Glucose-ATP Phosphotransferases During H Epatocarcinogenesis'

Glucose-ATP Phosphotransferases During H epatocarcinogenesis'

RAJANI MANJESHWAR SHARMA,2 CHAKRAVARTHI SHARMA, ANDREW J. DONNELLY, HAROLD P. MoRRIs, AND SIDNEY WEINHOUSE (The Fele Research Institute and the Department of Chemistry, Temple University School of Medicine; The Institute for Cancer Research, Philadelphia, Penmsylvania; and the Laboratory of Biochemistry, The National Cancer Institute, Bethesda, Maryland)

SUMMARY

The 2 glucose-ATP phosphotransferases normally present in rat liver were assayed in the â€œpreneoplasticâ€•liverof azo dye-fed rats, in regenerated liver, in primary cho langio- and hepatocarcinomas resulting from azo dye feeding, and in a series of trans plantable rat hepatomas. In the â€œpreneoplasticâ€•liver there was a progressive rise in hexokinase, the low glucose Km enzyme, to levels 5 to 6 times higher than normal and to a 5-fold lowering of glucokinase, the high glucose Kmenzyme, during an 11-week period of feeding 3'-methyl-dimethylaminoazobenzene (3'-Me-DAB) in an otherwise normal diet. These changes were correlated with bile duct cell proliferation and marked decreases in parenchymal cells. No marked changes were found in regen erated rat liver after partial hepatectomy, except for an early small increase in hexo kinase and lowering of glucokinase during the first 4 days after hepatectomy. All but one of 4 cholangiocarcinomas that developed after dimethylaminoazobenzene (DAB) feeding had no glucokinase and all had high hexokinase activities, whereas 3 out of 5 hepatocarcinomas had moderate glucokinase and all had moderate hexoki nase activities. Of 8 hepatocarcinomas or mixed hepatocholangiocarcinomas that developed on 3'-Me-DAB feeding, 4 had zero or borderline glucokinase activity, and 4 had moderate glucokinase activity. All 8 had moderate to high hexokinase ac tivity. Of 10 transplantable liver tumors ranging widely in transplant generation and in growth rate, 8 had zero or borderline glucokinase and low to high hexokinase activi ties. Two tumors had low but significant glucokinase activity. One of these, the Morris 3683 hepatoma, had a very high hexokinase activity and a rapid growth rate; the other, the 7794A, grew slowly and had a moderate hexokinase activity. One tumor, studied in the 3d and 4th transplant generations, a highly differentiated and extremely slow-growing hepatocarcinoma, the Morris 7787, had a low hexokinase and a moderate-to-high glucokinase activity; this is the only tumor found thus far that has a phosphotransferase pattern similar to that of normal rat liver. The pres ence or absence of the two hepatic glucose-ATP phosphotransferases cannot be cor related with the histologic type of either primary or transplantable liver tu mors, though cholangiocarcinomas are likely to contain no glucokinase.

Although the neoplastic process is commonly assumed cal composition of DNA or in one of the â€œregulatorâ€• to represent an alteration in the genetic apparatus of the substances remains uncertain (3, 6). If there is a specific cell, the question whether this change occurs in the cherni site of cancer initiation in the genome, the neoplastic transformation should be associated with specific bio 1 This work was supported by grants AM-05487 and CA-07174 chemical aberrations. If such exist, they have not yet from the National Institutes of Health; and Grants P119 and been uncovered. Perhaps the closest approach to a bio P202 from the American Cancer Society. chemical phenotype of cancer is the loss of those functional 2 Part of a thesis to be submitted by Rajani Manjeshwar Sharma activities that characterize the normal, differentiated tissue to the Graduate Council of Temple University in partial fulfill ment of the requirements for the M.A. Degree. of origin (7, 8, 17). However, such â€œdeletionsâ€•have Received for publication August 19, 1964. generally been observed in tumors transplanted many 193

Calbiochem, Inc. The carcinogenic diets were prepared as described previously (13) and were fed to 8- to 10-week old male rats of a pathogen-free (CFN) strain obtained from Carworth Farms. Control rats were maintained at the same time and under the same conditions on exactly the same diet without the carcinogen. At intervals, as designated in the description of the individual experiments, the rats were killed by decapi tation, the livers or tumors were excised, and after removal of small samples for histologic examination (13) the remainder was prepared for enzyme assay. Care was taken in the dissection of the primary tumors to remove as completely as possible any liver tissue; and with liver, to exclude any grossly visible tumor-containing material. Weeks on 3'-Me-DAB Diet This was generally not too difficult, since under the con CHART 1.â€”Hepatic glucose-ATP phosphotransferases in rats ditions of tumor development, many of the tumors grow fed 3'-Me-DAB. Bar at extreme left denotes average hexokinase as discrete, large masses, which can be dissected grossly and glucokinase levels in livers of 9 control rats, one each week, at the same time assays were made on 3 carcinogen-fedrat livers. away from the adjacent tissue. Tissues were excluded Whole bar represents total activity; shaded bar, hexokinase; and from the tables if the histologic examination revealed any clear section, glucokinase. Vertical lines indicate ranges. Each appreciable number of normal liver cells. Tumors that value is the mean of 3 separate assays on 3 rats. were so intermixed with normal liver tissue as to be non separable were not assayed; and only those assays were times, thus leaving unsettled the question of which of included in the tables in which the histologic examination these activities are lost in the initial transformation step revealed tumor cells of unquestionable hepatic or bile duct and which are lost during subsequent cell division. For origin, or were mixtures of these types (Fig. 1). a proper understanding of the significance of the loss of The transplanted tumors, except the Novikoff, were functional activities in neoplasia it is necessary to have propagated in inbred Buffalo strain rats, or in F1 hybrids much more data than are now available concerning which of the Buffalo and Lewis strains, usually by I.P., but also functional activities are lost and which are retained, and by I.M. or S.C. injection of tumor â€œmashes.â€•Trans of the former, which are lost initially and which are lost plantation was carried out in Bethesda and rats were subsequent to the initial transformation. shipped to Philadelphia. Tumor growth was followed by As part of such studies now under way in our laboratory palpation, and when the tumors reached a size of about we have chosen in the present investigation to examine 2 cm in diameter, the animals were utilized for enzyme the glucose phosphorylation system of the liver, and of assays. The tumor was dissected free of any adhering primary and transplanted liver tumors. Recent studies normal tissue, cut open, and blood, necrotic tissue, or from our own and other laboratories have established that other debris carefully removed by blotting, wiping, or rat liver contains two glucose-ATP phosphotransferases.1 scraping. Every attempt was made to exclude all but One, designated hexokinase, is similar in its properties to relatively firm, viable tissue. After weighing and re other, nonhepatic phosphotransferases, whereas the other, moving a sample for formalin-ethanol fixation, the re designated glucokinase, is evidently unique to liver. Its mainder was homogenized as described for liver and properties indicate that it plays an important role in liver primary tumors. Inasmuch as part of the tumor phospho function (1, 12, 14, 15). The object of the present investi transferases are in the particulate fraction of the cells, gation was to determine the activities of each of these it was found advisable for complete extraction of the enzymes in the liver of rats undergoing hepatic neoplasia enzymes to add Triton X 100 to the homogenizing medium by feeding of carcinogenic azo dyes, and to determine to to give a final concentration of 0.3%. what extent each enzyme is deleted or is retained in The Novikoff tumor was propagated in our laboratory primary and transplanted rat liver tumors induced by by I.P. transplantation in female rats of the Sprague these chemical carcinogens. Inasmuch as the two phos Dawley strain, obtained from the Holtzmann Company. photransferases may exist, respectively in different liver Enzyme assays were conducted exactly as described cell types, it was of added interest to correlate the enzyme previously (12) using the Zeiss PMQ spectrophotometer activities with changes in liver cell population during for measurement of TPNH formation at 340 m@. Though â€œpreneoplasiaâ€•and with the histologic type of the tumors. the temperature was not controlled, all assays were done in an air-conditioned room in which the temperature re MATERIAlS AND METHODS mained between 22Â°and 25Â°C. Some of the more recent DAB and 3'-Me-DAB were obtained from the Eastman assays of transplanted tumors were carried out with the Kodak Company, the ingredients of the diet were ob Beckman spectrophotometer equipped with the Gifford tained from Nutritional Biochemicals Corporation, and automatic cuvette positioner and direct recording. reagents used in the enzyme assays were obtained from RESULTS $ The enzymes under consideration in this paper are : hexo kinase, E. C. 2.7.1.1. ATP: D-hexOse-6-phOsphOtransferase; and Phosplwtransferase ratio in 3'-Me-DAB-fed rat liver. g!ucokinase, E. C. 2.7.1.2. ATP: D-glucOse-6-phOsphotransferase. Our first efforts were directed to determining whether the

TABLE 1 TABLE 2 GLVCOSE-ATP PEOSPHOTRANSFERASES IN GLUCOSE-ATPPHOSPHOTRANSFERASESOFLIVER AND REGENERATED RAT LIVERa LIVER TuMoRs AFTER AZO-DYE FEEDINGa

TISSUEDAYS DAYSTuMoR TISSUELzvza DAYSATrER HEPATECTOMYPHOSPBOTPANS1'ERASEHexokinaseGlucokinaseTotal2 ONDYEUNTILTUMOR DEVEL nueDAB102181C1.6000.250.44102182C1.480â€”0.72102189C1.2800.270.68102208C2.280.480.211.13102253HC1.490.250.190.71102259H0.890.450.251.1863270H0.390c63416H0.950.310.330.8163422H0.290.540.231.2063533H0.780.040.190.583'-MeDAB52172HC0.6800.150.2752174H2.7700.230.9352181HC0.870.150.231.1452178HC1.100.330.250.5635351HO0.290.330.230.9335355H1.160.240.330.6635391H0.700.500.231.0135525H0.350.100.140.80OPEDHHexoki naseGlucoki naseHexoki nueGlucoki Â±0.01 Â±0.04 Â±0.04 3 0.37 Â±0.06 0.54 Â±0.11 0.91 Â±0.06 4 0.33 Â±0.02 0.84 Â±0.08 1.17 Â±0.07 7 0.26 Â± 0.05 0.94 Â± 0.05 1.20Â± 0.05 14 0.23 Â± 0.03 1.04Â± 0.08 1.27Â± 0.11 Control (4 rats) 0. 19 Â±0.02 1.33 Â±0. 30 1.52 Â±0.27 7 (fasted48hr.) 0.33 Â± 0.05 0.75 Â± 0.10 1.08Â± 0.06 7 (fasted48hr., 0.20 Â± 0.020.281.44 Â±0.270.631.64 Â±0.25 then re-fed 24 hr.)0.34 aPartialhepatectomywasperformedonmaleratsweighing 240-250 gm; they were then fed ad lib on commercial checkers and 1% NaC1 solution. Each value is the mean Â±the average devia tion of three separate assays on three animals. Values are given in units per gram liver. inclusion in the diet of a carcinogen such as 3'-Me-DAB would alter the ratio of hexokinase to glucokinase in rat liver. Previous results made it evident that neither the total activities of the glucose-ATP phosphotransferases, nor the response to glucose-feeding in 24-hr. fasted rats was affected in the â€œpreneoplasticâ€•liver (13). In the aAzodye-fedratsweregiventhediet describedpreviously experiment recorded in Chart 1 rats were fed the carcino (15) with the addition of the carcinogen, for the designated period, gen, 3'-Me-DAB, in a dosage of 0.06 %, and control rats after which they were fed the commercial checker diet until a were given the same diet without the carcinogen, as tumor could be palpated, at which time the rat was killed. Each described in the experimental section. At weekly intervals value represents a single assay and is given in units per gram 3 dye-fed and 1 control rat were killed and the livers tissue. assayed. Throughout the 11-week feeding period the b Tumors designated C are cho!angiocarcinomas with minimal control rats exhibited no significant alteration from the proportions of hepatic cells ; H are hepatocarcinomas with mini normal levels, nor did total phosphotransferase activities ma! bile duct cells; and HC are mixtures of both cell types. change greatly with dye-feeding. However, the dye-fed C Analysis was lost. rats showed, in general, marked increases of hexokinase activity beginning at about 4 weeks and corresponding the early periods following hepatectomy, and receding decreases in glucokinase activity. By 10 or 11 weeks, toward normal in 1 or 2 weeks. The glucokinase level when liver disease was grossly evident, and where micro was low during the early period but reached the essentially scopic examination revealed extensive fibrosis and early normal range in 4 to 7 days. Animals that fasted 2 days foci of hepatocarcinoma, hexokinase was highest and after 7 days of liver regeneration showed relatively little glucokinase was lowest. Correlation with the histologic loss of glucokinase activity compared with fasted normal estimation of bile duct proliferation was moderately good. animals; however, the response to glucose feeding was In every instance in which the hexokinase activity was qulte comparable with normal rats (12). Glucose feeding high, moderate to marked bile duct hyperplasia was seen; after 2 days starvation also markedly lowered the hexo however, in no instance was the proportion of bile duct kinase level, from 0.33 to 0.20 unit/gm. Thus the data cells estimated to be higher than about 15 %. Those clearly show no appreciable abnormality of the amounts samples of liver examined at 10 and 11 weeks were grossly or ratios of the enzyme activities or in the response of abnormal, with extensive fibrosis and irregular patches of glucokinase to fasting or carbohydrate feeding. early hepatocarcinoma. Hence the high hexokinase levels Phosphotransferases in liver and liver tumors after inter in these livers cannot be attributed simply to changes in rupted feeding of azo dyes. In a previous study (13) we normal liver cell distribution. found that liver tumors induced by azo dyes had high, but Phosphotransferases in regenerated liver. Since cell re variable, levels of total glucose phosphotransferase ac generation is presumed to be occurring constantly as a tivities. At that time, an analytic procedure for separate response to the toxic effects of the carcinogen (4, 16), we determination of hexokinase and glucokinase was not were prompted to study the phosphotransferase levels in available; however, we found that all but one of these liver regenerated after partial hepatectomy. Only the tumors had an enzyme activity that was saturated with regenerated portions were assayed. The results shown in glucose at < 10@ M, thus indicating the absence of any Table 1 demonstrate an elevated level of hexokinase during appreciable hepatic type glucokinase, which has a glucose

TABLE 3 GLUCOSE-ATP PHOSPHOTRANSFERASEACTIVITIESIN TRANSPLANTEDLIVER TUMORS

TumorGenerationHexokinaseGlucokinaseTotal36832801342.32

0.13Novikoffa1541.74 Â± 0.100.23 Â± 0.052.55 Â± 0.355123TC252830.49 Â± 0.350.001.74 Â± 0.06H-3STC193540.33 Â± 0.060.03 Â± 0.010.53 Â± 0.037288C264942.15 Â± 0.030.000.33 Â± 0.35779598730.60 Â± 0.370.10 Â± 0.072.25 Â± 0.047800138830.47 Â± 0.040.10 Â± 0.030.70 Â± 0.137794A1011230.89 Â± 0.150.09 Â± 0.030.55 Â± 0.467316A1114530.99 Â± 0.270.28 Â± 0.091.18 Â± 0.085123 Â± 0.050.13 Â± 0.041.13 Â± (A,B, C)30â€”43100â€”16050.41 Â±0.050.07 Â±0.020.48 Â±0.05 aNotknownbutwe!!over500.

Km of 0.01 to 0.02 M (1). Since all of these tumors were feeding also had low to moderate hexokinase levels; one cholangiocarcinomas, it appeared likely that the bile duct had no glucokinase and the other three had low but cell contains a phosphotransferase different from that in definite glucokinase activities. the parenchymal cell. To shed further light on this The data thus obtained allow no categorical conclusions matter, we used a milder exposure to the carcinogen, in concerning the exclusive presence of a particular phospho order to promote the induction of hepatocellular tumors. transferase in a specific cell type. Reasoning from our The carcinogens, DAB and 3'-Me-DAB, were fed to the previous data (13) and the results of this study, cholangio rats for limited periods, and they were then maintained on carcinomas are not likely to have glucokinase and should an adequate, carcinogen-free diet, a dietary regime similar have moderate to high levels of hexokinase. There is, of to that employed by Morris et al. (5) for the induction of course, one glaring exception in line 4 of the table. How the so-called â€œminimal deviationâ€• tumors. Groups of ever, it is possible that, since only a minor portion of the rats were fed DAB for either 102 or 63 days, and 3'-Me whole tumor was taken for histologic examination, and DAB for either 52 or 35 days. The results of the assays the major portion used for the enzyme assay, the latter carried out on the tumors which developed under these may have contained hepatomatous tissue. Of the fourteen conditions are displayed in Table 2. hepatocarcinomas or mixed tumors, three had no giuco Of the 6 tumors occurring in rats fed DAB for 102 days, kinase, and two had borderline levels. In the others, the development times ranged from 181 to 270 days, 4 were levels were definitely above the experimental error and strictly cholangiocarcinomas, 1 was mixed, and 1 was a were in the range of values observed in fasting rat liver. hepatocarcinoma. Three of the 4 cholangiocarcinomas It would thus appear that the hepatocellular carcinomas had only hexokinase, the low Km enzyme, and 1 had both that develop on feeding of azo dyes are likely to, but will high hexokinase and low glucokinase activity. The 1 not necessarily, contain glucokinase as well as hexokinase mixed tumor also had a high hexokinase and a low gluco activity. kinase activity. The 1 clear-cut hepatocellular tumor in Phosphotransferases in livers of tumor-bearing rats. The this group had a moderate hexokinase and a moderate interruption of carcinogen feeding allowed sufficient time glucokinase activity, the latter being at about the level for the reversal of the usual pathologic changes occurring usually found in a fasted rat liver. in the liver during carcinogen feeding, and in every in A striking dichotomy was observed between the tumor stance the livers had a normal gross appearance, despite types that developed on DAB feeding. The 4 that de the presence of one or more tumors. Histologically the veloped earliest were cholangiocarcinomas, whereas all liver tissue was fairly normal, with the normal ratio of those that required 259 days or longer for development hepatic cell types, except that several of the livers showed were hepatocarcinomas. Table 2 shows that all 4 of the slight to moderate bile duct hyperplasia. All of the livers hepatomas that arose after 63 days of carcinogen feeding had normal hexokinase levels and all but 2 had glucokinase required 270 days or more for development ; they had low activities within the normal range of the fed rats. The 2 to moderate hexokinase levels, and only 2 of these had (lines 1 and 11) that had low glucokinase levels were appreciable glucokinase. normal histologically. Apparently, the enzyme pattern Because of the higher carcinogenicity of the 3'-Me-DAB, of reversed hexokinase and glucokinase that was found for shorter dietary regimes were employed. Apparently as a the livers during carcinogen-feeding, as shown in Chart 1, result of this, all of the tumors that arose in animals which does not persist after removal of the carcinogen from the were fed the drug for either 35 or 52 days were hepato diet, and a normal enzyme ratio accompanies an essentially carcinomas, or were mixed hepato- and cholangiocarci normal histologic appearance. nomas. Only one of these, an hepatic cell tumor, had Phosphotransferasesintraneplantedlivertumors. Extend very high hexokinase activity and no glucokinase. The ing this study to a wide variety of transplanted hepatic other three hepatomas had low to moderate glucokinase tumors of varying growth rates, glycolytic rates, and total levels. The four mixed tumors arising from 3'-Me-DAB hexokinase activities (2), data on the 2 hepatic phospho

TABLE 4 differentiated trabecular carcinomas,45 point to the need GLUCOSE-ATP PHOSPHOTRANSFERASE ACTIVITY IN for the development of, and examination at, the molecular MORRIS HEPATOMA 7787 level of many more hepatomas in our efforts to detect key alterations involved in the mechanism(s) of carcinogenesis. GenerationHexokinaseGlucokinaseTotal32980.200.580.7832980.101.531.6342200.200.500.7042120.140.500.6441850.360.400.7641850.280.520.824a1850.380.440.824a1850.460.400.86 DISCUSSION Phosphotransferase in relation to liver cell population. As suggested earlier (12, 13) the possibility was entertained that of the two glucose-ATP phosphotransferases of rat liver, one may be restricted to bile duct cells and the other to the parenchyrnal cells. Data on phosphotrans ferase activities of the livers of animals fed 3'-Me-DAB support this possibility. Along with the extensive early bile duct proliferation, there was a reversal in the phospho a Fasted 48 hr. transferase pattern ; hexokinase increased with bile duct proliferation, whereas glucokinase decreased as the paren chymal cell population decreased. However, this pattern transferases are displayed in Table 3. Two of these did not extend unequivocally to the primary tumors. tumors grow very rapidly, have a high rate of glycolysis Although all of the cholangiocarcinomas had moderate to and a high total hexokinase activity. One, the 3683 high hexokinase activities, one of these had a high gluco tumor, has low, but significant, glucokinase activity, kinase activity as well. Although glucokinase activity whereas the Novikoff tumor has none. The low total was observed in most of the hepatocarcinomas, 5 of these glucokinase activities of the 5123 tumors, as well as some tumors had little or no glucokinase ; in every one there was others, such as the H-35 and 7795 hepatomas, were re moderate to high hexokinase activity. Clearly, if these ported previously. The present data make it clear that hepatocellular carcinomas arise from parenchymal liver these tumors have little or no significant activities of the cells, they must carry with them at least the genetic high Km enzyme, the values of 0.07 to 0.13 units/gm apparatus for hexokinase formation. shown in Table 3 being the borderline of measurement. Phosphotransferases in transplanted tumors. A similar The only other tumor of this group that had significant ambiguity exists with respect to the loss or retention of glucokinase activity was the 7794A hepatoma. these enzymes in transplanted tumors. It is not surprising Recently there became available a new hepatic tumor, to find no glucokinase in the Novikoff hepatorna, since 7787 hepatoma, which is characterized by extremely slow this tumor has apparently lost all resemblance to its growth and a high degree of differentiation. This is the parent tissue (16) ; however, glucokinase is lost also in only tumor thus far examined which has activities corn several of the â€œminimaldeviationâ€• tumors, such as the parable with liver; namely, a low hexokinase and a moder 5123 and H-35, which have retained certain resemblances ate to high glucokinase (Table 4). Comparison of the to liver (5, 16). Further detracting from any correlation data on tumors of the 3d and 4th generations already between glucokinase activity and hepatic characteristics is indicates a tendency toward higher hexokinase and lower the fact that a very rapidly growing tumor, namely, the glucokinase with successive transplantation, a possible 3683, which is not of the minimal deviation type, also molecular counterpart of the well known phenomenon of contains glucokinase. It is perhaps of some significance tumor progression (17). If substantiated for subsequent that an early generation, highly differentiated hepato transplant generations, this observation would represent carcinoma, the 7787, does retain a hepatic pattern of these a partial deletion, after a few transfers, of an important enzymes ; a serial study of succeeding generations of this phosphotransferase. tumor may shed light on the question whether glucokinase, The 7787 tumor was induced in a female rat following an enzyme closely related to hepatic function, but which ingestion of N-2-fiuorenylphthalamic acid (2-FPA) under can survive the neoplastic transformation, will be lost in conditions similar to those used for the induction of subsequent transplantations. hepatoma 5123 (5). The primary 5123 hepatoma (5) was initially transplanted 8 months after withdrawal of 2-FPA, REFERENCES whereas 7787 was transplanted 3.6 months after with 1. Di PIETRO, D. L. ; SHARMA,C. ; ANDWEINHOTJSE,S. Studies on drawal of the carcinogen. The growth rate of 7787 is Glucose Phosphorylation in Rat Liver. Biochemistry, 1:455â€” 62, 1962. approximately one third that of 5123. The amount of 2. ELWOOD,J. C.; LIN, Y. C.; CRISTOFALO,V. J.; ANDMoRRIs, 2-FPA ingested by rat no. 5123 was 1.12 gm, and for rat H. P. Glucose Utilization in Homogenates of the Morris no. 7787 it was 1.18 gm over the same period of time, in Hepatoma 5123 and Related Tumors. Cancer Research, 23:906â€” diets of the same composition. The amount of 2-FPA 13, 1963. ingested would not appear to be significantly different in 3. JACOB,F., ANDMONOD,J. On the Regulation of Gene Activity. the induction of these two hepatomas. It is suggested that different alterations of normal liver cells leading to 4 Miyaji, H. Histological Study of Some Primary and Trans. neoplastic cells have occurred in these two hepatomas. plantable Slow-Growing Hepatic Tumors. To be published. 6 Morris, H. P., and Wagner, B. P. Induction of Transplant The differences in glucokinase activity in these two able Hepatic Rat Tumors of Different Growth Rate. To be pub hepatomas, both tumors designated histologically as well lished.

Cold Spring Harbor Symposium on Quantitative Biology, 11. SALAS,M. ; VINu@t, E. ; ANDSoLs, A. Insulin-Dependent 26:193â€”209,1961. Synthesis of Liver Glucokinase in the Rat. J. Biol. Chem., 4. MILLER,J. A., AND MILLER,E. C. The Carcinogenic Amino 238:3535-38, 1963. Azo Dyes. In: J. P. GREENSTEINand A. HADDOw(eds.), Ad 12. Sa&itM@&,C.;MANJESHWAR,R.; AND WEINHOUSE,S. Effects vances in Cancer Research, 1:339â€”396, New York: Academic of Diet and Insulin on Glucose-ATP-Phoaphotransferases of Press, Inc., 1953. Rat Liver. Ibid., 238:3840-45, 1963. 5. MoRRIs, H. P. ; SIDRANBKY,H.; WAGNER,B. P. ; AND DVER, 13. SHATTON,J.; DONNELLY,A. J. ; ANDWEINROUSE,S. Metabo H. M. Some Characteristics of Transplantable Rat Hepatoma lism of Neoplastic Tissues. XVI. GlucokinaseActivity and @ 5123 Induced by Ingestion of N(2-fiuorenyl) phthalamic Glycogen Levels During Hepatocarcinogenesis by Azo Dyes. acid. Cancer Research, 20:1252â€”54,1960. Cancer Research, 22 :1372â€”80, 1962. 6. PITOT, H. C., ANDHEIDELBERGER,C.Metabolic Regulatory 14. VINu@m@,E.; SAi1&s,M. ; ANDSoz@s,A. Glucokinase and Hexo Circuits and Carcinogenesis. Ibid., 23:1694â€”1700,1963. kinase in Liver in Relation to Glycogen Synthesis. J. Biol. 7. POTTER,V. R. Transplantable Animal Cancer, the Primary Chem., 238:PC1175,1963. Standard. Ibid., 21:1331-33,1961. 15. WALKER,D. G., ANDRAG, S. The Role of Glucokinase in the 8. . New Aspects in Biochemistry. In: G. WEBER (ed.), Phosphorylation of Glucoseby Rat Liver. Biochem.J., 90360- Advances in Enzyme Regulation, 1:279-308. New York: The 68, 1964. Macmillan Company and Oxford: Pergamon Press. 1963. 16. WEBER, G. Behavior of Liver Enzymes in Hepatocarcino 9. Po@rrsa, V. R.; PITOT, M. C.; ONo, T.; AND Moams, H. P. genesis. In: A. HADDOWandS. WEINHOUSE(eds.), Advances The Comparative Enzymology and Cell Origin of Rat Hepa in Cancer Research, 6:403â€”94. New York : Academic Press, tomas. I. Deoxycytidylate Deaminase and Thymidine Degra Inc., 1963. dation. Cancer Research, 20:1255-61, 1960. 17. WEINHOUSE,S. Enzyme Activities and Tumor Progression. 10. REID, E. Significant Biochemical Effects of Hepatic Carcino In: J. T. EDSALL(ed.),AminoAcids,ProteinandCancerBio gens in the Rat. A Review. Ibid., 22:398-430, 1962. chemistry, pp. 109-19. New York: Academic Press, Inc., 1960.

FIG. 1.â€”Liver tumor with fairly typical cholangiocarcinoma. H. & E., x 170. FIG. 2.â€”Liver tumor with typical hepatocarcinoma. H. & E., x 170. FIG. 3.â€”Liver tumor in which poorly differentiated cholangio carcinoma is the dominant histopathologic pattern. H. & E., x 170.

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199

Rajani Manjeshwar Sharma, Chakravarthi Sharma, Andrew J. Donnelly, et al.

Cancer Res 1965;25:193-199.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/25/2_Part_1/193

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