Cell cycle dependent regulation of IMP dehydrogenase activity and effect of tiazofurin.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.     

Down-regulation of c-myc and c-Ha-ras gene expression by tiazofurin in rat hepatoma cells

There was an overexpression of the c-myc gene (11-fold) and of the c-Ha-ras gene (2-fold) in rat hepatoma 3924A cells compared to normal rat liver as measured by dot-blot analysis of total cytoplasmic RNA. The overexpression of c-myc was attributed to a 10- to 14-fold amplification and rearrangement of the c-myc sequences as determined by Southern blot analysis. The expression of the c-myc also was dependent upon the proliferative state of the hepatoma cells. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide; NSC 286193), an inhibitor of the activity of IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of GTP biosynthesis, resulted in a rapid drop (less than 1 h) to 50% of control in the target enzyme activity in the hepatoma cells and in a subsequent marked decrease to 55% in GTP concentration. These events were followed at 12 h of tiazofurin treatment by a 3-fold reduction in the expression of the c-myc gene and a 9-fold decline in that of the c-Ha-ras gene. These results in the hepatoma cells provide evidence in support of the earlier demonstrated correlation in K562 cells between GTP concentration and expression of c-myc and c-ras genes (Olah et al., 1989). These genes might depend on GTP for their expression in hepatoma cells and they might cooperate in a signal pathway that controls cell proliferation.     

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.     

Synergistic action of tiazofurin and difluorodeoxycytidine on differentiation and cytotoxicity.

Tiazofurin (TR), an inhibitor of IMP dehydrogenase, causes remissions and induced differentiation in human leukemia through lowering the concentrations of GTP and dGTP. A deoxycytidine analog, difluorodeoxycytidine (DFDC), is an anti-tumor agent phosphorylated by deoxycytidine kinase, resulting in decreased concentration of dCTP, leading to inhibition of DNA synthesis. In HL-60 cells DFDC induced differentiation and inhibited proliferation in a dose-dependent manner (IC50 = 4 nM); TR provided synergism with DFDC. DFDC inhibited proliferation in OVCAR-5 human ovarian carcinoma cells (IC50 = 25 nM) and colony formation in PANC-1 human pancreatic carcinoma cells (IC50 = 2 nM) and rat hepatoma 3924A cells (IC50 = 22 nM). TR and DFDC are synergistically cytotoxic in hepatoma cells and additive in PANC-1 cells. The two drugs together should be helpful in treating leukemias and solid tumors in humans.     

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.     

Purification of IMP dehydrogenase from rat hepatoma 3924A

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Synergistic cytotoxic effect of tiazofurin and ribavirin in hepatoma cells

Tiazofurin, an anti-cancer drug, which induces remissions in human leukemia, and ribavirin, an anti-viral agent, bind at separate sites (NADH and IMP-XMP sites, respectively) on the target enzyme, IMP dehydrogenase. Now we show that the binding to IMP dehydrogenase of these drugs at two separate sites is translated into synergistic inhibition of de novo guanylate biosynthesis and synergistic toxicity in rat hepatoma 3924A cells. These results may be utilized in the chemotherapy of neoplastic diseases and in the treatment of hepatitis virus infection and hepatocellular carcinoma.     

The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase

It is not yet clear to what extent depletion of intracellular GTP pools contributes to the antiviral activity of ribavirin. Therefore, the antiviral activities of (i) ribavirin, (ii) its 5-ethynyl analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), and (iii) mycophenolic acid (MPA) (a compound that inhibits only cellular IMP dehydrogenase activity) were studied on the replication of flaviviruses and paramyxoviruses. In addition, the effects of these three compounds on intracellular GTP pools were assessed. A linear correlation was observed over a broad concentration range between the antiviral activities of ribavirin, EICAR, and MPA and the effects of these compounds on GTP pool depletion. When the 50% effective concentrations (EC50s) for the antiviral activities of ribavirin, EICAR, and MPA were plotted against the respective EC50 values for GTP pool depletion, a linear correlation was calculated. These data provide compelling evidence that the predominant mechanism of action of ribavirin in vitro against flavi- and paramyxoviruses is based on inhibition of cellular IMP dehydrogenase activity.     

Mechanism of the potentiating effect of ribavirin on the activity of 2',3'-dideoxyinosine against human immunodeficiency virus

Ribavirin enhances the anti-human immunodeficiency virus activity of 2',3'-dideoxyinosine (ddIno) in MT-4, CEM and peripheral blood lymphocyte cells. Ribavirin causes an increase in the levels of IMP, the presumed phosphate donor for the conversion of ddIno to ddIMP by 5'-nucleotidase. Consequently, ribavirin stimulates the conversion of ddIno to its antivirally active metabolite ddATP. Ribavirin also causes a marked depletion of the guanine nucleotide pools. The increase in IMP pool levels may result from (i) a direct inhibitory effect of ribavirin 5'-monophosphate on IMP dehydrogenase (which converts IMP to XMP) and (ii) an indirect inhibition of adenylosuccinate synthetase by the decreased GTP and dGTP pools (since GTP is an obligatory cofactor in the conversion of IMP to succinyl AMP). GTP depletion plays a key role in the accumulation of IMP and the resultant higher rate of ddIno phosphorylation to ddIMP and eventually ddATP. Our findings are in agreement with the observations that guanosine and 2'-deoxyguanosine, but not 2'-deoxyadenosine, reverse (i) the stimulatory effect of ribavirin on the anti-human immunodeficiency virus activity of ddIno and (ii) the accumulation of endogenous IMP pools as well as accumulation of [3H]IMP from exogenous [3H]hypoxanthine in ribavirin-treated cells.     

Partial purification, properties and regulation of inosine 5''phosphate dehydrogenase in normal and malignant rat tissues.

IMP dehydrogenase (EC 1.2.1.14) was purified 180-fold from rat liver and from the transplantable rat hepatoma 3924A. The enzymes from the two sources were apparently identical; they exhibited hyperbolic saturation kinetics and an ordered, sequential mechanism, and were subject to inhibition by a number of purine nucleotides. Km values for the substrates, IMP and NAD+, were 12 and 24 micrometer respectively. IMP dehydrogenase activity in a spectrum of rat hepatomas was increased, relative to normal liver, by 2.5--13-fold; these increases correlated with tumour growth rate. Activity in two rat kidney tumours was increased 3-fold relative to that in normal renal cortex; control of activity of this enzyme is apparently altered in neoplastic cells. After partial hepatectomy, IMP dehydrogenase activity began to rise 6 h after operation, reaching a peak of 580% of normal activity by 18 h. Activity in neonatal liver, however, was only slightly higher than that in the adult. Organ-distribution studies showed highest enzyme activities in spleen and thymus. In livers of rats starved for 3 days, where all enzymes, except those involved in gluconeogenesis, showed decreased activity IMP dehydrogenase activity was increased; this change was accompanied by a rise in hepatic GTP concentrations. It is concluded that IMP dehydrogenase is a key enzyme in the regulation of GTP production, and thus involved in regulation of nucleic acid biosynthesis. The increased activity of IMP dehydrogenase in liver of starved rats may be related to the requirements for GTP for gluconeogenesis.     

Detection of apoptosis and phagocytosis in vitro in C6 rat glioma cells treated with tiazofurin

Tiazofurin, an anticancer drug which inhibits IMP dehydrogenase activity and decreases GTP concentration in various malignant cells, induced inhibition of growth and apoptosis in C6 rat glioma in vitro. The effects of tiazofurin were significantly blocked by addition of exogenous guanosine, suggesting the role of decreased GTP in triggering specific signal transduction pathways involved in apoptosis of C6 cells. The most interesting result of this study was the evidence of phagocytosis of apoptotic cells in vitro by neighbouring cells, a phenomenon considered to occur only in apoptosis in vivo. The possibility of observing phagocytosis in C6 glioma cells suggests that this cell system could be a good model for studying mechanisms of phagocytosis in vitro.     

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase

The inhibitory mechanisms of ribavirin 5'-monophosphate (RMP) and thiazole-4-carboxamide adenine dinucleotide (TAD), the active forms of the antimetabolites ribavirin and tiazofurin, were investigated in IMP dehydrogenase purified to homogeneity from rat hepatoma 3924A. The hepatoma IMP dehydrogenase has a tetrameric structure with a subunit molecular weight of 60,000. For the substrates IMP and NAD+, Km's were 23 and 65 microM, respectively. Product-inhibition patterns showed an ordered Bi-Bi mechanism for the enzyme reaction where IMP binds to the enzyme first, followed by NAD+; NADH dissociates from the ternary complex first and then XMP is released. XMP interacts with the free enzyme and competes for the ligand site with IMP, while NADH binds to the enzyme-XMP complex. RMP exerted the same inhibitory mechanisms as XMP, and the inhibition by TAD was similar to that by NADH. However, the Ki values for RMP (0.8 microM) and TAD (0.13 microM) were orders of magnitude lower than those of XMP (136 microM) and NADH (210 microM). Thus, the drugs interact with IMP dehydrogenase with higher affinities than the natural substrates and products, RMP with the IMP-XMP site and TAD with the NADH site. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner. The enzyme was protected from this inactivation by IMP or XMP. These results provide a biochemical basis for combination chemotherapy with tiazofurin and ribavirin targeted against the two different ligand sites of IMP dehydrogenase.     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Antineoplastic activity of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC50 of 19.2 microM. TAD content in K 562 cells (1.3 nmol/10(9)/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.     

IMP dehydrogenase and action of antimetabolites in human cultured blast cells

Tiazofurin was demonstrated to be an effective inhibitor of the growth of human cultured blast cells, and the high specific activities of IMP dehydrogenase (EC 1.1.1.205) were observed in all the cell extracts tested. IMP dehydrogenase has been purified to homogeneity from MOLT 4F human T-lymphoblast, and the Km values for IMP and NAD were 29 and 54 microM, respectively. The inhibitory mechanisms of thiazole-4-carboxamide adenine dinucleotide (TAD) and ribavirin 5'-monophosphate (RMP), the active forms of the antimetabolites tiazofurin and ribavirin, were investigated on the purified enzyme. RMP inhibits competitively with respect to IMP as well as XMP, and the inhibition by TAD was similar to that by NADH, which was uncompetitive with NAD. However, the Ki values of RMP (0.58 microM) and TAD (0.075 microM) were several orders of magnitude lower than those of XMP (85 microM) and NADH (94 microM). Thus, the drugs interact with the two distinct sites of IMP dehydrogenase with much higher affinities than the natural substrates and products. Preincubation of the purified enzyme with RMP enhanced its inhibitory effect in a time-dependent manner, and the enhancement was further increased by the addition of TAD. The combination of tiazofurin and ribavirin exerted a synergistic effect on the growth inhibition in MOLT 4F cells.     

Unique bioactivation of tiazofurin--studies with resistant cells

Using resistant cells mechanism of action of new oncolytic nucleoside, tiazofurin (2-beta-D-ribofuranosyl thiazole-4 carboxamide, RTC) was studied in tissue cultured cells of Chinese Hamster Ovary cells (CHO-cells). Tiazofurin got converted in CHO-cells to tiazofurin-monophosphate and to NAD-analogue, a potent inhibitor of inosinate dehydrogenase. Resistant cells produced tiazofurin-5'-monophosphate in vitro but had a much reduced capacity to produce NAD-analogue, indicating absence of any effect of tiazofurin on incorporation of [14C]formate into guanine, inhibition of inosinate dehydrogenase as well as GTP levels in resistant cells. Using competition with various possible substrates it is found that the initial tiazofurin metabolism is catalysed by nicotinamide nucleoside kinase and NAD-analog formation is mediated by NAD-pyrophosphorylase. Decreased activity of the latter enzyme found in tiazofurin resistant cells not only inhibited the NAD analog formation from tiazofurin-5'-monophosphate but also the NAD-formation from nictotinamide-5'-monophosphate.     

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells.

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.     

Tiazofurin: biological effects and clinical uses

Inosine 5'-phosphate dehydrogenase (IMPDH) activity is increased in all cancer cells. It is the rate-limiting enzyme of guanosine triphosphate (GTP) biosynthesis, and therefore, a sensitive target of chemotherapy. Tiazofurin selectively blocks IMPDH activity. Tiazofurin was found to have an antiproliferative effect on tumor cells in vitro and in the murine system. Based on these findings, Phase I trials were started elsewhere in patients with solid tumors, but were discontinued because of toxicity. In leukemic patients, we were able to demonstrate a good correlation between biochemical parameters (i.e., decline in IMPDH activity and GTP concentrations in blast cells) and clinical response. The most consistent responses to therapy were seen in patients with myeloid blast crisis of chronic myeloid leukemia. Severe toxicity was seen in the earlier patients in the study. However, better patient selection, limitation of treatment duration and earlier recognition and treatment of complications have now made it possible to administer tiazofurin without undue toxicity.     

Compartmentation of guanine nucleotide precursors for DNA synthesis.

We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo----dGMP----dGDP----dGTP and secondly: Gua----GMP----GDP----dGDP----dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.     

Comparative in vitro studies of Tiazofurin and a selenazole analog

2-beta-D-Ribofuranosylselenazole-4-carboxamide, a selenazole analog of the antitumor agent Tiazofurin, is severalfold more cytotoxic to murine tumor cells in culture than Tiazofurin. Like Tiazofurin, the cytotoxicity of the selenazole analog is reversed by exogenous guanosine, and both nucleosides specifically inhibit IMP dehydrogenase activity in cultured P388 cells. The dose-dependency for this inhibition correlates with the relative cytotoxicities of both drugs, indicating that a more potent inhibition of IMP dehydrogenase by the selenazole analog is primarily responsible for its increased cytotoxicity. The specific inhibition of the isolated enzyme by potential metabolites of the selenazole analog is discussed.     

Methotrexate decreases thymidine kinase activity.

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.