Biochemical Programs of Slowly and Rapidly Growing Human Colon Carcinoma Xenografts

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[CANCER RESEARCH 41, 854-859, March 1981] 0008-5472/81 /0041-0000$02.00 Biochemical Programs of Slowly and Rapidly Growing Human Colon Carcinoma Xenografts

George Weber,1 Jean C. Hager,2 May S. Lui, Noemi Prajda,3 Diana Y. Tzeng, Robert C. Jackson,4 Eiji Takeda,5 and John N. Eble

Laboratory for Experimental Oncology. Indiana University School of Medicine, Indianapolis, Indiana 46223 [G. W., M. S. L., N. P., D. Y. T., R. C. J., E. T.. J. N. E.Â¡, and Department of Medicine. Roger Williams General Hospital, Brown University School of Medicine, Providence. Rhode Island 02903 [J. C. H.]

ABSTRACT There was a marked enlargement of the pools of adenylates, guanylates, uridylates, and cytidylates in the rapidly growing The purpose of this investigation was to elucidate the en- neoplasm, in line with the increased enzymic capacities. Par zymic programs of pyrimidine, carbohydrate, and purine me ticularly marked rises were observed in the concentrations of tabolism and the pattern of pyrimidine and purine ribonucleo- xanthosine 5'-phosphate, uridine 5'-diphosphate, cytidine 5'- tides in two lines of human colon carcinoma xenografts of phosphate, and cytidine 5'-diphosphate (11- to 18-fold). different growth rates. The slower-growing colon tumor line The high activities of both de novo and salvage pathways of was well differentiated; the more rapidly growing line was a pyrimidine biosynthesis and the elevated enzymic capacities in poorly differentiated one. The carcinoma xenografts were car carbohydrate catabolism and in purine biosynthesis, along with ried in nude (athymic) mice. the large pools of pyrimidine and purine ribonucleotides, may The increased malignancy and growth rate of the rapidly account for, in part at least, the clinical difficulties encountered growing colon tumor were characterized by a markedly ampli in the chemotherapy of human colon neoplasia. fied imbalance in the enzymic programs and nucleotide pat The results indicate the applicability of the molecular corre terns. In the rapidly growing carcinoma line, the activities of lation concept to human colon neoplasia and should be helpful enzymes of the pyrimidine de novo biosynthesis (cytidine 5'- in the rational design of enzyme pattern-targeted chemo triphosphate synthetase, orotidine 5'-monophosphate decar- therapy of colon tumors. boxylase, orotate phosphoribosyltransferase) and those of the salvage pathways (thymidine kinase, uracil phosphoribosyl INTRODUCTION transferase, uridine kinase) were markedly higher than those in the slower-growing tumor line. The activities of the glycolytic In this laboratory, studies guided by the molecular correlation enzymes (hexokinase, phosphofructokinase, pyruvate kinase) concept led to the discovery of an integrated pattern of enzymic and of those of pentose phosphate production (glucose-6- imbalance that was linked with transformation and progression6 phosphate and 6-phosphogluconate dehydrogenases, transal- in a series of chemically induced, transplantable, solid rat dolase) were also elevated in the rapidly growing neoplasm. hepatomas of different growth rates (13, 14). Recently, we also The activity of the enzyme that converts ribose 5-phosphate reported that the levels of certain purine and pyrimidine ribo into phosphoribosylpyrophosphate (phosphoribosylpyrophos- nucleotides and deoxyribonucleoside triphosphates in the hep phate synthetase) was also augmented. In the purine metabo atomas also were linked with transformation and progression lism of the rapidly growing carcinoma, there was increase in (4, 5, 19). the activity of the first enzyme committed to de novo biosyn The purpose of this investigation was to test the applicability thesis (glutamme phosphoribosylpyrophosphate amidotrans- of the molecular correlation concept to human colon tumor ferase); by contrast, the activities of the opposing purine cat- xenografts of different growth rates and differentiation. The abolic enzymes (xanthine oxidase, inosine phosphorylase) studies also were planned to determine whether or not there were decreased. The activities of the enzyme that produces inosine 5'-phosphate from adenylates (adenosine 5'-phos- are any enzymic activities or nucleotide concentrations that might serve as biochemical markers of growth rate and degrees phate deaminase) and of the rate-limiting enzyme of guanylate biosynthesis (inosine 5'-phosphate dehydrogenase) also were of differentiation in the human colon tumors. It was also pos tulated that a deeper insight into the enzymic programs and elevated in the rapidly growing colon tumor lines. nucleotide patterns might illuminate the biochemical pharma The enzymes that were identified as progression linked in cological basis of the difficulties encountered in the drug treat the rat hepatoma system were also progression linked in the ment of human colon tumors. two human colon carcinomas of different growth rates (cytidine The results showed that the approaches of the molecular 5'-triphosphate synthetase, thymidine kinase, hexokinase, phosphofructokinase, pyruvate kinase, inosine 5'-phosphate correlation concept of neoplasia did apply to the human colon dehydrogenase, and adenosine 5'-phosphate deaminase). xenografts of different growth rates. The activities of certain key enzymes in pyrimidine, carbohydrate, and purine metabo ' Recipient of USPHS Grants CA-13526 and CA-05034. To whom requests lism provided markers for the growth rate and degrees of for reprints should be addressed. malignancy of the human colon tumor xenografts. The increase 2 Present address: Department of Immunology, Michigan Cancer Foundation, in the pools of pyrimidine and purine ribonucleotides also Detroit, Mich. 48201. 3 Permanent address: National Cancer Institute, Budapest, Hungary. provided a biochemical indicator of the degrees of malignancy. ' Recipient of USPHS Grant CA-18129. 5 Permanent address: Department of Pediatrics, Tokushima University School 6 In this context, the graded expression of the neoplastic program in the of Medicine. Tokushima City, Japan. different tumor lines in a spectrum of neoplasms of the same cell type is termed Received July 7, 1980; accepted November 11, 1980. "progression" (13).

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The clinical difficulties in the treatment of colon tumors with tumor cells. The clone A carcinoma was more pleomorphic, single agents that inhibit solely the de novo synthesis [e.g., had an increased nucleancytoplasmic ratio, had an increased pyrazofurin, A/-(phosphonacetyl)-L-aspartic acid] or utilize the mitotic rate, showed decreased mucus production, and had salvage pathway for activation (e.g., 5-fluorouracil) might be fewer glandular structures than the DLD-1 nude mouse tumor. accounted for, in part at least, by the presence of strong The clone A tumor was considered, on this basis, a poorly activities of the salvage enzymes and of the de novo synthetic differentiated adenocarcinoma. An inoculum of 1 x 106 cells enzymes of uridylate and cytidylate production. These obser produced tumors in nude mice in approximately 2 weeks. The vations should be helpful in the design of rational chemotherapy cell line in culture had a doubling time of approximately 20 hr. of human colon carcinoma. Development of the tissue culture cell lines, the conditions for in vitro culture, passage of cells into athymic mice, and in MATERIALS AND METHODS vitro characteristics of the cell lines were described elsewhere by Dexter ef al. (2) and Hager et al. (3). Preparation of Tumor Extracts. Clone A cells were injected Nude (athymic) mice were inoculated s.c. with the tumor 3 weeks prior to testing, and DLD-2 tumor cells were inoculated xenografts at 4 sites on the flanks. The mice were housed 5/ 7 weeks prior to the experiment. The tumor-bearing mice were cage in air-conditioned rooms that were illuminated daily from shipped from Brown University, Providence, R. I., to the Lab 6 a.m. to 7 p.m. Purina laboratory chow and water were oratory for Experimental Oncology, Indiana University School available ad libitum. Mice were killed between 9 and 10 a.m. of Medicine, Indianapolis, Ind., and experiments were carried The animals were autopsied, and the stomach contents were out 2 to 7 days later, when tumors were 1.0 cm in diameter. examined to confirm that all mice were healthy and well fed The mice were stunned, decapitated, and exsanguinated. during the night before death. The xenograft tumors were quickly excised and placed in Biological Systems: Origin, Histology, and Biological Prop beakers embedded in crushed ice. The neoplasms were dis erties of the Human Colon Carcinoma Xenografts (2, 3) sected free of macroscopically visible necrotic, hemorrhagic, and normal tissues. The careful selection and utilization of only The DLD-2 Human Colon Carcinoma Xenograft (Slowly the viable carcinomatous tissue was important in ensuring the Growing Line). The DLD-2 line was established from a well- to comparability of neoplasms in the different shipments. moderately differentiated adenocarcinoma of the human colon resected surgically at one of the Brown University Affiliated Biochemical Procedures Hospitals. The original neoplasm penetrated the muscularis Enzyme Assays. From the tissues, 20% homogenates were propria, invaded lymphatics, and was inflamed. It grew mainly prepared, and the 100,000 x g supernatant was obtained as as glands and had no solid areas. The glands were medium described elsewhere (18). The resulting clear supernatant fluid sized, well defined, and arranged in a back to back pattern; some were cystically dilated and filled with mucus. The cancer was used for the enzyme assays. cells were large and cylindrical-cuboidal, with brush borders The methods for determination of the enzymes of pyrimidine, purine, carbohydrate, and pentose phosphate metabolism were and mucin vacuoles. The nuclei were large and oval or round, as cited elsewhere (9, 13-16, 18). The enzyme assays were with clefts and folds. Most nuclei contained one or more large adapted to the conditions of the colon tumors by working out eosinophilic nucleoli. Mucicarmine staining showed the cells to optimum substrate and activator concentrations and optimum be 3 to 4 + in intracellular and intraglandular mucin, mainly supranuclear, and also along brush borders; 90% of the tumor pH. As a result, enzyme measurements were carried out under linear kinetic conditions where the enzyme activity was pro stained positively. An inoculum of 1 to 2 x 106 cells produced palpable tumors portionate to the amount of enzyme added and with time in nude mice in about 4 to 5 weeks. The cell line in culture had elapsed. Metabolic Assays. The concentrations of ribonucleotides a doubling time of about 48 hr and did not produce colonies in were measured as reported earlier (4, 5) in samples obtained soft agar. by the freeze-clamp method adapted to the conditions of the The Clone A Human Colon Carcinoma Xenograft (Rapidly tumor-bearing animals (19). Growing Line). The clone A cell line was derived from another surgical specimen, the DLD-1 tumor. This neoplasm was also Protein content was determined by the routine method (10). a well- to moderately differentiated colonie adenocarcinoma Expression and Evaluation of Results with (a) small closely packed glands lining a large space, cystically dilated, filled with mucus or (b) numerous medium Enzymic activities were calculated in nmol substrate metab and small glands, back to back with prominent mucus produc olized per hr per mg of protein (specific activity). Nucleotide tion. The cells were of moderate to large size and of variable concentrations were given in nmol/g (wet weight) of tissue. contour. The nuclei were medium to large and oval, with some The results were subjected to statistical evaluation by means indentation and folding. The cytoplasm was eosinophilic and of the f test for small samples. Differences between means frequently contained mucous vacuoles. One or more nucleoli giving a probability of less than 5% were considered as statis were present in each nucleus. Mucicarminophilic intracyto- tically significant. plasmic vacuoles and luminal secretions were present through out the neoplasm. RESULTS AND DISCUSSION The clone A cell line was obtained by selecting colonies growing in soft agar. The tumor produced by injection of clone Histological examination of both the slowly (DLD-2) and the A cells into athymic mice consisted of many solid nests of rapidly (clone A) growing transplanted colon carcinoma lines

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. G. Weber et ai. as xenografts was done to provide a morphological character enzyme of de novo purine biosynthesis (glutamine PRPP ami- ization of the tumors on which the biochemical studies were dotransferase) was increased, and concurrently the activity of carried out. the opposing, rate-limiting, purine catabolic enzyme (xanthine oxidase) was decreased. The activity of the purine catabolic Histology of the Slowly Growing Carcinoma (OLD-2) as Xen enzyme (inosine phosphorylase) was also decreased. Conse ogran quently, the ratio of the activities of the synthetic to those of the catabolic purine enzymes was markedly elevated in the Microscopically, the tumors grew as circumscribed nodules rapidly growing colon carcinoma line. composed of neoplastic glands producing abundant mucus The activity of the enzyme that is rate limiting in channeling and arranged on delicate fibrovascular septa. Individual cells IMP to guanylate biosynthesis (IMP dehydrogenase) and the had increased ratios of nucleus to cytoplasm, increased density activity of the enzyme that produces IMP from AMP (AMP of nuclear chromatin, and abnormal variability of nuclear size deaminase) were markedly elevated in the rapidly growing and shape. The cytoplasmic contours were often columnar but colon tumor line. also were variably polygonal and ill defined. Mucus goblet cells were present; mitotic figures were seen frequently. The histo- Purine and Pyrimidine Ribonucleotides in Slowly and Rapidly pathological diagnosis was moderately differentiated adeno- Growing Human Colon Carcinoma Xenografts carcinoma with prominent mucus secretion (Figs. 1 and 2). The concentrations of adenylates, guanylates, uridylates, Histology of the Rapidly Growing Carcinoma (Clone A) as and cytidylates were compared in freeze-clamped preparations Xenograft obtained from the slowly and rapidly growing human colon carcinoma xenograft lines (Table 2). The concentrations of Microscopically, the neoplasm grew as circumscribed nod adenylates were higher than those of the other nucleotides. ules consisting of sheets of malignant cells. These cells had The highest concentrations were observed for ATP, whereas large vesicular nuclei, often with prominent nucleoli. The cy those for IMP, XMP, GMP, CDP, and CMP were the lowest toplasm was sparse and its borders were ill defined. The nuclei among the nucleotides. were quite crowded together and many mitotic figures were observed. A few poorly formed glands were present, identified Table 1 by the peripheral location of the nuclei and central disposition Enzymic programs of slowly and rapidly growing human colon carcinoma xenografts of the cytoplasm of their cells. Mucicarmine-stained sections Slow (nmol/ showed mucin in the lumens of some of these glands and hr/mg pro- Rapid (% tein) of slow)3 mucin-containing vacuoles in some of the cells. The histopath- Enzymes EC No. ological diagnosis was poorly differentiated adenocarcinoma Pyrimidine (Figs. 3 and 4). Synthetic CTP synthetase 6.3.4.2 6.5 186 Thymidine kinase 2.7.1.75 0.8 1,409 Enzymology of Slowly and Rapidly Growing Human Colon Orotidine 5'-monophosphate 4.1.1.23 14.2 302 Carcinoma Xenografts decarboxylase Orotate phosphoribosyl 2.4.2.10 13.9 269 transterase The specific activities of key enzymes of pyrimidine, carbo Uracil phosphoribosyl trans- 2.4.2.9 5.9 266 hydrate, and purine metabolism in the slowly and more rapidly ferase growing colon carcinomas are compared in Table 1. Uridine kinase 2.7.1.48 141.0 261 Catabolic Pyrimidine Metabolic Enzymes. In the rapidly growing car Uridine phosphorylase 2.4.2.3 235.4 50 cinoma line, the activities of the enzymes of de novo biosyn Uridine kinase:phosphorylase 0.6 525 thesis (CTP synthetase, orotidine 5'-monophosphate decar- Glycolytic boxylase, orotate phosphoribosyltransferase) were markedly Hexokinase 2.7.1.1 262 756 increased above those of the slowly growing neoplasms. The Phosphofructokinase 2.7.1.11 9,100 159 activities of the salvage enzymes (thymidine kinase, uracil Pyruvate kinase 2.7.1.40 50,000 498 phosphoribosyltransferase, uridine-cytidine kinase) were also Pentose phosphate elevated. Glucose-6-phosphate dehydro- 1.1.1.49 539 807 genÃ¤se Carbohydrate Metabolic Enzymes. In the rapidly growing 6-Phosphogluconate dehydro- 1.1.1.44 1,860 163 carcinoma, the activities of the key glycolytic enzymes (hexo- genÃ¤se kinase, phosphofructokinase, pyruvate kinase) were increased Transaldolase 2.2.1.2 3,490 318 PRPP synthetase 2.7.6.1 40 155 above those of the slowly growing carcinoma xenograft. The activities of the enzymes involved in the biosynthesis of pentose Purine phosphate (glucose-6-phosphate dehydrogenase, 6-phospho- Synthetic IMP dehydrogenase 1.2.1.14 8 175 gluconate dehydrogenase, transaldolase) were elevated above AMP deaminase 3.5.4.6 2,480 659 those of the slowly growing colon tumor line. The activity of the Adenosine deaminase 3.5.4.4 1,670 193 enzyme that converts ribose 5-phosphate to PRPP7 (PRPP Glutamine PRPP amidotrans- 2.4.2.14 35 489 ferase synthetase) was also increased. Catabolic Purine Metabolic Enzymes. In the rapidly growing human Xanthine oxidase 1.2.3.2 514 17 Inosine phosphorylase 2.4.2.1 7,700 29 colon carcinoma xenografts, the activity of the first committed Amidotransferase:xanthine 0.07 2,592 oxidase 7 The abbreviations used are: PRPP, phosphoribosylpyrophosphate; XMP, " All values are significantly different from those of the slowly growing tumor xanthosine monophosphate. (p < 0.05).

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Table 2 evated, and that of the opposing catabolic enzymes, inosine Purine and pyrimidine ribonucleotide pattern in human colon tumor xenografts phosphorylase and xanthine oxidase, was decreased. Nucleotides Slow (nmol/g) Rapid (nmol/g) i of slow Certain aspects of the ribonucleotide patterns of the hepa Â±16Â°145 ATPADPAMPTotal Â±127793 tomas were applicable to the human colon cancer xenografts, Â±1273 Â±186446 with some important differences (Table 2). Earlier work indi Â±20392 Â±672653 adenylatesNAD*IMPXMPGTPGDPGMPTotalÂ±2978 Â±40031 cated that the pool of ATP underwent a growth rate-related depletion (5, 19). However, in the colon carcinomas, the ade- Â±816 0Â±5391 nylate pools were elevated. The overall orders of magnitude in Â±104 Â±2245 the relationships of adenylates, guanylates, cytidylates, and Â±234 Â±20336 uridylates appeared to be roughly similar in colon tumor xen

Â±629 Â±39243 ografts and in hepatomas. Among the nucleotides, IMP, XMP, Â±710 Â±9243 GMP, CDP, and CMP pools were the lowest in human colon Â±473 Â±14622 tumor xenografts as was observed earlier in hepatomas (5). It guanylatesUTPUDPUMPUDP-glucoseUDPAGTotalÂ±1055 Â±120395 is striking that the expansion of the pools in rapidly growing Â±824 Â±84303 human colon carcinoma xenografts uniformly occurred in all Â±S18 Â±92119Â± Â±967 20412 purine and pyrimidine ribonucleotides. This is in contrast to the Â±1332 Â±29143Â± elevations observed in hepatomas which occurred selectively Â±7196 371372 for GMP, CTP, CMP, and the total amount of cytidylates. uridylatesCTPCDPCMPTotal Â±3527 Â±133164 Markers of Malignancy in Colon Tumor Xenografts. The Â±56 Â±151 progression-linked enzymic markers that might be the best Â±43 10 Â±2040 indicators of the growth rate and degree of differentiation in Â±336 Â±8320 cytidylates174 Â± 121414 Â± 418135476116773975691125988718430852718126067061944770060718301530889these tumors are the activities of CTP synthetase, thymidine All values are significantly different from values of slowly growing tumor (p kinase, hexokinase, pyruvate kinase, IMP dehydrogenase, and < 0.05). AMP deaminase. The profound elevations observed in concen b Means Â±S.E. of 5 to 7 determinations. trations of XMP, UDP, CMP, and CDP should also be helpful markers in malignancy in the human colon tumors. In the rapidly growing colon carcinoma line, the concentra tions of all purine and pyrimidine ribonucleotides were elevated Biochemical Imbalance in Human Colon Tumor Xenografts: 3- to 18-fold. The most marked enlargement of the pools was Selective Advantages Conferred and Reasons for Difficulties observed in the concentrations of those nucleotides that were in Chemotherapy of These Neoplasms particularly low in the slowly growing neoplasm. For instance, XMP, CMP, and CDP levels were increased 11-, 15-, and 18- The presence of a large noncycling fraction in primary human colon carcinomas contributes to the low sensitivity of these fold, respectively. The enlargement in the pools of the ribonu tumors to drug action. The profound biochemical imbalance cleotides corresponds with the elevated capacity of the purine observed in colon tumors throws light on some of the biochem and pyrimidine synthetic enzymes. For instance, the increased concentrations of XMP should reflect the elevated IMP dehy- ical reasons for difficulties encountered in any single drug treatment protocol for these neoplasms. The heightened activ drogenase activity. The increased concentrations of uridylates ities of both de novo and salvage pathway enzymes and the and cytidylates probably are reflections of the elevated activi remarkably enlarged pools of purine and pyrimidine ribonucle ties of the de novo and salvage enzymes in these metabolic otides should confer selective advantages for replication on areas. The increased concentration of IMP may well be the result of the elevated activities of AMP deaminase and gluta- the human colon carcinoma cell. Single agents that might inhibit the activity of the de novo pathway [pyrazofurin, N- mine PRPP amidotransferase. (phosphonacetyl)-L-aspartic acid] could fail to provide thera- Applicability of the Molecular Correlation Concept to Human peutically significant inhibition of growth (1, 12) because of the Colon Tumors operation of the high activities of the salvage enzymes, partic ularly that of uridine kinase. Inhibition of CTP synthetase (by The increased enzymic activities (Table 1) demonstrate that glutamine antagonists, e.g., azaserine, etc.) might be only the enzymic and metabolic imbalance, originally discovered in partially effective because the marked activity of uridine-cyti- chemically induced, transplantable hepatomas of different dine kinase should provide salvage of cytidine which is beyond growth rates in rat (13, 14), was applicable to human colon the block for CTP synthetase activity. In the design of chemo carcinoma xenografts of different growth rates. This is partic therapy, an enzyme pattern-targeted approach (7, 8, 11) will ularly clear in the behavior of activities of enzymes that were have to be designed that takes cognizance of the enzymic and observed to increase with hepatoma growth rates. Thus, the nucleotide pattern now revealed in these human colon tumor activities of CTP synthetase (20), thymidine kinase (18), hex- xenografts. For this purpose, enzymic activities and metabolic okinase, phosphofructokinase, pyruvate kinase (20), AMP de functions restricted to or characteristic of human colon mucosa aminase, and IMP dehydrogenase (6) that were identified as cells should be identified and their behavior in neoplasia should progression linked in the rat hepatomas were also progression be elucidated. Through such an approach, a selective effect of linked in the human colon carcinoma xenografts. cancer drugs may be achieved that might be specific to human The reciprocal relationship of the activities of the synthetic colon neoplasia. and catabolic enzymes, first recognized in the hepatomas (13, The importance of the observations outlined in this investi 14), also applied to the colon carcinomas as the activity of the gation is emphasized by our current studies in 9 patients (1 7) synthetic enzyme, glutamine PRPP amidotransferase, was el- that have confirmed, by enzymic studies of human colon mu-

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1981 American Association for Cancer Research. G. Weber et al. cosa and primary colon adenocarcinomas, the relevance to on rat hepatoma cells in vitro. Biochem. Pharmacol., 25. 2613-2618, 1976. 8. Jackson, R. C., Williams, J. C., and Weber, G. Enzyme pattern-directed human primary colon tumors of the enzymic alterations re chemotherapy: synergistic interaction of 3-deazauridine with o-galactosa- ported in the present work. mine. Cancer Treat. Rep., 60: 835-843, 1976. 9. Kizaki, H., Morris, H. P., and Weber, G. Behavior of inosine phosphorylase (EC 2.4.2.1) activity in normal, differentiating, regenerating, and neoplastic ACKNOWLEDGMENT liver. Cancer Res., 40: 3339-3344, 1980. 10. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein The authors thank Dr. Daniel L. Dexter, Brown University School of Medicine, measurement with the Folin phenol reagent. J. Biol. Chem., 793: 265-275, for his role in providing the colon carcinoma xenografts. 1951. 11. Lui, M. S., Jackson, R. C., and Weber. G. Enzyme pattern-directed chemo REFERENCES therapy. Effects of anti-pyrimidine combinations on the ribonucleotide con tent of hepatomas. Biochem. Pharmacol., 28: 1189-1195, 1979. 1. Cadman, E. C., Dix, D. E., and Handschumacher, R. E. Clinical, biological, 12. Ohnuma, T., Roboz, J., Shapiro, M. L., and Holland J. F. Pharmacological and biochemical effects of pyrazofurin. Cancer Res., 38: 682-688, 1978. and biochemical effects of pyrazofurin in humans. Cancer Res., 37: 2043- 2. Dexter, D. L., Barbosa, J. A., and Calabresi, P. W.N-Dimethylformamide- 2049, 1977. induced alteration of cell culture characteristics and loss of tumorigenicity 13. Weber, G. Enzymology of cancer cells, part 1. N. Engl. J. Med., 296: 486- in cultured human colon adenocarcinoma cells. Cancer Res., 39. 1020- 493, 1977. 1025, 1979. 14. Weber, G. Enzymology of cancer cells, part 2. N. Engl. J. Med., 296: 541- 3. Hager, J. C., Gold, D. V., Barbosa, J. A., Fligiel, Z., Miller, F., and Dexter, 551, 1977. D. L. N.W-Dimethylformamide-induced modulation of organ and tumor-as 15. Weber, G., Jackson, R. C., Williams, J. C., Goulding, F. J., and Eberts, T. J. sociated markers in cultured human colon carcinoma cells. J. Nati. Cancer Enzymatic markers of neoplastic transformation and regulation of purine Inst., 64: 439-446, 1980. and pyrimidine metabolism. Adv. Enzyme Regul., 75: 53-77, 1977. 4. Jackson, R. C., Boritzki, T. J., Morris, H. P., and Weber, G. Purine and 16. Weber, G., Kizaki, H., Tzeng, D., Shiotani, T., and Olah, E. Colon tumor: pyrimidine ribonucleotide contents of rat liver and hepatoma 3924A and enzymology of the neoplastic program. Life Sci., 23: 729-736, 1978. effect of ischemia. Life Sci., 19: 1531-1536, 1976. 17. Weber, G., Lui, M. S., Takeda, E., and DentÃ³n, J. E. Enzymic programs of 5. Jackson, R. C., Lui, M. S., Boritzki. T. J., Morris, H. P., and Weber, G. human colon tumors. Proc. Am. Assoc. Cancer Res., 27. 135, 1980. Purine and pyrimidine nucleotide patterns of normal, differentiating, and 18. Weber, G., Shiotani, T., Kizaki, H.. Tzeng, D., Williams, J. C., and Gladstone, regenerating liver and of hepatomas in rats. Cancer Res., 40: 1286-1291, N. Biochemical strategy of the genome as expressed in regulation of pyrim 1980. idine metabolism. Adv. Enzyme Regul., 76: 3-19, 1978. 6. Jackson, R. C., Morris, H. P., and Weber, G. Enzymes of the purine 19. Weber, G., Stubbs, M., and Morris, H. P. Metabolism of hepatomas of ribonucleotide cycle in rat hepatomas and kidney tumors. Cancer Res., 37: different growth rates in situ and during ischemia. Cancer Res., 37: 2177- 3057-3065. 1977. 2183, 1971. 7. Jackson, R. C.. and Weber, G. Enzyme pattern-directed chemotherapy: the 20. Williams, J. C., Kizaki, H., Morris, H. P., and Weber, G. Increased CTP effects of combinations of methotrexate. 5-fluorodeoxyuridine and thymidine synthetase activity in hepatomas. Nature (Lond.), 277: 71-73, 1978.

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â€¢â€¢^"v'\ ^.^div ÃŽV"V% '

:V- Ali- WÃr^vÃÂ«Ã1%%&r5 V'Z*?*-Â«Â«.**>,Ly \ \_ >u*j ***"'â€¢! ^ Â»**i Â«i.'â€¢ â€¢*â€¢â€¢**Â« i!'4.t* i â„¢ , !kÂ«,* " ' *ri5l^,V^- ^ Tiw<Â».wt * ^B ' ' i Ftg. 1. Photomicrograph of the DLD-2 human colon carcinoma as xenograft. H & E, x 40. Fig. 2. Photomicrograph of the DLD-2 human colon carcinoma as xenograft. H & E, x 63. Fig. 3. Photomicrograph of the clone A human colon carcinoma as xenograft. H & E, x 40. Fig. 4. Photomicrograph of the clone A human colon carcinoma as xenograft. H & E, x 63.

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George Weber, Jean C. Hager, May S. Lui, et al.

Cancer Res 1981;41:854-859.

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