Biomedical subjects

D G Johns

Publications and source records attributed to D G Johns.

At least 37 records · Page 2Linked to original sources

PubMed1998

Is ceramide signaling a target for vascular therapeutic intervention?

Today's society is plagued by cardiovascular diseases such as hypertension and atherosclerosis. Research devoted to the study of these diseases has focused, in part, on physiological phenomena responsible for the development and progression of the disease. Peripheral vascular function is one such focal point of research, and identification of cellular mechanisms that regulate vascular contractility and/or cellular proliferation is crucial for the development of new therapeutic interventions to combat these diseases. This review evaluates a new signaling mechanism, the ceramide signaling pathway as a potential target for therapeutic intervention. Special attention is given ceramide signaling in the contexts of vascular reactivity and cell proliferation in the vasculature. While ceramide signaling is a nascent area of vascular research, a growing body of evidence from other physiological systems implicates this new pathway as a potential regulator of contractile and cell proliferative functions within the blood vessel wall.

Arteriosclerosis↗

PubMed1997

Ceramide: a novel cell signaling mechanism for vasodilation.

Ceramide is a lipid second messenger generated by membrane hydrolysis of sphingomyelin by sphingomyelinase, but a role for this novel signaling pathway in vascular smooth muscle has not been elucidated. Based upon observations of cytokine-induced increases in sphingomyelinase activity, we hypothesized that ceramide plays a cell signaling role in vasodilation. Here, we demonstrate that ceramide is present at significant basal levels in cultured vascular smooth muscle cells and that these levels may be increased using exogenous sphingomyelinase. We also report that both exogenously added ceramide and sphingomyelinase cause dose-dependent relaxation in phenylephrine-contracted endothelium-denuded rat thoractic aortic rings. We conclude that the ceramide signaling pathway represents a novel signal transduction mechanism for vasodilation.

Animals↗

PubMed1997

Mechanism of 'bystander effect' killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment.

'Bystander' killing of adjacent wild-type tumor cells was seen when tumors transduced with the herpes thymidine kinase gene were treated with the antiviral agent ganciclovir (GCV). Some tumors were 'bystander-sensitive' while others were 'bystander-resistant'. Mixtures of different 'sensitive' tumor lines showed cross-transfer of bystander killing, while in mixtures of 'resistant' with 'sensitive' tumors, the resistant phenotype was predominant. Using 3H-GCV with 'sensitive' mixtures, phosphorylated 3H-GCV was found in both herpes thymidine kinase transduced and unmodified cells, while 'resistant' cell combinations showed little or no transfer of phosphorylated GCV between cells. The capacity of intracellularly produced nucleotide toxin to spread from cell to cell within a tumor mass effectively amplifies the apparent efficiency of gene transfer in the tumor and makes feasible the use of this system for therapy of localized cancer.

Adenocarcinoma↗

PubMed1997

Antiproliferative effects of cyclopentenyl cytosine (NSC 375575) in human glioblastoma cells.

Cyclopentenyl cytosine (CPEC) exerts an antiproliferative effect against a wide variety of human and murine tumor lines, including a panel of human gliosarcoma and astrocytoma lines. This effect is produced primarily by the 5'-triphosphate metabolite CPEC-TP, an inhibitor of cytidine-5'-triphosphate (CTP) synthase (EC 6.3.4.2). Because previous studies with human glioma cell lines utilized cells in long-term tissue culture, we have undertaken to determine whether the activity of CPEC in such model systems is also demonstrable in freshly excised human glioblastoma cells. Glioma cells obtained at surgery and in log phase growth were exposed to the drug at levels ranging from 0.01 to 1 microM for 24 h, and CPEC-TP and CTP levels were determined by HPLC. Dose-dependent accumulation of CPEC-TP was accompanied by a concomitant decrease in CTP pools, with 50% depletion of the latter being achieved at a CPEC level of ca. 0.1 microM. Human glioma cell proliferation was inhibited 50% by 24-h exposure to 0.07 microM CPEC. Postexposure decay of CPEC-TP was slow, with a half-time of 30 h. DNA cytometry showed a dose-dependent shift in cell cycle distribution, with an accumulation of cells in S-phase. The pharmacological effects of CPEC on freshly excised glioblastoma cells are quantitatively similar to those seen in a range of established tissue culture lines, including human glioma, colon carcinoma, and MOLT-4 lymphoblasts, supporting the recommendation that the drug may be advantageous for the treatment of human glioblastoma.

Adult↗

PubMed1997

Decay rates of anti-HIV dideoxynucleotides in tissue culture systems: a simple correction for the effect of cell replication.

Measurement of intracellular drug levels in cell culture systems can be of predictive value in establishing rational clinical dosage schedules. Such in vitro measurements carried out with anti-HIV agents of the 2',3'-dideoxynucleoside (ddN) class have shown that many of the pharmacologically active ddNTP metabolites of these agents have relatively long intracellular half-lives and little or no host-cell cytotoxicity. As a consequence, replication of drug-exposed cells continues at an unperturbed rate so that a systematic dilution error occurs in the measurement of ddNTP decay half-times. The aim of this study is to present a simple general formulation for the correction of measured t1/2-values for ddNTPs and for other agents with similar intracellular pharmacokinetic properties. Two factors of practical interest emerge: first, the error is greater for agents with slow intracellular clearance rates than for agents with rapid rates; and second, for cell lines with long doubling times, the measured t1/2-values approach more closely to the true t1/2-values, until with the extreme case (quiescent or "G(o)" cells), the observed and true decay times are identical. The greatest dilution errors are seen with adenodine-based agents such as ddATP and 2'-F-ddATP, while the smallest errors are seen with rapidly cleared agents of the dideoxythymidine class.

Adenosine Triphosphate↗

PubMed1996

Comparison of the DNA incorporation in human MOLT-4 cells of two 2'-beta-fluoronucleosides, 2'-beta-fluoro-2',3'-dideoxyadenosine and fialuridine.

Incorporation of 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ddA), a recently developed anti-HIV agent, into the cellular DNA of human MOLT-4 cells has been compared with the DNA incorporation seen with fialuridine (FIAU; 1-[2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl]-5-iodouracil), a potent anti-hepatitis B (anti-HBV) nucleoside analogue recently found to cause severe hepatic toxicity in human subjects. At equimolar concentrations (10 microM), incorporation of F-ddA was less than 1% of that for FIAU, a difference attributable to the lack of a 3'-hydroxyl group in the former compound and a consequent inability of F-ddA, unlike FIAU, to form DNA internucleotide linkages.

Antiviral Agents↗

PubMed1996

2',3'-Didehydro-3'-deoxythymidine: regulation of its metabolic activation by modulators of thymidine-5'-triphosphate biosynthesis.

The anti-human immunodeficiency virus (anti-HIV) agent 2',3'-didehydro-3'-deoxythymidine (D4T), like other 2',3'-dideoxynucleosides, requires conversion to its 5'-triphosphate to exert its pharmacological effect. Although D4T-triphosphate is unusually potent as an inhibitor of HIV-1 reverse transcriptase, the phosphorylation of the drug at low dose levels is inefficient because of its low affinity as an alternate substrate for the initial phosphorylation enzyme thymidine kinase. Because thymidine kinase is under feedback regulatory control by the physiological deoxynucleoside-5'-triphosphate dTTP, we examined the effect on D4T phosphorylation and thus, potentially, on its antiviral activity, of a variety of agents that lower intracellular dTTP pools. We found that agents that inhibit the de novo pyrimidine biosynthetic pathway have the ability to increase D4T phosphorylation, the most effective being two inhibitors of thymidylate formation, methotrexate and 5-fluoro-2'-deoxyuridine, compounds that block the enzymes dihydrofolate reductase and thymidylate synthetase, respectively. Because HIV itself lacks the capacity to synthesize dTTP and the other deoxynucleoside triphosphates essential for viral replication, combinations of D4T with modulatory agents that deplete host-cell dTTP, unlike conventional anti-HIV drug monotherapy directed solely at viral enzymes, have the ability to inhibit replication of mutant HIV strains as well as of wild-type virus.

Amides↗

PubMed1995

Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

We and other groups have recently reported the potentiation by ribonucleotide reductase inhibitors such as hydroxyurea of the anti-human immunodeficiency virus type 1 (HIV-1) activity of purine and pyrimidine 2',3'-dideoxynucleosides in both resting and phytohemagglutinin-stimulated peripheral blood mononuclear cells. Little agreement prevails, however, as to the mechanism of the synergistic effects described. We report here that in phytohemagglutinin-stimulated peripheral blood mononuclear cells, two mechanisms exist for the potentiation of the anti-HIV-1 activity by low-dose hydroxyurea of the purine-based dideoxynucleoside 2',3'-dideoxyinosine and the pyrimidine-based dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine. For 2',3'-dideoxyinosine, the enhancement arises from a specific depletion of dATP by hydroxyurea, resulting in a favorable shift of the 2',3'-dideoxyadenosine 5'-triphosphate/dATP ratio. For the pyrimidine dideoxynucleosides 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, the more modest anti-HIV enhancement results from hydroxyurea-induced increases of pyrimidine kinase activities in the salvage pathway and, hence, increased 5'-phosphorylation of these drugs, while depletion of the corresponding deoxynucleoside 5'-triphosphates (dTTP and dCTP) plays no significant role.

Adenosine Kinase↗

PubMed1995

Enhancement by hydroxyurea of the anti-human immunodeficiency virus type 1 potency of 2'-beta-fluoro-2',3'-dideoxyadenosine in peripheral blood mononuclear cells.

Ribonucleotide reductase inhibitors such as hydroxyurea (HU) and related compounds, at low, non-toxic doses, enhance the anti-human immunodeficiency virus type 1 (HIV-1) potency of both purine and pyrimidine 2',3'-dideoxynucleosides (ddNs) in human lymphocytes and macrophages. The most marked enhancement of inhibition of HIV-1 replication reported to date has been seen with the purine ddN 2',3'-dideoxyinosine (ddIno): a low level of HU (0.1 mM) permitted a 4.5-fold reduction in optimal ddIno dosage with no decrease in therapeutic effect or increase in toxicity. We report here even more marked enhancement by HU of the potency of the purine ddN 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo), where the addition of 0.1 mM HU permitted a 7.1-fold reduction in the optimal dose of 2'-beta-F-ddAdo in the phytohemagglutinin-activated peripheral blood mononuclear cell HIV-1 test system.

Antiviral Agents↗

PubMed1995

In situ cyclopentenyl cytosine infusion for the treatment of experimental brain tumors.

Cyclopentenylcytosine (CPEC; NSC 375575) is a pyrimidine nucleoside analogue that has potent antitumor effects when tested in vitro and also when tested in experimental tumors outside the central nervous system. CPEC exerts its antiproliferative effect through inhibition of CTP synthetase and consequent depletion of CTP and dCTP pools required for cell replication. Due to its poor penetration of the bloodbrain barrier, CPEC has failed to demonstrate therapeutic efficacy in experimental brain tumors after systemic administration. We therefore examined the in vivo activation, distribution, and antitumor effect of CPEC after long-term regional infusion of the drug directly into experimental brain tumors in rats. HPLC analysis of CPEC incubated with homogenized human brain and brain tumor tissue showed minimal degradation of the drug over 24 h. Analysis of rat cerebral 9L gliosarcoma infused with tritium-labeled CPEC demonstrated intratumoral accumulation of the active metabolite CPEC-triphosphate and concomitant depletion of CTP to a much greater extent in tumor tissue than in the adjacent brain. Tumor tissue UTP also decreased, but no significant effects on other ribonucleoside triphosphates were detected. Only trace amounts (< 1%) of CPEC and its metabolites reached peripheral sites, including the liver and kidneys, after intratumoral infusion. Rats treated with continuous intratumoral infusion of CPEC for 4 weeks using s.c. implanted osmotic pumps survived significantly longer than control rats receiving intratumoral saline or i.p. CPEC (P < 0.0001). Long-term intratumoral infusion of CPEC was not associated with any detectable toxicity. Our results support the feasibility of using intratumoral administration of CPEC as a regional therapy for malignant brain tumors.

Animals↗

PubMed1995

Reversal by cytidine of cyclopentenyl cytosine-induced toxicity in mice without compromise of antitumor activity.

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1-25 microM, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most abundant CPEC metabolite, CPEC-5'-triphosphate, is a potent [K1 approximately 6 microM] inhibitor of CTP synthetase (EC 6.3.4.2). Accumulation of this inhibitor resulted in a depletion of CTP levels to 17% of their original cellular concentration. Exogenous cytidine reversed CPEC-induced cellular cytotoxicity by suppressing the formation of CPEC-5'-triphosphate by 70%, and by partially replenishing intracellular CTP to at least 60-70% of its original concentration. In vivo, cytidine (500 mg/kg) administered intraperitoneally 4 hr after each daily dose of CPEC (LD10-LD100) for 9 days reduced the toxicity and abolished the lethality of CPEC to non-tumored mice. Of greater practical importance is the finding that, under these experimental conditions, cytidine did not curtail the antineoplastic properties of CPEC in L1210 tumor-bearing mice. Moreover, the concentration range over which CPEC exhibited antineoplastic activity was extended with cytidine administration.

Animals↗

PubMed1994

Effect of anti-HIV 2'-beta-fluoro-2',3'-dideoxynucleoside analogs on the cellular content of mitochondrial DNA and on lactate production.

Many dideoxynucleosides that are effective against human immunodeficiency virus (HIV) also are potent inhibitors of mitochondrial DNA (mtDNA) synthesis, and the resulting mtDNA decrease could be responsible for the delayed clinical toxicity sometimes observed with these drugs. The following compounds have been examined for their toxicity to human lymphoid CEM cells, and their ability to suppress mtDNA content: 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyinosine (ddI) and 2',3'-dideoxyguanosine (ddG); and their 2'-beta-fluoro analogs; beta-F-ddC, beta-F-ddA, beta-F-ddI and beta-F-ddG. Two other fluoro analogs, 5-F-ddC and 2'-beta,5-di-F-ddC were also examined. The ratio of C-IC50 (concentration that inhibited cell growth by 50%) to mt-IC50 (concentration that inhibited mtDNA synthesis by 50%) was determined for each compound. The rank-order of this ratio was ddC > 5-F-ddC >> ddA > ddI > ddG > beta-F-ddC > beta-F-ddA > beta-F-ddG with the highest ratios indicating the greatest potential for delayed toxicity. In comparison with ddC, beta-F-ddC and beta-F-ddA were 5,000 and 22,000 times less potent, respectively, in suppressing cellular mtDNA content, while their anti-HIV potencies were decreased only modestly relative to their unfluorinated parent compounds. beta-F-ddI and 2'-beta,5-di-F-ddC produced neither cellular toxicity nor mtDNA suppression at concentrations of 500 and 1000 microM, respectively. Lactic acid, the product of compensatory glycolysis that results from the inhibition of mitochondrial oxidative phosphorylation, was measured after cells were treated with these compounds. There appears to be a concentration-related correlation between the increase of lactic acid and the extent of mtDNA inhibition for the compounds examined.

Antiviral Agents↗

PubMed1994

Enzymatic assay for quantification of deoxynucleoside triphosphates in human cells exposed to antiretroviral 2',3'-dideoxynucleosides.

Quantification of intracellular 2'-deoxynucleoside-5'-triphosphates (dNTPs) is of importance in studies of antiretroviral 2',3'-dideoxynucleoside analogs (ddNs) and a highly sensitive enzymatic assay for dNTPs has frequently been used for this purpose. However, the susceptibility of the assay to interference from the corresponding substrate analogs, ddNTPs, is still undefined. Ideally, DNA polymerases used in the assay should meet at least two criteria: (i) high fidelity to the template even in the presence of ddNTPs and (ii) low affinity for ddNTPs. None of the currently used exonuclease-free Klenow and Sequenase enzymes met both criteria. However, Sequenase had higher fidelity to the template than did the Klenow enzyme in the presence of pyrimidine-ddNTPs, and its reaction followed first order kinetics. We have, therefore relying primarily on Sequenase, designed a dNTP proportional reduction assay to correct the ddN-induced deviation in the enzymatic assay. With the use of high-fidelity exonuclease-free DNA polymerase and the application of correction factors, we now can accurately quantify dNTPs with a minimum detection limit as low as 0.1 pmol, using as few as 1 x 10(4) peripheral blood mononuclear cells. The method described should be useful in the study and development of antiretroviral ddNs.

Antiviral Agents↗

PubMed1994

Quantitation of mitochondrial DNA in human lymphoblasts by a competitive polymerase chain reaction method: application to the study of inhibitors of mitochondrial DNA content.

With increasing awareness of the mitochondrial toxicity associated with certain 2',3'-dideoxynucleosides used in anti-human immunodeficiency virus therapy, procedures for quantitative analyses of drug effects on mitochondrial DNA (mtDNA) have assumed enhanced importance. For this reason we have developed a method to measure the copy numbers of mtDNA in cultured MOLT-4 cells. First a hybrid competitive DNA template was synthesized by conventional polymerase chain reaction (PCR), using two custom-synthesized 40-mer composite primers incorporating mitochondrial displacement loop sequences linked by a non-mitochondrial cDNA template (a 76-base pair sequence from the tat/rev region of human immunodeficiency virus cDNA). For the competitive assay, increasing known copy numbers of the hybrid competitive template were added as an internal control to samples containing total cellular DNA. With this approach, two competitive PCR products were generated, 1) a mitochondrial displacement loop-derived fragment (182 base pairs) and 2) a competitive DNA template-derived fragment (156 base pairs). Absolute quantitation was achieved by radiometric comparison of the relative amounts of the two products. To test the versatility of this method, varying amounts of competitive template (6.6 x 10(4) to 6.6 x 10(9) copies) were used with a fixed quantity of total cellular DNA taken from cells cultured for 9 days in the presence or absence of selected pyrimidine and purine dideoxynucleosides. The results showed that the copy number of cellular mtDNA is 823 +/- 71 copies/cell in MOLT-4 cells. Little selective depletion of mtDNA, compared with total cellular DNA, was seen with the purine dideoxynucleosides examined; however, when the cells were exposed to the pyrimidine dideoxynucleoside 2',3'-dideoxycytidine (50 nM) for 9 days, mtDNA content was specifically depleted, although total cellular DNA decreased by only 10%. Thus, in addition to the presently used methods of assessing mitochondrial impairment, i.e., Southern blot analysis and electron microscopy, the competitive PCR method provides a third and convenient assay, with particular applicability to determination of mtDNA in very small numbers of cells.

Base Sequence↗

PubMed1994

Effects of IMP dehydrogenase inhibitors on the phosphorylation of ganciclovir in MOLT-4 cells before and after herpes simplex virus thymidine kinase gene transduction.

We have undertaken to characterize the role of cytoplasmic 5'-nucleotidase (EC 3.1.3.5) in the phosphorylation of the anti-herpes simplex virus and anti-human cytomegalovirus agent ganciclovir (GCV) in MOLT-4 cells, a human T cell line adapted to grow in suspension culture. The rate of formation of GCV triphosphate was found to be approximately doubled by preincubation of nontransfected MOLT-4 cells with agents that cause the accumulation of IMP, such as ribavirin (20 microM) and mycophenolic acid (1 microM), and the reaction rate was found to be unaffected by high levels of thymidine (100 microM). With herpes simplex virus-1 thymidine kinase (HStk) gene-transduced MOLT-4 cells, the rate of GCV phosphorylation was approximately 40-fold faster than that in uninfected cells and, in marked contrast to uninfected cells, the reaction was significantly inhibited both by IMP dehydrogenase inhibitors and by thymidine. These latter effects appear to be the result of 1) the accumulation of high levels of dTTP in IMP dehydrogenase inhibitor-treated cells, with consequent feedback inhibition of HStk, and 2) direct competitive substrate inhibition by thymidine of the HStk-catalyzed phosphorylation of GCV. Thus, agents that enhance 5'-nucleotidase-catalyzed phosphorylation of GCV in uninfected cells do not play a similar role in HStk-transfected cells, a consequence of the quantitative predominance of the viral thymidine kinase-catalyzed reaction over that attributable to endogenous cytoplasmic 5'-nucleotidase.

Cell Line↗

PubMed1994

Anti-human immunodeficiency virus type 1 activity of hydroxyurea in combination with 2',3'-dideoxynucleosides.

The effects of hydroxyurea (HU), an inhibitor of ribonucleotide reductase, on the replication of human immunodeficiency virus type 1 (HIV-1) in activated peripheral blood mononuclear cells were studied. The inhibition of HIV-1 replication by HU alone was dose dependent, with a 90% inhibitory concentration of 0.4 mM, a plasma concentration tolerated by patients with oncological diseases. HU at lower concentrations (< 0.1 mM) was found to potentiate the antiviral activity of 2',3'-dideoxyinosine (ddl), 3'-azido-2',3'- dideoxythymidine, and 2',3'-dideoxycytidine against HIV-1, with the potentiation being ddl greater than 3'-azido-2',3'- dideoxythymidine = 2',3'-dideoxycytidine. In the presence of 0.1 mM HU, the 90% inhibitory concentration of ddl was reduced by 6-fold in activated peripheral blood mononuclear cells. The potentiating effect of HU on ddl action was time dependent, with the greatest inhibition of HIV-1 growth being seen when HU was present during and after virus adsorption, i.e., apparently coinciding with the time of proviral DNA synthesis. A brief incubation of activated cells with HU and ddl at low concentrations before virus exposure reduced p24 production by > 50%. Analyses using high performance liquid chromatography and enzymatic assays suggested that the greater degree of potentiation by HU of the action of ddl, compared with the other dideoxynucleosides, is due to the more effective inhibition by HU of dATP synthesis, compared with the synthesis of the other deoxynucleoside triphosphates (dGTP, dTTP, and dCTP). The present study suggests that, for appropriate agents, pharmacological reduction of deoxynucleoside triphosphate levels represents a potential therapeutic approach for inhibition of HIV-1 replication.

Antiviral Agents↗

PubMed1994

Enhancement by 2'-deoxycoformycin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of 2',3'-dideoxyadenosine and 2'-beta-fluoro-2',3'-dideoxyadenosine.

The anti-human immunodeficiency virus agents 2',3'-dideoxyadenosine (ddAdo) and 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo) are rapidly converted, both in vitro and in vivo, to the corresponding inosine analogs by the widely distributed enzyme adenosine deaminase (EC 3.5.4.4). We have determined the effects of the potent adenosine deaminase inhibitor 2'-deoxycoformycin (2'-dCF) on ddAdo and 2'-beta-F-ddAdo metabolism in MOLT-4 cells and on ddAdo antiviral activity in the ATH8 test system. At levels as low as 5 nM in the incubation medium, 2'-dCF effectively blocks the extracellular deamination of both agents, thus permitting their rapid cellular uptake as the unchanged parent compounds, rather than as the less lipid-soluble 2',3'-dideoxyinosine or 2'-beta-fluoro-2',3'-dideoxyinosine. The result is a significant increase in intracellular levels of the pharmacologically active forms 2',3'-dideoxyadenosine-5'-triphosphate and 2'-beta-fluoro-2',3'-dideoxyadenosine-5'-triphosphate. The effect becomes maximal over the range of 50-250 nM 2'-dCF and declines to control levels when extracellular 2'-dCF levels exceed 1 microM. This decrease in ddAdo and 2'-beta-F-ddAdo phosphorylation with higher levels of the inhibitor appears to result from intracellular penetration of 2'-dCF and consequent inhibition of intracellular deamination, a critical step in the activation of both agents through the 5'-nucleotidase pathway. In anti-human immunodeficiency virus assays, a 2.2-fold increase in ddAdo antiviral potency was seen at 2'-dCF levels of 20 and 50 nM.

Adenosine Deaminase Inhibitors↗

PubMed1993

Resistance to cyclopentenylcytosine in murine leukemia L1210 cells.

Cyclopentenyl cytosine (CPEC) exhibits oncological activity in murine and human tumor cells and has now entered Phase I clinical trials. Its mode of action as an antitumor agent appears to be inhibition by its triphosphate (CPEC-TP) of CTP synthase, the enzyme which converts UTP to CTP. In an attempt to elucidate the mechanism of resistance to CPEC, a murine leukemia cell line resistant to CPEC (L1210/CPEC) was developed by N-methyl-N-nitro-N-nitrosoguanidine-induced mutagenesis and subsequent selection by cultivation of the L1210 cells in the presence of 2 microM CPEC. Resistant clones were maintained in CPEC-free medium for 6 generations before biochemical studies were performed. The resistant clone selected for further studies was approximately 13,000-fold less sensitive to growth inhibition by CPEC than the parental cells, and the concentration of CPEC required to deplete CTP in the resistant cells was 50-fold higher than in the sensitive cells. A comparison of the kinetic properties of CTP synthase from sensitive and resistant cells indicated alteration in the properties of the enzyme from the latter; the median inhibitory concentration for CPEC-TP increased from 2 to 14 microM, Km for UTP decreased from 126 to 50 microM, and Vmax increased 12-fold from 0.2 to 2.3 nmol/mg/min. Northern blot analyses of polyadenylated RNA from the resistant and sensitive cells indicated a 3-fold increase in transcripts of the CTP synthase gene in the resistant line. Consistent with these alterations in the properties of the enzyme, the resistant cells exhibited significantly expanded CTP and dCTP pools (4- 5-fold) when compared with the sensitive cells. No change was observed, however, in the properties of uridine-cytidine kinase, the enzyme responsible for the initial phosphorylation of CPEC; despite this, however, cellular uptake of CPEC was greatly decreased, and phosphorylation of CPEC and its incorporation into RNA were 10-fold less than in the parental cells. These latter observations are most readily explained by feedback inhibition by the increased CTP levels of the resistant cells of uridine-cytidine kinase and/or of the membrane transport process used for initial entry of CPEC.

Animals↗