tretinoin and tiazofurin

New light was thrown on the action of tiazofurin in the treatment of end-stage leukemic patients and in leukemic cells in tissue culture. 1. In a population of 21 consecutive patients 50% responded to tiazofurin treatment, confirming the usefulness of this therapy in end-stage leukemia. 2. In leukemic patients treated with tiazofurin and allopurinol reciprocal action was manifested in the increase in hypoxanthine and the decrease in uric acid concentrations in the plasma. On discontinuation of allopurinol, hypoxanthine levels steeply declined but uric acid concentration increased slowly, taking days to reach pretreatment level. 3. With a new and sensitive method the concentration of the active metabolite of tiazofurin, TAD, was measured in the mononuclear cells of tiazofurin-treated patients. Approximately 5 to 13% of the plasma tiazofurin level was observed as TAD in the mononuclear cells. This TAD concentration was sufficient to account for the inhibition of IMP DH in these cells. 4. Tiazofurin or retinoic acid caused differentiation of HL-60 leukemic cells and inhibition of cell proliferation. 5. By treating leukemic cells incubated with tiazofurin or retinoic acid also with guanosine it was elucidated that the mechanism of the two drugs differed since only the tiazofurin effects were counteracted by guanosine. 6. Tiazofurin and retinoic acid together in HL-60 cells provided synergistic impact on differentiation and cytotoxicity. 7. Tiazofurin resulted in down-regulation of the expression of ras and myc oncogenes in three systems: K562 human erythroleukemic cells, rat hepatoma 3924A cells and human HL-60 leukemia cells. 8. Because both tiazofurin and retinoic acid are licensed drugs, their potential use in combination chemotherapy may have clinical relevance in the treatment of end-stage leukemia where our earlier studies have demonstrated the usefulness of tiazofurin.

Topics: Animals; Antineoplastic Agents; Cell Line; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Leukemia; Oncogenes; Proto-Oncogenes; Ribavirin; Ribonucleosides; Tretinoin; Tumor Cells, Cultured

Human IMP dehydrogenase (IMPDH; EC 1.1.1.205) was recently found to consist of two molecular species (types I and II) with high expression of type II isozyme in leukemic cells. Here we report that the low level of type II mRNA in normal lymphocytes was up-regulated by phytohemagglutinin stimulation (3.2-fold) and Epstein-Barr viral transformation (5.7-fold). The type II mRNA expression in quiescent HL-60 cells was also elevated 2.8-fold by serum stimulation. Conversely the enhanced level of type II IMPDH mRNA in HL-60 cells was down-regulated to less than 5% along with differentiation induced by retinoic acid (1 microM), phorbol-12-myristate-13-acetate (33 nM), or dimethyl sulfoxide (1.25%) independent of end-stage phenotype. By contrast, type I IMPDH mRNA was expressed constitutively in the various states of proliferation and differentiation. The type II IMPDH stringently linked with cell proliferation should be a crucial target for antileukemic and immunosuppressive chemotherapy.

Topics: Blotting, Northern; Cell Division; Dimethyl Sulfoxide; Gene Expression; Guanosine; Humans; IMP Dehydrogenase; Leukemia, Myeloid; Lymphocyte Activation; Lymphocytes; Ribavirin; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

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.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Differentiation; Cell Line; Cell Survival; Deoxycytidine; Dose-Response Relationship, Drug; Drug Synergism; Female; Gemcitabine; Humans; IMP Dehydrogenase; Kinetics; Leukemia, Promyelocytic, Acute; Liver Neoplasms, Experimental; Models, Biological; Ovarian Neoplasms; Pancreatic Neoplasms; Rats; Ribavirin; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Stem Cell Assay

Tiazofurin, a potent inhibitor of inosine 5'-phosphate dehydrogenase, depletes guanine nucleotide pools and induces granulocytic maturation of HL-60 leukemia cells. These effects are reversed when cells exposed to this agent for 24 h are washed and placed in tiazofurin-free medium. HL-60 cells treated with tiazofurin for a 24 h period, retain a precommitment memory that lessens the time interval necessary for cells to express the mature phenotype upon re-exposure. That protein synthesis was required for maintaining the expression of memory was demonstrated by the finding that memory was blocked when primed cells were exposed to cycloheximide during the intervening inducer-free interval, but not during the priming or subsequent drug exposure periods. The findings have significance with respect to the sequence of events required for commitment to a differentiation pathway.

Topics: Antimetabolites, Antineoplastic; Cell Cycle; Cell Differentiation; Cell Line; Cycloheximide; DNA Replication; DNA, Neoplasm; Guanosine; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Ribavirin; Ribonucleotides; Thymidine; Tretinoin

Tiazofurin and retinoic acid synergistically induced differentiation and inhibited colony formation in HL-60 human promyelocytic leukemia cells in cell culture. The synergism was the result of different mechanisms of action, since the effect of tiazofurin, unlike that of retinoic acid, was prevented by addition of guanosine. Since it has been shown that tiazofurin down-regulated the expression of c-Ki-ras oncogene, and retinoic acid that of the myc oncogene, the joint impact of these drugs is of clinical interest particularly in end-stage leukemia where the therapeutic usefulness of tiazofurin has recently been demonstrated.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Dose-Response Relationship, Drug; Drug Synergism; Guanosine; Humans; IMP Dehydrogenase; Leukemia, Promyelocytic, Acute; Molecular Structure; Phenotype; Ribavirin; Ribonucleosides; Time Factors; Tretinoin; Tumor Cells, Cultured

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.

Topics: Biomarkers; Gene Expression Regulation, Neoplastic; Guanosine; Humans; IMP Dehydrogenase; Melanins; Melanoma; Monophenol Monooxygenase; Mycophenolic Acid; Neoplasm Proteins; Ribavirin; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The impact of tiazofurin on inhibition of IMP dehydrogenase was discussed at the clinical and molecular levels. 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. The argument for expecting an impact of tiazofurin in human myelocytic cells was provided. 3. Similarity of the kinetics of human leukemic cell IMP dehydrogenase to the rat hepatoma enzyme was documented. 4. New evidence was provided for the role of salvage in chemotherapy and the function of hypoxanthine in inhibiting guanine salvage. 5. The action of tiazofurin and retinoic acid was reported in HL-60 leukemic cells. 6. The effect of tiazofurin and retinoic acid on proliferation and cytotoxicity was outlined for hepatoma 3924A cells. 7. The effect of guanine on induced differentiation by tiazofurin and retinoic acid was examined. 8. Biochemical basis was provided for the lack of development of resistance in patients treated with tiazofurin. 9. Presumptive evidence was provided that tiazofurin treatment induced differentiation of leukemic cells in the patients. 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.

Topics: Animals; Antimetabolites, Antineoplastic; Cell Differentiation; Cell Division; Cell Line; Guanosine; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Leukemia, Promyelocytic, Acute; Liver Neoplasms, Experimental; Rats; Ribavirin; Ribonucleosides; Tretinoin; Tumor Cells, Cultured

Some of the current critical issues in the tiazofurin treatment of end-stage leukemia were presented and discussed. 1. Tiazofurin infusions (daily X 10 to 15) provided remissions in 50% of end-stage leukemic patients. The remissions, of 1 to 10 months' duration, varied from antileukemic effect or hematologic improvement to complete response and complete remission. The total survival of the responding patients was from about 1 to 15 months. 2. Our administration of tiazofurin in a 60-min infusion by pump decreased the incidence and severity of toxicity. 3. It was shown that tiazofurin dose does not need to be escalated at each relapse. Depending on the biochemical and hematological response in this novel protocol, 2,200 to 4,400 mg/m2 tiazofurin appeared to be sufficient to provide remissions. 4. A new role was identified for allopurinol, originally given to decrease uric acid in the plasma. Allopurinol markedly increased plasma hypoxanthine concentrations which competitively inhibited the activity of the salvage enzyme, guanine phosphoribosyltransferase, in the blast cells. Thus, the elevated hypoxanthine plasma levels inhibited guanine salvage. To maintain high hypoxanthine levels allopurinol (100 mg) was given every 4 to 6 hr. This provided combination chemotherapy with tiazofurin which inhibited IMP dehydrogenase activity and blocked the de novo biosynthesis of guanylates in the blast cells. 5. Preliminary evidence was obtained in the patients that tiazofurin induced differentiation of the bone marrow. Recent studies also showed that tiazofurin down-regulated the expression of the c-Ki-ras oncogene in K562 erythroleukemic cells. Therefore, tiazofurin treatment provides an impact by chemotherapy, induced differentiation, and, if applicable, through down-regulation of the ras oncogene. 6. Novel aspects of tiazofurin treatment include rational targeting and a continuously monitored trial by measurement of the activity of IMP dehydrogenase and of GTP and TAD concentrations in blast cells and of tiazofurin and hypoxanthine in plasma. 7. Since tiazofurin has not yet achieved lasting remissions in patients nor terminal differentiation of leukemic cells it probably will be advantageous to combine tiazofurin with other drugs to provide synergism. In preclinical tissue culture studies in HL-60 cells synergy was observed with retinoic acid. This may be of interest because retinoic acid also caused differentiation and down-regulation of the myc oncogene.

Topics: Adult; Aged; Aged, 80 and over; Allopurinol; Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cell Differentiation; Down-Regulation; Drug Interactions; Female; Genes, ras; Guanine; Humans; Hypoxanthine; Hypoxanthines; Leukemia; Liver Neoplasms, Experimental; Male; Middle Aged; Ribavirin; Ribonucleosides; Tretinoin; Tumor Cells, Cultured; Uric Acid

We have shown previously that induced maturation of the human myeloid leukemia cell line, HL-60, is associated with a selective down-regulation of guanine ribonucleotide synthesis and depletion of intracellular guanosine triphosphate (GTP) and guanosine diphosphate (GDP) pools. We showed, furthermore, that inhibitors of the enzyme, inosine monophosphate (IMP) dehydrogenase, which catalyzes the initial rate-limiting step of guanylate synthesis from the central intermediate IMP, are potent inducers of myeloid maturation in these cells. We now show that induced maturation of HL-60 cells is prevented or impaired if intracellular concentrations of guanine ribonucleotides are maintained at high levels. HL-60 cells can utilize exogenous guanine and guanosine to maintain GTP and GDP pools through a salvage pathway that bypasses guanylate synthesis from IMP. Moreover, incubation of HL-60 cells with guanosine or guanine (10(-6) to 10(-4) mol/L) prevents both the depletion of intracellular guanine ribonucleotides and the induction of myeloid maturation caused by the IMP dehydrogenase inhibitor, tiazofurin. These findings provide strong additional support for the concept that terminal myeloid differentiation is influenced by a guanine ribonucleotide-dependent regulatory system.

Topics: Cell Differentiation; Cell Line; Dimethylformamide; Guanine; Guanine Nucleotides; Guanosine; Hematopoiesis; Humans; IMP Dehydrogenase; Leukemia, Myeloid, Acute; Ribavirin; Tretinoin