APOPTOSIS RESISTANCE IN HIV-1-PERSISTENTLY INFECTED CELLS AS A MODEL OF HIV SURVIVAL IN RESERVOIR CELLS

P. N. FERNÁNDEZ LARROSA; D. A. RIVA; D.O. CROCI; M. BIBINI; R. LUZZI; M. SARACCO; G.A. RABINOVICH; S. E. MERSICH; L. A. MARTÍNEZ PERALTA

Sidney, Australia

Conferencia; 4th IAS Conference on HIV Pathogenesis, Treatment and Prevention; 2007

Objectives: In order to analyse the effects of HIV-1 over reservoir cells, persistently infected cells were treated with different apoptosis inducers and results were compared with those of uninfected cells. Methods: Uninfected H9, Jurkat and U937 cell lines and persistently infected H9/HTLVIIIB, J1.1 and U1 cell lines were treated with different concentrations of H2O2 or staurosporine (STS) and collected 24 hours post treatment. Non-treated controls (NT) were performed simultaneously. Cell death parameters were evaluated by MTT, and dual staining with annexin-V-FITC and Propidium Iodure (PI), or APO-BRDU (revealed by FACS). P24 antigen production was quantified. Western Blot analyses were performed to evaluate apoptosis proteins. Results: When treated with both inducers, persistently infected cells showed significantly lower apoptosis levels than uninfected cells. Infected cells showed significant decrease in the levels of p24 production when subjected to both treatments. Induction of viral production in J1.1 with TNF-a and U1 with PMA did not show significant differences in cell viability with respect to unstimulated cells. Western Blot analysis revealed no differences in BCL-2 expression in H9 or H9/HTLVIIIB cells, treated or not with H2O2 or STS. However, Bax was decreased by 40% (H2O2) or 70% (STS) in H9/HTLVIIIB cells treated with apoptosis inducers in comparison with their NT control but only 20% (H2O2) or 40% (STS) in uninfected cells (H9). Besides, H9 cells treated with STS showed great decrease of procaspase-3 (70%), indicating high levels of cleavage; while H9/HTLVIIIB cells shown much lower decrease (40%). Conclusions: Our results suggest that, when treated with H2O2 and STS, persistently infected cell lines were resistant to dying by apoptosis compared with uninfected cell lines, and this effect was independent of active viral replication. This resistance could be regulated at the mitochondrial level via Bcl-2/Bax balance. This in vitro resistance could help to understand the in vivo HIV reservoir persistence.