Y-Glutamyltransferase in Putative Premalignant Liver Cell Populations During Hepatocarcinogenesis1

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[CANCER RESEARCH 38, 823-829, March 1978] y-Glutamyltransferase in Putative Premalignant Liver Cell Populations During Hepatocarcinogenesis1

Ross Cameron,2 John Kellen, Arnost Kolin, Aaron Malkin, and Emmanuel Farber

Department of Pathology. University of Toronto. Toronto U5G 1L5 Â¡R.C., A. K., E. F.], and Department of Clinical Biochemistry, Sunnybrook Medical Centre, Toronto M4N 3U5 [J. K., A. M.Â¡,Ontario Canada

ABSTRACT the fetus but very low activity in the adult. In primary tumors induced by aflatoxin B,, 3'-Me-DAB, N-OH-2-AAF, and 2- The activity of y-glutamyltransferase, as measured AAF and in transplantable hepatomas of rats, a 30- to 50- quantitatively and by histochemical staining, was studied fold increase in GGT activity has been observed (8, 9, 11, in different cell populations during the induction of liver 34). GGT activity of whole liver showed a 5-fold increase as cancer with 2-acetylaminofluorene (2-AAF) or diethylni- early as 4 weeks after 2-AAF treatment, by 6 weeks after 3'- trosamine and compared with findings in fetal and in Me-DAB, and by 8 weeks after N-OH-2-AAF (9, 11). Since intact and regenerating adult liver. The enzyme activity is duct epithelial cells show the highest concentration of GGT 20-fold higher in 12-week nodules than in control livers in normal liver as judged by histochemistry and since and 30-fold higher in 20-week nodules than in controls. A proliferation of these cells ("oval cells"), often to striking similar 30-fold increase in activity relative to control is degrees, is a common reaction in the liver during carcino- present in hepatomas, induced by either 2-AAF or dieth- genesis with many chemicals, the interpretation of the ylnitrosamine, and in fetal hepatocytes. The enzyme increase in GGT activity in homogenates of whole liver is shows increases in activity in foci of very early putative complicated. Histochemically, GGT is demonstrable in pro preneoplastic hepatocytes induced by a single dose of liferating bile duct cells [oval cells (5)] after 4 weeks of N- diethylnitrosamine and selected by low doses of 2-AAF hydroxy-A/-2-fluorenylacetamide treatment (11) and in hy- plus partial hepatectomy. By 7 days, the foci show a 4- perplastic nodules induced by aflatoxin B, (15) and N-OH-2- fold increase in enzyme activity, and by 3 weeks they are AAF (11). These observations suggested that GGT may be a 40-fold higher than in the control liver. Histochemically, marker not only for neoplastic hepatocytes but also for the foci are strongly positive for y-glutamyltransferase, preneoplastic hepatocytes (15). especially in the bile canaliculi. By 21 days, the ductular A number of hepatocyte populations have been deline (oval) cells induced by 2-AAF have disappeared. When ated as possible precursor lesions for liver cancer induced stained for the enzyme activity, the foci stand out clearly by chemical carcinogens (7). Recently, in this laboratory, a against the negative background of the liver, allowing putative premalignant population of hepatocytes, "the fo easy quantitation. It appears that y-glutamyltransferase cus," has been identified morphologically within 1 week is a useful marker for preneoplastic hepatocytes. after carcinogen treatment (29). These proliferating foci of hepatocytes emerge as early as 30 hr after partial hepatec INTRODUCTION tomy in the livers of rats treated with a single dose of diethylnitrosamine i.p. to "initiate" and with a diet contain Tissue antigens and enzymes that will serve as pheno- ing a low level of 2-AAF to "select" the initiated 2-AAF- typic markers for malignant cells are becoming increasingly resistant hepatocytes during the regeneration period after important as diagnostic aids (2, 20, 27). Some of these hepatectomy (29, 30). These foci have been produced with markers may appear in the blood and other body fluids and a number of hepatocarcinogens as initiators, namely, dieth allow for early identification of the presence of neoplastic ylnitrosamine, A/-hydroxy-/V-2-fluorenylacetamide, dimeth- cells (11, 27). In the liver, a-fetoprotein, preneoplastic ylnitrosamine, and aflatoxin B,. The foci show a functional antigen, and GGT3 have been identified as possible positive resistance to the cytotoxic effects of carcinogens, a prop markers for preneoplastic hepatocytes (4). GGT has been erty found in hyperplastic nodules and hepatomas (1, 6, studied both biochemically and histochemically in many 10). In order to confirm the possibility that GGT could be a tissues. GGT is found in a number of epithelial cells includ marker for premalignant hepatocytes, putative premalig ing those located in jejunal villi, pancreatic acini, bile ducts, nant hepatocyte populations including early hyperplastic seminal vesicles, and proximal convoluted tubules (23, 25), nodules, late hyperplastic nodules, and foci were examined as well as in lymphocytes, the retina, and choroid plexus for GGT activity, both biochemically and histochemically. (19, 22). In the liver, hepatocytes show high GGT activity in The striking increase in activity of this enzyme in such hepatocytes in comparison with those in normal and regen 1 This research was supported in part by grants from the National Cancer erating liver is the subject of this communication. Institute of Canada and Medical Research Council of Canada and the Connaught Development Fund of the University of Toronto. 2 Research Fellow of the National Cancer Institute of Canada (1974 to MATERIALS AND METHODS 1976). 3 The abbreviations used are: GGT, -y-glutamyltransferase (EC 2.3.2.2); 3'- Me-DAB, 3'-methyldimethylaminoazobenzene; N-OH-2-AAF. N-hydroxy-2- Animals and Carcinogenic Diet Regimens. For produc acetylaminofluorene; 2-AAF, 2-acetylaminofluorene. tion of foci, male Fischer 344 rats (Charles River, Wilming Received September 6. 1977; accepted December 13, 1977. ton, Mass.) weighing 100 to 200 g were given 1 dose of

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1978 American Association for Cancer Research. R. Cameron et al. diethylnitrosamine, 100 or 200 mg/kg i.p. Two weeks later, to 10 min. Protein was determined according to the method the rats were fed a standard basal diet (24% protein) of Lowry ef al. (18). containing 0.02% 2-AAF (Bioserv, Frenchtown, N. J.) for 1 â€¢y-Glutamyl-p-nitroanilide, glycylglycine, and bovine se week and were then subjected to two-thirds partial hepatec- rum albumin were obtained from Sigma Chemical Co., St. tomy (12). 2-AAF was continued 1 week after hepatectomy Louis, Mo. All other chemicals were from other commercial (Fig. 1), at which time multiple foci are visible grossly in the sources except as specified. liver as small, grayish white nodules about 1 mm in diameter GGT Histochemistry. The GGT was detected according (29). Essential controls were treated as shown in Chart 1. to the method described by Rutenberg ef al. (26). Slices of For chronic feeding experiments, the basal diet containing liver with foci, nodules, hepatomas, or control liver were 0.02% 2-AAF was fed to rats for 20 weeks continuously, and serially cut in a cryostat (5 nm), air dried, fixed in ice-cold then the rats were given Purina rat chow. For diethylnitros- acetone for 30 min, and incubated in freshly prepared amine-induced hepatomas, diethylnitrosamine was given in medium containing y-glutamyl-4-methoxy-2-naphthylamide the drinking water continuously (50 ppm) for 36 weeks. (Vega-Fox Biochemicals, Tucson, Ariz.) as substrate and Biochemistry. All animals were sacrificed by cervical Fast Blue BB salt (GURR-Searle Diagnostic, High Wy- fracture after a 24-hr period of fasting. Grossly visible combe, Buckinghamshire, England) as coupling agent (26). nodules and foci were shelled out. Where sufficient tissue Spare sections were stored at 4Â°and retained their enzy (about 0.5 g) could be obtained, biochemical analysis was matic activity unchanged for at least 6 months. Control performed on tissue from each animal separately; other sections incubated in a similar medium without substrate wise, nodules or foci and surrounding nonnodular liver were negative. tissue were pooled. Careful histological and histochemical studies were performed on all tissues used for biochemical assay. The tissue was fixed in Carnoy's solution for histo RESULTS logical examination and stained with hematoxylin and eosin. Histology. Animals fed the continuous 0.02% 2-AAF regi Gray-white hyperplastic nodules were identified as early men develop grossly visible gray-white nodules within 6 to as 6 to 8 weeks in livers of animals fed the 0.02% 2-AAF diet 8 weeks after beginning the diet. Such nodules, microscop continuously, and at 12 weeks they were sufficiently large ically, are typical hyperplastic liver nodules (3). Seven for gross isolation for discrete biochemical and morpholog months after the beginning of 2-AAF feeding, a few rats ical study. Late liver-colored hyperplastic nodules develop develop hepatocellular carcinomas and by 1 year most rats by 16 to 20 weeks on a continuous 2-AAF diet (3, 6). have hepatomas. With diethylnitrosamine given continu Assay for GGT. Tissue was homogenized in 10 volumes ously in the drinking water, rats first develop hepatomas at of ice-cold 0.25 M sucrose with 10 strokes of a Thomas about 10 months following the beginning of carcinogen homogenizer. The homogenate was centrifuged at 1,000 x treatment, and by 1.5 years most rats have hepatomas. With g for 10 min, and the pellet containing nuclei and debris a single dose of diethylnitrosamine (250 to 300 mg/kg Â¡.p.), was discarded. The supernatant was further centrifuged at some rats develop hepatomas by 12 to 15 months. All the 100,000 x g for 60 min (International IEC B60 centrifuge hepatomas were typical trabecular hepatocellular carcino with Rotor A169) to pellet the nonnuclear particulate frac mas. tion. This pellet was resuspended in 5 volumes of 0.1 M Foci appear very early following initiation with a single Tris-HCI (pH 7.6) containing 0.01 M MgCL and used for dose of diethylnitrosamine and selection with 2-AAF com assay. bined with partial hepatectomy (29) (Fig. 1). Beginning GGT activity was assayed with y-glutamyl-p-nitroanilide about 30 hr after operation, multiple focal islands of prolif as substrate and glycylglycine as acceptor, according to a erating, intensely basophilic hepatocytes are seen distrib modification (31) of the method of Szasz (32). Tissue ali- uted randomly throughout the liver. They grow rapidly, and quots of 0.5 ml were incubated with 1.5 ml of substrate by 7 days they are visible grossly as gray-white nodules mixture in a cuvet at 37Â°. The standard final reaction about 1 mm in diameter. No such foci were seen in control mixture contained 4 HIM -y-glutamyl-p-nitroanilide, 10 mM animals 4 weeks after diethylnitrosamine alone, after treat MgCL, and 100 mM Tris-HCI (pH 7.6). The reaction rate was ment with diethylnitrosamine and 2-AAF without hepatec followed in a recording spectrophotometer at 405 nm. After tomy, and with 0.9 NaCI solution in place of diethylnitrosa 3 min, 50 Â¿ilof glycylglycine were added (final concentra mine, or with diethylnitrosamine and hepatectomy but no tion in reaction mixture, 50 mM), and the reaction was AAF (29) (Chart 1). The number of foci induced by diethyl recorded for another 5 min. One unit of enzyme activity (ID) nitrosamine varies approximately linearly with the dose. is equivalent to 1 /Â¿moleof substrate transformed per min With 200 mg/kg, there were 50 foci/sq cm or about 1000/5 at 37Â°(31, 32) and is calculated with the molar extinction g liver (29). For biochemical study, a 200-mg/kg dose of coefficient forp-nitroaniline as 10,820 x 10". At the concen diethylnitrosamine was used for maximum yield of nodule tration of acceptor glyclyglycine used (50 mM), the side tissue. For histochemical studies diethylnitrosamine, 100 reactions such as substrate hydrolysis are repressed in mg/kg, was used to achieve a wider separation of the GGT- favor of the transfer of the -y-glutamyl residue from the positive foci against the negative background. The foci substrate to glycylglycine (31, 32). The relationship between continue to grow in size, but not in number, following the the enzyme concentration and enzyme activity was linear treatment regimen, so that by 8 weeks the foci have become within the range of total activity used (0 to 180 ID). The large hyperplastic nodules indistinguishable from those enzyme activity was also linear as a function of time for up induced by chronic feeding of hepatocarcinogens. Under

824 CANCER RESEARCH VOL. 38

1st week 2nd week 3rd week 4th week fold greater than that found in control untreated liver and 5- DEN to 10-fold higher than in nonnodular surrounding liver (Table 1). "Nonnodular surrounding" liver often contains Experimental [_ DEN small, grossly undetected nodules and therefore is usually a mixture of nonnodular and nodular liver. Late hyperplastic Control Cl /Ã•MfMÃ•um1 fJ> } nodules and hepatomas induced by 2-AAF show a 20- to 30- 11 ,l\L1"l fold increase in GGT activity over control livers in untreated Control C2 1"l rats of a similar age. A 25- to 30-fold increase in activity is l found in liver 100,000 x g pellets of diethylnitrosamine- Control C3 [ _L induced hepatomas (Table 1). The 100,000 x g pellets prepared from whole livers Chart 1. Diagrammatic representation of experimental and control (C1, C2, and C3) regimens. Open bars, Basal diet; hatched bars, basal diet containing foci at 7 days following hepatectomy show containing 0.02% 2-AAF; SAL, 0.9% NaCI solution; PH, two-thirds partial roughly a 4-fold increase in GGT activity relative to controls hepatectomy; DEN, diethylnitrosamine; SH, sham hepatectomy; large arrow, and, by 14 to 21 days, a 10- to 20-fold greater activity than time of injections; first small arrow, time of hepatectomy; second small arrow, end of experimental diets and start of control diet. in controls (Table 2). Foci at 21 days after hepatectomy are large enough to shell out from the liver, and GGT activities these conditions, the first hepatomas appear by about 8 in these are about 35-fold higher and at 28 days as high as months (30). 50-fold greater than in control liver 100,000 x g pellets Histochemistry. GGT activity in the control adult liver is (Table 2). The nonuniform changes in whole liver between localized in the bile duct cells in the portal areas and, most 14 and 28 days are probably due, at least in part, to the strongly, on the luminal surface of these cells. Hepatocytes progressive proliferation and subsequent disappearance of show no staining with GGT, except for 1 or 2 hepatocytes in ductular or oval cells during this period. The variation in immediate proximity to portal areas in about 1 in 10 liver their numbers seriously complicates the interpretation of acini. Hepatocytes in hyperplastic nodules, induced by the significance of any increase in GGT activity in prepara continuous 2-AAF, are GGT positive with strong positive tions made from whole liver. staining of the bile canaliculi and more diffuse staining in GGT activity in regenerating liver and in a variety of the cytoplasm. In hepatomas induced by either 2-AAF or control, carcinogen-treated liver and carcinogen-treated diethylnitrosamine, hepatocytes show positive staining out regenerating liver, in the absence of the appearance of lining the plasma membrane and more diffusely in the nodules, is uniformly low (Table 3). Fetal liver shows very cytoplasm. high GGT activity, which drops to adult levels by 6 days Foci containing GGT-positive hepatocytes are readily after birth (Chart 2). Even with only 1 value at each point, apparent as discrete areas microscopically by 3 days after the transition from fetal to adult is striking. Fetal liver partial hepatectomy. At 7 days after hepatectomy, the foci 100,000 x g pellets, in fact, show a 30-fold increase in GGT have enlarged considerably and are of sufficient size to activity compared to controls, which is a level of activity allow macroscopic quantitation of the number of foci (Fig. 1). The GGT-stained foci are easily counted because of their prominence against the negative background of the imme Table 1 diate surrounding liver. All of the hepatocytes within the GGT activity in hyperplastic nodules and hepatomas during 2-AAF foci stain positively for GGT, with especially strong linear or diethylnitrosamine carcinogenesis and in control livers staining in the bile canaliculi, and more diffuse staining in activity (IU/ the cytoplasm (Fig. 2). For 1 week following the end of 2- mg protein) in TissueNoduleNoduleHepatoma''Hepatoma''Control100,000 x gpellet70 AAF treatment, oval cell proliferation is evident (3, 29) near 12wk2-AAF, Â±35*^(S)"125 the portal areas, and these are also GGT positive (Fig. 2). 20wk2-AAF, Â±20f(5)70 This is especially prominent in this model, where the prolif basaldiet,21 wk, and Â±14r(4)110Â±30C eration of the vast majority of hepatocytes is inhibited by 17wkDiethylnitrosamine the 2-AAF. The proliferating ductular (oval) cells, however, indrinking (3)3Â± are easily distinguished on microscopic examination from wkBasal water, 36 liverControl wkBasaldiet, 12 2(6)4 the random foci of proliferating hepatocytes. By 21 days liverSurrounding wk2-AAF,diet, 20 Â± 2(5)11 following hepatectomy, the oval cells are no longer promi liver,nonnodularDiet"2-AAF, 12 weeksGGT Â± 8 (3) nent and large GGT-positive foci of hepatocytes stand out distinctly (Fig. 3). " The rats were fed 0.02% 2-AAF continuously in basal diet for Regenerating liver studied at 2, 3, 5, and 7 days shows 12 or 20 weeks. Control rats were fed basal diet. Diethylnitrosa- some diffuse GGT activity in the cytoplasm of a few peripor- mine-ifiduced hepatomas were induced by feeding 50 ppm diethyl nitrosamine in the drinking water for 36 weeks. tal cells on Days 2 and 3. At 5 and 7 days following partial 6 Mean Â±S.D. of 3 or more separate experiments. Each experi hepatectomy, positive staining for GGT was found only in ment represents the liver of 1 rat. In the case of early nodules, the bile duct cells, as in intact controls. Neonatal livers show a nodules'' The valuesof 2 or for 3 rats nodules were and occasionally hepatomas pooled. were statistically highly strong positive reaction for GGT in all of the hepatocytes, again especially intense in the area of the bile canaliculi significant when compared to controls (p < 0.001) and nodule compared to surrounding tissue (p < 0.001). (Fig. 4). '' Numbers in parentheses, number of experiments. Biochemistry. GGT activity in the 100,000 x g pellets of ' All hepatomas were classified as trabecular hepatocellular early hyperplastic nodules induced by 2-AAF is 20- to 30- carcinomas.

MARCH 1978 825

Table 2 elevated in liver during carcinogenesis induced by diethyl GGT activity in liver and in foci induced by diethylnitrosamine, 2- nitrosamine or 2-AAF confirms previous work by other AAF, and partial hepatectomy at various times after the hepatec- investigators using azo dyes (9, 34). However, as clearly tomy indicated by the histochemical findings, the interpretation GGT activity in 100,000 x g pellet III/ of the elevation in activity during carcinogenesis is subject mg protein to considerable difficulty, since the enzyme activity appears Days following partial in 2 sites, namely, proliferating ductular cells and hepato- hepatectomy" Whole liver Foci' cytes. With the majority of hepatocarcinogens, ductular 7142128Control Â± 5r-"(5)62 Â±18''(4)35 (oval cell) proliferation is commonly seen early during Â± 5"(3)51 Â±1416-'16*(4)(1) carcinogenesis (5), and these retain the enzyme activity Â±14"(6)3 seen in the ductular epithelium of resting normal liver. With (untreated)12 Â± 2 (6)106 most carcinogens the ductular cells remain as such or " The rats were given diethylnitrosamine, 200 mg/kg body disappear during carcinogenesis and have no direct rela weight i.p., in 1 dose followed by 2 weeks of 0.02% 2-AAF in the tion to the genesis of hepatocellular carcinoma (5). With the diet and subjected to partial hepatectomy as outlined in the protocol (Fig. 1). Control rats were fed basal diet. carcinogenic azo dyes, the ductular cells have a special life * Foci were shelled out of the liver, and the foci of 3 rats were history and show conversion to hepatocytes as 1 source of pooled for each experiment. hepatocellular carcinoma (see Ref. 5 for references). ' Mean Â± S.D. of 3 or more experiments. Each experiment This study indicates that diethylnitrosamine and 2-AAF, represents the liver of 1 rat. '' The values for experimental were statistically highly significant like aflatoxin B, (15) and N-OH-2-AAF (11), induce a new when compared to controls (p < 0.001). hepatocyte population that shows levels of GGT activity ' Numbers in parentheses, number of experiments. equal to or greater than those seen in hepatomas. Thus, 1 This value involved only 1 experiment. changes in total activity of GGT or any other enzyme must be carefully related to the type of cellular response if they Table 3 are to be interpreted realistically. GGT activity in regenerating liver, carcinogen-treated liver, and The most important findings in this study are those regenerating carcinogen-treated liver relating to the quantitative and histochemical observations GGT activity (III/ on GGT activity in the various hepatocyte populations that mg protein) in Tissue 100,000 x g pellet occur prior to the development of cancer with several Regenerating liver" chemicals. Clearly, GGT activity increases markedly in very 18 hr 3Â±2"(3)r early hepatocytes induced by diethylnitrosamine and se 24 hr 4 Â±1 (3) lected for resistance to the inhibition of hepatocyte prolif 48 hr 3 Â±1 (3) eration imposed by low levels of 2-AAF (29). The enzyme 3 days 4 Â±1 (2) activity levels rapidly approach those seen in hepatocellular 5 days 3 Â±1 (7) carcinoma and fetal liver, such that by 3 weeks they are in 7 days 3 Â±1 (5) Carcinogen-treated liver'' the same range. This is not a properly of regenerating liver, 14 days of 2-AAF 2 Â±1 (2) which remains in the range of the normal liver. Thus, the 14 days after diethylnitrosamine 2Â±1 (5) brief exposure to diethylnitrosamine induces a change in 4 wk after diethylnitrosamine + 2 wk of 2- 3 Â±1 (6) scattered, isolated, apparently random hepatocytes that is AAF (Chart 2, Control C1) 12 wk of 2-AAF (surrounding liver) 11 Â±8 (3) expressed not only as resistance to some cytotoxic effects Regenerating carcinogen-treated liver of another carcinogen but also as the appearance of a large 2-AAF for 2 wk + partial hepatectomy 2 Â±1 (2) amount of enzyme activity that is characteristic of fetal liver (Chart 2, Control C3) (9, 33). Another early developmental component, the ÃŸ- 2-AAF -i- partial hepatectomy + 7 days 6 Â±1 (2) basal diet (Control C3 + 7 days basal diet) 110 " These rats were subjected to two-thirds partial hepatectomy 100 (12) and sacrificed at the different times following the procedure. 90 Mean Â±S.D. of 2 or more separate experiments. Each experi l 80 ment represents the liver of 1 rat. ' Numbers in parentheses, number of experiments. I 70 '' The rats were given 0.02% 2-AAF in the diet and/or diethylni i 60 trosamine, 200 mg/kg body weight i.p. Ãˆ 50 similar to that seen in foci, nodules, and hepatomas (Tables 1 and 2).

DISCUSSION 14 20 B 2 2 4 6 12 17 42 The results of this study, showing elevated levels of GGT days days hr days days days days days days in hepatomas induced by diethylnitrosamine or 2-AAF, FETAL NEONATAL ADULT confirm in principle those of other investigators who Chart 2. GGT activity [lU/mg protein (mg. prof.)] in 100.000 * g pellet at showed a similar elevation in several rat hepatomas induced various times before and after birth (B) in normal liver from fetal, neonatal, by 3'-Me-DAB, N-OH-2-AAF, and 2-diacetylaminofluorene and adult Fischer 344 rats. Values represent the mean of 2 or more determinations on samples from a single experiment. For the fetal and early (8, 11, 34). In addition, the observation that GGT activity is neonatal rats, livers from 3 or more rats were pooled for each experiment.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1978 American Association for Cancer Research. GGT in Premalignant Hepatocytes subunit of chorionic gonadotropi!"!, or an antigenically re 3879-3887, 1976. lated substance, also appears very early in altered hepato- 7. Farber, E., Hartman, S. P., Solt, D.. and Cameron, R. Precancerous Liver Cell Populations and Their Identification. In: P. N. Magee et al. cytes (J. A. Kellen, A. Malkin, A. Kolin, R. Cameron, and E. (eds.). Fundamentals in Cancer Prevention, pp. 71-87. Tokyo: University Farber, unpublished observations). Thus, at least 3 proper of Tokyo Press, 1976. 8. Fiala, S., Fiala, A. E., and Dlxon, B. Gamma-glutamyl Transpeptidase in ties of embryonic or fetal tissue, GGT, chorionic gonadotro- Transplantable, Chemically Induced Rat Hepatomas and "Spontaneous" pin, and low levels of some activating enzymes (1) become Mouse Hepatomas. J. Nati. Cancer Inst., 48: 1393-1401. 1972. 9. Fiala, S., Mohindru, A., Kettering. W. G.. Fiala, A. E., and Morris, H. P. evident very early in a small population of hepatocytes Glutathione and Gamma-glutamyl Transpeptidase in Rat Liver during during liver carcinogenesis. These changes are seen long Chemical Carcinogenesis. J. Nati. Cancer Inst., 57: 591-598, 1976. before any behavioral properties phenotypically character 10. GrÃ¡vela, E., Feo, F.. Canuto, R. A., Garcea R., and Gabriel, C. Func tional and Structural Alterations in Liver Ergastoplasmic Membranes istic of malignant neoplasia, such as autonomous or even during DL-Ethionine Hepatocarcinogenesis. Cancer Res., 35. 3041-3047, semiautonomous growth, invasion, or metastasis, or even 1975. atypical cytological aberrations, are apparent. It is tempting 11. Harada. M.. Okabe. K Shibata, K. Masuda. H Miyota, K and Enomoto, M. Histochemical Demonstration of Increased Activity of to consider that these and perhaps other associated Gamma-glutamyl Transpeptidase in Rat Liver during Hepatocarcino changes may appear as a "functional package," conceiva genesis. Acta Histochem. Cytochem. 9: 168-179, 1976. bly due to some permanent change in 1 or more regulatory 12. Higgins, G. M., and Anderson, R. M. Experimental Pathology of the Liver. 1. Restoration of the Liver of the White Rat following Partial genes or gene clusters, and may have some role to play in Surgical Removal. Arch. Pathol., 12: 186-202. 1931. the progressive evolution to malignant neoplasm (13). 13. Hirai, H. Summation: Model Systems for the Study of Oncodevelopmen- Whether a-fetoprotein (28) is also intimately associated with tal Gene Expression â€”Neoplastic Transformation. Cancer Res., 36: 4276-4277, 1976. this early altered hepatocyte population remains to be 14. Holley, R. W. A Unifying Hypothesis Concerning the Nature of Malignant studied. Growth. Proc. Nati. Acad. Sei. U. S.. 69: 2840-2841. 1972. 15. Kalengayi. M. M. R., Ronchi, G., and Desmet, V. J. Histochemistry of Also interesting in this context is the apparent localization Gamma-glutamyl Transpeptidase in Rat Liver during Aflatoxin B,-in- of the new GGT activity to, or near, the bile canaliculus. duced Carcinogenesis. J. Nati. Cancer Inst., 55: 579-588, 1975. This, likewise, might indicate a change in a segment of the 16. Laishes, B. Gamma-glutamyl Transpeptidase: A Positive Marker for Cultured Rat Hepatocytes Derived from Putative Premalignant and membrane of the cell that could be related to the ultimate Malignant Lesions. Proc. Am. Assoc. Cancer Res., 18: 140, 1977. development of neoplasia, since membrane changes are 17. Laishes, B., and Farber. E. Properties of Carcinogen-Altered Hepato cytes in Cell Culture. Lab. Invest.. 36: 29, 1977. most easily related theoretically to neoplastic behavior (21). 18. Lowry, 0. H., Rosebrough, N. J. Farr, A. L., and Randall, R. J. Protein The functional biochemical significance of the elevated Measurement with the Folin Phenol Reagent. J. Biol. Chem., 793: 265- GGT levels in the early putative preneoplastic hepatocytes 275, 1951. 19. Meister, A. Glutathione: Metabolism and Function via the Gamma- is unknown. GGT has been postulated to function in 1 glutamyl Cycle. Life Sci., 15: 177-190, 1974. mechanism of amino acid transport into cells, as part of the 20. Munjal, D., Chawla, P. L., Lokich, J. J., and Zamchech, N. Carcmoem- y-glutamyl cycle (19, 24). Holley (14) has proposed that bryonic Antigen and Phosphohexose Isomerase, Gamma-glutamyl Transpeptidase and Lactate Dehydrogenase Levels in Patients with and accelerated or altered uptake of amino acids and other without Liver MÃ©tastases. Cancer, 37: 1800-1807, 1976. metabolites may play an essential role in the genesis of 21. Nicolson, G. Trans-membrane Control of the Receptors on Normal and Tumor Cells. II. Surface Changes Associated with Transformation and neoplasia. The availability of at least 2 grossly identifiable Malignancy. Biochim. Biophys. Acta, 458: 1-71. 1976. populations in the same liver, 1 very low and 1 very high in 22. Novogrodsky, A., TÃ¤te, S. S., and Meister, A. Gamma-glutamyl Trans GGT activity, makes this system a potentially analyzable peptidase, a Lymphoid Cell-surface Marker: Relationship to Blastogen- esis. Differentiation and Neoplasia. Proc. Nati. Acad. Sei. U. S.. 73. one for the study of the relationship between GGT and 2414-2418, 1976. amino acid uptake. If changes in the appropriate direction 23. Orlowsky, M., and Meister, A. Isolation of Gamma-glutamyl Transpepti dase from Hog Kidney. J. Biol. Chem., 240: 338-347, 1965. are observed, the possible relationship of such changes to 24. Orlowski, M., and Meister, A. The Gamma-glutamyl Cycle: A Possible neoplasia could then be explored. Transport System for Amino Acids. Proc. Nati. Acad. Sei. U. S., 67: Finally, from a pragmatic viewpoint, the establishment of 1248-1255, 1970. 25 Rosalki, S. B. Gamma-glutamyl Transpeptidase. Adven. Clin. Chem., 17: marked elevation of GGT as a marker for very early, as well 53-107. 1975. as late, putative liver preneoplastic and neoplastic cells 26. Rutenberg, A. M., Kim. H., Fischbein, J. W., Harper, J. S., Wasserburg, opens up many new possibilities to follow the fate of new H. C., and Seligman, A. M. Histochemical and Ultrastructural Demon stration of Gamma-glutamyl Transpeptidase Activity. J. Histochem. Cy populations, both in vivo and in vitro (16, 17), in relation to tochem., 77: 517-526, 1969. cancer. 27. Schwartz, M. K. Enzymes in Cancer. Clin. Chem., 79: 10-22, 1973. 28. Sell, S., Becker, F. F., Leffert, H. L., and Watabe, H. Expression of an Oncodevelopmental Gene Product (a-fetoprotein) during Fetal Develop REFERENCES ment and Adult Oncogenesis. Cancer Res., 36: 4239-4249, 1976. 29. Solt, D., and Farber, E. A New Principle for the Sequential Analysis of 1. Cameron. R.. Sweeney. G. D., Jones, K., Lee, G., and Farber, E. A Chemical Carcinogenesis Including a Quantitative Assay for Initiation in Relative Deficiency of Cytochrome P-450 and Aryl Hydrocarbon Hydrox- Liver. Nature, 263: 701-703, 1976. ylase Â¡nHyperplastic Nodules Induced by 2-Acetylaminofluorene Â¡nRat 30. Soll, D. B., Mediine, A., and Farber, E. Rapid Emergence of Carcinogen- Liver. Cancer Res., 36. 3888-3893, 1976. Induced Hyperplastic Lesions in a New Model for the Sequential Analysis 2. Chaimoff, C., Wolloch, Y., Eicchorn. F., and Dintsman. M. Gamma- of Liver Carcinogenesis. Am. J. Pathol., 88: 595-618, 1977. glutamyl Transpeptidase as an Aid in the Diagnosis of Liver MÃ©tastases. 31. Strommer, J. H., and Theodorsen, L. Gamma-glutamyl Transferase: Israel J. Med. Sci., 11: 781-784, 1975. Substrate Inhibition, Kinetic Mechanism, and Assay Conditions. Clin. 3. Farber, E. Hyperplastic Liver Nodules. Methods Cancer Res., 7: 345- Chem.,22: 417-421, 1976. 375. 1973. 32. Szasz, G. A Kinetic Photometric Method for Serum Gamma-glutamyl 4. Farber, E. Putative Precursor Lesions: Summary and Some Analytical Transpeptidase. Clin. Chem., 75: 124-136, 1969. Considerations. Cancer Res.. 36. 2703-2705, 1976. 33. Taniguchi, N., Saito, K., and Takakuwa, E. Gamma-glutamyl Transferase 5. Farber, E. The Pathology of Experimental Liver Cell Cancer. In: H. M. from Azo Dye Induced Hepatoma and Fetal Rat Liver. Similarities in Cameron, D. A. Linsell, and G. P. Warwick (eds.), Liver Cell Cancer, pp. Their Kinetic and Immunologie Properties. Biochim. Biophys. Acta, 397: 243-277. Amsterdam: Elsevier Scientific Publishing Co., 1976. 265-271. 1975. 6. Farber, E., Hartman. S., and Gruenstein, M. Resistance of a Putative 34. Taniguchi, N., Tsukada, Y., Mukuo, K., and Hirai, H. Effect of Hepato- Pre-Malignant Liver Cell Population, the Hyperplastic Nodule, to the carcinogenic Azo Dyes on Glutathione and Related Enzymes in Rat Acute Cytotoxic Effects of Some Hepatocarcinogens. Cancer Res., 36: Liver. Gann. 65: 381-387, 1974.

MARCH 1978 827

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Fig. 1. Section of liver at 7 days following partial hepatectomy in carcinogen-treated rat. Multiple GGT-positive foci of hepatocytes are clearly evident and distributed randomly throughout the liver, x 6. Fig. 2. Ten days following partial hepatectomy, GGT-positive foci are large and periportal bile duct (oval) cell proliferation, also positive for GGT, is prominent, x 65.

828 CANCER RESEARCH VOL. 38

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Fig. 3. GGT-positive 21-day foci stand out clearly against the negative background of the surrounding liver. Oval cells are not prominent at this time, x 65. Fig. 4. Section of neonatal liver (Day 1) shows a strong positive reaction in all hepatocytes for GGT, especially in the bile canalicoli, x 125.

MARCH 1978 829

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1978 American Association for Cancer Research. γ-Glutamyltransferase in Putative Premalignant Liver Cell Populations During Hepatocarcinogenesis

Ross Cameron, John Kellen, Arnost Kolin, et al.

Cancer Res 1978;38:823-829.

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