Disulfiram reactivates latent HIV-1 expression through... : AIDS (original) (raw)
Introduction
Although combination antiretroviral therapy (cART) can efficiently reduce plasma HIV-1 levels in infected individuals to below the limit of detection (<50 copies/ml), it has no impact on the latent reservoir that resides in resting CD4+ T cells [1]. In this reservoir, the integrated provirus remains transcriptionally silent as long as the host cell is in a resting state. However, upon cellular activation, HIV-1 RNA is transcribed and virus is produced, providing a source for recrudescent infection upon cessation of cART. The latent reservoir in resting CD4+ T cells is extremely stable with an estimated half-life of approximately 44 months, which effectively means that more than 60 years of therapy would be required to eliminate it [2,3]. A prevailing hypothesis in the field is that intervention strategies that reactivate latent HIV-1 infection will purge this reservoir by inducing transcription of the latent provirus, thereby causing cells to undergo apoptosis. Ongoing cART will prevent infection of new cells with the net result being reduction in the latent reservoir. Antiviral immune responses may also aid in the clearance of infected cells, and prevent the spread of infection to other cells. With regard to inducing HIV-1 transcription, Xing _et al._[4] demonstrated in a primary CD4+ T-cell model that disulfiram (DSF), a United States Food and Drug Administration approved drug used to treat chronic alcoholism, reactivates latent HIV-1 without global T-cell activation. On the basis of this finding, an ongoing clinical trial is determining whether a 2-week course of DSF will reduce the HIV-1 latent reservoir in patients on cART (ClinicalTrials.gov identifier: NCT01286259). Preliminary results from this trial revealed that DSF administration resulted in a small transient increase in viral load in some trial participants that was not statistically significant. Furthermore, a modest 14% decline in the size of the latent reservoir was noted [5]. Despite its evaluation in clinical trials, the mechanism(s) by which DSF reactivates latent HIV-1 expression has not been elucidated. As such, the primary objective of the current study was to address this important knowledge gap.
Methods
Materials
DSF was obtained from Sigma-Aldrich (St Louis, Missouri, USA). The phosphatase and tensin homolog (PTEN), phospho-Akt and Akt antibodies were obtained from Cell Signaling Technology (Boston, Massachusetts, USA). The β-actin antibody was obtained from Abcam (Cambridge, Massachusetts, USA). The Akt inhibitor VIII, cyclosporine A, SP600125 and Go6983 were purchased from Sigma Aldrich. Potassium bisperoxo (1,10-phenanthroline) oxovanadate [bpV(phen)] and the NF-κB activation inhibitor were acquired from EMD Biosciences (San Diego, California, USA). DNA primers were synthesized by Integrated DNA Technologies (San Diego, California, USA). Cellular proteins were separated using 4–12% SDS-PAGEs from Invitrogen (Grand Island, New York, USA). Immunodetection was performed using the Millipore SNAP i.d. (Billerica, Massachusetts, USA).
Cells and cell culture
ACH2 and U1 cells were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (T. Folks, contributor) [6–8]. J89GFP cells were a kind gift from D. Levy [9]. All three cell lines were maintained in RMPI 1640 medium supplemented with 10% fetal bovine serum, 0.3-mg/ml L-glutamine, 100-U/ml penicillin and 100-μg/ml streptomycin at 37°C in humidified air with 5% CO2.
Isolation of resting CD4+ T cells from HIV-negative donors
One hundred milliliter of blood was obtained from healthy HIV-negative volunteers. Donation was approved by the University of Pittsburgh Institutional Review Board. Written informed consent was provided for all volunteer donors. Resting CD4+ T cells were isolated from peripheral blood mononuclear cells by magnetic bead selection (Miltenyi Biotech, Cambridge, Massachusetts, USA) as described previously [10].
Small interfering RNA knockdown
Small interfering RNAs (siRNAs) targeting PTEN, as well as a control scrambled sequence control siRNA, were purchased from Qiagen (SA Biosciences, Qiagen Inc., Valencia, California, USA). U1 cells were transfected with 80-nmol/l siRNA using the Neon Transfection System from Invitrogen, according to the manufacturer's protocol. The efficiency of gene knockdown was assessed by determining mRNA levels and by western blot analyses of protein expression.
Quantitative analysis of gene transcripts
RNA was extracted from cells using the RNeasy Plus RNA extraction kit (Qiagen Inc.) according to the manufacturer's protocol. RNA was quantified using a Nanodrop 2000, and 400 ng of total RNA was used in each reverse transcription (RT) PCR reaction. Amplification was performed using the QuantiTect SYBR Green RT-PCR kit (Qiagen Inc.) and the DNA Engine Opticon system (Bio-Rad Hercules, California, USA). Initiated and elongated HIV-1 transcripts were assessed as described previously [11].
Results and discussion
Disulfiram reactivates HIV-1 expression in the U1 but not ACH2 or J89GFP cell line models of latency
We first assessed the ability of DSF to induce HIV-1 expression in three different cell line models of HIV-1 latency, namely ACH2, J89GFP and U1. The ACH2 and J89GFP cell lines are chronically HIV-1-infected T-lymphocytic cells. ACH2 cells are a subclone of A3.01, which is derived from CEM, a human T-cell line originally isolated from a 4-year-old female with acute lymphoblastic lymphoma, whereas the J89GFP cell line was established from the peripheral blood of a 14-year-old male with T-cell leukemia. By contrast, U1 cells are an HIV-1-infected monocytic cell line, subcloned from the U937 cell line, which was isolated from a pleural effusion of a 2-year-old male with diffuse histiocytic lymphoma. Reactivation of latent HIV-1 expression in each of these cell lines was measured by quantitative assessment of early and late viral RNA transcripts (Fig. 1a; Supplementary Table 1, https://links.lww.com/QAD/A230). DSF was found to reactivate latent HIV-1 RNA expression in the U1 cell line, but not in the ACH2 or J89GFP cell lines. To investigate further this finding, we searched for functional differences in the three cell lines that could potentially account for the differential activity of DSF.
Effect of disulfiram on reactivation of latent HIV-1 and phosphatase and tensin homolog expression.(a) Disulfiram (DSF)-induced HIV-1 expression in ACH2, J89GFP and U1 cells was assessed by quantitative PCR analysis of early and late viral RNA transcripts. Data are the average ± SD of at least three separate experiments. (b) Western blot analysis of phosphatase and tensin homolog (PTEN) expression in the ACH2, J89GFP and U1 cell lines, and in primary resting CD4+ T cells isolated from HIV-1-negative donors. (c) Ten-micromoles per liter DSF depletes PTEN in the U1 cell line. Ten-nanograms per milliliter tumor necrosis factor - alpha (TNFα) was used a negative control. (d) Western blot analysis of PTEN and phosphorylated Akt as a function of time following addition of 1-μmol/l DSF to resting CD4+ T cells purified from HIV-negative donors. (e) DSF (10 μmol/l) does not increase phosphorylated Akt in ACH2 or J89GFP cells. Ten-nanograms per milliliter TNFα, which activates the Akt pathway, was used a positive control.
The ACH2 and J89GFP cell lines lack the phosphatase and tensin homolog
PTEN is a tumor suppressor gene that is frequently mutated in many different cancers. It negatively regulates intracellular levels of phosphatidylinositol-3,4,5-trisphosphate (PIP3) in cells and functions as a tumor suppressor by negatively regulating the Akt signaling pathway. The Jurkat T-cell line is PTEN-null [12–14]. Similarly, CEM cells have a deletion in exons 2–5 of the PTEN gene [12] that corresponds to the phosphatase domain and renders the protein unstable [15]. By contrast, PTEN is expressed in the U1 cell line and in resting CD4+ T cells isolated from HIV-negative donors (Fig. 1B).
Disulfiram depletes phosphatase and tensin homolog and activates the Akt signaling pathway
A prior study showed that DSF led to decreased expression of PTEN and activation of Akt in a dose-dependent and time-dependent manner in human breast cancer cells [16]. Therefore, we assessed whether DSF treatment in both U1 cells and in resting CD4+ T cells impacted PTEN expression and/or activation of Akt. We found that DSF treatment depleted PTEN levels and increased Akt phosphorylation in both cell types (Fig. 1c, d). Interestingly, quantitative PCR analyses revealed that DSF did not impact PTEN expression at the RNA level (data not shown). Furthermore, DSF did not trigger Akt activation in either ACH2 or J89GFP cells, which lack PTEN (Fig. 1e).
Disulfiram reactivates latent HIV-1 expression via the Akt signaling pathway
To determine whether PTEN depletion and Akt activation contributed to the latent HIV-1 reaction phenotype of DSF in U1 cells, we assessed its activity in combination with inhibitors that targeted Akt or NF-κB (a downstream target of Akt). The Akt inhibitor VIII reduced the activity of DSF in a dose-dependent manner (Fig. 2a). Similarly, the NF-κB activation inhibitor completely negated DSF activity. By contrast, inhibitors of the nuclear factor of activated T cells, protein kinase C (PKC) and the c-Jun N-terminal kinase did not affect DSF activity. Of note, a control experiment was also performed using the PKC agonist prostratin instead of DSF. Consistent with its mechanism of action, the latent reactivation activity of prostratin was only diminished by a PKC inhibitor. To directly determine the role of PTEN in maintaining the HIV-1 latent phenotype in U1 cells, we assessed the ability of a high-affinity PTEN inhibitor [bpV(phen)] [17] to stimulate HIV-1 RNA expression. The bpV(phen) was found to be a potent activator of HIV-1 expression in the U1 cell line (Fig. 2b). Furthermore partial knockdown of PTEN expression by siRNA also resulted in reactivation of latent HIV-1 expression in the U1 cell line (Fig. 2c). Taken together, these data demonstrate that DSF reactivates latent HIV-1 via the Akt signaling pathway through depletion of PTEN. They also provide strong evidence that PTEN is a key regulator of HIV-1 latency.
Disulfiram reactivates latent HIV-1 expression via the Akt signaling pathway.(a) The Akt inhibitor VIII (used at 5, 10 and 15 μmol/l) and the NF-κB activation inhibitor (used at 20 μmol/l) diminish the ability of disulfiram (DSFO to reactivate latent HIV-1 expression in U1 cells. By contrast, inhibitors of nuclear factor of activated T cell (NFAT) (1-μmol/l cyclosporine A), c-Jun N-terminal kinase (JNK) (1.0-μmol/l SP600125) and protein kinase C (PKC) (0.5-μmol/l Go6983) did not impact DSF activity. Prostratin, a PKC agonist, was also included as a control in these experiments. (b) The phosphatase and tensin homolog (PTEN) inhibitor potassium bisperoxo (1,10-phenanthroline) oxovanadate [bpV(phen)], used at concentrations of 5, 10 and 15 μmol/l, reactivates latent HIV-1 expression in U1 cells. Forty-micromoles per liter DSF and 10-ng/ml TNFα were used as positive controls. Data are the average±SD of at least three separate experiments. (c) mRNA expression of PTEN in U1 cells after knockdown by siRNA. A siRNA with a scrambled sequence was used as a control. (d) Reactivation of latent HIV-1 in the U1 cells after knockdown of PTEN. The scrambled sequence siRNA was again used as the control.
Conclusion
PTEN dephosphorylates PIP3, resulting in the biphosphate product phosphatidylinositol 4,5-bisphosphate. This dephosphorylation is important because it mediates inhibition of the Akt signaling pathway that controls multiple cellular processes including transcription. In this study, we show that DSF also reactivates latent HIV-1 expression via the Akt signaling pathway through depletion of PTEN. This mechanism is unrelated to the activity of DSF as an inhibitor of aldehyde dehydrogenase, which is the basis for its clinical use to prevent alcohol intake. Of note, prior studies have demonstrated that both histone deacetylase inhibitors [11,18] and hexamethylene bisacetamide [19] reactivate latent HIV-1 through activation of the Akt signaling pathway. In this regard, activated Akt phosphorylates hexamethylene bis-acetamide inducible 1 (HEXIM1), which results in the subsequent release of the active positive transcription elongation factor b (p-TEFb) from its transcriptionally inactive complex with HEXIM1 and the 7SK small nuclear protein. As a result, p-TEFb is recruited to the HIV-1 promoter to stimulate transcription elongation and viral production [19]. Importantly, our findings suggest that PTEN could be targeted to reverse HIV-1 latency. They also suggest that Akt phosphorylation and/or PTEN depletion could serve as a pharmacodynamic marker of DSF activity in vivo. Finally, our research highlights the limitations of studying HIV-1 latency in cell lines. Many of these cell lines were isolated from human cancers and have mutations and/or deletions in genes encoding key enzymes/proteins involved in signal transduction pathways that may play important roles in regulating HIV-1 latency.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
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Keywords:
Akt; disulfiram; HIV-1; latency; phosphatase and tensin homolog
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