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[CANCERRESEARCH39, 3495-3501, September19791 0008-5472/79/0039-0000$02.00 Differential Expression of a-Fetoprotein and y-Glutamyltranspeptidase in Chemical and Spontaneous Hepatocarcinogenesis Hannu Jalanko1 and Erkki Ruoslahti2 Division of Immunology, City of Hope National Medical Center, Duarte, California 91010 ABSTRACT The expression of two markers of fetal liver, a-fetoprotein (AFP) and -y-glutamyltranspeptidase (GGT), was studied in chemical and spontaneous hepatocarcinogenesis in mice. Se rum AFP concentration increased within 3 weeks from the commencement of feeding of o-aminoazotoluene. This early elevation subsided about 3 months after the beginning of the administration of the carcinogen. A new, sustained elevation of the serum AFP level followed at 5 to 6 months accompanied by the appearance of liver tumors. In immunofluorescence, some small oval cells and scattered adult-type hepatocytes contained AFP during the early stage of chemical carcinogenesis. During the later phase, AFP was detected in a few of the nodular areas, in solitary hepatocytes, and in groups of carcinoma cells. GGT activity in the liver increased within 1 week after the carcinogen regimen was started, preceding the early increase of AFP production. At the final stage, the chemically induced hepatomas contained about 80 times more GGT than did normal liver. In histochemical staining, proliferating oval cells and small areas of hepatocytes stained for GGT during the early weeks, and later most nodules, small areas of nonnodular parenchyma, and carcinomas contained GOT. During spontaneous carcinogenesis in male C3HeB/FeJ mice, premalignant lesions, accompanied by a slight increase of serum AFP, precede the appearance of liver tumors. No cells staining for AFP were detected during this early stage. Once overt liver cancers had developed, AFP was readily detectable in the tumors and was localized to some but not all carcinoma cells. The corresponding serum AFP levels were highly elevated. In contrast to the high levels of GOT found during chemical carcinogenesis, no elevation of GOT was found in livers at various stages of spontaneous carcinogene sis, including cancers in eight individual mice. These results indicate that the production of AFP and GOT is not turned on as a single ‘ ‘genetic package,' ‘ and that these two markers differ in their behavior in liver carcinogenesis. in the circulation and of GOT in the lesions of the liver serve as useful markers for the progress of the carcinogenic process (1, 4, 8, 22). In rats and mice, liver cell cancer (hepatocellular carcinoma, hepatoma) can be induced by various chemicals. Several stud ies have previously shown that in rats serum AFP concentration increases early during the chemical hepatocarcinogenesis, and the elevation shows a biphasic pattern (14, 15, 26, 31 ). The first, transient increase coincides with the appearance of small oval cells; some of these cells contain AFP as shown by immunofluorescence and immunoperoxidase stainings (16, 18, 25, 30). The second increase of serum AFP concentration is believed to indicate appearance of neoplastic cells (14, 16). GOT is a membrane-bound enzyme which catalyzes the transfer of the -y-glutamyl moiety of glutathione to an amino acid acceptor (10). Normal adult hepatocytes contain only small amounts of GGT activity, but in the fetal and neonatal rat liver GOT activity is substantially higher (4). In contrast to normal hepatocytes, neoplastic and preneoplastic liver cells seem to contain increased amounts of GOT as studied in rats during chemical hepatocarcinogenesis (4, 7, 8, 13, 28). While the expressions of both AFP and GOT have been studied separately, no studies are available on simultaneous measure ment of both these markers. Two models for hepatocarcinogenesis, chemically induced tumors and spontaneous hepatomas, are available in mice. Some strains of mice spontaneously develop hepatocellular carcinomas with a high frequency, and this process is associ ated with an increase of the concentration of serum AFP (3, 12). In this report, we show that the expression of GOT and AFP, while similar in chemical carcinogenesis, differs in spon taneous hepatocarcinogenesis. MATERIALS AND METHODS Mice. Female C3H/A/BOM mice from Cl. Bomholtgard, Ltd. (Ry, Denmark) were used for the study of chemical hepatocar cinogenesis. In our hands, females of the C3H/A/BOM strain have had a relatively low frequency of spontaneous hepatomas. Of 20 mice observed by repeated determinations of AFP level INTRODUCTION and autopsy at sacrifice, until the age of 20 months, one In experimental hepatocarcinogenesis, the liver undergoes developed a hepatoma. Twenty-two of the 54 female C3H/A/ a series of morphological and biochemical alterations leading BOM mice used in the feeding experimentswere control mice ultimately to overt liver cell cancer (6). The increases of AFP3 from an immunotherapy experiment (1 1). They were immunized with complete or incomplete Freund's adjuvant without any antigen added, once a month as detailed in Ref. 11. Such the Finnish Cancer Society. To whom requests for reprints should be addressed, immunization has been found to affect neither the serum AFP at the Department of Bacteriology and Immunology, University of Helsinki, 00290 level nor the histology of the liver of C3H mice (12). Since no Helsinki 29, Finland. 2 Supported by Grants CA 1 9894 and CA 1 6434 from National Cancer differences were seen in the kinetics of serum AFP during Institute, Department of Health, Education, and Welfare. carcinogenesis or in the appearance of GOT in the liver be 3 The abbreviations used are: AFP, a-fetoprotein; GGT, y-glutamyltranspepti tween the immunization control mice and mice that did not dase; 0-AT, o-aminoazotoluene. Received January 17, 1979: accepted June 7. 1979. receive any additional treatments, the results from the 2 groups 1 Recipient of grants SEPTEMBER1979 from the Orion Foundation, the Lääke Foundation, and 3495 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1979 American Association for Cancer Research. H. Ja!anko and E. Ruos!ahti have been pooled. Male C3HeB/FeJ mice from The Jackson Laboratory (Bar Harbor, Maine) were used for the study of spontaneous liver cancer. The mice were toe-marked individ ually, kept in plastic cages, and fed standard mouse pellets and water ad !ibitum. Carcinogen Feeding. o-AT (ICN Pharmaceuticals, Plainview, N. V.) was dissolved in maize oil and mixed with standard laboratory meal (Sotka, OTK, Finland) to obtain a diet contain ing 0.06% (w/w) o-AT. Feeding of the carcinogen was started when the mice were 4.5 months old. The feeding was continued for 8 months until the animals were 12.5 months old. Bleedings. The mice were bled from the upper tail vein at 2to 6-week intervals. Blood (100 @@l) was collected in calibrated capillary tubes and diluted with 0.4 ml of phosphate-buffered saline (0.01 55 M NaH2PO4.H2O,0.01 55 M NaHPO4.7H2O, pH 7.4). Quantitation of AFP. Measurement of serum AFP was done by competitive double-antibody radioimmunoassay (20, 21). The monitoring of AFP to obtain livers with premalignant and malignant lesions during spontaneous hepatocarcinogenesis in male C3HeB/FeJ mice has been described previously (12). Immunofluorescence. The mice were exsanguinated under pellet was suspended in 0.6 ml of 100 m@iTris-HCI (pH 7.6) containing 10 mM MgCl2, and used for assay. GOT was assayed with ‘y-glutamyl-p-nitroanilide (Sigma) as substrate and glycylglycine as acceptor using previously pub lished procedures with slight modifications (19, 27). Tissue aliquots of 0.25 ml were incubated with 0.75 ml of substrate mixture at 37°for 15 mm. The final reaction mixture contained 4 mM L-'y-glutamyl-p-nitroanilide, 50 mM glycylglycine, 10 mt.i MgCI2, and 100 mM Tris-HCI (pH 7.6). The reaction was ter minated by addition of 3 ml of 1.3 M acetic acid, and the solution was clarified by centrifugation at 105,000 x g for 15 mm. Enzyme activity (nmol of p-nitroaniline liberated per mg of protein per 15 mm) was calculated using a molar extinction coefficient of p-nitroaniline at 405 nm of 9900 M1 cm1. Sample aliquots to which acetic acid was added before sub strate solution were used as blanks. Protein was determined according to the method of Lowry et a!. (1 7). Normal and pathological tissue samples were measured simultaneously in every assay. RESULTS ether anesthesia, and a careful autopsy was performed on each mouse. The liver was sliced, and macroscopic tumors and suspect areas were separately processed. For immunoflu orescence staining, each block was fixed in 95% cold ethanol containing 1% acetic acid and processed according to the Serum AFP during Chemical Hepatocarcinogenesis. Dur ing chemical carcinogenesis in female C3H/A/BOM mice, elevated serum AFP was first observed at 3 weeks after com mencement of o-AT feeding (Chart 1). This early increase method of Sainte-Marie (24). The sections were labeled with months. At the first peak, AFP levels ranged from 295 to 2,600 the use of a double-antibody ng/mI and, ultimately, from 600 up to 34,000 ng/mI. The AFP procedure with goat anti-mouse AFP as the first antibody and IgO of a horse anti-goat IgO serum labeled with fluorescein (Sigma Chemical Co., St. Louis, Mo.) as the second antibody. A serial section was labeled with rabbit anti-mouse lgG as the first antibody and fluorescein subsided and was followed by a sustained increase at 5 levels prior to the carcinogen treatment varied between 10 and 280 ng/ml. All but 2 of the 54 mice treated with the carcinogen 10 labeled goat anti-rabbit lgG as the second antibody to control for nonspecific staining caused by the presence of serum proteins in the sections. The same or serial sections were 5.0 afterwards stained with hematoxylin and eosin for histological study. GGT Histochemistry. GOT was detected according to the 2.5 method of Autenberg et a!. (23). Small tissue samples were frozen in liquid nitrogen, cut in a cryostat into 5-@sections, air dried, fixed in cold acetone for 30 mm, and incubated in a freshly prepared solution containing L-y-gIutamyl-4-methyl-f@naphthylamide (Vega-Fox Biochemicals, Tucson, Ariz.) as the substrate, Fast Blue BB salt (Sigma) as the diazonium coupling reagent, and glycylglycine (Fluka AG, Buchs SG, Switzerland) 0.50 as the acceptor. For histological study, the same or serial sections were later fixed with 10% formalin and stained with hematoxylin and eosin. In some cases, a Carnoy fixation followed by staining with toluidine blue (pH 5) was used (5). Assay for GGT. Tissue samples were frozen in liquid nitrogen and stored at —20°. In livers containing large macroscopic tumors, these tumors were first isolated and assayed sepa rately. For the assay, samples were homogenized with 12 strokes of a Teflon homogenizer in 5 ml of an ice-cold solution containing 0.25 M sucrose, 100 mM Tris-HCI (pH 7.6), and 10 mM MgCI2. The homogenate was centrifuged at 9,1 50 x g for 10 mm, and the supernatant was collected. This was further centrifuged at 105,000 x g for 60 mm (Beckman Model L5-65 centrifuge) to pellet the microsomal membrane fraction. The 3496 I 0.25 0.10 0 1 2 3 4 5 6 7 8 9 10 11 MONTHS Chart 1. Serum AFP concentration during chemical hepatocarcinogenesis in female C3H/A/BOM mice. Feeding of 0.06% o-AT was started immediately after the first bleeding and continued for 8 months. AFP levels were monitored for the first 5 months in 54 mice and thereafter in 27 mice. The line was drawn using the mean values: bars, SE. The elevations of the AFP levels at the first peak and from the fifth month on are statistically highly significant ( p < 0.001). CANCER RESEARCH VOL. 39 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1979 American Association for Cancer Research. AFP and GOT in Hepatocarcinogenesis in Mice showed a value higher than 300 ng/ml at some point between 3 and 10 weeks of the treatment, and 70% of the mice showed a greater than 5-fold increase of serum AFP level during this period. Chemically Induced Tumors. After 8 months with carcino gen followed by 2.5 months without carcinogen, 25 of the 28 (89%) of the female C3H/A/BOM mice observed for this length contrast to the findings in chemical carcinogenesis, the sur rounding parenchyma was relatively normal, and no AFP-con taming cells could be observed in these areas. Staining for AFP in the early phase of spontaneous carcinogenesis did not reveal positive cells. The livers of 4 old (1 4 to 18 months) male C3HeB/FeJ mice with slightly elevated serum AFP levels (380 to 760 ng/ml), but no macroscopic tumor, were cut in small surface of the liver was covered by 10 to 50 small nodules with pieces, and several sections from different pieces were studied. Histologically, areas of hyperplasia, groups of atypical hepa tocytes, and in one case strong inflammation were found, but diameters ranging from barely visible to 3 mm. In addition to no cells positive for AFP were present. of time had grossly visible liver tumors. In all tumor mice, the liver contained multiple tumors. The this, 1 1 livers (39%) contained one large hepatoma varying from 5 to 12 mm in diameter. Two livers (7%) contained 2 large tumors each. Ten of the 13 large tumors (77%) were invasive, showed areas of papilliform and adenoid growth, necrotic areas, abundant mitoses, and various nuclear abnormalities. The remaining 3 large tumors and about 80% of the smaller tumors were well differentiated. In these tumors, the normal architectural structure of the liver was preserved, no invasion of the surrounding parenchyma was observed, and the cells looked relatively normal. Many tumors contained alternating areas with malignant and benign appearance. Microscopic nodular areas, variation of cell size, nuclear abnormalities, and GGT in the Liver. Biochemical determination of GOT from tissue homogenates was performed at different stages of chem cal and spontaneous hepatocarcinogenesis. GOT activity was low in normal mouse liver (Table 1). Fetal liver contained from 100 to 1000 times more GOT than did adult liver. During carcinogenesis induced by o-AT, a significant increase was found within 1 week after the carcinogen regimen had been started. This increase preceded the elevation of the serum AFP level. In the late phase, GOT activity was on the average of 80 times higher than in normal liver, both in carcinomas and in livers showing various premalignant lesions. In histochemical staining for GGT, small proliferating oval mitoses were present in the parenchyma surrounding the tu mors. No clear-cut correlation was found between the circulat ing AFP level and the pathology of the liver lesions. cells and small areas of normal-looking hepatocytes (Figs. 9 and 10) were positive during the early weeks of chemical AFP in the Liver. During the early weeks of chemical hepa tocarcinogenesis, damage of hepatocytes, cell hyperplasia, areas stained for GOT, either partially or completely (Figs. 1 1 and 12). Small GOT-positive areas were found also in the nonnodular parenchyma in this late phase (Fig. 13). Some carcinomas stained uniformly with GOT (Fig. 14); in others, variation in the intensity of staining was observed. In contrast to the marked increase of GGT activity found in and appearance of small, proliferating oval cells were seen histologically. Immunofluorescence staining revealed AFP in some of the oval cells. This is similar to previous findings in rats (16, 18, 25, 30). Some scattered cells, not clearly different from normal adult hepatocytes, and 2). also stained for AFP (Figs. 1 During the second increase of serum AFP, when nodular areas, bizarre cytological changes, and frank carcinomas ap peared, various patterns of AFP staining were observed. Stain ing for AFP was found in the nonnodular parenchyma in small groups of cells or in solitary hepatocytes (Figs. 3 and 4). A vast majority of nodular areas were negative for AFP, but in about 10% of the nodules in the late phase, all or some cells con tamed AFP (Figs. 5 and 6). The cells in the AFP-positive nodules showed cytological changes, but no clear histological difference was found between the few nodules containing AFP positive cells and the well-differentiated nodules in which no staining for AFP was observed. The large carcinomas resulting from carcinogen treatment were positive for AFP. There were groups of cells which stained more brightly than the rest of the tumor. In one of the mice, an infiltrating reticuloendothelial neoplasm was observed. This tumor was negative for AFP. Hepatomas that developed spontaneously in male C3HeB/ FeJ mice also contained AFP. Eight macroscopic tumors, which varied from 1 to 12 mm in diameter, were studied. Histologi cally, 3 of these tumors were clearly invasive, had lost the normal architectural structure, and showed cellular atypia. In 5 tumors, the cells looked less abnormal and the normal architectural structure of the tissue was preserved. AFP-con taming cells were found in both types of tumors. The intensity of the staining for AFP varied in different parts of the tumors, and the AFP-positive cells were mainly found in groups (Figs. 7 and 8) as was the case with chemically SEPTEMBER 1979 induced tumors. In cardinogenesis. Later on, approximately 80% of the nodular the liver lesions during carcinogenesis induced by o-AT, no increase of GOT activity was found in spontaneous liver lesions whether malignant or benign. All the 8 macroscopic tumors described above were negative for GOT, both biochemically and histochemically (Figs. 15 and 16; Table 1). The livers of the 4 mice with benign lesions and slightly elevated serum AFP levels also had normal GOT levels. DISCUSSION The behavior of 2 carcinofetal markers, AFP and GGT, has been compared in chemical and spontaneous hepatocarcino genesis in mice. We found that, while the levels of both of these markers increased early during the process of chemical carci nogenesis, only AFP was present in spontaneous liver card nomas. Both AFP and GGT were present in some oval cells and adult-type hepatocytes during the early stage of chemical carcinogenesis. For technical reasons, it was not possible to obtain evidence on the question of whether the same cells contained both AFP and GGT during this or any other situation where both markers were present. Due to leakage of AFP, staining for AFP could not be done reliably on frozen sections, although we attempted this using several different fixatives. GOT, on the other hand, became inactivated under conditions where AFP could be stained. Differences in the expression of AFP and GOT became apparent during the later phases of chemical carcinogenesis. A great majority of nodules with no signs of invasiveness contained large amounts of GOT, 3497 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1979 American Association for Cancer Research. H. Ja!anko and E. Ruos!ahti @ Table 1 Expression of AFP and GGT during chemical and spontaneous hepatocarcinogenesis in mice Chemical carcinogenesis was induced in female C3H/BOM mice by feeding the mice 0.06% o-AT for 8 months. Early phase means here the early weeks of the carcinogen feeding, when serum AFP concentration first became increased. Late phase means the time from 5 months on (Chart 1) when a sustained increase of serum AFP level occurred. Spontaneous carcinogenesis was followed in male C3HeB/FeJ mice by monitoring the serum AFP concentration and sacrificing the mice either soon after increase of AFP was observed (early phase) or later when carcinomas were found in the liver (late phase). Fetal blood and livers were taken from fetuses 1 to 3 days before estimated time of delivery. Normal samples were obtained from adult C3HeB/FeJ and C3H/A/BOM mice. Measurements and stainings Methods.AFPGOTType were done as described in Materials and in liveraCells in serum (@zg/ml)Cells of liver RangeNormal0.18 tissueAmount containing AFPAmount GOTMeanRangeMean containing 0.01—0.28 (25)bNo staining0.40—2.8 1100—4900 (4)Hepatocytes436250—623 cellsFetal1970 (1 1)bScattered (5/2)Ductal ductal cells HepatocytesChemicalcarcino genesisEarly cellsLate phase0.77 0.29—2.6 (54)Some of small oval cells60.3—1 hepatocytes (27)Scattered Some late nodular areas Solitary hepatocytes and groups of hepatocytes outside the nodules Groups of carcinoma cells31 0.24—34 phase3.70 7 —32 2731 5—87 cellsSpontaneouscarcino genesis Early phase Late phase0.42 0.27—i.4 (19)e staining observedt 0.05—420 (30)@NoGroups of carcinoma cells0.8 1.30 (5)Most of small oval areas of hepatocytes Most nodular areas Small groups of hepatocytes outside the nodules (5/3f (7/3)C. dSmall Uniform or uneven staining of carcinoma .6 (4/2)C 0.5—0.6 (8/3)CNo 0.50—1 staining of hepatocytes No staining of carcinoma cells a Nmol of p-nitroaniline liberated per mg protein per 15 mm. b Numbers in parentheses, C Individual samples d Isolated a Some I Four large of the livers number were pooled of mice to yield tested. enough tissue for the assay. The number of mice used as source of samples/number of pooled samples tested is shown. carcinomas. AFP from values mice were with obtained elevated from serum Ref. 12. AFP concentration were stained. Hyperplasia, cell atypia, and in one case strong inflammation were observed histologically. whereas only few nodules in the late phase stained for AFP. In addition to the lack of AFP staining in most nodules, a major dissimilarity in the expression of AFP and GOT concerns the sion of such tumors during their maintenance by transplantation (9). One could argue that the expression of GOT in chemically staining patterns observed.The benign and malignant lesions tended to stain uniformly with OGT, clearly contrasting the induced tumors may be due to their more malignant nature and/or faster proliferation rate as compared to spontaneous hepatomas, rather than to difference in the etiology of the tumor. However, many of the spontaneous tumors are histolog pathological lesion against the surrounding parenchyma, whereas AFP was mostly found in scattered cells or in groups of cells within the lesions. This difference may at least partly depend on the fast secretion of AFP and its dependence on the phase of the cell cycle (26). It is also possible that the ically poorly differentiated, produce tumors when transplanted to syngeneic animals (2), and, judged from the changes in serum AFP level (12), progress with a speed at least equal to production of the 2 markers is associated with different stages of cell differentiation. that of chemically induced tumors. The main finding of this study concerns the different expres sion of GOT in chemical and spontaneous carcinogenesis. in spontaneous carcinogenesis, it is possible that the induction of GOT is mainly associated with the exposure of these animals Since the expression of GOT does not seem to be increased localization of AFP did not reveal any to chemical carcinogens. An increase of GGT activity can be differences between the chemically induced and spontaneous liver tumors. In both cases, patches of AFP-positive cells were seen in the liver within 24 to 48 hr after a single large dose of a carcinogen (8), and it is known that neoplastic liver cells are seen. In contrast to this, we found GOT to increase only during more resistant to toxic effects of carcinogens than are normal liver cells (6). This is in agreement with the role GOT is thought to play in the detoxification of compounds such as carcinogens (29). The increased resistance to the toxicity of the carcinogen Immunohistochemical chemical hepatocarcinogenesis. No increase of GOT activity was seen during the early stages of spontaneous carcinogen esis or in the resulting hepatomas. It seems that increased GOT is rarely, if ever, a characteristic of primary spontaneous hep atomas in C3H mice. Fiala et a!. (7) reported increased GOT activity in 1 of 4 transplantable tumors, originally obtained from spontaneous hepatomas in C3H x V mice. However, primary hepatomas such as the ones we have studied are likely to be more representative of spontaneous hepatomas than are trans planted tumors since changes may occur in the gene expres 3498 could convey a selective advantage to the cells containing GOT and could explain the presence of GOT in the proliferative lesions induced by carcinogens. However, it is obvious that the presence of large quantities of GOT in fetal liver cells cannot be explained on the basis of the above considerations. Also, the fact that we have studied spontaneous hepatomas in only one highly inbred mouse strain does not warrant general con CANCERRESEARCHVOL. 39 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1979 American Association for Cancer Research. AFP @ clusions on the role of GOT in malignant liver cells. How the expression of AFP and GOT is regulated is not 14. known, but the present results indicate that these 2 carcinofetal markers are not turned on simultaneously as a single “genetic package. ‘ ‘ They possibly reflect different aspects of malig nancy or the development of liver carcinomas through different pathogenetic processes (25). The difference in the expression 16. of AFP and GOT in chemical and spontaneous carcinogenesis, if true in other species, might allow conclusions to be made on 17. the etiology (chemical carcinogenesis versus other causes) of a given liver tumor. 18. REFERENCES 19. 1. Abelev, 0. I. a-Fetoprotein as a marker of embryo-specific differentiations in normal and tumor tissues. Transplant. Rev., 20: 3—37,1974. 20. 2. Andervont,H. B., and Dunn,T. B. Transplantationof spontaneousand 21 . 3. 4. 5. 6. induced hepatomas in inbred mice. J. NatI. Cancer Inst., 13: 455-503, 1952. Becker, F. F., Stillman, D., and Sell, S. Serum a-fetoprotein in a mouse strain (C3H-A'@fB)with spontaneous hepatocellular carcinomas. Cancer Aes., 37: 870—872,1977. Cameron, A., Kellen, J., Kolin, A., Malkin, A., and Farber, E. @Glutamyltrans ferase in putative premalignant liver cell populations during hepatocarcino genesis. Cancer Res., 38: 823—829,1978. Daoust, A., and Calamai, R. Hyperbasophilic foci as sites of neoplastic transformation in hepatic parenchyma. Cancer Aes., 31: 1290—1 296, 1971. Farber, E. The pathology of experimental liver cell cancer. In: M. H. Cameron, D. A. Linsell,and0. P. Warwick(ed). LiverCellCancer,Vol. 1, pp. 243277. Amsterdam: Elsevier Press, Inc., 1976. 7. Fiala, S., Fiala, A. E., and Dixon, B. y-Glutamyl transpeptidase in transplant able,chemicallyinducedrat hepatomasand spontaneousmousehepato mas. J. Natl. Cancer Inst., 48: 1393-1401 , 1972. 8. Fiala, S., Mohindru, A., Kettering, W. 0., Fiala, A. E., and Morris, H. P. Glutathione and gamma-glutamyl transpeptidase in rat liver during chemical carcinogenesis. J. NatI. Cancer Inst., 5 7: 591 —598,1976. 9. Hancock, R. L., and Grunau, J. Soluble RNA methylase activity of transplant able mouse hepatomas. J. NatI. Cancer Inst., 45: 971 —978,1970. 10. Hanes, C. S., Hird, F. J. A., and Isherwood, F. A. Synthesis of peptides in enzymic reactions involving glutathione. Nature (Lond.), 166: 288—292, 1950. 11. Jalanko, H., Engvall, E., and Ruoslahti, E., Immunochemical properties of alpha-fetoprotein (AFP) and autologous antibodies to AFP. Immunol. Com mun. 7: 209—222,1978. 12. Jalanko, H., Virtanen, I., Engvall, E., and Ruoslahti, E. Early increase of serum alpha-fetoprotein in spontaneous hepatocarcmnogenesisin mice. lnt. J. Cancer,21: 453-459, 1978. 13. Kalengayi, M. M. A., Ronchi, 0., and Desmet, ‘I. J. Histochemistry of and GGT in Hepatocarcinogenesis in Mice gamma-glutamyl transpeptidase in rat liver during aflatoxin B-induced car cmnogenesis.J. NatI. Cancer Inst., 55: 579—588,1975. Kitagawa, T., Yokochi, T., and Sugano, H. Alpha-fetoprotein and hepato carcinogenesis in rats fed 3'-methyl-4-dimethylammnoazobenzeneor N-2fluoroenylacetamide. lnt. J. Cancer, 10: 368—381 , 1972. Kroes, A., Sontag, J. M., Sell, S., Williams, 0. M., and Wiesburger, J. H. Elevated concentrations of serum alpha-fetoprotein in rats with chemically induced liver tumors. Cancer Res., 35: 1214—1 217, 1975. Kuhlmann, W. 0., Localization of alpha,-fetoprotein and DNA-synthesis in liver cell populations during experimental hepatocarcmnogenesisin rats. Int. J. Cancer, 21: 368—380,1978. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, A. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265—275, 1951. Onoe, T., Kaneko, A., Dempo, K., Ogawa, K., and Minase, T. Alpha-fetopro tom and early histological changes of hepatic tissue in DAB-hepatocarcino genesis. Ann. N. V. Aced. Sci., 259: 168—180,1975. Orlowski, M., and Meister, A. Isolation of y-glutamyl transpeptidase from hog kidney. J. Biol. Chem., 240: 338-347, 1965. Pihko, H., and Ruoslahti, E. High level of alpha-fetoprotein in the sera of adult mice. Int. J. Cancer, 12: 354-360, 1973. Ruoslahti, E. Antigen-antibody precipitates and immunoadsorbents: appli cation to purification of alpha-fetoprotein. Scand. J. lmmunol., Suppl. 3, 3649, 1976. 22. Ruoslahti,E., andSeppálä, M. Alpha-fetoprotein in cancerandfetaldevel opment. Adv. Cancer Res., in press, 1979. 23. Rutenberg, A. M., Kim, H., Fischbein, J. W., Hank, J. S.. Wasserkrug, H. L., and Seligman, A. M. Histochemical and ultrastructural demonstration of ‘y glutamyl transpeptidase activity. J. Histochem. Cytochem., 17: 51 7—526, 1969. 24. Sainte-Marie, 0. A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem., 10: 250—256,1962. 25. Sell, S. Distribution of a-fetoprotein- and albumin-containing cells in the livers of Fischer rats fed four cycles of N-2-fluorenylacetamide. Cancer Res., 38: 3107-31 13, 1978. 26. Sell, S., Becker, F. F., Leffert, H., and Watabe, H. Expression of an oncodevelopmental gene product (a-fetoprotein) during fetal development and adult onco9enesis. Cancer Res., 36: 4239—4249,1976. 27. Szasz,0. A kineticphotometricmethodfor serumgamma-glutamyl trans peptidase. dIm. Chem., 15: 124-136, 1969. 28. Taniguchi, N., Tsukada, Y., Mukuo, K., and Hirai, H. Effect of hepatocarcin ogenic azo dyes on glutathione and related enzymes in rat liver. Gann, 65: 381-387, 1974. 29. Tate, S. S., Thompson, G. A., Miester, A. Recent studies on gamma-9lutamyl transpeptidase. In: I. M. Arias and N. B. Jakoby (eds.), Glutathione: Metab olism and Function, pp. 45—55.New York: Raven Press, 1976. 30. Tchipysheva, T. A., Guelstein, V. I., and BAnnikov, G. A., Alpha-fetoprotein containing cells in the early stages of liver carcmnogenesisinduced by 3'methyl-4-dimethylaminoazobenzene and 2-acetylaminofluorene. lnt. J. Can cer (Phila.), 20: 388-393, 1977. 31 . Watabe, H. Early appearance of embryonic a-globulin in rat serum during carcinogenesis with 4-dimethylaminoazobenzene. Cancer Aes., 3 1: 1192— 1194, 1971. Fig. 1. AFP-containing hepatocytes in the fifth week of chemical hepatocarcinogenesis in female C3H/A/BOM mice. All the AFP-positive cells in this figure and in Figs. 3, 5, and 7 were negative when stained with anti-mouse IgG. lmmunofluorescence, x 400. Fig. 2. The same section as in Fig. 1. The AFP-positive cells are histologically indistinguishable from normal adult type hepatocytes. H & E, x 400. Fig. 3. A solitary AFP-positive hepatocyte in the 8th month of carcino9enesis induced by o-AT. Immunofluorescence, x 400. Fig. 4. Same area as in Fig. 3 showing the atypical AFP-positive hepatocyte (arrow) in nonnodular parenchyma. H & E, x 250. Fig. 5. Nodular area of AFP-positive cells during ninth month of chemical carcinogenesis. Immunofluorescence, x 100. Fig. 6. Same area as in Fig. 5 with higher magnification showing the small nodular area (arrows). Cellular hypertrophy, mitoses, and bizarre cytological changes are seen. H & E, x 250. Fig. 7. Group of AFP-positive cells in spontaneously developed hepatoma in a male d3HeB/FeJ mouse. lmmunofluorescence, x 400. Fig. 8. The same area as in Fig. 7, showing the well-differentiated carcinoma. H & E, x 400. Fig. 9. Small GOT-positive area in the fourth week of chemical hepatocarcinogenesis. GGT staining, x 250. Fig. 10. Same area as in Fig. 9 of a serial frozen section. GOT-positive area corresponds to the small group of hepatocytes (arrow). H & E, x 250. Fig. 11. GOT-positive area in eighth month of chemical hepatocarcinogenesis. GOT staining, x 200. Fig. 12. Same area as in Fig. 11 of a serial frozen section. The nodulus (arrows) is composed of 2 types of cells. The left part is made of small basophilic hepatocytes expressing GGT activity (Fig. 11), and the right half is formed by vacuolated clear hepatocytes not staining with GGT. Toluidine blue, x 100. Fig. 13. Three GGT-positive areas of different sizes (arrows) in the late phase of chemical hepatocarcinogenesis. GOT staining, x 250. Fig. 14. Edge of a poorly differentiated hepatoma induced by o-AT showing uniform high GOT activity. GOT staining, x 100. Fig. 15. GOT staining of spontaneously developed hepatoma in a male C3HeB/FeJ mouse. No activity observed. x 100. Fig. 16. The same moderately differentiated carcinoma as in Fig. 15. H & E, x 200. SEPTEMBER 1979 3499 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1979 American Association for Cancer Research. @ @ @ •: .@ @-.@@‘@f! @ @ @ @ @@/ @‘ @: @‘ @e:.i :@ I C H. Jalanko and E. Ruos!ahti .@ @ .: Os I 1@ @. @ . . . @, C (@) ... C 4@ tl. .jQ @: 1@ ;@ %, -* 0 @ ,r @ .@ @r!‘ ; . @ .@ .. @ ‘ @. . - @*i―@@ . i. ‘ @ - . a. I •.i@.' @,: . .@ ‘@-@‘ ‘ ,@) j@, _ ó:@ . . .. - • w .‘ _•@@ @ @ ., I' . :a@r@ -‘S @‘ ;, . .. -@ I ,:@c ; • .@ c@ ‘r ‘@T @ @! @f4@ •, . , @f , ,@1 @ @ ;@ I've':@).@‘2,4@1' ‘ “I .@. .. . ,, @.. I :; :: . ;r.J t4' “ • :‘ @. C 4 @ @ , :@ - ,@ 9 •:- p !@ a4) -@ @ ..@ @ e_) , @ :@ . @ . C,. . •I@' @ . @ 0 0. . 0 .@ 0 (• ff@ a' @‘è' 0 • . 0 . @, 0 0 • C 0 @ • —I. 3500 •@ . C • •4II@' CANCERRESEARCHVOL. 39 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1979 American Association for Cancer Research. @ @ @h ‘@ @. *@ :@p@' @ . I AFP and GGT @I@$ in Hepatocarcinogenesis @ in Mice :‘@ @ C @ @r@%.1r@ I 1@ Ti@;@1. @ @ ‘ @ p @ .9 ?‘p , @I “ • •‘@@r(. . ‘0' *@ ‘P e@ # , @: ,‘:_@. :@ c @ .:@ @ •-@-@ 11 @‘ .- I 4; •@.i@ I @:12 C V ‘@ @ ,, @ @ -.. .,,@, % @ ‘&, .@ . @ @S4@ , ‘t':@@ ‘@.$@‘ I@ 4. @ I.. 15 I@ ,‘•“_ ‘@ ‘: ‘@? . @ ! .: @ I , .,.@ L@v@;..@I.%A@#; @,. . .1 .,‘ ‘ ..@:“ ‘ @%, 4. : . I, @ ‘.@ ,.. . @‘‘@‘ @i*@,,;; @ @ @I@'4'• . . @1- @. >- ... , ‘. . •/•@... . .. @*.. . -‘ SEPTEMBER1979 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1979 American Association for Cancer Research. 3501 Differential Expression of α-Fetoprotein and γ -Glutamyltranspeptidase in Chemical and Spontaneous Hepatocarcinogenesis Hannu Jalanko and Erkki Ruoslahti Cancer Res 1979;39:3495-3501. 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