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Recent work in this Laboratory showed increased activity of PI 4-kinase, PIP kinase and PLC in various cancer cells, indicating a stepped-up capacity for signal transduction. This elevated potential was paralleled with increased concentration of the end product of signal transduction, IP3. Current investigations showed that in normal cells the activities of the specific phosphatases (which degrade PIP2 and PIP and oppose those of the synthetic enzymes) were 4 to 5 orders of magnitude higher than those of the synthetic kinases. In hepatoma cells the specific phosphatase activities markedly decreased. Thus, in cancer cells the marked elevations in activities of the synthetic enzymes were opposed by a reduction in the activities of the degradative specific phosphatases. This enzymic imbalance is responsible, in part at least, for the elevated capacity of signal transduction and IP3 concentration. Since the enzymic activities measured were proportionate with time elapsed and amount of enzyme added, the alterations in activities should reflect changes in enzyme amounts. These alterations indicate a reprogramming of gene expression which should confer selective advantages to the cancer cells, marking out the elevated synthetic enzyme activities as potentially sensitive targets for drug treatment. We showed earlier that tiazofurin, which curtailed the biosynthesis of enzymes with short half-lives such as PI and PIP kinases, down-regulated signal transduction and brought down IP3 concentration. Quercetin and genistein chiefly inhibited PI-4 kinase and PIP kinase, respectively, and as a result reduced IP3 concentration in cancer cells. Current studies reveal that tiazofurin with quercetin, tiazofurin with genistein, and quercetin with genistein were synergistic in killing human cancer cells and in reducing signal transduction activity. In estrogen receptor-negative MDA-MB-435 human breast carcinoma cells which have elevated signal transduction activity, tamoxifen caused IC50S for growth inhibition and cytotoxicity of 12 and 0.7 microM, respectively. When tiazofurin was added to breast carcinoma cells, followed 12 hr later by tamoxifen, synergism was observed in growth inhibition, in clonogenic assays and in the reduction of IP3 concentration. The synergistic action of tiazofurin and tamoxifen and the other synergistic drug interactions outlined above may have implications in the clinical treatment of neoplasias.

A single treatment with quercetin (5.5 microM), a plant flavonoid, activated both apoptosis and differentiation programs in K562 human leukemia cells. K562 cells expressed commitment to apoptosis after 1 h exposure, however, at least 12 h of drug exposure was needed to induce differentiation. Early (1 h) down-regulation of the c-myc and Ki-ras oncogenes and rapid reduction of inositol-1,4,5-trisphosphate (IP3) concentration (IC50 = 9 microM, 1 h incubation) are part of the antiproliferative action of quercetin and appear to relate to induction of differentiation and/or apoptotic program of K562 leukemia cells treated with quercetin.

The purpose of this paper was to clarify critical aspects of the behavior of signal transduction activity in normal and cancer cells. 1. 1. Signal transduction activity in the conversion of phosphatidylinositol through PI and PIP kinases and PLC to IP3 is regulated at multiple sites. In liver, hepatomas and human carcinomas PIP kinase is the rate limiting enzyme and PLC activity is present in great excess. 2. 2. The steady-state signal transduction activity as measured by the three enzyme activities and IP3 concentration was markedly up-regulated in rat hepatomas of different growth rates. The steady-state specific activities of the three signal transduction enzymes were elevated in ovarian carcinomas as compared to normal ovary. Increased enzyme activities were also observed in human breast carcinoma cells as compared to normal human breast parenchymal cells. In breast, ovarian and rat hepatoma cells as they go through lag, log and plateau phases, IP3 concentration in the early lag phase increased 4.5- to 20-fold and PI and PIP kinase activities peaked in mid-log phase. These events returned to baseline levels in the plateau phase. PLC activity did not change. 3. 3. The bone marrow PI and PIP kinase activities in 3-day starvation were decreased to 13% and IP3 concentration was reduced to 24%; at 1-day refeeding they returned to normal. PLC activity changed little. These alterations are in line with the rapid t12 degradation rates (12 min) of PI and PIP kinases observed in studies with cycloheximide. By contrast, PLC has a long half-life. 4. 4. The molecular action of tiazofurin entails inhibition of IMP DH activity, decrease in GTP and IP3 concentrations, reduction of ras and myc oncogene expression, and signal transduction enzyme activities. These events are followed by induced differentiation and apoptosis. There are also decreases in enzyme activities which have rapid turnover, including TdR kinase, dTMP synthase, and GPRT. In vitro studies indicated that these events are abrogated by addition of guanine which restores GTP concentration. Therefore, most or all these events were brought about by the reduced GTP concentration in the tiazofurin target cells. 5. 5. Quercetin and genistein are able to inhibit PI and PIP kinase activities and reduce IP3 concentration in vivo and in tissue culture systems. These flavonoids are also inhibitors of cell proliferation and clonogenic ability in rat hepatoma 3924A and in human OVCAR-5 and MDA-MB-435 cells. Quercetin down-regulated the expression of c-myc and Ki-ras oncogenes and led to induced differentiation and apoptosis in K562 cells. Genistein reduced IP3 concentration in vivo and in the tissue culture system. Genistein is anti-proliferative and has cytototoxicity in human carcinoma cells. All three drugs, tiazofurin, quercetin and genistein, act, in part at least, through depression of cellular IP3 concentration although the mechanisms may not be identical. 6. 6. Quercetin and genistein, which attack different targets and different phases of the cell cycle, proved to be synergistic in OVCAR-5 cells. The impact of tiazofurin, genistein and quercetin is of interest because the drugs crucially inhibit the display of the neoplastic program of cells and lead to induced differentiation and apoptosis.

In cancer cells, particularly in leukaemic cells, guanylate biosynthesis is up-regulated as shown by the increased activities of IMP dehydrogenase, the rate-limiting enzyme of de novo GTP biosynthesis, and of the salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). In enzyme pattern-targeted chemotherapy, tiazofurin inhibits IMP dehydrogenase activity in cancer cells and allopurinol-induced high serum hypoxanthine levels inhibit HGPRT activity. A triad of responses was observed in the blast cells of patients treated with tiazofurin infusions: chemotherapy, induced differentiation, and down-regulation of c-Ki-ras and c-myc oncogenes. Tiazofurin was synergistic in cytotoxicity and in causing differentiation with ribavirin, retinoic acid, and gemcitabine [corrected]. Induced differentiation plays an important role in the overall impact of antipurine agents.

1. (1)|The currently used clinical anti-metabolites are targeted against key enzymes of de novo purine and pyrimidine biosynthesis. However, the activities of salvage enzymes in each of the biosynthetic segments are markedly higher than those of the rate-limiting enzymes of de novo biosynthesis. Enzyme-pattern-targeted chemotherapy has been suggested to overcome the circumvention activity of salvage. Combination of inhibition of de novo and salvage pathways does provide a synergistic impact. Examples that enzyme-pattern-targeted drug treatment yields synergism include the following: tiazofurin (against IMP DH) and allopurinol (by raising serum hypoxanthine levels it inhibits GPRT); methotrexate or 5-FU lead to inhibition of the dTMP synthase reaction and AZT (a competitive inhibitor of thymidine kinase) or dipyridamole (a nucleoside transport inhibitor); acivicin, an inhibitor and inactivator of glutamine-utilizing enzymes in the de novo pathways of purine and pyrimidine biosynthesis, and dipyridamole. 2. (2)|Administration of MTX, 5-FU, tiazofurin or acivicin causes inhibition and/or inactivation of target enzymes. That these drugs are effective in spite of the presence of highly active salvage enzymes is now accounted for, at least in part, by new observations showing that these drugs markedly reduce (but do not eliminate) the activities (amounts) of CdR and TdR kinases, dTMP synthase and GPRT. This action is attributed to the rapid decay rate of these enzymes. 3. (3)|Studies on the bone marrow enzymic programs indicate that there is a window of opportunity for strengthening therapy and for the protection of bone marrow by administering salvage metabolites when the salvage enzymes are still present in high enough activities, i.e., 2–6 hr after administration of the blockers of de novo enzyme activities. 4. (4)|These results are a strong argument for discovering and utilizing inhibitors of purine and pyrimidine salvage enzymes to achieve more successful enzyme-pattern-targeted chemotherapy and to avoid development of resistant clones of cancer cells. 5. (5)|These approaches provide greater explanatory coherence than the previous accounts because recognition of (a) the importance of salvage and (b) rapid decay of key and salvage enzymes reveals a paradigm shift. The problem-solving process in chemotherapy should now be not only data-driven but also explanation-driven.

1. (1) Deoxycytidine kinase activity increased in a transformation- and progression-linked fashion in rat hepatomas of different proliferation rates (1). The activity also increased and was growth rate-linked in a series of tissue culture cell lines of human and animal tumors. 2. (2) Deoxycytidine kinase activity was stringently linked with expression of the neoplastic proliferative program as it sharply increased in log phase in tissue culture cells of hepatoma 3924A and several human carcinoma strains. 3. (3) Deoxycytidine kinase is subject to nutritional and hormonal regulation. On starvation the activity in liver decreased and on refeeding it returned to normal. Steroid hormone increased liver enzymic activity. Deoxycytidine kinase is substrate-inducible, since deoxycytidine injections in rat led to a 2- to 3-fold increase in hepatic enzyme activity. 4. (4) Actinomycin or cycloheximide treatment blocked the increase in liver deoxycytidine kinase activity induced by steroid or deoxycytidine treatment. Therefore, it is assumed that the rise in deoxycytidine kinase activity requires new RNA and protein synthesis. 5. (5) Cycloheximide treatment of rats carrying hepatomas yielded a t 1 2 = 3.4 hr in the tumor for deoxycytidine kinase activity which was the shortest among the examined enzymes of purine and pyrimidine biosynthesis. 6. (6) Actinomycin treatment of rats carrying hepatomas yielded a t 1 2 of 5.8 hr for deoxycytidine kinase activity in the tumor which was one of the shortest in the examined enzymes of purine and pyrimidine biosynthesis. 7. (7) Difluorodeoxycytidine (DFDC) is a competitive inhibitor ( K i = 7 − 28 μ m ) of deoxycytidine kinase from rat hepatoma and from human pancreatic carcinoma and ovarian carcinoma cells in culture.

Cancer cells have an increased ability to synthesize GTP (guanosine triphosphate) because of increased activity of IMP DH (inosine 5'-phosphate dehydrogenase, EC 1.1.1.205). Because IMP DH activity is rate limiting for de novo biosynthesis of GTP, this enzyme was suggested as a sensitive target for chemotherapy. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is converted in the cells into the active metabolite, TAD, (thiazole-4-carboxamide adenine dinucleotide) which potently inhibits IMP DH activity. By adding TAD to tissue extracts one can determine the extent of inhibition of IMP DH. We applied the IMP DH assay method to extracts of normal ovaries (N = 11) and epithelial ovarian carcinomas (N = 10). The IMP DH activity (mean +/- SE) in ovarian carcinoma was 21.1 +/- 5.8 which was markedly higher than that observed in normal ovaries (2.9 +/- 0.7 nmol/hr/mg protein) (P < 0.05%). The inhibition by TAD of IMP DH activity in ovarian carcinomas (N = 4) was 81%. The results indicate that IMP DH activity is elevated sevenfold in ovarian carcinomas as compared to normal ovary and can be inhibited by exposure to tiazofurin (TAD). Similar high IMP DH activity and inhibition of the activity by TAD was observed in patients with chronic granulocytic leukemia in blast crisis among whom 70 to 80% remissions were reported. Since there is increased IMP DH activity in human ovarian carcinomas and in OVCAR-5 cells and tiazofurin and TAD inhibit IMP DH activity of these cells and the proliferation of human ovarian carcinoma xenografts in the mouse, tiazofurin may merit serious consideration for a Phase II trial for patients with recurrent/refractory epithelial ovarian carcinoma.

MTX cytotoxicity is not fully explained by its well-known inhibition of dihydrofolate reductase activity which leads to a decrease in the dTMP synthase reaction, since TdR kinase which converts TdR to dTMP could readily circumvent MTX action through this salvage activity. TdR kinase is of particular significance, since in various types of carcinoma cells its activity is orders of magnitude higher than that of dTMP synthase. To throw light on this problem, we tested the hypothesis that the impact of MTX treatment might in fact involve an inhibition or decrease in TdR kinase activity. Injection in rat of MTX (i.p.) decreased TdR kinase activity in a time- and dose-dependent fashion in liver (t1/2 = 46 h; IC50 = 95 mg/kg), bone marrow (t1/2 = 10 h; IC50 = 5 mg/kg) and rapidly growing transplantable hepatoma 3924A (t1/2 = 56 h; IC50 = 5 mg/kg). Injection in rat of cycloheximide (15 mg/kg, i.p.), an inhibitor of protein biosynthesis, rapidly decreased TdR kinase activity in the hepatoma (t1/2 = 3.6 h); activities of other purine and pyrimidine synthetic enzymes, dTMP synthase, IMP dehydrogenase, GMP reductase and GMP synthase, declined at a markedly slower rate (t1/2 = 11, 11.6, 12 and 22 h, respectively). MTX, by curtailing purine and pyrimidine biosynthesis, limits product of TdR kinase which is more sensitive to unopposed protein degradation than other enzymes of nucleic acid biosynthesis. TdR kinase is a newly discovered target of MTX treatment.

(1) In all examined rat and human tissues and cells, PIP kinase activity was rate-limiting and PLC activity was present in great excess. (2) The steady-state activities of the signal transduction enzymes, PI kinase, PIP kinase and PLC, and the concentration of the end product, IP3, were determined in rat liver and hepatomas of different malignancies. The activities of all three enzymes were elevated in the hepatomas in a non-random fashion. A generalization emerged that the enzyme with the lowest activity in liver, PIP kinase, increased to the highest extent and the enzyme with the highest activity in liver, PLC, increased to the smallest extent in rapidly growing hepatomas. The IP3 concentration in the hepatomas was elevated in a progression-linked fashion. (3) The three signal transduction enzyme activities were elevated in human ovarian carcinoma samples and in human breast carcinoma cells. (4) When human breast carcinoma MDA-MB-435 cells were allowed to go through lag, log and plateau phases, the IP3 concentration reached a 20-fold peak at 12 hr after plating. The elevation in IP3 concentration preceded the rise in PI and PIP kinase activities which increased 11-fold in the log phase. The IP3 concentration and PI and PIP kinase activities returned to their baseline levels when the plateau phase was reached. The PLC activity did not change significantly during the whole period. (5) Administration of cycloheximide i.p. in rats revealed short half-lives in the bone marrow for the two kinases (8 min) and a long half-life for PLC (> 6 hr). In a group of 10 enzymes, the half-lives of the kinases were the shortest. In cycloheximide-injected rats, the bone marrow IP3 concentration was reduced to about 50% in 30 min. The reduction of IP3 concentration is attributed to the decline to 15 and 12%, respectively, in PI and PIP kinase activities since PLC activity did not change. (6) In 3-day starved rats, the bone marrow PI and PIP kinase were reduced to activities (13%) that were markedly lower than the decrease in the protein concentration (to 55%). By contrast, the PLC activity was preferentially maintained (to 78%) over the protein level. Under starvation, the IP3 concentration decreased (to 24%), indicating that starvation can markedly disrupt IP3 homeostasis. Refeeding returned the enzymic activities and the IP3 concentration to the normal level in bone marrow in 24 hr. (7) Comparison of the absolute activities of PI and PIP kinases and PLC showed that PLC is present in an excess; therefore, it does not appear to have a rate-limiting action in cycloheximide treated rats or in starvation. (8) Whereas PI and PIP kinases have short half-lives and apparently rapid synthetic rates, PLC has high activity, a long half-life and responds to starvation with only a small decrease. (9) The gain in function manifested in the over-expressed capacity for signal transduction confers growth advantages to cancer cells. These increased activities, particularly those of PI and PIP kinases, should be sensitive targets for chemotherapy.

Signal transduction activity was markedly elevated in cancer cells as shown by the increased activity of enzymes utilizing 1-phosphatidylinositol, PI (PI 4-kinase and PI-4-phosphate 5-kinase) for the production of the second messenger inositol 1,4,5-trisphosphate, IP3, in rat hepatomas (Cancer Res. 54: 2611;5574, 1994) and in human ovarian and breast carcinoma cells (Life Sci. 55:1487, 1994). Quercetin, a flavonoid, in human breast carcinoma MDA-MB-435 cells produced growth inhibition (IC50 = 55 microM) and cytotoxicity (LC50 = 26 microM). Quercetin inhibited PI kinase activity in extracts of breast carcinoma cells (IC50 = 6 microM) and in cultured cells (IC50 = 10 microM) with a minor inhibition of PIP kinase activity. IP3 concentration decreased in parallel with PI kinase activity. In time sequence studies quercetin in breast carcinoma cells brought down PI kinase and IP3 concentration in 60 min to 5 and 6%, respectively; PIP kinase activity was at 63% of controls. The results demonstrate for the first time in proliferating human breast carcinoma cells a reduction by quercetin of the increased capacity for signal transduction, thus providing a novel and sensitive target in cancer cells.

Quercetin inhibited 1 -phosphatidylinositol (PI) 4-kinase, EC 2.7.1.67 (PI kinase) activity in a concentration-dependent manner (IC 50 = 4 μ M) in participate extracts from human ovarian carcinoma. In OVCAR-5 cells quercetin produced growth inhibition (IC 50 = 63 μ M) and cytotoxicity (LC 50 = 17 μ M). The growth inhibition by quercetin was accompanied by an 80% decrease in PI kinase activity and a 65% decrease in the concentration of the second messenger, inositol 1,4,5-trisphosphate (IP 3 ). When human OVCAR-5 cells were plated and expressed their neoplastic proliferative program in the log phase, PI kinase and PI 4-phosphate 5-kinase, EC 2.7.1.68 (PIP kinase) activities coordinately increased to a peak of 5.8- and 4.5-fold, respectively. The results demonstrate for the first time inhibition by quercetin of the enhanced capacity for operation of signal transduction in human ovarian carcinoma cells, thus providing a novel target in cancer cells.

In evolution two pathways emerged for the biosynthesis of pyrimidine and purine nucleotides. The de novo biosynthetic pathway assembles nucleotides from small building blocks. By contrast, the salvage pathways provide mechanisms to recycle nucleosides and bases from the dead cells of tissues and from the blood stream1. In some lower organisms only one of these pathways operates2. However, in mammalians both de novo and salvage pathways function in all tissues. It has been recognized in the past 10 years that the activities of salvage enzymes in each of the biosynthetic segments of metabolism are markedly higher than those of the rate-limiting enzymes of de novo biosynthesis1.

1. 1. A systematic study is reported on the control of 1-phosphatidylinositol 4-kinase (PI kinase) and PI 4-phosphate 5-kinase (PIP kinase), enzymes of the phosphatidylinositol phosphorylation pathway which leads to the production of second messengers, IP3 and DAG. In liver of normal male, adult, fed Wistar rats the steady state activity of PI kinase was 0.5 ± 0.01 and that of PIP kinase was 0.046 ± 0.003 nmol/hr/mg protein. The concentration of IP3 was 1.8 ± 0.1 pmol/mg protein. 2. 2. That the two kinases have short half-lives was observed in starvation, where in the rat liver or bone marrow activities rapidly decreased and on refeeding were restored in a day. Injection to rats of the protein synthetic inhibitor, cycloheximide, yielded t 1 2 = 8 min for the two enzymes in bone marrow and t 1 2 = 80 min in liver. 3. 3. Linkage of the signal transduction enzymes with proliferation was shown by the high activities as compared to liver of these enzymes in rat organs of high cell renewal capacity, e.g., thymus, bone marrow, spleen and testes. 4. 4. Linkage with malignant proliferation was indicated by the observation that in rat hepatomas the enzyme activities increased 5- to 9-fold and were highest in rapidly growing hepatoma 3924A (29- and 45-fold). 5. 5. In human primary ovarian carcinoma PI and PIP kinase activities were elevated 4.4 and 2.9-fold, respectively, and in OVCAR-5 cells, 32- and 11-fold, respectively. Similar increases were observed in MDA-MB-435 human breast carcinoma cells in comparison with normal breast parenchymal cells. 6. 6. The linkage of signal transduction enzyme activities with malignant proliferation was also observed in experiments when human breast carcinoma cells were plated in flasks and expressed their proliferative capacity in the log phase. PI and PIP kinase activities steadily and coordinately increased to a peak 11-fold rise in mid-log phase. In late log and plateau phases the kinase activities gradually declined to the starting level. Similar observations were made for the two enzymes in human ovarian carcinoma OVCAR-5 cells and in rat hepatoma 3924A cells in tissue culture. 7. 7. In animals injected with cycloheximide the bone marrow PI and PIP kinase activities exhibited t 1 2 = 0.12 hr , the shortest decay rate in comparison with 8 enzymes of purine and pyrimidine biosynthesis with t 1 2 = = 0.6 to 4.3 hr . 8. 8. Injection of tiazofurin decreased PI and PIP kinase activities in the bone marrow with t 1 2 = 82 and 78 min , respectively. IP3 concentration decreased with a t 1 2 = 23 min . 9. 9. Injection of quercetin decreased PI kinase activity in the bone marrow with t 1 2 = 17 min ; however, PIP kinase activity only decreased to 70% and activity leveled off at this value for 24 hr. The IP3 concentration followed the decline in PI kinase activity with t 1 2 = 40 min . 10. 10. Addition of quercetin to OVCAR-5 cells in culture showed that different concentrations of this drug have little effect on PIP kinase activity; however, PI kinase activity decreased in a dose-dependent fashion with IC50 = 44 μ m . The IP3 concentration sharply declined with an IC50 of 46 μ m and correlated best with the decrease in PI kinase activity. 11. 11. Quercetin inhibited the proliferation of OVCAR cells with an IC50 of 63 μ m and of human breast carcinoma cells with IC50 = 55 μ m . The decline of PI kinase activity preceded the inhibition of cell proliferation whereas PIP kinase activity was inhibited only to a minor extent. 12. 12. In vivo studies showed that in the bone marrow IP3 concentration could be depleted by only 40–60% by injection of cycloheximide, tiazofurin or quercetin. Therefore, we postulated that there might be a salvage pathway that could replenish or maintain IP3 concentration in a homeostatic adaptation. This hypothesis is strengthened by reports in the literature indicating that IP3 may be generated by degradation of IP6 and observations that addition of IP6 to cancer cells yields IP3 provide support for this hypothesis for a salvage pathway for IP3 production.

In ovarian and pancreatic carcinoma cell lines, the activity of the salvage enzyme, thymidine kinase (EC 2.7.1.21), was 2- to 13-fold higher than that of the key enzyme of thymidylate de novo biosynthesis, thymidylate synthase (dTMP synthase, EC 2.1.1.45). AZT (3'-azido-3'-deoxythymidine, zidovudine) competitively inhibited thymidine kinase activity in extracts of human ovarian and pancreatic carcinoma cells, with Dixon plots yielding Ki = 1.1 microM in both cell lines. AZT (20 microM) yielded synergistic cytotoxicity with methotrexate (0.4 microM) in human pancreatic carcinoma cells in clonogenic assay and also with methotrexate (0.02 microM) in human ovarian carcinoma cells, as measured by cell counts. Thymidine (10 microM) and hypoxanthine (100 microM) reversed these inhibitions. AZT (20 or 40 microM) also provided synergistic cytotoxicity with 5-fluorouracil (0.5 and 1.0 microM) in human pancreatic carcinoma cells in clonogenic assay. These studies suggest a new role for AZT, which, as an inhibitor of thymidine salvage, should be useful as a biochemical response modifier to provide a synergistic clinical anticancer impact on de novo biosynthesis of thymidylates in conjunction with methotrexate or 5-fluorouracil.

The modes of action of azaserine and acivicin were compared. The results were evaluated by assessing the impact of these drugs on primary targets, the activities of key enzymes, and on secondary and tertiary targets, the concentrations of pools of ribonucleotides and deoxyribonucleotides. 1. 1. It was observed that both drugs act as competitive inhibitors for glutamine-utilizing enzymes involved in the biosynthesis of purines and pyrimidines, but in addition acivicin exerts a direct inactivating effect (probably by alkylation) on the enzymes. 2. 2. The different tissues examined displayed varying sensitivity to the drugs which may be attributed in part at least to the tissue glutamine content. 3. 3. Acivicin markedly depleted the CTP pools, but ATP and UTP were unaffected. It also decreased the concentration of all 4 deoxynucleoside triphosphates. 4. 4. These biochemical targets serve as indicators of acivicin action in cancer cells and should also be helpful in the design of combination chemotherapy. 5. 5. On the basis of the biochemical action of acivicin, actinomycin and dipyridamole were selected for testing in combinatino chemotherapy. Both drugs acted synergistically with acivicin.

The impact of tiazofurin on inhibition of IMP dehydrogenase was discussed at the clinical and molecular levels. 1. 1. Evidence was provided for the role of IMP dehydrogenase and guanylates in the expression of the neoplastic program in cancer cells with particular relevance to human leukemic cells. 2. 2. The argument for expecting an impact of tiazofurin in human myelocytic cells was provided. 3. 3. Similarity of the kinetics of human leukemic cell IMP dehydrogenase to the rat hepatoma enzyme was documented. 4. 4. New evidence was provided for the role of salvage in chemotherapy and the function of hypoxanthine in inhibiting guanine salvage. 5. 5. The action of tiazofurin and retinoic acid was reported in HL-60 leukemic cells. 6. 6. The effect of tiazofurin and retinoic acid on proliferation and cytotoxicity was outlined for hepatoma 3924A cells. 7. 7. The effect of guanine on induced differentiation by tiazofurin and retinoic acid was examined. 8. 8. Biochemical basis was provided for the lack of development of resistance in patients treated with tiazofurin. 9. 9. Presumptive evidence was provided that tiazofurin treatment induced differentiation of leukemic cells in the patients. 10. 10. The molecular biology of tiazofurin-induced differentiation in K-562 cells was reviewed with the possible relevance to clinical treatment that tiazofurin might also act through down-regulation of ras oncogene.

The behavior of the activity of the rate-limiting enzyme of the biosynthesis of purines, amidophosphoribosyltransferase (EC 2.4.2.14), and of the catabolism, xanthine oxidase (EC 1.2.3.2), was elucidated in primary renal cell carcinomas in human and in chemically-induced, transplantable renal cell carcinomas in rat. Enzyme activities were measured in the supernatant fluid prepared by centrifugation of 5% homogenates at 100,000 X g for 30 min. The activities in human and rat kidney for amidotransferase were 2.0 ± 0.2 and 8.9 ± 0.4 and for xanthine oxidase 0.4 ± 0.09 and 5.5 ± 0.3 μmol per hr/per mg protein x 10−2, respectively. In the human and rat tumors the activities of amidotransferase increased 1.5− to 2.7-fold and of xanthine oxidase decreased to 25 to 69% of those of the respective controls. The ratios of the activities of amidotransferase/xanthine oxidase were increased 2.1− and 5.3-fold in the tumors. Since amidotransferase activity increased and xanthine oxidase decreased in all examined kidney tumors, the alterations in the activities of these enzymes appeared to be linked with neoplastic transformation. With the reciprocal alterations in activity of the synthetic enzyme, amidotransferase, and the concurrent decrease in that of the catabolic enzyme, xanthine oxidase, the reprogramming of gene expression resulted in an imbalance that favors the synthetic over the degradative capacity. These results indicate the applicability of the pattern of enzymic imbalance discovered in rat hepatomas to human and rat kidney neoplasia.

The biochemical strategy of the cancer cell was examined in terms of malignant transformation-linked enzymatic imbalance. Previous work established that the activities of two of the key enzymes of the oxidative and non-oxidative synthetic pathways of pentose phosphate production were increased in all hepatomas. Thus, the activities of glucose-6-phosphate dehydrogenase and transaldolase were increased in all liver tumors irrespective of growth rate and degree of differentiation. The present work reports that amidophosphoribosyltransferase (glutamine PRPP amidotransferase, EC 2.4.2.14) and UDP kinase (ATP:UDP phosphotransferase, EC 2.7.4.6) were markedly increased in all hepatomas. In studying the behavior of amidophosphoribosyltransferase the kinetic parameters in normal liver and rapidly growing hepatomas were compared. For both normal liver and hepatoma 3924-A apparent K m 's for glutamine and magnesium were 1.7 and 0.4 m m , respectively. In contrast, the liver enzyme exhibited sigmoid properties toward the substrate, PRPP, but the enzyme in hepatoma 3924-A did not. In liver and hepatoma the apparent K m 's for PRPP were 0.9 and 0.4 m m , respectively. In the standard assay that was developed, proportionality was achieved for enzyme activity with amount of enzyme added and with time elapsed in normal and neoplastic livers. In all hepatomas amidophosphoribosyltransferase activity was increased 1.7- to 3.1-fold over the normal liver values. The hepatoma enzyme was less sensitive to inhibition by AMP than the liver enzyme. Studies in kidney tumors of different growth rates indicated that amidotransferase activity was markedly increased over the values of normal rat kidney cortex. The enzyme activity was also increased in human primary hepatomas. Since the markedly increased amidotransferase activity and the hyperbolic affinity for PRPP were present only in tumors and not in rapidly growing normal differentiating or regenerating liver, the altered activity of this key purine-synthesizing enzyme appears to be specific to the neoplastic transformation. The increased amidotransferase activity, the enhanced affinity of the hepatoma enzyme to the substrate, PRPP, and the decreased sensitivity to inhibition by AMP confer selective biological advantages to the cancer cell. In examining the behavior of UDP kinase it was observed that the kinetic parameters were similar for normal and neoplastic liver. The apparent K m values for UDP, ATP and magnesium were 0.5, 3.0 and 3.0 m m , respectively. Through kinetic studies a standard assay was developed where good proportionality was achieved for enzyme activity with amount of enzyme added and time elapsed in both normal and neoplastic liver. The UDP kinase activities were approximately 2- to 4-fold of those observed in the liver of control normal rats. Studies in two different primary hepatomas from man also indicated that UDP kinase activity was increased 2- to 3-fold above the activities observed in normal human liver. The increased UDP kinase activity occurred only in liver tumors, and the enzyme activity did not rise in rapidly growing normal, differentiating or regenerating liver; therefore, the increased activity of this UTP-synthesizing enzyme appears to be characteristic of the neoplastic transformation in the liver. As the hepatomas contain markedly more UDP kinase activity than the normal liver the cancer/cells possess an increased capacity for UTP biosynthesis. This alteration in UDP kinase activity indicates a reprogramming of gene expression that should confer selective biological advantages to the neoplastic cells. Since the marked increases in activities of amidophosphoribosyltransferase, UDP kinase, glucose-6-phosphate dehydrogenase and transaldolase occur in all hepatomas, including even the slowest-growing and most-differentiated tumors, these alterations indicate reprogramming of gene expression that is connected with the malignant transformation per se .

On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.

The in vivo actions of two antimetabolites, acivicin (NSC-163501) and tiazofurin (NSC-286193), were examined on the enzymic programs of rat bone marrow. From the bone marrow of the femurs, 100,000 g supernatant fractions were prepared; enzymic activities were measured by isotopic assays, and cellularity was determined. In the normal bone marrow, the specific activities of pyrimidine de novo synthetic enzymes, CDP reductase, dTMP synthase, CTP synthase, carbamoyl-phosphate synthase II (synthase II), orotidine 5'-phosphate decarboxylase and aspartate carbamoyltransferase, were 1, 2.7, 5, 10, 63 and 601 nmol/hr/mg protein, respectively, whereas those of the salvage enzymes, deoxycytidine, thymidine, cytidine and uridine kinases were 3, 43, 149, and 367 nmol/hr/mg protein, respectively. In purine biosynthesis, the activities of the de novo synthetic enzymes, IMP dehydrogenase, fonnylglycinamidine ribonucleotide (FGAM) synthase, GMP synthase, amidophosphoribosyitransferase (AT) and adenylosuccinate synthase were 16, 8, 107, 78 and 124 nmol/hr/mg protein, respectively, and those of the salvage enzymes, adenine, hypoxanthine and guanine phosphoribosyl-transferases, were 340, 407, and 1018 mol/hr/mg protein, respectively. The sequence of events was elucidated after a single i.p. injection of acivicin (5 mg/kg) or tiazofurin (200 mg/kg). Within 2hr after acivicin injection, CTP, GMP and FGAM synthases lost 85–90%, while AT and synthase II lost 50 and 80%, respectively, of their activities. The activities rose to near normal range by 72–96 hr. The bone marrow cellularity decreased, reaching a nadir at 24 and 48 hr, and returning to normal range by 72 and 92 hr; thymidine kinase activity followed a similar pattern. Tiazofurin injection depressed IMP dehydrogenase activity to 20% by 2 hr with a rebound to normal range by 48 and 72 hr. The cellularity decreased more slowly, reaching its lowest point at 24 hr and returning to normal range at 72 hr. For acivicin the marked depletion of the activities of the glutamine-utilizing enzymes and for tiazofurin that of IMP dehydrogenase might account, in part at least, for the bone marrow toxicity of these antimetabolites. Because of the presence in the bone marrow of high activities of purine and pyrimidine salvage enzymes, it should be possible to design methods utilizing nucleosides and nucleobases to protect the bone marrow from the action of antimetabolites.

The purpose of this investigation was to test the concept that key enzymes of IMP synthesis, degradation and utilization may show characteristic patterns in proliferative conditions. In the synthetic pathway of IMP the first enzyme committed to de novo purine biosynthesis, glutamine PRPP amidotransferase, was measured. In the pathway of IMP catabolism the rate-limiting enzyme, xanthine oxidase, and uricase, the final enzyme, were examined. In the synthetic utilization of IMP, IMP dehydrogenase, the enzyme involved in channeling IMP into the production of XMP and adenylosuccinate synthetase, the enzyme channeling IMP into SAMP biosynthesis, were studied. Investigations were also carried out on GMP synthetase and adenylosuccinase. The enzymes of IMP metabolism were examined in normal and neoplastic liver proliferation, such as in differentiating and regenerating liver and in a spectrum of hepatomas of different growth rates. The following main observations were made. 1. 1.|In differentiating liver the specific activities of IMP dehydrogenase and amidotransferase were high after birth and decreased during development to the levels observed in the adult rat liver. In contrast, the adenylosuccinate synthetase and xanthine oxidase activities were low after birth and rose to the adult levels during development. 2. 2.|In a spectrum of hepatomas of different growth rates the specific activity of IMP dehydrogenase was 2- to 3-fold increased in the slow growing hepatomas and it increased in parallel with tumor growth rates, reaching 12- to 13-fold increases in the most rapidly growing neoplasms. The behavior of IMP dehydrogenase belongs to Class 1 as grouped by the molecular correlation concept. 3. 3.|The activities of adenylosuccinate synthetase and glutamine PRPP amidotransferase were increased in all hepatomas to approximately 2- to 4-fold, and those of the adenylosuccinase 1.2- to 2-fold of the levels observed in the corresponding normal rat livers. In contrast, the activities of the catabolic enzymes, xanthine oxidase and uricase, were decreased in all hepatomas to 2- to 10-fold of the activity of the relevant control rat livers. The ratios of amidotransferase/xanthine oxidase were markedly increased in all hepatomas and they reached 20- to 27-fold increases in those of rapid growth rate. The behavior of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase belongs to Class 2 of the molecular correlation concept where enzymes are grouped that exhibit alterations that occur in all hepatomas irrespective of growth rate and degree of differentiation. 4. 4.|Since the alterations in the activities of adenylosuccinate synthetase, adenylosuccinase, amidotransferase, xanthine oxidase and uricase are expressed in all the hepatomas, even in the slowest growing, most liver-like tumors, this reprogramming of gene expression appears to be linked with the malignant transformation per se . However, these enzyme activities do not signal a linkage with the degrees in the expression of malignancy and growth rate. In contrast, the activity of IMP dehydrogenase is an indicator both of malignant transformation and growth rate. 5. 5.|In the regenerating liver IMP dehydrogenase specific activity rapidly increased to 500% in 6 to 18 hours; it slowly returned towards normal range in 96 hours after operation. Amidotransferase specific activity reached a peak of 165% of the sham-operated value at 48 hours after operation and then returned to normal range by 96 hours. The activities of SAMP synthetase, adenylosuccinase, xanthine oxidase and uricase did not change in regeneration. Thus, IMP dehydrogenase appears to be the earliest enzyme to rise in the regenerating liver, whereas the elevation of amidotransferase is a delayed and a minor one. The increase in activities of these two enzymes in the regenerating liver is in the range of the activity observed in the newborn rat liver. However, the activities are markedly lower than those observed in the very malignant hepatomas that exhibit the same growth rate as the regenerating liver. 6. 6.|The discriminating power of the biochemical pattern of enzymes of purine metabolism indicates that the imbalance in the pattern of alteration of the key enzymes of IMP synthesis, degradation and utilization is specific to malignancy. The pattern of these enzyme activities also provides quantitative and qualitative discriminants for the identification of regenerating and differentiating liver and for distinguishing the enzyme patterns from those observed in normal and rapidly growing neoplastic liver. 7. 7.|These experiments indicate the operation of an antagonistic behavior for the key synthetic enzymes, glutamine PRPP amidotransferase and IMP dehydrogenase, that are high in differentiation, regeneration and neoplasia as compared to the rate-limiting catabolic enzyme, xanthine oxidase, that is low in differentiation and in neoplasia and is unaltered in the regenerating liver. These observations are in line with earlier studies that demonstrated an antagonistic behavior for the opposing key enzymes of gluconeogenesis and glycolysis and of the synthetic and catabolic pathways of the de novo and salvage pathways of pyrimidine metabolism.

Xanthine oxidase was decreased 2- to 10-fold in all examined rat hepatomas irrespective of the malignancy; growth rate and degrees of histological differentiation of the neoplasms. The affinity to substrate (KM=6-8 muM) and the pH optimum (8.0) of the liver and hepatoma enzymes were the same. The reprogramming of gene expression, as manifested in the decreased activity of this key purine metabolizing enzyme, appears to be specific to neoplastic transformation. Since glutamine PRPP amidotransferase activity was increased but the opposing enzyme, xanthine oxidase, was decreased in all the hepatomas, the reprogramming of gene expression results in an imbalance that favors synthesis against catabolism. This enzymatic imbalance should confer selective advantages to the cancer cells.