TY - JOUR
T1 - 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
AU - Taddeo, Brunella
AU - Nickoloff, Brian J.
AU - Foreman, Kimberly E.
PY - 2000
Y1 - 2000
N2 - Objectives.-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. Design.-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 μg/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 μg/mL, cyclosporin A strongly inhibited HIV-1 replication. Retinoic acid between 0.01 and 1 μg/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. Conclusion.-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.
AB - Objectives.-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. Design.-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 μg/mL, HIV-1 replication was further enhanced above the solvent effect, but at 1 μg/mL, cyclosporin A strongly inhibited HIV-1 replication. Retinoic acid between 0.01 and 1 μg/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. Conclusion.-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.
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M3 - Article
C2 - 10656733
AN - SCOPUS:0033961147
SN - 0003-9985
VL - 124
SP - 240
EP - 245
JO - Archives of Pathology and Laboratory Medicine
JF - Archives of Pathology and Laboratory Medicine
IS - 2
ER -