 15) and the sialoprotein nature of the placental isoenzyme (16). Immunologic studies (17, 18) have shown lines of identity between alkaline phosphatase antigens of placental and tumor tissue and the corresponding

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295

rabbit antisera. Moreover, the antigen-antibody complexes so produced exhibit identical losses in the enzyme activity of the antigen as a function o f dilution of antiserum. The retarded migration rate of such antigen-antibody complexes has been taken advantage of in developing qualitative tests for Regan isoenzyme (1, 19). A summary of the properties of the four recognized isoenzymes of alkaline phosphatase is given in Table 1. TABLE 1. PROPERTIES OF ALKALINE PHOSPHATASES OF LIVER, BONE, INTESTINE AND PLACENTA

Alkaline phosphatase of

Inhibition by L-phenylalanine(%) Inhibition by L-homoarginine(%) Heat-inactivation (%) Anodal migration on starch gel (cm) Effect of pre-treatment with neuraminidase Reaction with dilute antisera to placental isoenzyme Reaction with dilute antisera to liver isoenzyme Reaction with dilute antisera to intestinal isoenzyme

Liver

Bone

Intestine

Placenta

References

0-10

0-10

75

75

(8)

78 50-70

78 90-100

5 50-60

5 0

(20), (21) (8)

4.4-5.0

4.0-6.0

3.0

3.84.2

(1)

+

+

0

+

(1)

0

0

+

(19)

+

0

0

(22)

0

+

(23), (24)

Kang et al. (25) confirmed the production of placental type alkaline phosphatase from lung-cancer tissue. A poorly differentiated adenocarcinoma yielded an alkaline phosphatase preparation which behaved exactly like placental isoenzyme with regard to inhibition by L-phenylalanine, heat inactivation, and urea; to enzyme kinetics (Michaelis constants), to gel electrophoresis, and to immunologic tests. Examination of the activities of a few other enzymes, including acid phosphatase, GOT, G P T and L D H showed no difference between lung cancer and its metastases and normal lung and liver. NAGAO ISOENZYME Nakayama et aL (26) discovered a L-leucine and L-phenylalanine-sensitive heat stable alkaline phosphatase isoenzyme in a patient (Mr. Nagao) with

296

w. H. FISHMAN

pleuritis carcinomatosa. The pleura1 fluid enzyme was stable at 65 ° for 10 rain, migrated to a position between alpha-2 and beta globulin and it and placental isoenzyme were identical immunologically. Unlike Fishman's experiences, urea inactivation was the same from 1 M to 8 M. L-Leucine (0.4 mM) inhibited 50 ~ of neoplastic enzyme activity and only 6 ~o of placental alkaline phosphatase. "Similar results were obtained with other aliphatic amino acids such as isoleucine or valine." Next, 0.4 mM EDTA inhibited neoplastic isoenzyme 5 0 ~ and placental not at all. Km with phenylphosphate was 0.26 mM for neoplastic as compared to 2.2 mM for placental isoenzyme. N o t e ! agar gel electrophoresis with napththol AS-MX phosphate showed identical electrophoretic mobility of the two phosphatases. (Data in the present paper show a difference with starch gel electrophoresis.) More recently, Nakayama et al. (27) have demonstrated the Nagao isoenzyme in the tumor tissue of a patient with primary adenocarcinoma of the tail of the pancreas. Confirmation has come from the studies of Jacoby and Bagshawe (28) who made a butanol extract of liver metastases from an adenocarcinoma of the bile duct, precipitated the enzyme with cold acetone and chromatographed the solution of the precipitate on a DEAE cellulose column. In the presence of L-phenylalanine (5 mM) the activities of both tumor and placental alkaline phosphatases were inhibited around 90 ~ but in the presence of L-leucine (0.5 mM), the tumor enzyme was inhibited 60 ~o while the placental one, only 15 ~. Similarly, the tumor enzyme was more strongly inhibited by EDTA. Moreover, the tumor enzyme was heat-labile at pH 11.0 (56°1 hr) in contrast to the placental isoenzyme. Using vertical flat-bed acrylamide gel electrophoresis at pH 9.5, the placental phosphatase appeared as three closely spaced bands while the tumor showed a more diffuse band at the same position. Both isoenzymes were precipitated by rabbit antiserum to human placental isoenzyme. On Ouchterlony plates, tumor, placental and intestinal enzymes showed a definite interaction.

REGAN VARIANT (WARNOCK AND REISMAN)

Warnock and Reisman (29) found a Regan isoenzyme variant in eight of ten patients with hepatocellular cancer and two of sixty control livers. This variant moved faster than liver in starch gel electrophoresis, was moderately heat-stable and was inhibited by L-phenylalanine. It was immunologically distinct from ~-fetoprotein. Neuraminidase cleaved the variant. Recently, Higashino et al. (30) have demonstrated this isoenzyme was immunologically identical to the placental isoenzyme and it was L-leucinesensitive as well. A summary of known Regan isoenzymes appears in Table 2.

20

75 79 90 92

Hepatoma (29) Pleuritis carcinomatosa (26)

Liver metastases Adenocarcinoma bile duct (28) 62

n.d. 70

n.d.

78

L-leucine 0.4 mM

Squamous cell cancer of the lung (variant D) (17, 2) Adenocarcinoma of lung (25)

Tumor

Inhibition L-phenylalanine ( ~ ) 5 mM placental phenotype placental phenotype (polyacrylamide) pre-albumin placental pbenotype (agar gel) placental phenotype (polyacrylamide)

Electrophoresis corresponds to

Properties

-I-

n.d. +

+

+

Immunologic test with anti-P1

TABLE 2. REGAN ISOENZYMES OF ALKALINE PHOSPHATASE

inhib. unlike P1 inhib. unlike P1

inhib. like P1

n.d.

EDTA

stable at neutral pH; sens. pH 11.0

stable

stable

stable

Heat

0.26 mM (phenylphosphate)

2.27 mM (phenylphosphate)

0.55 m M

Km

IO -..j

O rn Z N

rrl :Z

z

298

w.H. FISHMAN HELA CELL ALKALINE PHOSPHATASE

HeLa cells are a human cell line derived from cancer of the cervix. Certain sublines do show a placental-type of alkaline phosphatase (1, 31). Spencer and Macrae (32) confirmed the observation (33) that in HeLa cells nuclei possess 6 4 ~ of the alkaline phosphatase activity and found, in addition, that 29 ~ of the total activity is located in the mitochondrial fraction. Beryllium inhibition studies showed that HeLa enzyme is more sensitive than non-HeLa alkaline phosphatase. "When 10-SM beryllium was added to monolayer cultures, enlarged nuclei and no cell division were observed by phase contrast microscopy after 2 days. At 10-3 M beryllium cell death occurred within one day." Earlier Chevremont and Firket (34) proposed that inhibition of cell division by beryllium is mediated via alkaline phosphatase. Spencer and Macrae found that nuclear alkaline phosphatase is inhibited more completely than mitochondrial enzyme by L-phenylalanine, while the reverse is true for beryllium. Mitochondrial enzyme is slightly heat labile. Previously, Cox and McLeod (35) had suggested that alkaline phosphatase production by cultured human cells could provide an enzyme marker which might be useful in studying regulation by mammalian genes. They discovered the ability of prednisolone, added to the culture medium of HeLa cells, to enhance alkaline phosphatase activity (36). This system was next studied by Griffen and Cox (37), who examined the properties of the base level and corticoid-induced enzymes. Most recently, Cox et al. (38) have published evidence indicating that the increase in enzyme activity may be due to an increase in the catalytic efficiency of the enzyme. Elson and Cox (31) and Fishman et al. (I) reported that HeLa cells in culture exhibit an alkaline phosphatase of placental type. Studies in our laboratory by Kelly et al. (39) have centered on another heteroploid human cell line (C-sp) which exhibits a high titer of only Regan isoenzyme. This single isoenzyme of alkaline phosphatase was a consistent feature of the C-sp cells even after 350 generations. Originally, the C-sp cells appeared as loci of epitheliaMike cells in one lot of human diploid Wl-38 fibroblast cells. If the C-sp cells were products of transformation of the fibroblasts, then such transformants should be recognizable in the Wl-38 cell population by their cytochemically positive alkaline phosphatase. This was not the case. The first C-sp cells were identified by morphology alone, the alkaline phosphatase test being negative. However, in later passages, these epithelioid cells acquired intense alkaline phosphatase activity. It was then found that upon mixing Wl-38 cells with C-sp cells the alkaline phosphatase cytochemical activity became visibly diminished, the effect being most pronounced at high ratios of W1-38 to C-sp cells. Later experiments utilizing a double culture technique with a single communication

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299

showed that the reduction in enzyme activity did not require direct cell contact. Evidence that a repressor substance was produced by the W1-38 cells was obtained. Finally the effect of the W1-38 repressor extract was reversible upon transferring the C-sp cells to fresh medium. These results are suggestive of the action of the repressor substance at the translational level of phenotype expression. NON-REGAN ISOENZYME IN HUMAN CANCER Stimulated by the discovery of Regan isoenzyme in the first such case in cancer of the lung, Timpefley (40) examined a number of lung cancers and found one which produced a non-Regan isoenzyme. This alkaline phosphatase was heat labile and L-phenylalanine-insensitive. Later, Timpedey and Warnes (41) found a similar non-Regan isoenzyme in five meningiomas and in two craniopharyngiomas (42). In the latter tumor extracts, a main broad band (acrylamide gel) was seen to migrate to a slower position than liver. Ectopic production of non-Regan alkaline phosphatase was reported by Romslo et al. (43) in a patient with widespread skeletal metastases from a ventricular mucoid adenocarcinoma. Six months earlier a Krukenberg tumor of the ovary of the same histologic type had been removed. The enzyme was characterized as heat labile, L-phenylalanine-sensitiveand different from liver, intestine, placenta and bone. The difference from bone resided in Km values, inhibition by L-arginine and L-histidine, electrophoresis and Sephadex column gel filtration. A summary of the known neoplastic non-Regan isoenzymes appears in Table 3. Tumors may show alkaline phosphatase in the noncancer cell moieties as, for example, in Jensen and Schiodt's study (44) of breast carcinoma and fibroadenomatosis. Here, little enzyme activity was noted in the tumor cells but the surrounding stroma often showed reaction zones containing proliferating fibroblasts with high phosphatase activity, the incidence of which increased with the degree of anaplasia of the tumors. These authors consider that TABLE3. NON-REGANISOENZYMESOFALKALINEPHOSPHATASE Properties Tumor Craniopharyngiomas, meningiomasand cancer of lung (40-42) Choriocarcinoma(17)

Inhibition by L-phenylalanine

Heat-inactivation

Electrophoresis

none 14.5%

ioo% 90%

bonetype ?