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.
OBJECTIVE
To determine the relationship, if any, between reagents that modulate survival of T-cells and replication of human immunodeficiency virus 1 (HIV-1) and to determine the effects of the solvent dimethyl sulfoxide (DMSO) and drugs such as cyclosporin A and all-trans retinoic acid on HIV-1 replication.


METHODS
To first establish the direct effects of solvent alone (ie, DMSO) at various concentrations on HIV-1 replication, followed by the ability of various compounds such as the caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethylketone (z-VAD-fmk), cyclosporin A, and all-trans retinoic acid on HIV-1 replication. Next, to determine if HIV-1 induces T-cell apoptosis using TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assays and DNA fragmentation and poly-(ADP-ribose)-polymerase (PARP) cleavage, and then to examine how the various compounds influence T-cell survival after HIV-1 exposure.


METHODS
The human T-cell line, CEM cells, were exposed to HIV(IIIB) and viral replication monitored using reverse transcription assays at 3, 6, and 9 days following infection. Cells were pretreated with various compounds dissolved 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 DNA fragmentation (laddering). Furthermore, PARP cleavage implicated in the apoptotic 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 cyclosporin A, such as 0.01 microgram/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 microgram/mL, cyclosporin A strongly inhibited HIV-1 replication. Retinoic acid between 0.01 and 1 microgram/mL did not influence HIV-1 replication. In addition, a discrepancy was noted in that HIV-1-infected T-cells were TUNEL positive, indicating DNA strand breaks; however, more complete DNA fragmentation was not detected nor was PARP cleavage identified. 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 observed increase in HIV-1 replication.


CONCLUSIONS
Low levels of DMSO and cyclosporin A can enhance HIV-1 replication in CEM cells. At higher levels, cyclosporin A inhibits HIV-1 replication with no significant effects by all-trans retinoic acid. No evidence for classic 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 this solvent on HIV-1 replication.
Taddeo
B
B
Department of Pathology, Skin Cancer Research Laboratories, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, Illinois, USA.
Nickoloff
B J
BJ
Foreman
K E
KE
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D061945:Caspase Inhibitors; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D013601:T-Lymphocytes / Q000473:pathology* / Q000821:virology; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014779:Virus Replication / Q000187:drug effects*
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D014212:Tretinoin; D016572:Cyclosporine; D004121:Dimethyl Sulfoxide