[44] regarding the effects of Aza-CdR in activating latently infected cells. four different Jurkat T cell-derived J-Lat cell lines (6.3, 8.4, 9.2 and 10.6), which have a latent HIV provirus in which GFP replaces Nef coding sequence, and ACH-2 and J1.1 (T cell-derived), and U1 (promonocyte-derived) cell lines with full-length provirus. We found that Aza-CdR plus TNF activated HIV at least twice as well as TNF alone for almost all J-Lat cells, as previously described, but not for J-Lat 10.6, in which TNF plus Aza-CdR moderately decreased activation compared to TNF alone. Surprisingly, a much greater reduction of TNF-stimulated activation with Aza-CdR was detected for ACH-2, J1.1 and U1 cells. Reaching the highest reduction in U1 cells with a 75% reduction. Interestingly, Aza-CdR not only decreased TNF induction of HIV expression in certain cell lines, but also decreased activation by TSA. Since DNMT inhibitors reduce the activity of provirus activators in some HIV latently infected cell lines the use of epigenetic modifying agents may need to be carefully optimized if they are to find clinical utility in therapies aimed at attacking latent HIV reservoirs. Findings Despite the effectiveness of UNC1079 Highly Active Antiretroviral Therapy (HAART) for Human Immunodeficiency Virus type 1 (HIV-1) infection, patients cannot be cured due to the persistence of long-lived reservoirs of cells latently infected with HIV ([1-5] and reviewed in [6-11]). Much interest has focused on attacking this reservoir of HIV latently infected cells. A potentially useful strategy, sometimes termed “shock and kill” [12], aims to attack the latent reservoir treating patients with HIV-activating agents to stimulate HIV replication in the latently infected cells while blocking new infection of cells with antiretrovirals. One activation approach employs agents like phorbol esters (e.g 12-O-Tetradecanoylphorbol-13-acetate (TPA), prostratin) [13], interleukins (IL-2, IL-7) [14-18] and cytokines (e.g. tumor necrosis factor alpha (TNF)) [19] that directly activate HIV gene expression via well-known transcriptional activation pathways, like NF-B, but many UNC1079 such agents are toxic or incompletely effective em in vivo /em [14,16,20,21]. Other approaches target the provirus’ epigenetic environment, employing histone deacetylase (HDAC) inhibitors (HDACIs) like trichostatin A (TSA) [22,23], suberoylanilide hydroxamic acid (SAHA) [22,24], sodium butyrate [25,26], and valproic acid [27,28], and/or DNA methyltransferase (DNMT) inhibitors (DNMTIs) like 5-aza-2’deoxycytidine (Aza-CdR) [29], with some strategies combining approaches [30] (and reviewed in [6,31]). In addition, to increase the efficiency of CT96 viral activators, combining the use of activators with compounds aimed at limiting the toxicity of the activator, as shown with buthionine sulfoximine [32] may play a major role in optimizing treatment strategies. Nucleosomes occupy specific positions on the HIV LTR [22,33,34], with the nucleosome occupying the position termed ‘nuc-1′ at a regulatory region of the LTR 610 to 720 bp 3’ to the transcription start site having significant inhibitory effects on HIV expression. Chromatin condensed around nuc-1 in its deacetylated form presents a block to HIV expression. Following hyperacetylation and chromatin remodeling, mediated by the recruitment of histone acetylases by transcription factors binding directly or indirectly to the LTR, or by Tat, the nuc-1 block is removed [22,33,35-37]. HDACIs, by shifting histones to a more acetylated state facilitate remodeling and removal of the block [38,39]. DNMTIs likely activate HIV because, at least in some cells, the LTR contains two CpG islands (particularly island 2) that can be hypermethylated and the hypermethylated DNA can recruit methyl-CpG binding domain (MDB) protein family members, notably MDB2. MDB2 recruited to the LTR can serve a bridging function between DNA and chromatin-modifying factors, such as HDACs [29]. Several studies linked retroviral promoter CpG methylation with transcription inactivation [40-42] and cellular gene silencing [43]. DNMTIs can moderately activate HIV alone, but in some systems they significantly enhance agents, like TNF, that directly activate the HIV LTR, probably because activation is limited due to UNC1079 proviral DNA hypermethylation [29,44,45]. While DNMTIs and HDACIs significantly enhance HIV activation in some latently infected cells, their ability to enhance activation across a wider range of latently infected cells is currently unknown. The breadth of activating ability is important, since complete or close-to complete eradication of latently infected cells may be required to cure HIV infection. To better assess the breadth of activity of activators representative of both direct activators and activators acting via epigenetic effects we surveyed the ability of three prototypical HIV activating agents, the DNMTI Aza-CdR (2.5 M), the HDACI TSA (1.5 M), and TNF (10 ng/ml), activating via NF-B, in a panel of.
