Figure 4. Counteracting effect of ALP compounds against the cytoprotective phenotype of HIV-1 or Tat expressing CHME5 cells. (A) Death of CHME5 cells untreated (‘‘Control’’) or treated with LPS (50 mg/ml) and CHX (10 mg/ml) (‘‘LPS/CHX’’) was determined by staining with ethidium homodimer (red) at 24 hours post treatment. CHME5 cells were also transduced by DHIV-GFP (‘‘D3’’, .90% transduction), and treated with LPS/CHX in the presence and absence of 25 mM edelfosine (‘‘EF’’) or miltefosine (‘‘MF’’) at 48 hours post transduction. The dead cells (red) among the transduced cells (green) were determined to calculate the cell death percentage. (B) CHME5 (2) and (+) Tat subline cells were treated with LPS/CHX for 24 hours in the presence or absence of edelfosine (25 mM), perifosine (25 mM) or miltefosine (50 mM). Dead cells were stained with Trypan blue. (C) Dose- dependent cell death of the (+) Tat CHME5 subline by edelfosine, perifosine, and miltefosine treatment in the presence of LPS (50 mg/ml) and CHX (10 mg/ml). The percent cell death of the (+) Tat cells treated with both LPS/CHX and different concentrations of the ALP compounds Targeted HIV-1 Reservoirs were determined and compared to the percent cell death of the (2) Tat control cells treated with LPS/CHX alone, which gave 39% cell death. doi:10.1371/journal.pone.0013121.g004 HIV-1 infection in CHME5 cells. Importantly, edelfosine and miltefosine alone did not significantly induce cell death in the transduced cells cultured in the absence of LPS/CHX, suggesting that these compounds do not induce nonspecific cytotoxicity, at least at the concentrations used in Figure 4. However, upon treatment with both edelfosine or miltefosine and LPS/CHX, a high proportion of the transduced cells underwent cell death, suggesting that the ALP compounds effectively abolished the cytoprotective effect of HIV-1 transduction in CHME5 cells. Next, we tested if the ALP compounds also block the Tat- induced cytoprotective phenotype of CHME5 cells, which was previously reported [5]. In this experiment, cell death of these two sublines was monitored after treatment of perifosine, edelfosine, and miltefosine in the presence and absence of LPS/CHX with a Trypan blue exclusion assay. As shown in Figure 4B, the (2) Tat control CHME5 cells showed a significant cell death (,40%) by the LPS/CHX treatment, whereas the (+) Tat CHME5 cells displayed a strong protection (,5% cell death) against the LPS/ CHX insults, supporting the cytoprotective effect of Tat in CHME5 cells. When treated with perifosine, edelfosine, or miltefosine alone (Figure 4B), both subline cells displayed a background level of cell death, suggesting that these three ALP compounds do not induce nonspecific cytotoxicity in this cell line system at the concentrations used in this experiment. However, upon treatment with both LPS/CHX and the ALP compounds (Figure 4B), the (+) Tat cells lost the Tat-induced cytoprotective effect against the LPS/CHX insult and became sensitive to the LPS/CHX treatment. These data support that all three ALP compounds were able to block the cytoprotective function of Tat in CHME5 cells. Interestingly, the (2) Tat control subline treated with both ALP compounds and LPS/CHX still showed the same high cell death as observed with the treatment of LPS/CHX alone, and thus this indicates that, in contrast to the (+) Tat cells, the ALP compounds do not sensitize the (2) Tat cells to the LPS/ CHX insult. Next, ALP compound-induced sensitization of the (+) Tat CHME5 subline to LPS/CHX was tested for concentration dependent effects. The amount of cell death of (+) Tat cells was determined at 0, 5, 15 and 25 mM of perifosine or edelfosine in the presence of the fixed amount of the LPS/CHX stress. As shown in Figure 4C, the amount of death of the (+) Tat subline cells gradually increases when the concentrations of perifosine and edelfosine increase, and at 25 mM, the ALP compounds re- sensitize the Tat (+) CHME5 cells to the LPS/CHX. Thus, the data shown in Figure 4 support that the ALP compounds induce the loss of the cytoprotective phenotype of both HIV-1 transduced and Tat expressing CHME5 cells. Induction of death in HIV-1 infected primary human macrophages by perifosine Next, we examined if perifosine can also counteract the cytoprotective phenotype of primary human macrophages infected with HIV-1. Importantly, we previously reported that indeed, HIV-1 infected macrophages display activation of Akt (GSK3b phosphorylation and Akt migration to the membrane), and miltefosine was able to effectively counteract the pro-survival phenotype of the HIV-1 infected macrophages [5]. In addition, in this investigation, we chose perifosine (PF) for its clinical significance; PF has been tested through Phase 2 trials for its anti-cancer effect [21]. PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e13121