B. Gröschel

Human Immunodeficiency Virus Resistance to AZT in MOLT4/8 Cells is Associated with a Lack of AZT Phosphorylation and is Bypassed by AZT-Monophosphate SATE Prodrugs

Human T lymphoid MOLT4/8 cells were grown continuously for more than 2 years in a medium containing 3′-azido-2′,3′-dideoxythymidine (zidovudine; AZT) at a concentration of 250 μM. These cells, designated MOLT-4/8rAZT250, were used to test the cytotoxic and antiviral activity of AZT. Intracellular accumulation of AZT, expression of the multidrug resistance 1 (MDR-1) gene, thymidine kinase (TK) gene and activity of the TK enzyme in cellular extracts were measured. The results showed that both the cytotoxic and antiviral activity of AZT were significantly lower in MOLT4/8rAZT250 than in MOLT4/8 cells; concentrations required to inhibit 50% production of the p24 human immunodeficiency virus type 1 (HIV-1) antigen of two laboratory strains were at least 100-fold higher in resistant cells. The MDR-1 gene was not expressed in the resistant cells. TK mRNA expression was significantly lower in the resistant than in the sensitive cells. TK enzymatic activity for deoxythymidine phosphorylation was impaired in MOLT4/8rAZT250 cells compared to the sensitive cells. AZT was phosphorylated only in the sensitive cells whereas no phosphorylation of AZT was found in the resistant cells. We tested whether several AZT-monophosphate triesters, which bypass cellular TK, could overcome resistance to the cytotoxic and antiviral activity of AZT. The bis(t-butylSATE) phosphotriester derivative of AZT showed comparable cytotoxic and antiviral activity in sensitive and resistant cells. The results demonstrated that MOLT4/8rAZT250 cells exert resistance to the anti-HIV activity of the drug mainly owing to the lack of AZT phosphorylation and that resistance may be bypassed by using AZT-monophosphate SATE prodrugs.

Effects of cycloSal-D4TMP Derivatives in H9 Cells with Induced AZT Resistance Phenotype

Cytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9rAZT250). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9rAZT250 cells, which exert decreased thymidine kinase (TK) gene expression.

S-acyl-2-thioethyl (SATE) pronucleotides are potent inhibitors of HIV-1 replication in T-lymphoid cells cross-resistant to deoxycytidine and thymidine analogs

The biological evaluation of mononucleotide prodrugs (pronucleotides) of various nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), zalcitabine (ddC) and lamivudine (3TC) was reported in human T-lymphoid MOLT-4/8 cells which were grown continuously for more than 1 year in a medium containing cytarabine (Ara-C). In this cell line, expression of deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) was decreased in comparison to parental cells (3.8 and 2.9-fold, respectively). The lower mRNA level of TK1 correlated significantly with lower enzyme activity, whereas no dCK activity was detectable. In Ara-C-resistant cells, anti-HIV-1 effects of ddC, 3TC and AZT were more than 100-fold lower compared with parental cells. In contrast, the corresponding mononucleoside phosphotriesters bearing S-acyl-2-thioethyl (SATE) groups as biolabile phosphate protection retained anti-HIV-1 activity due to their ability to bypass the first monophosphorylation step catalyzed by dCK or TK1. The results demonstrate that in vitro selection of T-lymphoid cells in the presence of Ara-C results in cross-resistance to deoxycytidine (ddC, 3TC) and thymidine (AZT) analogs and that these cellular resistance mechanisms can be bypassed by the use of bis(SATE) pronucleotides.

Activity of cellular thymidine kinase 1 in PBMC of HIV-1-infected patients: novel therapy marker.

SummaryCellular cytoplasmatic thymidine kinase 1 (TK1) catalyzes the intracellular phosphorylation of anti-HIV-1 nucleoside analogs zidovudine (AZT) and stavudine (d4T) to the corresponding monophosphate form. In HIV-1-infected patients, treated with combination therapy including one of these compounds for more than 1 year, enzymatic activity of TK1 in peripheral blood mononuclear cells (PBMC) was determined by radioactive assay. TK1 activity in PBMC of HIV-1-infected patients correlated with CD4 cell count (r = 0.4, p < 0.05) and HIV-1 RNA copy number (r = 0.4, p < 0.05), being lower in patients with decreased CD4 cell count and high viral load. Furthermore, TK1 activity differs between HIV-1-infected individuals treated for more than 6 months (13.5 pmol/mg/h) compared to patients treated for less than 6 months (28.1 pmol/mg/h; p < 0.05) with chemotherapeutic agents including thymidine analogs. The results demonstrate that TK1 deficiency in PBMC of HIV-1 infected patients may develop due to continuous treatment with thymidine analogs and correlates with a more progressed stage of disease expressed as diminished CD4 cell count and increased viral load.

Zidovudine (AZT) resistance in H9 cells due to decreased TK expression is associated with hypermethylation of TK gene.

AZT resistant human T-lymphoid H9 cells, deficient in TK gene expression, re-expressed TK mRNA and regained the ability to metabolize AZT by exposure to the demethylation agent azacytidine (AzaCd). Cytotoxic and anti-HIV-1 effects of AZT were increased in H9 AZT resistant cells treated with AzaCd when compared to untreated cells. This leads to the assumption that drug induced DNA hypermethylation was involved in the TK gene-silencing mechanism. Our results suggest approaches using modulation of gene methylation for increasing antiviral efficiency of drugs.

Cytarabine treatment of human T-lymphoid cells induces decreased HIV-1 receptor expression and reduced HIV-1 susceptibility.

Continuous cultivation of T-lymphoid C8166 cells in the presence of pharmacological relevant concentration of cytarabine (Ara-C) results in significantly decreased expression of CD4 and CXCR4 molecules, the major cellular receptor and co-receptor of T-lymphotropic HIV-1 isolates. This change in receptor expression leads to decreased susceptibility of Ara-C resistant cells to HIV-1 infection demonstrated by reduced binding and penetration of HI-virus.

Cytotoxic Activity and Pharmacology of an Ara-CMP Prodrug in T-leukemic Sublines Resistant to Ara-C

Treatment with Ara-C in patients with acute leukemia may result in development of resistant cell populations. For the study of these cellular resistance mechanisms we have used T-leukemic C8166 sublines resistant to Ara-C, established by incubation with increasing concentrations of Ara-C. C8166rAra-C5 and C8166rAra-C250 were grown for more than two years in medium containing 5 μM or 250 μM Ara-C, respectively. Cytotoxic measurements showed, that cytotoxicity of Ara-C is strongly diminished in Ara-C resistant cell sublines in comparison to parental cells. Pharmacokinetic studies demonstrated that the ability of resistant cells to accumulate Ara-C to its triphosphate is abolished. Ara-CTP levels in Ara-C resistant sublines, after incubation with a biolabile bis(tBuSATE)Ara-CMP prodrug are higher than in parental cells. However, Ara-CMP prodrug is able to bypass only partly these cellular resistance mechanisms, as demonstrated by cytotoxic assay. These results showed that C8166 cells developed resistance to Ara-C probably due to deoxycytidine kinase deficiency, but other mechanisms may be also involved. C8166rAra-C5 and C8166rAra-C250 are novel T-leukemic cell sublines which may be valuable models for the investigation of Ara-C resistance.

Zidovudine Induces Resistance to Antineoplastic Agents and Alterations in Apoptosis in T-Cell Lymphoma Cells

Prolonged treatment of leukemic cell lines with antiretroviral agents such as nucleoside analogues may result in the development of cell resistance against antiviral agents. We tested whether prolonged treatment of H9 cells (T-cell lymphoma cell line) with 3′-azido-3′-deoxythymidine (AZT) interferes with sensitivity to antitumor agents commonly used for treatment of AIDS-associated malignancies. H9 cells grown for more than 2 years in medium containing 250 µM of AZT were at least 100-fold less sensitive to cytotoxic effects of AZT than parental H9 cell line. These cells designated H9rAZT250 were 5- to 20-fold less sensitive to toxic effects of antitumor agents including cisplatin (CP), vincristine (VIN), doxorubicin (DOX) and etoposide (VP-16). The resistance to these drugs was associated with inhibition of apoptosis as demonstrated by the terminal deoxynucleotidyl transferase-mediated nicked-end labelling (TUNEL) assay and DNA fragmentation assay. The expression of genes involved in regulation of apoptosis such bd-2 was enhanced in H9rAZT 250 cells. The results demonstrate that prolonged treatment of tumor cells with AZT may result in the development of resistance to antineoplastic agents due to the inhibition of apoptosis.