Konrad Pillwein

Salvage pathways as targets of chemotherapy

This paper discussed the significance of the activities of purine and pyrimidine salvage enzymes in cancer cells and the targeting against them of chemotherapy. 1. The activities of salvage enzymes in the rat liver were orders of magnitude higher than those of the rate-limiting enzymes of de novo biosynthesis. A similar relationship was observed in rat hepatomas of different growth rates and in primary colon carcinoma in human. 2. The concentrations of nucleosides and nucleobases were measured in plasma, liver and hepatoma 3924A in the rat. The freeze-clamp method was required to determine the concentrations of these precursors in rat liver and hepatoma in a reliable and precise fashion because ischemia markedly altered the concentrations of nucleosides, nucleobases and, as shown earlier, nucleotides in these tissues. The results indicated that the liver markedly concentrated the purine precursors, hypoxanthine, guanine and adenine, but not thymidine, which was one-third that of the plasma. Uridine and deoxycytidine occurred in the same concentration as in plasma, but cytidine was 3-fold higher in liver. In the hepatoma in comparison to the liver the concentrations of the nucleosides and bases were altered and for some of the changes the enzymic differences between liver and hepatoma appeared to be accountable. 3. Kinetic parameters for purine and pyrimidine synthetic enzymes and for the substrates and co-factors were determined in liver and hepatoma 3924A. When enzymic activities were calculated at the tissue steady-state concentrations of the various ligands, the activities of the salvage enzymes were markedly higher than those of the rate-limiting enzymes. 4. Hepatoma cells were highly sensitive to the action of the transport inhibitor, dipyridamole, in lag and log phases. However, plateau phase cells lost their sensitivity to dipyridamole. 5. Amphotericin B rendered plateau phase cells sensitive to the inhibitory action of dipyridamole for the incorporation of thymidine. 6. Amphotericin B enhanced cytotoxicity of dipyridamole in hepatoma and human colon cancer HT-29 cells. 7. In these studies we discovered the decreased responsiveness to dipyridamole of plateau phase cells and the ability of amphotericin B to restore the sensitivity. Moreover, dipyridamole and amphotericin B were synergistic in their cytotoxic action in rat hepatoma cells and human colon cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective sensitivity to tiazofurin of human leukemic cells

This study reports the selective sensitivity to tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC-286193) of human leukemic leukocytes as compared to normal ones in bone marrow and peripheral blood samples by comparing the production of the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), from labeled tiazofurin and the depression of GTP concentration. When labeled tiazofurin was incubated with leukocytes obtained from healthy volunteers or from leukemic patients (acute non-lymphocytic leukemia or acute lymphoblastic leukemia), the TAD production was 27.0 +/- 8.3, 551.3 +/- 71.8 and 755.9 +/- 94.1 pmoles/10(9) cells per hr, respectively. Thus, the leukemic cells produced over 20-fold higher concentrations of TAD than the normal leukocytes. Incubation with tiazofurin in leukemic leukocytes decreased the GTP pools (to 48-79%), whereas there was no change in the normal leukocytes. These results indicate a selectivity of response to tiazofurin in human normal and leukemic leukocytes. The procedure reported in this work may be suitable as a rapid predictive test for the sensitivity of leukemic leukocytes to tiazofurin. Such a diagnostic test should be helpful in identifying neoplastic cells sensitive to tiazofurin in the Phase II trials now being developed.

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.

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)

Targets and markers of selective action of tiazofurin

The molecular correlation concept proposed that IMP dehydrogenase activity should be a sensitive target of chemotherapy. This hypothesis received support from an array of evidence. IMP dehydrogenase has the lowest activity in purine biosynthesis; it is the rate-limiting enzyme in GTP production; the enzymic activity is transformation-and progression-linked; it is elevated in all examined animal and human neoplastic cells. The activity of GMP synthetase and the concentrations of GMP and dGTP were increased in cancer cells. Whereas guanine salvage has a high potential activity, the low guanine content may well curtail actual salvage capacity. Ribonucleotide reductase activity was two orders of magnitude lower than that of IMP dehydrogenase. Tiazofurin, a C-nucleoside, had marked cytotoxicity on hepatoma cells in vitro and was the first drug that as a single agent profoundly inhibited the proliferation of the subcutaneously inoculated solid hepatoma 3924A in the rat. The impact of tiazofurin administration in hepatoma cells was revealed in a cascade of biochemical alterations involving primary, secondary and tertiary targets and markers of this drug action. The primary target was IMP dehydrogenase where the active metabolite of tiazofurin, TAD, was thought to be absorbed to the NADH site of the enzyme. As a consequence, the enzymic activity declined rapidly to about 30-40% and returned to normal range by 36 to 48 hr after injection. The secondary targets and markers are the profoundly decreased pools of guanylates (GMP, GDP, GTP). Concurrently, the concentrations of IMP and PRPP were increased 8- to 15-fold. The elevated IMP pools were attributed to the de-inhibition of the AMP deaminase activity subsequent to the decline in GTP concentration. The rise in PRPP pools was attributed to the selective inhibition of GPRT and HPRT activities by the high IMP pool which did not affect APRT activity. This interpretation is supported by the 6- to 8-fold increase in the concentrations of guanine and hypoxanthine and the lack of change in the adenine pools inthe hepatomas after tiazofurin administration. The marked drop in NAD concentration which was drug dose- and time-dependent is attributed to the competition for NAD pyrophosphorylase activity by the precursors of NAD and tiazofurin monophosphate. The tertiary targets were dominated by the profound alterations in the concentrations of the dNTPs. This was characterized by a rapid and persistent drop (for 3 days) of the dGTP pool. The concentrations of dATP and dCTP also declined, but these alterations were less pronounced and the pools returned to normal after 2 days.(ABSTRACT TRUNCATED AT 400 WORDS)

Schedule-dependent synergistic action of tiazofurin and dipyridamole on hepatoma 3924A cells

SummaryTiazofurin is an oncolytic nucleoside analog that has shown therapeutic activity in end-stage acute non-lymphocytic leukemia and in chronic granulocytic leukemia in blast crisis. Tiazofurin is anabolized to the active metabolite, TAD, which inhibits IMP dehydrogenase activity, leading to a reduction in guanylate pools and to the cessation of neoplastic cell proliferation. The drug exhibits potent cytostatic and cytotoxic activity against hepatoma 3924A cells in culture. In growth-inhibition and clonogenic assays, the 50% inhibitory concentration of tiazofurin was 3.8 and 4.2 μm, respectively. Dipyridamole, an inhibitor of nucleoside transport, curtails the salvage of nucleosides and bases for nucleotide biosynthesis. Dipyridamole exhibited cytotoxicity against hepatoma 3924A cells, with an LC50 of 24 μm and an IC50 of 29 μm being recorded. A combination of tiazofurin and dipyridamole provided synergistic cytotoxicity in hepatoma 3924A cells in culture. This synergistic activity was dependent on the order of addition of the drugs. Simultaneous addition of the two drugs produced antagonism, whereas preincubation of cells with tiazofurin or dipyridamole followed by addition of the second drug resulted in synergy. TAD concentrations were significantly higher (129% and 135%) in cells that had been pretreated with tiazofurin or dipyridamole before the addition of the second agent as compared with cells that had been treated simultaneously (113%). These studies indicate the importance of the order of the addition of drugs to obtain a synergistic response in combination chemotherapy and suggest the need for a careful selection of drug modulation in clinical trials of tiazofurin and dipyridamole.

Oncolytic activity and mechanism of action of a novell-cystine derivative,l-cystine, ethyl ester,S-(N-methylcarbamate) monohydrochloride

SummaryA study on the oncolytic activity of thel-cysteine derivativel-cysteine, ethyl ester,S-(N-methylcarbamate) monohydrochloride (NSC 303861), revealed that the drug caused complete regression of the MX-1 human mammary tumor xenograft. The compound also exhibited moderate antitumor activity against murine leukemia P388 (T/C value of 169% at a daily dose of 400 mg/kg) and against M5076 sarcoma (T/C value of 135% at a daily dose of 600 mg/kg). The drug was inactive against B16 melanoma, Lewis lung, colon 38 and CD8F1 mammary carcinomas. The compound exhibited significant cytotoxicity against hepatoma 3924A cells in culture (LC50 = 6 µM). Studies on the mechanism of action revealed that the cytotoxicity of the drug could be partially abrogated by protecting hepatoma 3924A cells in culture withl-glutamine. At 6 h after an injection of the compound (400 mg/kg) into rats bearing hepatoma 3924A, the pools ofl-glutamine andl-glutamate in the tumor decreased to 33% and 71%, respectively, of control levels; the drug selectively inhibited the activities ofl-glutamine-requiring enzymes of purine nucleotide biosynthesis, amidophosphoribosyltransferase, FGAM synthase, and GMP synthase, to 21%, 1%, and 69%, respectively, without significantly altering the activities of pyrimidine biosynthetic enzymes, carbamoylphosphate synthase II and CTP synthase. Measurement of the nucleotide concentrations further corroborated the actions of the drug on the purine nucleotide biosynthetic enzyme activities. Drug injection (400 mg/kg) in the hepatoma 3924A-bearing rats reduced the concentrations of IMP in the tumor to 52%, those of total adenylates to 52%, those of total guanylates to 57%, and those of NAD to 73%, without significantly perturbing the pyrimidine nucleotide pools. Studies on the mechanism of action of thel-cysteine derivative suggested that the compound behaved as anl-glutamine antagonist, selectively acting on the enzymes of purine nucleotide biosynthesis.

Increased nucleoside concentrations in tumors as markers of tiazofurin action

Abstract Tiazofurin injection (150 mg/kg, i.p.) into rats bearing hepatoma 3924A increased in the tumor the pools of guanine, uridine, thymine, hypoxanthine, inosine, and thymidine 3.5-, 4.4-, 8.0- 18.4-, 18.7- and 42-fold over the controls. There were only minor changes in the host liver. This is the first report showing a selective action of tiazofurin in cancer cells on the concentrations of nucleosides and bases, indicating that these might be used as markers of the impact of tiazofurin in clinical trials.