9-aminoacridine Inhibition of HIV-1 Tat Dependent Transcription

ACH2 and J1.1 are latently HIV-1 infected T-cell lines. ACH2, J1.1, CEM, and Jurkat cells were grown in RPMI-1640 media containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% streptomycin/penicillin. TZM-bl cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 1% L-glutamine, and 1% streptomycin/penicillin. All cells were incubated at 37°C and 5% CO₂.

9-aminoacridine was obtained from Sigma, 2-aminoacridine and 4-aminoacridine from KaironKem, acridine hydrochloride from TCI, and 4-aminoquinoline from Tyger Scientific. p21WAF1, phospho-p21WAF1 (S146), cdk2, cyclin T, and actin antibodies were obtained from Santa Cruz Biotechnology. Cdk9 antibody was obtained from Biodesign International. p53, phospho-p53 (S15), AKT (pan), phospho-AKT (S473), phospho-AKT (T308), GSK3-β (pan), phospho-GSK3-β (S9) antibodies were obtained from Cell Signaling.

Plasmids (LTR-CAT and/or CMV-Tat) were transfected by electroporation using a Bio-Rad Gene Pulser (Bio-Rad, Richmond, CA) at 960 μF and 230 volts. Two hours after transfection drug treatment was initiated. After 48 hours, cells were lysed and chloramphenicol acetyltransferase (CAT) activity was determined. Briefly, a standard reaction was performed by adding the cofactor acetyl coenzyme A to a microcentrifuge tube containing cell extract (50 ug) and radiolabeled (¹⁴C) chloramphenicol in a final volume of 30 μl and incubating the mixture at 37°C for 1 hour. The reaction mixture was then extracted with ethyl acetate and separated by thin-later chromatography on silica gel plates (Baker-flex silica gel thin-later chromatography plates) in a chloroform-methanol (19:1) solvent. The resolved reaction products were then detected by exposing the plate to a PhosphoImager cassette.

TZM-bl cells were transfected with pc-Tat (0.5 ug) using the Attractene reagent (Qiagen) according to the manufacturers' instructions. TZM-bl cells contain an integrated copy of the firefly luciferase gene under the control of the HIV-1 promoter (obtained through the NIH AIDS Research and Reference Reagent Program). The next day, cells were treated with DMSO or the indicated compound. Forty-eight hours post drug treatment, luciferase activity of the firefly luciferase was measured with the BrightGlo Luciferase Assay (Promega). Luminescence was read from a 96 well plate on an EG&G Berthold luminometer.

ACH2 cells were treated with 2.5 uM 9AA and processed 48 hours later for ChIP. For ChIP, approximately 5 × 10⁶ cells were used per IP. Cells were cross-linked with 1.0% formaldehyde at 37°C for 10 minutes, pelleted, washed, and cells lysed using SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, one tablet complete protease inhibitor cocktail per 50 ml) on ice for 10 mins. Cells were sonicated on ice for 6 cycles to obtain an average DNA length of 500 to 1200 bp. Lysate was clarified by centrifugation at 14,000 rpm for 10 minutes at 4°C. Supernatant was then diluted 10 fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl) and pre-cleared with a mixture of protein A/G agarose (blocked previously with 1 mg/ml salmon sperm DNA and 1 mg/ml BSA) at 4°C for 1 hour. Pre-cleared chromatin was incubated with 10 μg of antibody at 4°C overnight. Next day, 60 μl of a 30% slurry of blocked protein A/G agarose was added and complexes incubated for 2 hours. Immune complexes were recovered by centrifugation and washed once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), twice with high salt buffer (0.1% SDS, 1% Triton X-100 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl), once with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and once with TE buffer. Immune complexes were eluted twice with elution buffer (1% SDS, 0.1 M NaHCO3) and incubating at room temperature for 15 minutes on a rotating wheel. Cross-links were reversed by adding 20 μl of 5 M NaCl and incubating elutes at 65°C overnight. The next day, proteinase K (100 μg/ml final concentration) was added and samples incubated at 55°C for 1 hour. Samples were extracted with phenol:chloroform twice and ethanol precipitated overnight. Pellets were then washed with 70% ethanol, dried, resuspended in 50 μl of TE, and assayed by PCR. Thirty-five cycles of PCR were performed in 50 μl with 10 μl of immunoprecipitated material, 0.1 μM of primers, 0.2 mM dNTPs, and 1.0 unit of Taq DNA polymerase. Finally, PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.

Cell extracts were resolved by SDS PAGE on a 4-20% tris-glycine gel (Invitrogen). Proteins were transferred to Immobilon membranes (Millipore) at 200 mA for 2 hours. Membranes were blocked with Dulbecco's phosphate-buffered saline (PBS) 0.1% Tween-20 + 3% BSA. Primary antibody against specified antibodies was incubated with the membrane in PBS + 0.1% Tween-20 overnight at 4°C. Membranes were washed two times with PBS + 0.1% Tween-20 and incubated with HRP-conjugated secondary antibody for one hour. Presence of secondary antibody was detected by SuperSignal West Dura Extended Duration Substrate (Pierce). Luminescence was visualized on a Kodak 1D image station.

Supernatants from ACH2 and J1.1 cells were collected to test for the presence of virus on day 7 post 9AA treatment. Viral supernatants (10 μl) were incubated in a 96-well plate with reverse transcriptase (RT) reaction mixture containing 1× RT buffer (50 mM Tris-HCl, 1 mM DTT, 5 mM MgCl₂, 20 mM KCl), 0.1% Triton, poly(A) (1 U/ml), pd(T) (1 U/ml), and [³H]TTP. The mixture was incubated overnight at 37°C, and 10 ml of the reaction mix was spotted on a DEAE Filtermat paper, washed four times with 5% Na₂HPO₄, three times with water, and then dried completely. RT activity was measured in a Betaplate counter (Wallac, Gaithersburg, MD).

GST tagged proteins were purified as described previously [46]. GST-p21 (1 μg), GST-p21 (N) (1 μg), GST-p21 (C) (1 μg), or GST (1 μg) proteins were added to 2 mg of CEM extracts from various cell lines and rotated overnight at 4°C. The next day complexes were washed twice with TNE₁₅₀ + 0.1% NP-40 and once with TNE₅₀ + 0.1%NP-40. Complexes were run on 4–20% Tris-glycine gel. Western blots were performed with anti-cdk9 (Biodesign), anti-cyclin T, and anti-cdk2 (Santa Cruz) antibodies.

Five thousand cells were plated per well in a 96-well plate and the next day cells were treated with various concentrations of compounds (1, 10, 50 μM) or DMSO. Forty-eight hours later, 10 μl MTT reagent (50 mg/ml) was added to each well and plates incubated at 37°C for 2 hours. Next, 100 μl of DMSO was added to each well and plate was shaken for 15 minutes at room temperature. The assay was read at 570 nM.

We have previously observed that 9AA inhibits viral replication [46]. We were therefore interested if 9AA could specifically inhibit Tat dependent transcription. To test this hypothesis, CEM cells were transfected with HIV LTR-CAT with and without Tat. Two hours after transfection, the cells were treated with various concentrations of 9AA or 100 nM of flavopiridol as a positive control for transcription inhibition. Cells were harvested 48 hours post treatment and CAT assays performed. As expected, Flavopiridol treatment decreased viral transcription (Figure 1A, lane 7). Interestingly, 9AA treatment also decreased viral transcription in a dose dependent manner (lanes 3–6). Based on this data we can estimate that the IC₅₀ of viral LTR Tat activated transcription inhibition by 9AA is approximately 250 nM. These results suggest that the observed inhibition of viral replication could be due at least in part to the ability of 9AA to inhibit viral transcription.

To determine if viral transcription inhibition also occurred on a fully chromatinized promoter, we utilized TZM-bl cells, which have an integrated LTR-luciferase reporter construct. TZM-bl cells were transfected with Tat and treated with various concentrations of 9AA or 100 nM flavopiridol the following day. Luciferase assays revealed that 9AA inhibited LTR transcription in a dose dependent manner, with 1 μM showing greater than 50% inhibition and 2.5 μM showing complete transcriptional inhibition (Figure 1B). We were interested if 9AA derivatives would display similar transcriptional inhibition ability. Four commercially available 9AA derivates were purchased (Figure 1C). Compounds 2-aminoacridine (2AA) and 4-aminoacridine (4AA) differ from 9AA only in the location of the amino moiety, while acridine hydrochloride (AH) lacks the amino group. 4-aminoquinoline (4AQ) retains the amino moiety in the same position as 9AA, but lacks the third aromatic ring. Luciferase assays performed with these compounds indicated that the presence and location of the amino moiety is critical for the activity of 9AA, as both AH and 4AA did not inhibit HIV-1 transcription (Figure 1D). Surprisingly, cells treated with 2AA exhibited an increase in viral transcription at both 1 μM and 10 μM. 4AQ treated cells showed limited viral transcription inhibition at 1 μM and approximately 50% inhibition at 1 μM. These results demonstrate the importance of both the presence and location of the amino moiety for the activity of 9AA and also show that, while improving 9AA activity, the third aromatic ring is not essential. The inhibition demonstrated by 9AA is specific to this class of compounds, as other amino acridines did not display activity to the same extent. In addition, they demonstrate that 9AA inhibits HIV-1 transcription in a specific manner.

To determine the contribution of p53 to 9AA mediated viral inhibition, viral replication was assessed in two different HIV-1 infected cell lines, J1.1 and ACH2, which vary in their p53 status. While there has been a discordance in the literature regarding p53 status in CEM cells [48, 49], which is the parental cell line of ACH2; in our hands we have observed p53 DNA binding and activation of downstream signals, therefore p53 is functional in these cells. Conversely, Jurkat cells (parental cell line of J1.1) are well accepted as being p53 mutant cells, with R196X, T256A, D259G, and S260A mutations [49]. Both of these cells produce little virus in the absence of stimulation and therefore, supernatants were collected seven days after treatment to measure viral replication. RT assays indicated that viral replication was inhibited in both J1.1 and ACH2 cells at 5.0 μM 9AA (Figure 2A). Cell viability at seven days was unaffected in J1.1 cells, but decreased by approximately 50% in ACH2 cells (Figure 2B). These results suggest that the influence of 9AA on cellular viability may be dependent on the status of p53 and/or cell type dependent. We next examined the levels of p21WAF1 in J1.1 cells upon 9AA treatment. Figure 2C indicates that even though there is no detectable p53 in J1.1 cells, p21WAF1 is still induced with low levels of 9AA, which is similar to the results observed with ACH2 cells [44]. These results are interesting as they indicated that 9AA is capable of inhibiting viral replication in p53 mutant cells and that p21WAF1 is still induced by 9AA in the absence of functional p53.

As HIV-1 replicates more efficiently in proliferating cells and inhibiting cellular proliferation of uninfected cells could result in toxicity, we accessed whether 9AA treatment could be inhibiting cell proliferation. To this end, MTT assays were performed (Figure 3). MTT assays measure the reduction of the yellow tetrazolium MTT reagent (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide). Reduction occurs only by metabolically active cells and thus a reduced reading indicates decrease proliferation and possibly reduced viability. MTT assays were performed on uninfected and infected cells treated with either 9AA (panel A), 2AA (panel B), 4AA (panel C), or AH (panel D). Results indicate that up to 50 μM of 9AA had little to no effect on uninfected cells, but decreased cellular proliferation at 10 and 50 μM was observed for HIV-1 infected cells. It is important to note that low 9AA concentrations have been used consistently to observed viral inhibition, which is below the level at which cellular proliferation is being inhibited. Interestingly, all of the 3-ring 9AA derivatives had no effect on cellular proliferation in either uninfected or infected cells (panels B-D). Therefore, these results indicate that 9AA does not inhibit cellular proliferation in uninfected cells. These results also confirm that the inhibition of proliferation observed in the infected cells is dependent on the presence and location of the amino moiety of 9AA.

Recently, Guo et al. demonstrated downregulation of p110γ, the catalytic subunit of the phosphoinositide 3-kinase (PI3K) family upon 9AA treatment of renal carcinoma cells [47]. Follow-up studies indicated that AKT and mammalian target of rapamycin (mTOR) signaling were inhibited, which contributed to p110γ downregulation, and possibly p53 and NF-κB alterations. Therefore, we were interested to see if similar events were occurring upon 9AA treatment in HIV-1 infected cells. Surprisingly, we observed a varying increase in AKT phosphorylation at both T308 and S473 in either ACH2 and J1.1 cells following 9AA treatment (Figure 4), indicating that AKT was activated upon 9AA treatment. C81 HTLV-1 infected cells are used as a positive control as AKT is active and phosphorylated in HTLV-1 infected cells in a Tax dependent manner [50]. These phosphorylation events were especially pronounced in J1.1 cells as compared to uninfected Jurkat cells (compare lanes 10 and 13 to lanes 14 and 17). AKT is phosphorylated at both T308 and S473 to induce enzyme activation, with T308 being phosphorylated by PDK1 [51, 52] and S473 by mTOR [53‐56]. To confirm that AKT was activated we examined a downstream substrate of AKT, GSK3-β [57]. GSK3-β is phosphorylated by AKT on S9, inhibiting the activity of GSK3-β and regulating downstream events such as glycogen synthesis and β-catenin signaling [58, 59]. Total GSK3-β levels remained constant in Jurkat and J1.1 cells, but decreased upon 9AA treatment in ACH2 and CEM cells. Interestingly, p-GSK3-β levels were increased in Jurkat, J1.1 and ACH2 cells, while CEM cells display no p-GSK-3β with or without 9AA treatment. We next examined an upstream event of AKT activation, PDK1 phosphorylation at S241, which is an autophosphorylation event necessary for PDK1 activation [60]. PDK1 exhibited increased phosphorylation and thus activation in J1.1 cells, but only a modest increase in CEM, ACH2, and Jurkat cells. Finally, we examined p21WAF1 phosphorylation on S146, which has been shown to be phosphorylated by AKT, resulting in increased p21WAF1 stability [61]. Both ACH2 and J1.1 cells displayed p21WAF1 phosphorylation following 9AA treatment. Collectively these results indicate that AKT activity is increased following 9AA treatment, resulting in increased p21WAF1 phosphorylation, which could aid in stabilization of p21WAF1. This phosphorylation was observed in both p53 wildtype and p53 mutant cells.

As 9AA treatment resulted in increased p21WAF1 expression and stability, we were interested to determine if p21WAF1 could bind to the cyclin T/cdk9 complex since this is one of the key factors regulating HIV-1 promoter activity. To this end, we performed a GST-pulldown assay. GST, GST-p21, GST-p21 C-terminus [GST-p21 (C)], and GST-p21 N-terminus [GST-p21 (N)] were incubated with CEM cellular extracts overnight at 4°C. The next day, complexes were bound to glutathione sepharose beads, washed with TNE buffer, analyzed by SDS-PAGE, and western blotted with anti-cyclin T and cdk9 antibodies. Full length p21WAF1 as well as the C- and N-terminal regions of p21WAF1 were observed in complex with cyclin T (Figure 5A). The strongest binding was observed with the N-terminal region. Interestingly, the N-terminal region of p21WAF1 was also observed in complex with cdk9. This data is in agreement with other studies indicating that the N-terminal of p21WAF1 contains cyclin and cdk binding domains, whereas the C-terminal only contains a cyclin binding site [62, 63]. As a positive control, we also tested for cdk2 and p21WAF1 binding (Figure 5B). Therefore, these results suggest that p21WAF1 is a component of the p-TEFb complex.

Due to the observed binding of p21WAF1 with the pTEFb complex and the importance of cdk9 in HIV-1 transcription, we performed ChIP experiments to determine if cdk9 binding at the LTR was altered upon 9AA treatment. ACH2 cells were treated with either DMSO or 9AA and collected 48 hours later. Results indicated that 9AA treatment results in a dramatic loss of cdk9 from the LTR (Figure 6). In addition, we observed a reduction in the levels of histone H3-phospho-S10 levels upon 9AA treatment. These results suggest that 9AA treatment alters LTR binding proteins to induce transcriptional inhibition.