Inhibition of NF-κB activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

In this study, we investigated molecular mechanisms underlying low susceptibility to apoptosis induced by the nucleoside analog azidothymidine (AZT) and the role of nuclear factor-κB (NF-κB) activation in these phenomena. A preliminary screening in different cell lines indicated U937 monocytic cell line as suitable to this purpose. Treatment of U937 cells even with suprapharmacological concentrations of AZT induced only moderate levels of apoptosis. Surprisingly, SuperArray analysis showed that AZT induced the transcriptional activity of both pro- and anti-apoptotic genes. Interestingly, moreover, several genes upregulated by AZT were NF-κB related. In fact, AZT, after an initial inhibition of NF-κB activation with respect to control, induced a transient, but consistent, increase in NF-κB-binding activity. Inhibition of NF-κB activation in U937 cells, stably transfected with a dominant-negative IκBα or by pharmacological treatment, sensitized them to apoptosis induced by AZT and impaired the upregulation of anti-apoptotic genes in response to AZT treatment, with respect to control cells. These results indicate that NF-κB activation by AZT has a role in protecting target cells from apoptotic cell death, improving our understanding of the toxicology and the therapeutic usage of this drug.

The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation.

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

Efficacy of 3'-azido 3'deoxythymidine (AZT) in preventing HTLV-1 transmission to human cord blood mononuclear cells.

The present study investigated the effect of 3'-azido 3'deoxythymidine (AZT) treatment on in vitro infection of human cord blood mononuclear cells (CBMCs) exposed to HTLV-1 by cocultivation with the MT-2 cell line. Cultures of CBMCs were grown in IL-2 and were either left untreated or were treated with concentrations of AZT ranging from 0.0078 to 32 microM. HTLV-1-infected cultures were monitored at different times of culture by evaluating proliferation activity, cell growth and the presence and expression of HTLV-1 genes. Results showed that untreated cultures infected with HTLV-1 were able to grow for several weeks, while those treated with AZT at 0.03 microM or higher concentrations were limited in their growth capacity. Moreover, the addition of AZT at the moment of infection significantly inhibited cell proliferation in a dose-dependent fashion. In the presence of AZT, detection of proviral DNA and, more remarkably, viral RNA expression were clearly reduced. In addition, treatment with AZT resulted in a noticeable decrease in Tax protein expression. Using treatment with relatively low doses of AZT, effective in exerting an antiviral action, cytotoxicity on CBMCs was not observed, whereas higher doses induced apoptosis in uninfected CBMCs. These data show that CBMCs are protected by AZT against HTLV-1 transmission even at low, non-toxic doses.

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells.

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.