APOPTOSIS RESISTANCE IN HIV-1 CHRONICALY INFECTED CELLS IS NOT ASSOCIATED TO VIRAL REPLICATION OR TAT EXPRESSION

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

Portoroz, Slovenia

Conferencia; 15th Euroconference on Apoptosis; 2007

HIV triggers the decline of CD4+ T cells and leads to progressive disfunction of cell-mediated immunity, mainly during the acute phase of HIV infection. On the other hand, chronically-infected macrophages and memory T-cells seem to be resistant to cell death, representing a potential viral reservoir. 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. Uninfected cell lines(H9, Jurkat, U937) and their HIV-1 persistently infected counterparts (H9/HTLVIIIB, J1.1, U1) were treated with H2O2 or staurosporine (STS) and collected 24 hours post treatment. Cells were treated with medium as controls (Ctrl). Tat-transfected Jurkat cells (Jurkat-tat) were also treated with inducers in order to evaluate the effect of tat over apoptosis susceptibility. Cell death parameters were evaluated by annexin-V/PI or APO-BRDU staining and FACS. P24 antigen production was quantified. Western Blot analyses were performed to evaluate apoptosis proteins. 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. Neither the induction of viral production in J1.1 with TNF-α and U1 with PMA, nor expression of tat, marked significant differences in cell viability with respect to uninduced cells. Bax levels were higher decreased (H2O2: 40%; STS: 70%) in H9/HTLVIIIB cells treated with apoptosis inducers than in H9 cells (H2O2: 20%; STS: 40%), while no difference in Bcl-2 expression was observed in both cell lines. Besides, H9 cells treated with STS showed an important decrease of procaspase- 3 (70%), indicating high levels of cleavage; compared to H9/HTLVIIIB (40%). Our results suggest that, when treated with H2O2 and STS, persistently infected cell lines were more resistant to dying by apoptosis compared with uninfected cell lines, and this effect was independent of active viral replication or tat expression. 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.