Effects of granulocyte-macrophage colony-stimulating factor on 3'-azido-3'-deoxythymidine uptake, phosphorylation and nucleotide retention in human U-937 cells

Previous studies have demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) both increases and decreases levels of 3'-azido-3'-deoxythymidine (AZT) nucleotides in certain human myeloid cells. The present studies have examined the effects of GM-CSF on AZT metabolism in U-937 cells. The results demonstrate that GM-CSF stimulated AZT nucleotide formation in these cells. This stimulation was detectable during concurrent exposure to GM-CSF and AZT or as a result of pretreatment with GM-CSF. The GM-CSF-induced enhancement in AZT nucleotide formation was associated with a 4-fold increase in AZT uptake. The finding that uptake of AZT into U-937 cells was only partially sensitive to 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) suggested a process primarily involving nonfacilitated diffusion. The results also demonstrate that treatment of U-937 cells with GM-CSF was associated with nearly a 2-fold increase in thymidine kinase activity. Moreover, the findings indicate that retention of AZT-MP and AZP-TP was prolonged significantly (P less than 0.05 and P less than 0.01 respectively) in association with GM-CSF treatment. Taken together, these results suggest that GM-CSF enhances the formation of AZT nucleotides by increasing AZT uptake and phosphorylation, as well as increasing retention of phosphorylated derivatives.     

3'-Azido-3'-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: a possible mechanism of AZT hepatotoxicity

3'-azido-3'-deoxythymidine (AZT) is a staple of highly active antiretroviral therapy (HAART). Prior to HAART, long-term use of high-dosage AZT caused myopathy, cardiomyopathy, and hepatotoxicity, associated with mitochondrial DNA depletion. As a component of HARRT, AZT causes cytopenias and lipodystrophy. AZT-5'-triphosphate (AZTTP) is a known inhibitor of the mitochondrial polymerase gamma and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart mitochondria, AZT phosphorylation beyond AZT-5'-monophosphate (AZTMP) was not detected. Rather, AZT was shown to be a more potent inhibitor of thymidine phosphorylation (50% inhibitory concentration (IC50) of 7.0+/-1.0 microM) than AZTTP is of polymerase gamma (IC50 of >100 microM), suggesting that depletion of mitochondrial stores of TTP may limit replication. This work has investigated whether an identical mechanism might account for the hepatotoxicity seen with long-term use of AZT. Isolated rat liver mitochondria were incubated with labeled thymidine or AZT, and the rate and extent of phosphorylation were determined by HPLC analysis of acid-soluble extracts of the incubated mitochondria. The results showed that in the phosphorylation of thymidine to TMP, liver mitochondria exhibit a higher Vmax and Km than heart mitochondria, but otherwise heart and liver mitochondria display similar kinetics. AZT is phosphorylated to AZTMP, but no further phosphorylated forms were detected. In addition, AZT inhibited the production of TTP, with an IC50 of 14.4+/-2.6 microM AZT. This is higher, but comparable to, the results seen in isolated rat heart mitochondria.     

Effects of granulocyte-macrophage colony-stimulating factor on 3′-azido-3′-deoxythymidine uptake, phosphorylation and nucleotide retention in human U-937 cells

Previous studies have demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) both increases and decreases levels of 3′-azido-3′-deoxythymidine (AZT) nucleotides in certain human myeloid cells. The present studies have examined the effects of GM-CSF on AZT metabolism in U-937 cells. The results demonstrate that GM-CSF stimulated AZT nucleotide formation in these cells. This stimulation was detectable during concurrent exposure to GM-CSF and AZT or as a result of pretreatment with GM-CSF. The GM-CSF-induced enhancement in AZT nucleotide formation was associated with a 4-fold increase in AZT uptake. The finding that uptake of AZT into U-937 cells was only partially sensitive to 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine (NBMPR) suggested a process primarily involving nonfacilitated diffusion. The results also demonstrate that treatment of U-937 cells with GM-CSF was associated with nearly a 2-fold increase in thymidine kinase activity. Moreover, the findings indicate that retention of AZT-MP and AZP-TP was prolonged significantly (P < 0.05 and P < 0.01 respectively) in association with GM-CSF treatment. Taken together, these results suggest that GM-CSF enhances the formation of AZT nucleotides by increasing AZT uptake and phosphorylation, as well as increasing retention of phosphorylated derivatives.     

3'-Azido-3'-deoxythymidine (AZT) is a competitive inhibitor of thymidine phosphorylation in isolated rat heart and liver mitochondria

Long-term use of 3'-azido-3'-deoxythymidine (AZT) is associated with various tissue toxicities, including hepatotoxicity and cardiomyopathy, and with mitochondrial DNA depletion. AZT-5'-triphosphate (AZTTP) is a known inhibitor of the mitochondrial DNA polymerase gamma and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart and liver mitochondria, AZT itself was shown to be a more potent inhibitor of thymidine phosphorylation (IC50 of 7.0+/-1.0 microM AZT in heart mitochondria and of 14.4+/-2.6 microM AZT in liver mitochondria) than AZTTP is of polymerase gamma (IC50 of >100 microM AZTTP), suggesting that depletion of mitochondrial stores of TTP may limit replication and could be the cause of the mitochondrial DNA depletion observed in tissues affected by AZT toxicity. The purpose of this work is to characterize the nature of AZT inhibition of thymidine phosphorylation in isolated rat heart and rat liver mitochondria. In both of these tissues, AZT was found to be a competitive inhibitor of the phosphorylation of thymidine to TMP, catalyzed by thymidine kinase 2. The inhibition constant (Ki) for heart mitochondria is 10.6+/-4.5 microM AZT, and for liver mitochondria Ki is 14.0+/-2.5 microM AZT. Since AZT is functioning as a competitive inhibitor, increasing thymidine concentrations may be one mechanism to overcome the inhibition and decrease AZT-related toxicity in these tissues.     

Effect of dipyridamole on transport and phosphorylation of thymidine and 3'-azido-3'-deoxythymidine in human monocyte/macrophages

Dipyridamole (DPM), a commonly used coronary vasodilator and antithrombotic drug, was shown recently to potentiate the antiviral effect of 3'-azido-3'-deoxythymidine (AZT) in HIV-1 infected human monocyte-derived macrophages (M/M) in vitro. We report in the present study that in uninfected M/M, DPM markedly inhibited cellular uptake of [3H]thymidine (dThd) and its incorporation into the nucleotide pools, particularly the dThd-triphosphate pool. In contrast, DPM did not affect cellular uptake and phosphorylation of [3H]AZT. Since dThd counteracts the phosphorylation and antiviral action of AZT, these findings support the hypothesis that the potentiation of the anti-HIV effect of AZT is due, at least in part, to differential inhibition of nucleoside salvage.     

Thymidine and zidovudine metabolism in chronically zidovudine-exposed cells in vitro.

Chronic exposure of H9 cells to 25 microM zidovudine (H9-AZT cells) causes a 2- to 3-fold increase in thymidine kinase (TK) activity (Agarwal RP, Int J Purines Pyrimidine Res, in press). The present study compared thymidine (TdR) and AZT anabolism in H9 and H9-AZT cells. After a 3.5-hr incubation with 10 microM TdR or AZT, the total intracellular accumulations of AZT (48.7 microM in H9 cells and 32.8 microM in H9-AZT cells) were 46.4% of TdR accumulation. Other major differences between TdR and AZT anabolism were: (i) the majority of TdR (84-87%) was incorporated into DNA compared to less than 1% of AZT; and (ii) whereas distribution of TdR in the nucleotides was TTP greater than TMP greater than TDP, zidovudine distributed was AZT-MP much greater than AZT-TP much greater than AZT-DP. Because of the poor substrate activity of AZT-MP for thymidylate kinase (TMP-kinase), most of the AZT (95-98%) remained as AZT-MP. TMP-kinase activities with TMP as substrate were 47.6 +/- 20.3 and 91.4 +/- 28.8 pmol/mg protein/min in H9 and H9-AZT cells, respectively. 5'-Nucleotidase activities with TMP as substrate were 428.9 +/- 37.8 and 255.9 +/- 28.7 pmol/mg protein/min in H9 and H9-AZT cells, respectively. Activities of these enzymes with AZT-MP as a substrate were very low. Despite an increase in TK and TMP-kinase, and a decrease in 5'-nucleotidase activities, the total intracellular accumulations of TdR and AZT were reduced significantly (P less than 0.05) to 67.5% in H9-AZT cells. Thymidine transport (0.66 to 0.68 pmol/sec/10(6) cells) was similar in both the cell lines. The severe reductions of TdR salvage caused by chronic exposure of cells to AZT, if it occurs in AIDS patients on AZT chemotherapy, may explain some of the long-term clinical toxicities of the drug.     

2',3'-Dideoxycytidine toxicity in cultured human CEM T lymphoblasts: effects of combination with 3'-azido-3'-deoxythymidine and thymidine

2',3'-Dideoxycytidine (ddCyd), a potent inhibitor of human immunodeficiency virus DNA replication, requires phosphorylation by cellular nucleoside kinases for antiviral activity. Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) is responsible for the formation of dideoxycytidine monophosphate and this enzyme is controlled by feedback regulation by the natural endproduct, dCTP. We have examined whether a decrease in intracellular dCTP levels affects the growth inhibition caused by ddCyd, as well as the capacity to accumulate dideoxycytidine triphosphate (ddCTP), using human T lymphoblast (CEM) cells in culture. Subtoxic concentrations of thymidine were used to decrease the dCTP pool. The effects of 3'-azido-3'-deoxythymidine (AZT), alone or in combination with ddCyd, on cell growth, DNA precursor pools, and accumulation of ddCTP were also studied. The combination of ddCyd and thymidine led to growth inhibition of CEM cells that was twice what would be expected from addition, whereas the combination of AZT and ddCyd showed an additive effect. CEM cells accumulated ddCTP efficiently, so that with 10 microM ddCyd (corresponding to the EC50 value) and a 6-hr incubation the ddCTP pool was 3-fold higher than the dCTP pool. Simultaneous addition of thymidine (10 microM) increased the dTTP pool 2-fold and gave a 50% reduction in the dCTP level but only a 10% increase in ddCTP accumulation. The presence of AZT (300 microM, corresponding to the EC50 value) led, in contrast, to an elevation of dCTP and no significant change in the other DNA precursor pools. With this high concentration of AZT, the accumulation of ddCTP decreased 42%. It was also found that ddCyd is metabolized into two additional compounds, besides the dideoxycytidine mono-, di-, and triphosphate, i.e., the liponucleotides dideoxycytidine diphosphate-ethanolamine and dideoxycytidine diphosphate-choline, constituting 45 and 6% of the total phosphorylated ddCyd metabolites, respectively, whereas the mono-, di-, and triphosphate corresponded to 3, 21, and 25% of the phosphorylated dideoxynucleotides. These results indicate that the formation of dideoxycytidine monophosphate is not rate limiting in the synthesis of ddCTP in human lymphoblasts, which clearly differs from what was observed earlier in mouse cells (Mol Pharmacol 32:798-806 1988). Furthermore, growth inhibition by ddCyd seems to be related to the ratio between dCTP and ddCTP. There was no direct interference between ddCyd and AZT metabolism in clinically relevant concentrations, which may encourage the use of combination of these compounds for anti-human immunodeficiency virus treatment.     

Intracellular metabolism of CycloSaligenyl 3'-azido-2', 3'-dideoxythymidine monophosphate, a prodrug of 3'-azido-2', 3'-dideoxythymidine (zidovudine)

The administration of CycloSaligenyl 3'-azido-2',3'-dideoxythymidine monophosphate (CycloSal-AZTMP) to CEM cells resulted in a concentration- and time-dependent conversion to the 5'-monophosphate (AZTMP), 5'-diphosphate (AZTDP), and 5'-triphosphate (AZTTP) derivatives. High ratios of AZTMP/AZTTP were found in the CEM cell cultures treated with CycloSal-AZTMP. The intracellular T(1/2) of AZTTP in CEM cell cultures treated with either AZT and CycloSal-AZTMP was approximately 3 h. A variety of human T- and B-lymphocyte cell lines efficiently converted the prodrug to the AZT metabolites, whereas peripheral blood lymphocytes and primary monocyte/macrophages showed at least 10-fold lower metabolic conversion of the prodrug. CycloSal-AZTMP failed to generate marked levels of AZT metabolites in thymidine kinase-deficient CEM/TK(-) cells, an observation that is in agreement with the substantial loss of antiviral activity of CycloSal-AZTMP in CEM/TK(-) cells. The inability of CycloSal-AZTMP to generate AZTMP in CEM/TK(-) cells is presumably due to a relatively high hydrolysis rate of AZTMP to the parent nucleoside AZT, combined with the inability of CEM/TK(-) cells to phosphorylate AZT to AZTMP through the cytosolic salvage enzyme thymidine kinase.     

Apoptosis inhibitory effect of the isothiourea compound, tri-(2-thioureido-S-ethyl)-amine

Sulfhydril compounds, some of them with immunomodulatory activity have also been shown to modulate the induction of apoptosis. This study was performed to assess the possible apoptosis inhibitory effect of the immunomodulatory compound tri-(2-thioureido-S-ethyl)-amine (K-1) in U-937 and HL-60 leukaemia cell lines as model systems. Treatment of U-937 and HL-60 cells with K-1 inhibited etoposide (ETO)-induced apoptosis in both cell lines in a dose-dependent manner, IC(50)=200 microg/ml. The results indicate that inhibition of ETO-induced apoptosis occurs downstream of ETO-mediated cleavable complex formation but early in, or at the level of the mitochondria events of the apoptotic pathway. Further, like other isothioureas, K-1 proved to be a potent inhibitor of nitric oxide synthase. This raises the possibility that where the activation of nitric oxide synthase is involved in apoptosis induction, K-1 might also be effective. These findings suggest that K-1 may serve as a potent inhibitor of apoptosis initiated by ETO or nitric oxide.     

The effects of chlorambucil on the utilization of exogenous thymidine by tumour cells.

Treatment of an alkylating agent-sensitive strain of the Yoshida ascites sarcoma with chlorambucil resulted in an inhibition of the incorporation of [³H]thymidine into DNA, which could be overcome by incubating cells in high extracellular concentrations of thymidine. Increase in cellular DNA content and the dilution of specific radioactivity in pre-labelled DNA indicated that DNA synthesis was continuing at times when [³H]thymidine incorporation was inhibited. Uptake and phosphorylation of thymidine were not impaired by the treatment and the reduced incorporation of [³H]thymidine into DNA is attributable to a block in the utilization of TTP derived from exogenous nucleoside.     

Differentiation-inducing effect of magnolialide, a 1 beta-hydroxyeudesmanolide isolated from Cichorium intybus, on human leukemia cells

Cichorium intybus contains two 1beta-hydroxyeudesmanolides, magnolialide and artesin, together with several constituents. Magnolialide inhibits the growth of several tumor cell lines and appears to induce differentiation of human leukemia HL-60 and U-937 cells to monocyte/macrophage-like cells. Another 1beta-hydroxyeudesmanolide, artesin, and other constituents were inactive. The content of magnolialide was shown to be highest in the leaves of Inje cultivar among the cultivars investigated in this study.     

Arabinose furanosyl thymidine: uptake, phosphorylation and incorporation into DNA of mammalian cells

The naturally occurring nucleoside analogue arabinosyl thymidine is known as an anti-herpes and anti-cancer agent. The biologically active form is arabinosyl thymidine triphosphate (Ara-TTP), which inhibits cellular and viral DNA-polymerases and thus interferes with DNA replication. Using two murine erythroleukemia cell lines, Friend cell clone F4-6 and F4-12N, the latter being thymidine kinase deficient (TK-) cells transformed to a TK+ phenotype with the HSV TK gene, we have determined 1) the role of cellular and herpes simplex virus thymidine kinase (HSV TK) in the uptake of Ara-T into the cells; 2) the subsequent phosphorylation of intracellular Ara-T to Ara-TMP, Ara-TDP and Ara-TTP; 3) the incorporation of Ara-TTP into the DNA. Incorporation into DNA was studied under different conditions, including selective inhibition of the different cellular DNA polymerases by aphidicolin (that inhibits polymerases alpha and delta) and dideoxythymidine (that preferentially inhibits polymerases beta and gamma). The uptake of Ara-T into the methanol soluble pool of the cells depends upon its phosphorylation to Ara-TMP, which is more efficiently performed by the HSV TK than by the cellular TK, thus explaining the sensitivity of HSV infected cells to Ara-T. However, using increasing concentrations of Ara-T, we have shown that phosphorylation also occurs in normal control cells due to the cellular thymidine kinase. More than 90% of Ara-T is phosphorylated in the cell, and more than 60% of total Ara-T(MP, DP, TP) exists in the triphosphate form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of thymidine phosphorylase expression by using an HSP90 inhibitor potentiates the cytotoxic effect of cisplatin in non-small-cell lung cancer cells

Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. In this study, we examined the role of TP expression in relation to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1650. Treatment with 17-AAG (0.1-1 μM) resulted in a decrease in cellular TP protein and mRNA levels, which was accompanied by a downregulation of phosphorylated MKK1/2-ERK1/2 and AKT protein levels. The 17-AAG treatment disrupted the interaction between HSP90 and TP and triggered TP protein degradation through the ubiquitin-26S proteasome pathway. Specific inhibition of TP expression by siRNA further enhanced the cell death and growth inhibition that had been induced by 17-AAG. An enhancement of ERK1/2 or AKT activation by transfecting the cancer cells with constitutively active MKK1/2 or AKT expression vectors significantly restored the 17-AAG-reduced TP protein levels as well as cell viability. In contrast, a combination of U0126 (MKK1/2 inhibitors) or LY294002 (PI3K inhibitor) further decreased the TP expression and cell viability induced by 17-AAG. Moreover, 17-AAG enhanced the cisplatin-induced cytotoxic effect through downregulation of the cisplatin-induced TP expression and ERK1/2 and AKT activation. Taken together, our results suggest that the down-modulation of TP protein induced by 17-AAG represents a key factor in enhancing the cytotoxic effects of cisplatin in NSCLC cells.     

Cross-resistance of dideoxycytidine-resistant cell lines to azidothymidine.

2',3'-Dideoxycytidine (ddC) and azidothymidine (AZT) inhibit HIV-1 replication and currently are used in AIDS therapy. Long-term use of the drugs is associated with the selection of drug-resistant HIV strains, thus limiting their effectiveness. Another mechanism, associated with their altered metabolism in host cells, also can cause "cellular" drug resistance. Human lymphocytic H9 cell lines (H9-ddC0.5w and H9-ddC5.0w) selected for ddC resistance by exposure to 0.5 and 5.0 microM ddC were found to be cross-resistant to AZT. Compared with controls, the thymidine kinase (TK) activities in H9-ddC0.5w and H9-ddC5.0w cells were 56.7 and 51.4% (with thymidine as a substrate) and 50.3 and 42% (with AZT as a substrate). Consequently the cellular incorporation of AZT and thymidine (24-hr incubation) also was reduced to 51.3 and 70.0% in H9-ddC0.5w cells and to 12.1 and 17.3% in H9-ddC5.0w cells. A 3-hr incubation with 25 microM AZT and ddC decreased their cellular incorporation to 50.5 and 76.15% in H9-ddC0.5w cells and to 12.95 and 47.8% in H9-ddC5.0w cells compared with H9 cells. Thus, the change in AZT accumulation did not correlate exactly with the decrease in TK activity and far exceeded the effect on ddC accumulation. Evidence is presented that ddC, in addition to deoxycytidine kinase, affected TK1 activity. The involvement of multidrug resistance proteins in the mechanism of the resistance was ruled out by the failure of trifluoperazine and verapamil to alter cellular accumulations of AZT, ddC, daunorubicin, and rhodamine-123. Development of cellular ddC and AZT cross-resistance may affect the therapeutic efficacy of these antiviral agents.     

Reduction of G protein-coupled receptor kinase 2 expression in U-937 cells attenuates H2 histamine receptor desensitization and induces cell maturation

Histamine and H2 agonists transiently induce an important cAMP response in promonocytic U-937 cells but fail to induce monocytic differentiation because of a rapid receptor desensitization mediated by G protein-coupled receptor kinases (GRKs). The aims of the present study were to investigate the participation of GRK2 in the desensitization mechanism of the H2 receptor in U-937 cells by reducing GRK2 levels through antisense technology and to evaluate the differentiating capacity of cells expressing lower GRK2 level, stimulated by H2 agonists. By stable U-937 cell transfection with a GRK2-antisense cDNA, we obtained D5 and A2 cell clones exhibiting a reduction in GRK2 expression and an H3 clone with no significant difference in GRK2 expression from control cells. The cAMP response induced by the H2 agonist in D5 and A2 but not in H3 cells was higher than in U-937 and persisted for a longer period of time, although the number of H2 receptors in D5 and A2 cells was lower than in U-937. Furthermore, D5 and A2 cells treated with H2 agonist showed patterns of c-Fos and CD88 expression consistent with monocytic differentiated cells. Overall, these results indicate a direct correlation between the expression of GRK2 and the desensitization of natively expressed H2 receptors in U-937 cells, suggesting that GRK2 plays a major role in the regulation of these receptors' response. In turn, desensitization process is a key component of H2 receptor signaling, determining the differentiation capability of promonocytic cells.     

The additive in vitro anti-HIV-1 effect of chloroquine, when combined with zidovudine and hydroxyurea

The 4-aminoquinoline chloroquine and its analogue hydroxychloroquine are endowed with anti-HIV-1 activity both in vitro and in vivo. We previously reported that the addition of CQ (chloroquine) to the combination of HU (hydroxyurea) and ddI (didanosine) provides additive anti-HIV-1 activity. We here extended this in vitro investigation by studying whether the addition of CQ also resulted in additive anti-HIV-1 activity when combined with HU plus AZT (zidovudine). The same effect was found, whether CQ was added to HU plus AZT or to HU plus ddI, in recently infected H-9 and U-937 cells or primary T cells and monocytes, as well as in immunologically or oxidatively stimulated ACH-2 and U-1 cells. At concentrations where CQ exerts its anti-HIV-1 effect in combination with the other drugs, CQ addition does not result in either cell toxicity or apoptosis.     

The NADPH oxidase inhibitor VAS2870 impairs cell growth and enhances TGF-β-induced apoptosis of liver tumor cells

Liver tumor cells show several molecular alterations which favor pro-survival signaling. Among those, we have proposed the NADPH oxidase NOX1 as a prosurvival signal for liver tumor cells. On the one side, we have described that FaO rat hepatoma cells show NOX1-dependent partial resistance to apoptosis induced by Transforming Growth Factor beta (TGF-β). On the other side, we have shown that FaO cells, as well as different human hepatocellular carcinoma (HCC) cell lines, are able to proliferate in the absence of serum through the activation of a NOX1-dependent signaling pathway. The aim of this work was to analyze the effects of NADPH oxidase pharmacological inhibition in liver tumor cells using the inhibitor VAS2870. This compound inhibits dose-dependently autocrine increase of cell number in FaO rat hepatoma cells, and almost completely blocked ROS production and thymidine incorporation when used at 25 μM. Such inhibitory effect on autocrine growth is coincident with lower mRNA levels of EGFR (Epidermal Growth Factor Receptor) and its ligand TGF-α (Transforming Growth Factor-alpha), and decreased phosphorylation of the EGFR itself and other downstream targets, such as SRC or AKT. Moreover, NADPH oxidase pharmacological inhibition also effectively attenuates serum-dependent growth and phosphorylation of AKT and ERK. Importantly, these inhibitory effects on either autocrine or serum-dependent cell growth are observed in several human HCC cell lines. Finally, we have observed that VAS2870 is also effective in enhancing apoptosis induced by a physiological stimulus, such as TGF-β. In summary, NADPH oxidase pharmacological inhibition could be considered a promising tool in the treatment of liver cancer.     

Cytotoxic effects of the mycotoxin beauvericin to human cell lines of myeloid origin.

Beauvericin, a cyclic hexadepsipeptide of potential importance to the health of humans and domestic animals, has been reported to exert cytotoxic effects on several mammalian cell types and to induce apoptosis. We investigated the cytotoxicity of this compound to two human cell lines of myeloid origin: the monocytic lymphoma cells U-937 and the promyelocytic leukemia cells HL-60. In some experiments HL-60 cells partially differentiated towards the eosinophilic phenotype were also used.Cultures of U-937 cells and HL-60 cells in stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 microM for periods of time of 4 and 24h, respectively. The effects of beauvericin on cell viability were assessed by the Trypan blue exclusion method. In another set of experiments, performed with U-937 cells, the mycotoxin was included in the culture medium at passaging, in order to assess its possible effects on cell growth.Viability of both U-937 cells and HL-60 cells was not affected by beauvericin at concentrations up to 3 microM, after 4h exposure, whereas a steady decline was seen at higher concentrations. Similarly, after an exposure time of 24h, a decline in viability was observed in cultures exposed to beauvericin at a concentration of 10 microM or higher. Thus, 50% cytotoxic concentrations at 24h of congruent with 30 microM and congruent with 15 microM were estimated for U-937 cells and HL-60 cells, respectively.Similar experiments were performed with cultures of HL-60 cells partially differentiated towards the eosinophilic phenotype, revealing that, in 4h exposure experiments (but not in 24h experiments), the viability of these cultures underwent a significantly less pronounced decline, in comparison to undifferentiated HL-60 cultures. Interestingly, when U-937 cells were allowed to proliferate in the presence of the mycotoxin, included in the culture medium at passaging, a substantial cytotoxicity was observed at lower concentrations, compared with prevalently resting, stationary phase cultures. Accordingly, a definite inhibition of the proliferative capability of the cells was detected.The information provided by this work may be useful in selecting appropriate myeloid cell models for the development of biossays aimed at detecting beauvericin (and, possibly, other mycotoxins) in foods and other commodities.     

3'-Deoxythymidin-2'-ene permeation of human lymphocyte H9 cells by nonfacilitated diffusion.

3'-Deoxythymidin-2'-ene (d4T) is a potent and selective inhibitor of human immunodeficiency virus replication in a variety of human cell types and is currently undergoing phase I clinical trials for the treatment of acquired immunodeficiency syndrome. As part of our ongoing studies of the cellular pharmacology of d4T, and in light of recent reports in which such nucleoside analogs as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyadenosine were shown to permeate cells by the unusual mechanism of nonfacilitated diffusion, we have investigated the uptake of d4T in the human lymphocyte cell line H9. Several lines of evidence suggest that d4T permeation of H9 cells occurs by nonfacilitated diffusion; 1) [3H]d4T influx was linear for the first 10 sec and was nonconcentrative, reaching equilibrium with the extracellular drug concentration in 2-3 min, 2) the initial rates of influx were a linear function of concentration over the range from 1 microM to 5 mM, with no sign of uptake by a saturable mechanism, and 3) the uptake of [3H]d4T was insensitive to the nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole, as well as a large molar excess of AZT, thymidine, or adenosine. The octanol/water partition coefficient of d4T was 0.179, intermediate between those of thymidine and AZT. Thus, d4T does not appear to be a substrate for the nucleoside transport system responsible for the uptake of physiological nucleosides as well as most nucleoside analogs, and it enters the cell by nonfacilitated diffusion.