tiazofurin and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H.N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c-myc (the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Blast Crisis; Blotting, Northern; Down-Regulation; Gene Expression Regulation; Genes, ras; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; IMP Dehydrogenase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Pilot Projects; Ribavirin; RNA; Uric Acid

The effect of Tiazofurin (TR) on the in vitro growth of bone marrow (BM) and peripheral blood (PB) leukemic progenitors was investigated in 29 patients. Nineteen of the patients were suffering the blast crisis of chronic myeloid leukemia (bcCML) and ten patients refractory acute myeloid leukemia (AML). PB and BM mononuclear cells were cultured in methylcellulose alone or with concentrations of TR ranging between 10 and 200 microM. TR produced a dose dependent inhibition of colony forming unit (CFU)-blast growth in all the samples tested from BM and PB. The most effective concentrations of TR used were 150 and 200 microM, while concentrations of less than 50 microM TR were not adequate for 50% inhibition of cell growth (IC50). Differences were found in the response of CFU-blasts to TR related to the type of underlying leukemia. Inhibition of CFU-blast growth was more pronounced in bcCML than in AML in both the BM and PB samples. The concentration of TR required to induce IC50 in bcCML was 50 microM, while the same effect in AML required a concentration of 150 microM. Analysis of the control samples also revealed that CFU-blasts from bcCML produced smaller numbers of colonies, though these differences were not statistically significant. It has therefore been demonstrated that TR has strong in vitro anti-leukemic activity, more pronounced in bcCML than in refractory AML. We thus feel this study gives further rationale for the clinical application of TR, and would strongly support this.

Topics: Adult; Aged; Antimetabolites, Antineoplastic; Blast Crisis; Blood Cells; Bone Marrow Cells; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Hematopoietic Stem Cells; Humans; Inhibitory Concentration 50; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Male; Middle Aged; Neoplastic Stem Cells; Ribavirin; Tumor Cells, Cultured; Tumor Stem Cell Assay

The ras oncogene product (p21ras, Ras) is a GTP-binding protein and is thought to transduce signals regulating cellular proliferation and differentiation. The active form Ras-GTP is inactivated by hydrolyzing bound GTP to GDP. Tiazofurin, a specific inhibitor of IMP dehydrogenase, decreased cellular GTP pools and downregulated c-ras gene expression, leading to differentiation (Olah, E. et al., Proc. Natl. Acad. Sci. USA 85: 6533-6537, 1988; Weber et al., Cancer Commun. 3:61-66, 1991). To clarify the link between the action of tiazofurin on metabolic alterations and the induction of differentiation, we examined the effect of tiazofurin on the ratio of active Ras-GTP to total Ras in K562 cells in culture. Cells were labeled for 6 h with [32P]Pi in phosphate-free RPMI 1640. Tiazofurin (100 or 200 microM) was added to cells, and samples were taken at 0, 2, 4, 6 and 12 h of incubation. Cell lysates were immunoprecipitated with monoclonal anti-p21 antibody (Y13-259), then developed on thin layer chromatography. GTP and GDP bound to Ras were visualized by autoradiography. Tiazofurin treatment decreased Ras-GTP concentration in a time- and dose-dependent fashion. In the untreated K562 cells the Ras-GTP concentration was 26.3 +/- 1.4, and tiazofurin (200 microM) decreased it at 6 h to 16.6 +/- 2.9 and at 12 h to 10.6 +/- 2.1%. Inhibition of the GTP salvage pathway with hypoxanthine (100 or 200 microM) enhanced the tiazofurin-induced decrease of Ras-GTP, whereas addition of guanosine (100 microM) prevented the Ras-GTP decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antimetabolites, Antineoplastic; Cell Differentiation; Guanosine; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Oncogene Protein p21(ras); Protein Binding; Ribavirin; Tumor Cells, Cultured

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.

Topics: Antineoplastic Agents; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Humans; IMP Dehydrogenase; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Molecular Structure; Nucleosides; Ribavirin; Ribonucleotides

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.

Topics: Antimetabolites, Antineoplastic; Cell Cycle; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Ribavirin; Tumor Cells, Cultured

Tiazofurin, 2-beta-D-ribofuranosylthiazole-4-carboxamide, is cytotoxic to murine and human tumor cells. In earlier Phase-I/-II trials performed in other centers in patients with solid tumors, the drug was given mainly as a 10-min bolus or as a continuous i.v. infusion for 5 days. These protocols were associated with serious side effects, including neurotoxicity, pleuropericarditis, and occasional myelosuppression. In our study, 26 patients with end-stage leukemia were treated with tiazofurin with 1-hr daily i.v. infusions, resulting in lower incidence and less severity of side effects. In this group, 7 attained complete remission and 7 showed hematologic responses. Out of 12 evaluable patients with myeloid blast crisis of chronic granulocytic leukemia, 10 (83%) responded to therapy, with 6 attaining complete response. We present pharmacokinetic parameters of our clinical study and examine some of the reasons for the lower toxicity found in our trials. In leukemic patients during and after infusion at doses of 1,100, 2,200 and 3,300 mg/m2 tiazofurin peak plasma concentrations were 245, 441 and 736 microM, respectively, values one-half of those calculated from other reports with a 10-min bolus administration. In our 1-hr infusion method, biphasic pharmacokinetics were noted with alpha t1/2 and beta t1/2 of 0.5 and 6.2 hr, and tiazofurin was eliminated at a faster rate than in previous trials with continuous infusion. The area under the curve with our 1-hr infusion was 52% of that reported for the same dose given by continuous infusion. Our 1-hr infusion method and prompt and effective treatment of side effects enabled us to administer higher doses and larger total amounts of tiazofurin in longer treatment cycles than in any previous trials elsewhere. Tiazofurin therapy using 1-hr infusion may be feasible for other carefully selected types of malignancies.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Blast Crisis; Drug Administration Schedule; Female; Humans; Infusions, Intravenous; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Male; Middle Aged; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Ribavirin

2-beta-D-Arabinofuranosylthiazole-4-carboxamide and 2-beta-D-xylofuranosyl-thiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Arabinonucleosides; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Humans; IMP Dehydrogenase; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nucleosides; Ribavirin

Topics: Adult; Aged; Allopurinol; Antineoplastic Agents; Female; Genes, myc; Genes, ras; Guanosine Triphosphate; Humans; IMP Dehydrogenase; In Vitro Techniques; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Ribavirin; Tumor Cells, Cultured

The physiologically active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], was found to inhibit erythroid differentiation of human leukemic K562 cells. Differentiation was induced by 1 mumol/L arabinocytosine (Ara-C), 40 mumol/L tiazofurin, 1 mumol/L aphidicolin, or 1 mumol/L hydroxyurea, and was monitored daily by the appearance of hemoglobin in an increasing proportion of cells. Pretreatment for 48 hours with 2.4 x 10(-8) mol/L 1,25(OH)2D3, a concentration that is also optimal for induction of monocytic differentiation of HL-60 cells, reproducibly inhibited subsequent induction of erythroid differentiation by all of the above inducers, and modified the morphologic changes that Ara-C produced in these cells. The inhibition of hemoglobinization was approximately 50% irrespective of the degree of differentiation produced by the various inducers, but growth inhibition associated with exposure to the inducers was not affected by 1,25(OH)2D3. Similar inhibition of differentiation by 1,25(OH)2D3 was observed in mouse erythroleukemia cells MEL-D1B treated with 5 mmol/L hexamethylenebisacetamide. The inhibitory effect of 1,25(OH)2D3 on erythroid differentiation of K562 cells was abrogated by cyclohexamide (20 micrograms/mL), an inhibitor of protein synthesis. The mRNA for 1,25(OH)2D3 receptor (VDR) was detected in K562 cells, and was downregulated by a 96-hour exposure to 1,25(OH)2D3 or a 48-hour exposure to Ara-C. The presence of VDR mRNA suggests a physiologic role for 1,25(OH)2D3 in K562 cells that are precursors of erythroid cells. This role is perhaps to shift the pathways of differentiation from the erythroid to the monocytic lineage.

Topics: Animals; Calcitriol; Cell Differentiation; Cell Division; Cell Line; Cytarabine; Humans; Kinetics; Leukemia, Experimental; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Receptors, Calcitriol; Receptors, Steroid; Ribavirin; RNA, Messenger; Time Factors

Tiazofurin through its active metabolite thiazole-4-carboxamide adenine dinucleotide (TAD) inhibits IMP dehydrogenase, the rate-limiting enzyme of GTP biosynthesis. IMP dehydrogenase activity in human leukemic cell extracts (33.4 +/- 0.1 nmol/h/mg protein) was increased 11-fold compared to normal leukocytes (3.1 +/- 0.5). Km values for IMP and NAD+ of leukemic IMP dehydrogenase were 22.7 and 44.0 microM, respectively. XMP inhibited competitively with IMP and noncompetitively with NAD+. NADH exerted mixed type inhibition with respect to both IMP and NAD+. The inhibitory pattern of TAD was quite similar to that of NADH; however, the affinity of TAD to leukemic IMP dehydrogenase (Ki = 0.1 microM) was three orders of magnitude higher than the natural product NADH (Ki = 150 microM). These results contribute to an understanding of the mechanism of action of tiazofurin in the treatment of leukemia.

Topics: Adenine Nucleotides; Antineoplastic Agents; Blast Crisis; Female; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Middle Aged; NADP; Ribavirin; Ribonucleosides