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.     

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.     

Ribavirin induced differentiation of murine erythroleukemia cells

Summary The synthetic nucleoside, ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide), a broad spectrum antiviral agent currently being tested in clinical studies with AIDS patients; and mycophenolic acid, a non-nucleoside inhibitor of inosinate (IMP) dehydrogenase, are effective inducers of terminal differentiation of Friend virus transformed murine erythroleukemia cells. The inhibition of cell division and the induced maturation produced by these agents appears to be a consequence of inhibition of IMP dehydrogenase, since growth inhibition is reversed and differentiation is prevented by the simultaneous exposure of cells treated with the agents to exogenous guanine or guanosine, which circumvents the effects of blockage of IMP dehydrogenase. However, while the effects mycophenolic acid, a pure IMP dehydrogenase inhibitor with no other biochemical effects, were completely reversed by guanine salvage supplies, cells exposed to ribavirin responded in a different manner. At levels of guanine salvage supplies below 50 M, growth inhibition and cell differentiation were partially reversed. At salvage supply concentrations greater than 50 M, while differentiation was completely blocked, the toxicity of ribavirin was increased and cell division was greatly diminished. These results indicate additional biochemical effects for ribavirin unrelated to the inhibition of IMP dehydrogenase, which may be related to its antiviral properties.     

3-Deazauridine triggers dose-dependent apoptosis in myeloid leukemia cells and enhances retinoic acid-induced granulocytic differentiation of HL-60 cells

Therapeutic nucleoside analogue 3-deazauridine (DU) exerts cytotoxic activity against cancer cells by disruption of DNA synthesis resulting in cell death. The present study evaluates whether DU alone at doses 2.5-15 microM or in combination with all trans retinoic acid (RA) or dibutyryl cAMP (dbcAMP) is effective against myelogenous leukemia. The data of this study indicate that DU induces dose-dependent cell death by apoptosis in myeloid leukemia cell lines HL-60, NB4, HEL and K562 as demonstrated by cell staining or flow cytometry and agarose gel electrophoresis. 24h-treatment with DU produced dose-dependent HL-60 cell growth inhibition and dose-independent S phase arrest that was not reversed upon removal of higher doses of DU (10-15 microM). Exposition to nontoxic dose of DU (2.5 microM) for 24h followed by RA or dbcAMP and 96 h-cotreatment with DU significantly enhanced RA- but not dbcAMP-mediated granulocytic differentiation. Cell maturation was paralleled with an increase in the proportion of cells in G1 or G2+M phase. We conclude that, depending on the dose or the sequence of administration with RA, an inhibitor of DNA replication, DU triggers a process of either differentiation or apoptosis in myeloid leukemia cells.     

Antileukemic activity of combined epigenetic agents, DNMT inhibitors zebularine and RG108 with HDAC inhibitors, against promyelocytic leukemia HL-60 cells

DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.     

Maturation of human promyelocytic leukemia cells induced by nicotinamide: Evidence of a regulatory role for ADP-ribosylation of chromosomal proteins

We have studied the role of ADP-ribosylation of chromosomal proteins in the regulation of myeloid cell maturation using the HL-60 cell line as a model. Nuclei isolated from this human promyelocytic leukemia cell line contained (ADP-ribose)_n synthetase activity, whereas little or no enzymatic activity was detectable in normal human blood neutrophils. Furthermore, the activity of (ADP-ribose)_n synthetase was decreased in HL-60 cells when they were induced to mature with retinoic acid (RA). To determine whether reduced (ADP-ribose)_n synthetase activity is simply a result of induced maturation or whether it is a necessary precedent event for the maturation process, we evaluated the effects of nicotinamide (NAm) and its methyl derivative, N′-methylnicotinamide (N′-Met-NAm), agents which decrease ADP-ribosylation. Treatment of HL-60 cells with these drugs caused the cells to undergo maturation and to acquire certain of the morphologic, functional, and biochemical characteristics of normal neutrophils. N′-Met-NAm was more potent than NAm in inducting maturation; at a concentration of 0.8 mM, it caused greater than 80% of the cells to mature, whereas a tenfold greater concentration of NAm was required to induce a similar degree of maturation. NAm and N′-Met-NAm also potentiated the maturation of HL-60 cells induced by RA. Exposure of cells to noninducing concentrations of these compounds caused a leftward shift in the dose-response curve for RA; maturation was observed at 10^?11 M RA in the presence of either 2 mM NAm or 0.2 mM N′-Met-NAm while 10^?9 M RA was required to induce maturation in their absence. A leftward shift in the dose response curve for maturation in the presence of low doses of NAm or N′-Met-NAm did not occur with another inducer, dimethyl formamide (DMF). Two enzymes, NAD glycohydrolase and tissue transglutaminase, that are abundant in macrophages, were induced by RA but not by NAm. N′-Met-NAm decreased by about 75% the amount of endogenous (ADP-ribose)_n in a selected fraction of chromosomal proteins which included histone H₁ and the nonhistone high mobility group proteins. The results of this study support the concept that ADP-ribosylation of chromosomal proteins influences the regulation of human myeloid cell maturation.     

Characterization of the metabolic forms of 6-thioguanine responsible for cytotoxicity and induction of differentiation of HL-60 acute promyelocytic leukemia cells

HL-60 human acute promyelocytic leukemia cells that lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity have been developed by mutagenization and selection. These cells exhibited markedly decreased sensitivity to the cytotoxic action of 6-thioguanine (TG) and, in contrast to parental HL-60 cells, had the capacity to undergo terminal granulocytic differentiation after treatment with this purine antimetabolite. Analysis of extracellular and intracellular metabolites of TG revealed negligible metabolism of TG in these HGPRT- HL-60 cells. These findings are consistent with the concept that inhibition of cellular replication requires generation of analog nucleotide and suggest that TG itself is capable of initiation of differentiation. 6-Thioguanosine (TGuo) had limited activity, while beta-2'-deoxythioguanosine (dTGuo) was inactive, as an inducer of maturation of HGPRT- HL-60 cells. These cells converted relatively large amounts of the nucleosides to the free base TG; the simultaneous exposure of cells to 8-aminoguanosine (AGuo), an inhibitor of purine nucleoside phosphorylase activity, decreased the degradation of TGuo and dTGuo to TG and promoted the intracellular accumulation of TG nucleotides, presumably through the action of nucleoside kinase activities. In a double mutant deficient in both HGPRT and deoxycytidine kinase (DCK) activities, dTGuo was devoid of cytotoxicity and was an effective inducer of maturation. The potency of dTGuo as an inducer in this system was not significantly affected by the presence of AGuo. These results suggested that dTGuo itself was also an active initiator of maturation. Thus, induction of differentiation appeared to be due to the free base, TG, as well as its deoxynucleoside form, dTGuo, whereas the formation of TG nucleotides appeared to antagonize maturation and produce cytotoxicity.     

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) exerts anticancer activity against HL-60 cells in vitro at clinically achievable concentrations

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) is an analogue of 1,25(OH)2D3 with reduced calcemic effects that is approved in the United States for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has anticancer activity in prostate cancer cells. We tested the effects of paricalcitol on the HL-60 leukemia cells, studying cellular differentiation, cell cycle changes, apoptosis and cellular proliferation. Paricalcitol at 10(-8)M concentration induced the maturation of HL-60 cells in a time-dependent manner, as shown by increased expression of CD11b differentiation surface antigen. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was markedly increased after exposure to paricalcitol at 10(-8)M for 72 h. Paricalcitol inhibited colony formation of HL-60 cells in a soft agar semisolid media after 10-day incubation (estimated IC50 of 5 x 10(-9) M. Exposure to 10(-8)M paricalcitol for 72 h increased the number of cells in G0/G1 phase, and decreased the number of cells in S phase, and significantly increased the number of HL-60 cells undergoing apoptosis. The concentration required to achieve inhibition of growth of HL-60 cells is comparable to clinically achievable levels. These findings support the clinical evaluation of paricalcitol as an antileukemia agent.     

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).     

Alteration of folate analogue transport following induced maturation of HL-60 leukemia cells. Early decline in mediated influx, relationship to commitment, and functional dissociation of entry and exit routes

During treatment of HL-60 myeloid leukemia cells in culture with polar solvents or retinoic acid at a concentration inducing terminal maturation in 90-95% of the cells, there is a rapid decline (within 2 h) in the Vmax for influx of the folate analogue, [3H]methotrexate. Following 24 h of exposure to these agents, there is no effect on growth, but influx Vmax is reduced by 70%. After 7 days of exposure, influx Vmax is reduced 90-95%. A similar time course was seen for the reduction in intracellular levels of dihydrofolate reductase, a marker of cellular proliferation. Both the extent of terminal maturation (as determined by the extent of nitro blue tetrazolium reduction) and decrease in influx showed the same dependence on the concentration of inducer. In contrast to the effect seen on influx Vmax, both influx Km and mediated efflux of [3H]methotrexate remained unchanged in HL-60 cells exposed to inducers of maturation. Finally, evidence is presented for the coupling of this alteration on [3H]methotrexate influx with commitment of HL-60 cells to terminal maturation. This evidence shows that the effect on folate analogue influx precedes commitment and documents the irreversible nature of the reduction in influx once the majority of the cells exposed to inducer were committed to the process of maturation. The possible relevance of these results to the process of neoplastic transformation is discussed.     

Expression of human IMP dehydrogenase types I and II in Escherichia coli and distribution in human normal lymphocytes and leukemic cell lines.

Two distinct cDNAs encoding proteins with 84% sequence identity have been isolated for human IMP dehydrogenase (EC 1.1.1.205) (Natsumeda, Y., Ohno, S., Kawasaki, H., Konno, Y., Weber, G., and Suzuki, K. (1990) J. Biol. Chem. 265, 5292-5295), an important target in antileukemic chemotherapy. We constructed expression plasmids containing these cDNAs in full length with pUC plasmids and produced lacZ'-IMP dehydrogenase fusion proteins in Escherichia coli. Both synthesized proteins exhibited IMP dehydrogenase activity and were partially separated from endogenous E. coli IMP dehydrogenase. By injecting the fusion proteins into mice we generated a polyclonal antibody specific to type I IMP dehydrogenase and an antibody which reacted with both types. Immunoblot analysis revealed that the total amounts of types I and II enzymes increased in human leukemic cell lines K562 and HL-60 in agreement with the increase in IMP dehydrogenase activity to 7.8- and 9.4-fold, respectively, above that of normal lymphocytes. The extent of expression of type I IMP dehydrogenase was similar in these cells, however, indicating that the increase in IMP dehydrogenase amount in leukemic cells was due to specific up-regulation of type II enzyme. Northern blot analysis also showed specific and predominant expression of type II in the leukemic cells. Thus, de novo GTP biosynthesis may be controlled differently in normal and neoplastic cells by different IMP dehydrogenase molecular species.     

Effects of inducers of differentiation on protein kinase C and CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase activities of HL-60 leukemia cells

Exposure of HL-60 leukemia cells to either 12-O-tetradecanoylphorbol-13-acetate (TPA), dimethylsulfoxide (DMSO), exogenous gangliosides GM3, GM1, or bovine brain ganglioside mixture (BBG) resulted in a marked inhibition of the growth of cells. The order of the inhibitory potency was TPA greater than GM3 greater than DMSO greater than BBG greater than GM1. In contrast, sulfatides were without effect on cellular replication. Treatment of HL-60 cells with TPA or GM3 induced differentiation along the monocyte/macrophage lineage, while treatment with DMSO induced maturation along the granulocytic pathway. These effects were accompanied by more than a twofold increase in protein kinase C (PKC) activity. In contrast, treatment with GM1, BBG, or sulfatides caused only a relatively small increase in PKC activity. The activity of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase (ST1), a key enzyme for membrane gangliosides synthesis, in HL-60 cells was also influenced by the exposure to TPA, GM3, DMSO, GM1, or sulfatides. The inducers of differentiation, TPA and DMSO, caused an increase in ST1 activity, whereas GM3, which also induced cellular differentiation, inhibited ST1 activity, perhaps through the action of end-product inhibition. The non-inducers of differentiation, GM1 and sulfatides, also increased the activity of ST1, but to a much lesser extent. The findings suggest that the direct or indirect modulation of PKC activity by some of these agents may be involved, at least in part, in the regulation of cellular growth and differentiation. Furthermore, it is conceivable that differences in PKC activity may be responsible for the changes in ST1 activity associated with cell differentiation and proliferation.     

Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition

The kinetic properties of highly purified human placental cytoplasmic 5'-nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo.     

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.     

3,4,4'-Trihydroxy-trans-stilbene, an analogue of resveratrol, is a potent antioxidant and cytotoxic agent

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a naturally occurring polyphenol widely distributed in food and dietary plants. This phytochemical has been intensively studied as an efficient antioxidant and anticancer agent, and a variety of substituted stilbenes have been developed in order to improve the potency of resveratrol. In this work, we described the synthesis of 3,4,4 -trihydroxy-trans-stilbene (3,4,4'-THS), an analogue of resveratrol, and studied its antioxidant and cytotoxic activity in vitro. 3,4,4 -THS was much more efficient than resveratrol in protecting against free radical-induced lipid peroxidation, photo-sensitized DNA oxidative damage, and free radical-induced hemolysis of human red blood cells. More potent growth inhibition in cultured human leukemia cells (HL-60) was also observed for 3,4,4 -THS. The relationship between the antioxidant efficiency and cytotoxic activity was discussed, with the emphasis on inhibition of the free radical enzyme ribonucleotide reductase by antioxidants. The result that this subtle structure modification of resveratrol drastically improves its bioactivity provides important strategy to develop novel resveratrol-based molecules.     

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.     

Evidence for the involvement of cytosolic 5'-nucleotidase (cN-II) in the synthesis of guanine nucleotides from xanthosine

In this paper, we show that in vitro xanthosine does not enter any of the pathways known to salvage the other three main natural purine nucleosides: guanosine; inosine; and adenosine. In rat brain extracts and in intact LoVo cells, xanthosine is salvaged to XMP via the phosphotransferase activity of cytosolic 5'-nucleotidase. IMP is the preferred phosphate donor (IMP + xanthosine --> XMP + inosine). XMP is not further phosphorylated. However, in the presence of glutamine, it is readily converted to guanyl compounds. Thus, phosphorylation of xanthosine by cytosolic 5'-nucleotidase circumvents the activity of IMP dehydrogenase, a rate-limiting enzyme, catalyzing the NAD(+)-dependent conversion of IMP to XMP at the branch point of de novo nucleotide synthesis, thus leading to the generation of guanine nucleotides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, inhibits the guanyl compound synthesis via the IMP dehydrogenase pathway but has no effect on the cytosolic 5'-nucleotidase pathway of guanine nucleotides synthesis. We propose that the latter pathway might contribute to the reversal of the in vitro antiproliferative effect exerted by IMP dehydrogenase inhibitors routinely seen with repletion of the guanine nucleotide pools.     

Leukemic cell maturation: variability of the myeloid leukemic cell phenotype

Leukemia is characterized by a proliferation of cells that exhibit an arrest in the normal differentiation sequence. The HL-60 promyelocytic leukemia is a useful model of the phenomenon of maturation arrest, particularly as modulated by inducers that partially restore myeloid differentiation. In this investigation, we report the development of two sublines of HL-60 and the acquisition of two others that are clonal variants of the parent cell line. Each of the sublines demonstrates an altered pattern of differentiation and resistance to one or more of the drugs that serves as an inducer of the parent line. The two cell lines developed in this study, HL-60S and HL-60I, are resistant to arabinosylcytocine (ARA-c); HL-60I is also resistant to PMA. A study of the phenotype as expressed by the granule-associated cytoplasmic enzymes revealed that each subline had a slightly different pattern of maturation in response to dimethylsulfoxide (DMSO) or ARA-c as inducers. The proliferative rate of all sublines was similar. These data demonstrate that the maturation arrest observed in this model is in part reversible. The maturation arrest observed in myeloid leukemias is due to a reversible block secondary to altered proliferative activity.