Taddeo B, Nickoloff B J, Foreman K E
Department of Pathology, Skin Cancer Research Laboratories, Cardinal Bernardin Cancer Center, Loyola University Medical Center, Maywood, Illinois, USA.
Arch Pathol Lab Med. 2000 Feb;124(2):240-5. doi: 10.5858/2000-124-0240-CIBHIV.
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.
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.
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.
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.
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.
确定调节T细胞存活的试剂与人类免疫缺陷病毒1型（HIV-1）复制之间是否存在关系，并确定溶剂二甲基亚砜（DMSO）以及环孢素A和全反式维甲酸等药物对HIV-1复制的影响。
首先确定不同浓度的单独溶剂（即DMSO）对HIV-1复制的直接影响，随后确定诸如半胱天冬酶抑制剂N-苄氧羰基-缬氨酸-丙氨酸-天冬氨酸-氟甲基酮（z-VAD-fmk）、环孢素A和全反式维甲酸等各种化合物对HIV-1复制的影响。接下来，使用TUNEL（末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记）检测法、DNA片段化检测以及聚（ADP-核糖）聚合酶（PARP）裂解检测来确定HIV-1是否诱导T细胞凋亡，然后研究各种化合物在HIV-1感染后如何影响T细胞存活。
将人T细胞系CEM细胞暴露于HIV（IIIB），并在感染后第3、6和9天使用逆转录检测法监测病毒复制。细胞用溶解于DMSO的各种化合物在广泛的浓度范围内进行预处理，同时也对DMSO本身进行检测。使用TUNEL染色检测3'-OH DNA链断裂，并用琼脂糖凝胶电泳检测DNA片段化（梯状条带）来评估T细胞死亡和凋亡。此外，还检测了参与凋亡过程的PARP裂解情况。
在极低水平（如0.002%）时，DMSO本身在感染后第6天和第9天似乎会增强HIV-1复制。在低水平的环孢素A（如0.01微克/毫升）时，HIV-1复制在溶剂影响的基础上进一步增强，但在1微克/毫升时，环孢素A强烈抑制HIV-1复制。0.01至1微克/毫升之间的维甲酸对HIV-1复制没有影响。此外，还发现一个差异，即HIV-1感染的T细胞TUNEL检测呈阳性，表明存在DNA链断裂；然而，未检测到更完全的DNA片段化，也未鉴定出PARP裂解。半胱天冬酶抑制剂z-VAD-fmk可阻断TUNEL阳性的出现，但DMSO或环孢素A不能。尽管z-VAD-fmk阻断了TUNEL阳性T细胞的出现，但并未始终观察到HIV-1复制增加。
低水平的DMSO和环孢素A可增强CEM细胞中的HIV-1复制。在较高水平时，环孢素A抑制HIV-1复制，全反式维甲酸无显著影响。HIV-1感染后，在CEM细胞中未检测到经典凋亡的证据，尽管TUNEL阳性显示可能存在DNA链断裂。HIV-1复制水平与T细胞存活或死亡之间没有相关性。HIV-1感染后T细胞死亡的机制需要进一步研究，添加溶解于DMSO的化合物的研究人员必须设置对照，以仔细检查即使是痕量水平的这种溶剂对HIV-1复制的直接影响。
