Annie Benoit

Induction of an error-prone mode of DNA repair in UV-irradiated monkey kidney cells.

The survival of UV-irradiated simian virus 40 (SV40) on UV-irradiated monkey kidney CV-1P cells at 33 degrees was increased over survival on unirradiated cells. During this process - called induced-virus reactivation - the progeny virus yielded by UV-irradiated cells had a much higher mutation frequency than did the progeny from unirradiated cells. Mutation rates were quantified by using phenotypic reversion towards wild-type growth of an early (tsA 58) or a late (tsB 201) temperature-sensitive SV40 mutant. Analysis of SV40 revertant genomes indicated that no detectable deletions of additions were responsible for the reversion process. These results suggest that enzymes from UV-irradiated cells are able to replicate UV-irradiated DNA by an error-prone mode of DNA repair. Induced virus reactivation and error-prone replication are probably one of the expressions of SOS functions in mammalian cells.

The proapoptotic effect of hepatitis B virus HBx protein correlates with its transactivation activity in stably transfected cell lines

The role of hepatitis B virus HBx protein in the carcinogenesis associated with chronic viral infection remains ill-defined. Indeed, pleiotropic effects have been ascribed to HBx: in addition to its well-documented ability to indirectly stimulate transcription, the protein has been reported to affect cell growth, signal transduction, DNA repair and apoptosis. In this work, we generated Chang (CCL-13)-derived cell lines constitutively expressing wild type or mutant HBx, as a model of HBx-host cell interaction closer to the chronic infection setting, than the classically used transient expression systems. We document the potentiation by HBx of the apoptotic cell death pathway in the recipient cells. This effect is unlikely to rely on p53 activity since the protein is functionally inactivated in CCL-13. In addition, anti-oxidants and cyclosporin A failed to reduce the apoptotic response back to the normal level, suggesting that production of reactive oxygen species and calcineurin activation are not directly involved in the proapoptotic effect of HBx. In contrast, our data show that transactivation and stimulation of apoptosis are tightly linked HBx activities. Finally, expression of transactivation-active protein did not result in detectable change in the pattern of MAP kinases phosphorylation nor did it affect the ability of the host cell to repair in vitro irradiated plasmid DNA.

Error-prone replication of ultraviolet-irradiated Simian Virus 40 in carcinogen-treated monkey kidney cells

To analyze the molecular mechanism of mutagenesis in carcinogen-treated mammalian cells, we developed a model system composed of various simian virus 40 (SV40) mutants as a biological probe to detect inducible DNA repair and mutagenesis in carcinogen-treated monkey kidney cells (CV1-P). Results have shown that treatment of cells with UV-light, acetoxy-acetyl-aminofluorene, or mitomycin C, increases the mutagenesis of UV-irradiated SV40 ts mutant measured as a reversion frequency from a thermosensitive phenotype toward a thermoresistant phenotype. This increased mutagenesis is not observed in the case of undamaged virus indicating that we are looking at targeted mutagenesis. The molecular analysis of several revertant genomes indicates that some DNA rearrangements may occur in the revertant genomes but in some cases the reversion site is a single basepair substitution located at positions different from the original thermosensitive mutation, which is still present. The general interpretation of our results leads to the conclusion that carcinogen treatment of monkey cells activates some kind of error-prone replication mode able to better replicate UV-damaged templates but leading to a higher level of mutagenesis. This activity may represent a SOS-like function in mammalian cells.

Stable SV40-transformation and characterisation of some DNA repair properties of fibroblasts from a trichothiodystrophy patient

To characterize nucleotide excision repair properties of cells from trichothiodystrophy (TTD) patients genetically-related to the xeroderma pigmentosum (XP) group D, TTD skin fibroblasts from two unrelated patients (TTD1VI and TTD2VI) belonging to the TTD/XPD group were transformed with a plasmid containing SV40 large T antigen-coding sequences and some DNA repair properties, such as unscheduled DNA synthesis (UDS), UV-survival, in vitro repair synthesis of cell extracts and reactivation of UV-irradiated reporter plasmid were studied. Results showed that: a) both untransformed and transformed TTD cells present a reduced UV-survival, compared to wild-type cells, but at significantly less reduced levels than XP-D cells; b) reduced repair activities were detected in both TTD and XP-D transformed cells by using in vitro cell free extract repair and reactivation of UV-irradiated plasmid procedures, and these relative reduced extents correlated with respective UV-survival; c) surprisingly, near wild-type UDS levels were detected in TTD2VILas transformed cells at different passages after the crisis, suggesting a phenotypic reversion of this transformed cell line; d) fluoro-cytometric analysis of TTD2VILas cells revealed a strong increase of a cell population containing a DNA amount more than twice as high than that of untransformed cells; finally, e) when UDS data were normalized to the DNA content in TTD2VILas cells, it appeared that the repair efficiency was only slightly higher than in untransformed cells. This implies that in transformed cells DNA repair properties should be evaluated, taking into account additional parameters. We obtained an immortalized TTD cell line which maintains DNA repair properties similar to those of parental untransformed cells and may be used to characterize the TTD defect at genetic, molecular and biochemical levels.