Mary A. Faderan

Salvage capacity of hepatoma 3924A and action of dipyridamole

The role and behavior of the salvage enzymes in the biosynthesis of purines (adenine and hypoxanthine-guanine phosphoribosyltransferases) and pyrimidines (uridine-cytidine, deoxycytidine and thymidine kinases) were elucidated. In liver purine metabolism the transferase activities were orders of magnitude higher than the activities of the enzymes of de novo biosynthesis. In both purine and pyrimidine biosynthesis the activities of the enzymes of the de novo pathways were low (23 pmol to 70 nmol/hr/mg protein), whereas those of salvage synthetic pathways ranged from 0.8 to 1,470 nmol/hr/mg protein. In purine metabolism the salvage enzymes had markedly higher affinity to the shared substrate PRPP (4 to 40 microM) than the rate-limiting enzyme of de novo synthesis, amidophosphoribosyltransferase (900 microM). In rapidly growing hepatoma 3924A the activities of the enzymes of de novo purine biosynthesis increased, whereas those of the salvage pathway changed little. However, the activities of the enzymes of the salvage pathways remained much higher than those of the enzymes of de novo purine production. In pyrimidine production in the hepatomas the activities of both de novo and salvage enzymes markedly increased. However, the activities of the salvage enzymes far outstripped those of the enzymes of the de novo pathways. To inhibit the operation of the salvage pathways, the action of the transport inhibitor, dipyridamole, was examined. In tissue culture, dipyridamole inhibited the transport of purine and pyrimidine nucleosides with an IC50 of 10(-6) or 10(-7) M. As measured by colony-forming assay, dipyridamole killed hepatoma cells with an IC50 of 20 microM. Dipyridamole markedly depressed the pools of ATP, GTP, CTP and UTP; in combination chemotherapy with acivicin, an anti-glutamine agent, synergistic action was observed on the pools of nucleotides in hepatoma 3924A in vivo. These investigations emphasize the importance of the capacity to utilize precursors by the salvage enzymes and may explain, in part at least, the failure of inhibitors of the de novo pathways to yield lasting chemotherapeutic results. Combination chemotherapy of inhibitors of the de novo pathways with an inhibitor of the salvage pathways (dipyridamole) should impact on our understanding of the contribution of salvage pathways and provide a rational basis for successful combination chemotherapy of neoplastic diseases.

Control of enzymic programs and nucleotide pattern in cancer cells by acivicin and tiazofurin

The mechanism of action of acivicin and tiazofurin was compared in hepatoma 3924A. 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. The action of acivicin entails inhibition and inactivation of the key enzymes of glutamine utilization in the biosynthesis of purines and pyrimidines. As a result, the GTP and CTP pools were markedly depleted, whereas those of ATP and UTP were unaffected. Acivicin also markedly decreased the concentrations of all 4 deoxynucleoside triphosphates. The nucleotide pools returned to normal or near normal range within 2 to 3 days after a single acivicin injection. The pharmacologic targets of acivicin in anticancer chemotherapy include prominently the activities of glutamine-utilizing enzymes and the pools of GTP and CTP and all 4 dNTPs. These biochemical targets also serve as indicators of acivicin action in cancer cells. The action of tiazofurin in hepatoma cells entails the primary target, IMP dehydrogenase. The subsequent effects include marked enlargement of IMP and PRPP pools and depletion of the pools of GDP and GTP. The increased IMP concentration selectively inhibited the activities of hypoxanthine-guanine phosphoribosyltransferase, but did not affect that of adenine phosphoribosyltransferase. The markedly decreased GTP pool de-inhibited the activity of AMP deaminase which permitted the channeling of AMP to IMP. An important indicator of tiazofurin action is the prolonged depletion of dGTP pools and similar but less pronounced declines in the pools of dCTP and dATP. In contrast, dTTP pools were increased. The crucial biochemical targets and indicators of tiazofurin action in sensitive cancer cells include inhibition of IMP dehydrogenase, a decrease in the concentrations of GDP, GTP, dGTP, dCTP, dATP and marked rise in the pools of IMP, PRPP and dTTP. Measurements of the molecular targets and indicators of drug action should be helpful in identifying cancer cells and tissues sensitive or resistant to the action of acivicin or tiazofurin. Identification of the targets and indicators should also be helpful in the design of frequency of administration of the drugs in combatting animal and human neoplasia.

Multi-enzyme-targeted chemotherapy by acivicin and actinomycin

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.

Mechanism of resistance to tiazofurin in hepatoma 3924A.

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC-286193) has shown potent cytotoxic and antitumor activity against hepatoma 3924A carried in the rat [Lui et al. J. biol. Chem. 259, 5078 (1984)]. However, eventually the tumor emerged, proliferated and killed the host. To throw light on the factors that play a role in the resistance to this drug, a tiazofurin-induced resistant hepatoma 3924A line in culture was produced, and its biochemical and pharmacological pattern was examined. Resistance in hepatoma cells was expressed by a reprogramming of gene expression that entailed the display of a program of multiple biochemical alterations. In the resistant cells the activity of IMP dehydrogenase, the target enzyme of tiazofurin, was increased 2- to 3-fold. The steady-state guanylate pools were elevated 3-fold, and there was a decrease in the de novo synthesis of guanylate. There was an expansion of guanylate salvage, which could circumvent inhibition of de novo guanylate synthesis by tiazofurin. For the first time in studies on the resistance of different cell lines to tiazofurin, reduced tiazofurin transport (to 50%) in resistant hepatoma cells was identified which might account for the decreased concentration (50%) of the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), in these cells. NAD pyrophosphorylase activity also decreased to 53% of that of the sensitive line, which was responsible, in part at least, for the decreased TAD concentration of the resistant cells. When resistant cells were cultured in the absence of tiazofurin, resistance to the drug gradually decreased, and by 50 passages sensitivity returned. Resistance to tiazofurin in hepatoma cells appears to be a drug-induced metabolic adaptation which involves alterations in the activity of the target enzyme, in the transport and concentration of the drug and the active metabolite, and an increase of guanylate concentration and guanine salvage capacity.

Molecular Targets of Anti-Glutamine Therapy with Acivicin in Cancer Cells

Systematic studies in this laboratory showed that there was an increase in the activities of enzymes of glutamine utilization of pyrimidine and purine biosynthesis in rat hepatomas of different growth rates and in other animal and human neoplasms. We also observed that there were increased concentrations of strategic nucleotides and deoxynucleotides in hepatomas and these elevations were linked with neoplastic transformation and progression (for overview see Weber [13]). Because of this increase in enzymic activities and nucleotide pools relating to glutamine utilization, an investigation was carried out to understand the impact of the anti-glutamine agent, acivicin, on the molecular targets, namely, key enzymic activities and concentrations of strategic nucleotides. These studies revealed a profound influence of the anti-glutamine agent on the enzymology and metabolism and survival of cancer cells [1, 3, 15].

Regulation of purine and pyrimidine metabolism by insulin and by resistance to tiazofurin

The purpose of this investigation was to elucidate the factors that regulate the pattern of gene expression in purine and pyrimidine metabolism in normal liver and hepatoma. For this purpose, the action of a hormone, insulin, and the development of resistance to a chemotherapeutic agent, tiazofurin, were studied. This investigation brought detailed evidence showing that in the rat insulin exerted a profound effect on liver purine and pyrimidine metabolism by regulating the concentrations of nucleotides through controlling the activities of strategic enzymes involved in their biosynthesis. When rats were made diabetic by alloxan treatment, in the average liver cell concentrations of ATP, GTP, UTP and CTP decreased to 66, 62, 54 and 63%, respectively, of those of normal liver. Administration of insulin for 2 days returned the hepatic nucleotide concentrations to normal range; further insulin treatment for an additional 5 days raised the concentrations of ATP, GTP, UTP and CTP to 197, 352, 412 and 792% of values observed in the liver of diabetic rats. In diabetic rats the hepatic activities of OMP decarboxylase, orotate phosphoribosyltransferase, uridine phosphorylase, uridine-cytidine kinase and uracil phosphoribosyltransferase decreased to 44, 48, 70, 36 and 41% of the activities of normal liver. Insulin treatment for 2 days returned activities to normal range. Continued insulin treatment for an additional 5 days increased the enzymic activities to 3.9- to 5.3-fold of those of the liver of the diabetic rats. The regulation by insulin treatment of the activities of enzymes of de novo and salvage synthesis of UMP should explain, in part at least, the decline and increase of the uridylate pool in diabetes and after insulin treatment. In the diabetic rat hepatic CTP synthetase, the rate-limiting enzyme of CTP biosynthesis, decreased to 53% and insulin administration for 2 days restored activity to normal range. Insulin treatment for an additional 5 days increased the synthetase activity to 4-fold of the values of the diabetic liver. Thus, the behavior of liver CTP synthetase activity is tightly linked with that of the CTP pool. In the diabetic rat liver, the activity of IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis, decreased to 24% of that of the normal liver. Insulin administration for 2 days returned the activity to normal range, yielding a 4.5-fold increase in the activity from the diabetic to the insulin-treated state.(ABSTRACT TRUNCATED AT 400 WORDS)

Tiazofurin-induced selective depression of NAD content in hepatoma 3924A

The NAD content in hepatoma 3924A was approximately 40% of that in the liver of ACI/N rats bearing this hepatoma. Treatment of tumor-bearing rats with tiazofurin decreased NAD pools in the hepatoma, but no change was apparent in the liver. In a dose-response study, injection of varying amounts of the drug decreased NAD pools in the hepatoma in a dose-dependent fashion. In time-sequence studies, a single drug dose (200 mg/kg) depressed NAD pools in the hepatoma from 2 to 24 h after injection to approximately 50% of control at the lowest point before returning to control range at 48 h. The tiazofurin-induced depletion of NAD pools in the hepatoma to approximately 20% of that of normal liver might play a role in the anti-cancer action and toxicity of this drug.

Expression of key enzymes of purine and pyrimidine metabolism in a hepatocyte-derived cell line at different phases of the growth cycle

SummaryThe effect of growth phase on enzymatic activities of the de novo and salvage pathways for purine and pyrimidine nucleotide synthesis was studied in a hepatocyte-derived cell line from the rat. The cells were in lag phase after plating for 36 h; log phase started at 48 h and persisted up to 120 h of culture. Then the cells stopped growing and entered into plateau phase (144 h). In non-proliferating cells (144 h of culture) the basal activities of the enzymes of purine de novo biosynthesis were 1.7- to 6.8-fold higher than in normal rat liver, those of pyrimidine de novo synthesis showed 0.6- to 30-fold increase in activity. The purine salvage enzymes were unchanged, and the pyrimidine salvage enzymes were 3.1- to 7.4-fold higher compared to normal liver. During the growth cycle all enzymes except the purine salvage enzymes, which did not change, showed a peak in activity at 72 h of culture (log phase). The increase in activity in log phase compared to plateau phase was 1.3- to 2.4-fold for purine de novo synthetic enzymes, 1.1- to 2.4-fold for pyrimidine de novo enzymes, and 1.4- to 4.7-fold for pyrimidine salvage enzymes. The specific activities of the enzymes in exponentially growing cells were comparable either to that in 24-h regenerating liver, or to that in hepatomas of low or medium growth rate. It was concluded that the enzymatic pattern and metabolic state of the cells shared some features with regenerating liver, others with tumors, although they were not tumorigenic after transplantation into athymic nude mice.