Caspase inhibitor blocks human immunodeficiency virus 1-induced T-cell death without enhancement of HIV-1 replication and dimethyl sulfoxide increases HIV-1 replication without influencing T-cell survival

Objectives.-To determine the relationship, if any, between reagents that mo dulate survival of T-cells and replication of human immunodeficiency virus 1 (HIV-1) and to determine the effects of the solvent dimethyl sulfoxide (D MSO) and drugs such as cyclosporin A and all-trans retinoic acid on HIV-1 r eplication. Design.-To first establish the direct effects of solvent alone (ie, DMSO) a t various concentrations on HIV-1 replication, followed by the ability of v arious compounds such as the caspase inhibitor N-benzyloxycarbonyl-val-ala- asp-fluoromethylketone (z-VAD-fmk), cyclosporin A, and all-trans retinoic a cid on HIV-1 replication. Next, to determine if HIV-1 induces T-cell apopto sis using TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assays and DNA fragmentation and poly-(ADP-ribose)-polymerase (PARP) cleavage, and then t o examine how the various compounds influence T-cell survival after HIV-1 e xposure. Methods.-The human T-cell line, CEM cells, were exposed to HIVIIIB and vira l replication monitored using reverse transcription assays at 3, 6, and 9 d ays following infection. Cells were pretreated with various compounds disso lved in DMSO over a wide range of concentrations, and DMSO itself was also examined. T-cell death and apoptosis were assessed using TUNEL staining to detect 3'-OH DNA strand breaks and agarose gel electrophoresis to detect DN A fragmentation (laddering). furthermore, PARP cleavage implicated in the a poptotic process was also examined. Results.-At very low levels, such as 0.002%, DMSO itself appears to enhance HIV-1 replication at 6 and 9 days after infection. At low levels of cyclos porin A, such as 0.01 mu g/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 mu g/mL, cyclosporin A strongly inhibited HIV -1 replication. Retinoic acid between 0.01 and 1 mu g/mL did not influence HIV-1 replication. In addition, a discrepancy was noted in that HIV-l-infec ted T-cells were TUNEL positive, indicating DNA strand breaks; however, mor e complete DNA fragmentation was not detected nor was PARP cleavage identif ied. The induction of TUNEL positivity was blocked by the caspase inhibitor z-VAD-fmk but not by DMSO or cyclosporin A. Even though z-VAD-fmk blocked the appearance of TUNEL-positive T-cells, there was not a consistently obse rved increase in HIV-1 replication. Conclusion.-Low levels of DMSO and cyclosporin A can enhance HIV-1 replicat ion in CEM cells. At higher levels, cyclosporin A inhibits HIV-1 replicatio n with no significant effects by all-trans retinoic acid. No evidence for c lassic apoptosis was detected in CEM cells after HIV-1 infection, although DNA strand breaks may be present as revealed by TUNEL positivity. There was no correlation between levels of HIV-1 replication and T-cell survival or death. The mechanism of T-cell death after HIV-1 infection requires further study, and investigators who add compounds dissolved in DMSO must include controls to carefully examine the direct effects of even trace levels of th is solvent on HIV-1 replication.