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Histone Deacetylase Inhibitor Scriptaid Reactivates Latent HIV-1 Promoter by Inducing Histone Modification in in Vitro Latency Cell Lines

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Histone Deacetylase Inhibitor Scriptaid Reactivates Latent HIV-1 Promoter by Inducing Histone Modification in in Vitro Latency Cell Lines 265-272.qxd 18/6/2010 08:23 Ì ™ÂÏ›‰·265 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 26: 265-272, 2010 265 Histone deacetylase inhibitor Scriptaid reactivates latent HIV-1 promoter by inducing histone modification in in vitro latency cell lines HAO YING, YUHAO ZHANG, SHIGUAN LIN, YEFEI HAN and HUAN-ZHANG ZHU State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, P.R. China Received March 2, 2010; Accepted April 13, 2010 DOI: 10.3892/ijmm_00000461 Abstract. Human immunodeficiency virus type 1 (HIV-1) completely eradicate HIV-1 (1,2). It is now widely believed latency remains a major problem for the eradication of viruses that the presence of a long-lived, stable population of latently in infected individuals undergoing highly active anti-retroviral infected resting memory CD4+ T cells is a major barrier to therapy. By inhibiting HIV-1 gene expression and virus virus eradication (3-5). These latently infected cells can go production, histone deacetylase (HDAC) may contribute to the dormant, and remain in the peripheral circulation and other quiescence of HIV-1 within resting CD4+ T cells. A novel tissues for years despite effective HAART (5). The decay rate HDAC inhibitor, Scriptaid, has been found to have robust of the HIV-1 latent reservoir has been found to be so low that activity and lower toxicity compared to trichostatin A (TSA). their eradication during a human lifespan is unlikely (6,7). We therefore investigated Scriptaid for its capability to reverse Therefore, any curative AIDS therapy should provide a solution HIV-1 latency by inducing HIV-1 activation in the Jurkat T to the HIV-1 latency hurdle (3,6). Under such a context, several cell line containing latent HIV proviruses. We found that ‘shock and kill’ strategies have been proposed to purge the Scriptaid can activate HIV-1 gene expression in these latent viral reservoirs by deliberately forcing HIV-1 gene expression infected cells by 2-15-fold over background levels, as analyzed in latent infected cells, along with HAART treatment to by flow cytometry. Chromatin immunoprecipitation (ChIP) prevent spreading the infection by newly synthesized viruses assays further revealed that the Scriptaid increased the (8-10). Some treatments being considered are able to reduce acetylation level of histones H3 and H4 at the nucleosome 1 the size of latent reservoirs by directly killing from the virus- site of the HIV-1 long terminal repeat compared to mock aroused cytopathic action or the intervenience from the treatment. In addition, Scriptaid can synergize with prostratin immune system. or tumor necrosis factor-· to activate the HIV-1 promoter, Mechanisms that allow HIV-1 to establish and maintain with relatively lower toxicity compared to TSA. These studies latency can be multi-factoral. Interestingly, several recent lines suggest the potential of Scriptaid in anti-latency therapies. of evidence suggest that histone deacetylases (HDACs) are critical regulators of HIV latency. Margolis and colleagues Introduction demonstrated that the transcription factor YY1 can act as a repressor of HIV transcription by recruiting HDAC-1 to the Human immunodeficiency virus type 1 (HIV-1) infection can provirus (11). Later studies demonstrated that nuclear factor now be treated effectively and become a chronic disease in (NF)-κB p50 homodimers (12), AP-4 (13), CTIP2 (14), Sp1 many patients, due to the advent of the highly active anti- and c-Myc (15), and CBF-1 (16) can participate in HDAC-1 retroviral therapy (HAART). Unfortunately, whereas HAART recruitment. HDAC-2 and -3 can also associate with the HIV regimens can significantly reduce levels of viral presence in long terminal repeat (LTR), and play a role in the repression plasma and lymphoid tissues, cessation of the therapies can of LTR expression (14,17). It has been shown that the quickly lead to the rebound of viral loads to pre-treatment disruption of HDAC-1 recruitment to LTR, resulting from the levels, indicating that HAART treatment alone cannot inhibition of HDAC activity by global HDAC inhibitors, leads to LTR activation and the escape of viral expression in both cell line models and primary cells obtained from patients _________________________________________ (10,18-26). Furthermore, HDAC inhibitor valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA; vorinostat) can Correspondence to: Dr Huan-zhang Zhu, State Key Laboratory induce viral outgrowth from resting CD4 T cells of aviremic of Genetic Engineering, Institute of Genetics, School of Life Sciences, patients undergoing HAART (27,28). These observations Fudan University, Shanghai 200433, P.R. China have led to the investigation of HDAC inhibition as a putative E-mail: [email protected] therapeutic approach to induce HIV from latency. Scriptaid, the 6-[1,3-dioxo-1H,3H-benzo(de)isoquinolin-2- Key words: human immunodeficiency virus type 1, latent reservoirs, yl]-hexanoic acid hydroxyamide, was identified by screening a histone acetylation, histone deacetylase inhibitors, Scriptaid library of 16,320 compounds (DiverSet, Chembridge, San Diego, CA) using a platform that contains a stably integrated 265-272.qxd 18/6/2010 08:23 Ì ™ÂÏ›‰·266 266 YING et al: Scriptaid REACTIVATES LATENT HIV-1 PROMOTER transcriptional reporter (29). Scriptaid has been reported to analyzed with FlowJo software (Tree Star, CA). Live cells were inhibit growth and induce differentiation and/or apoptosis in gated and two parameter analysis was used to differentiate a variety of cancer cells such as breast, endometrial, ovarian GFP-associated fluorescence from background fluorescence. A cancers, squamous carcinoma and colorectal cancer cell line total of 10,000 gated events were collected and data represent (29-33). The molecule is considered to be a novel HDAC the percentage of GFP-expressing cells in total gated events. inhibitor with robust activity and relatively lower toxicity than trichostatin A (TSA) (29). However, little is known about its Cytotoxicity assay. HEK 293, Jurkat cells, J-Lat Tat-GFP effect in inducing HIV expression in latently infected cells. Clone A7 cells were treated with or without Scriptaid or TSA This study aimed to investigate the effect of Scriptaid on the for 24 h, at of 25, 50, 100, 200 and 400 nM. To measure epigenetic change at HIV-1 LTR and the expression induction proliferation and viability in the presence of drugs, cells were of the latent viruses. The synergistical effect between Scriptaid subjected to an MTT assay (23). In brief, 3-(4,5-dimethyl- and tumor necrosis factor (TNF)-·/prostratin/5-azacytidine thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) (5-Aza) were analyzed in J-Lat Tat-GFP Clone A7 cells. In was placed in solution with PBS (5 mg/ml) and used to addition, the effects of Scriptaid on cell viability were also measure cellular proliferation. Cells (1x103) were incubated assessed in human embryonic kidney 293 cells and Jurkat cells. in 100 μl of culture medium for 48 h in 96-well plates, and Our results suggest that Scriptaid induces reactivation of latent 10 μl of MTT solution was added. After 4 h incubation, 50 μl HIV-1 transcription, increases acetylation of histones H3 and of solubilization solution (20% SDS) was added, and cells H4 at the nucleosome 1 (nuc-1) site of the HIV-1 LTR, and were then incubated at 37˚C for 16 h. In this assay, MTT was synergizes with prostratin or TNF-· to activate the HIV-1 cleaved to an orange formazan dye by metabolically active promoter in the Jurkat T cell line. As such, Scriptaid should be cells. The dye was directly quantified using an enzyme-linked further explored as a viable candidate for anti-latency therapies. immunoabsorbent assay reader at 540 nm. The 50% cyto- toxic concentration (CC50) was determined from the dose Materials and methods response curve. All experiments were performed independently at least three times in triplicate per experimental point. Chemical treatment and cell culture. Scriptaid, the 6-(1,3- dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid Western blotting. To determine the expression of various hydroxyamide, was purchased from Alexis Biochemicals proteins in A7 cells following various stimulations, Western (ALX-270-298). Recombinant human TNF-· was purchased blot analysis was performed as described previously (24). from Chemicon International. 5-Aza and TSA, were purchased Briefly, cells were harvested by trypsinization and lysed in lysis from Sigma (A1287, T8552). Prostratin was purchased from buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, LC laboratories (P-4462). Scriptaid, TSA, 5-Aza and prostratin 1 mM DTT, 0.1% SDS, 1% Nonidet P-40, 1 mg/ml leupeptin were dissolved in anhydrous dimethyl sulfoxide (DMSO) to and soybean trypsin inhibitor, 0.5 mM PMSF) for 30 min on a 100-mM stock solution. ice. Approximately 50-150 mg of protein extracts were loaded A7 cells, a latently infected Jurkat cell line encoding the on 12% polyacrylamide gel. Next, separated proteins were green florescence protein (GFP) as a marker for Tat-driven electroblotted from the gel onto nitrocellulose membrane and HIV LTR expression were kindly provided by the NIH then blocked with a blocking buffer (5% non-fat dry milk in 1x AIDS Research and Reference Reagent Program (from TBST, i.e. 20 mM Tris-HCl, pH 7.6 containing 0.8% NaCl and Dr Eric Verdin) (34,35). A7 cells, as well as Jurkat cells were 0.1% Tween-20) at room temperature for 1 h. The membrane grown in RPMI-1640 medium supplemented with 10% fetal was incubated with primary antibodies in blocking buffer, bovine serum (Gibco), 100 U/ml penicillin and 100 μg/ml of followed by incubation with second antibodies. Bands were streptomycin (Invitrogen) at 37˚C under 5% CO2. Human visualized using the ECL Western blotting system.
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