Despite its tight control in normal T cells, NF-κB is constitutively activated in both HTLV-I-transformed T-cell lines and freshly isolated ATL cells suggesting that activation of NF-κB is an important part of the oncogenic mechanism of HTLV-I. This pathologic action may largely rely on the viral transforming protein Tax, at least for many of the cell lines to date that are isolated for in vitro analysis and not necessarily are ATL samples, which also up-regulates the expressions and activities of cyclin E/CDK2 which is important in cell cycle transition from G₁ to S phase.

Most importantly, IKK has been established as a cellular target of Tax and an essential component in Tax-mediated NF-κB signaling in both canonical and non-canonical pathways. Therefore, we reasoned that the specific targeting of both the NF-κB signaling and cell cycle regulators with drugs might provide better insights into how to inhibit HTLV-1 infected cells. We sought to identify the targets of a range of NF-κB and CDK inhibitors in HTLV-1 infected and uninfected cells by culturing MT-2, MT-4, C8166, c10/MJ and uninfected CEM and Jurkat T-cells (0.5 × 10⁶ cells/well) in media with inhibitor concentrations ranging from 0, 0.01, 0.1, 1, and 10 μM. Cells were treated for 48 hours and the level of growth inhibition was estimated using trypan blue method. Results from 35 drugs that inhibit various CDKs and IKKs are shown in Table 1 where a number of drugs inhibited HTLV-1 infected cells much more efficiently than uninfected cells. Among the top two candidates that inhibited HTLV-1 infected cells were BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) and Purvalanol A. BMS-345541 is a selective inhibitor of IKKβ at IC₅₀ of 0.3 μM and to a lesser extent an inhibitor of IKKα at IC₅₀ of 4 μM 6465. All drugs were further tested at 10 μM concentration to effectively compare these different classes of inhibitors against one another. In Table 1, they are ranked as high, moderate, and poor inhibitors and the reported activities of these molecules against variety of CDKs and IKKs are indicated in the right-hand column. Collectively, these data indicate that initial cell based survival screening assays may be an effective tool in isolating drugs that are more selective against HTLV-1 infected cells as compared to control uninfected cells.

Resistance to cell apoptosis is one of the mechanisms that is important and is also required for the immortalization of T cells 6366. NF-κB signaling pathway is the survival pathway activated by HTLV-1 in order to keep the host cell active 6768. BMS-345541 targets IKKβ subunit which is responsible for activation of the NF-κB pathway 6465. To determine whether BMS-345541 can inhibit NF-κB pathway and induce apoptosis in HTLV-1 infected cells, we analyzed the level of apoptotic markers such as caspase-3 and PARP in both infected and uninfected cells. Caspase-3 is a member of cysteine protease and plays a key role in apoptosis 69. When apoptosis is activated, the inactive pro-caspase-3 is processed into active large (17 kD) and small (12 kD) subunits 70. PARP, poly(ADP-ribose) polymerase, is also an apoptosis marker that is cleaved from precursor form (116 kD) into active form (85 kD) by active caspase-3 during apoptosis 717273. Results in Figure 2A are Western blots that show titration of BMS-345541 in two infected and one uninfected cells. Samples were treated for 48 hours and extracts were made for Western blotting. The top panel shows the caspase Western and a gradual increase of p17 form in MT-2 cells as well as C8166 cells in concentrations between 0.5 and 1.0 μM. There was no change in the actin levels in any of the samples treated. Panel B shows the results of the Annexin V staining where live cells are represented at the bottom right corner box in each panel. All three samples were treated with 0.1 μM of BMS-345541 and stained for the presence of live and apoptotic cells. Interestingly both MT-2 and C8166 cells showed presence of few live cells as compared to CEM cells when treated with BMS-345541. Collectively, these data indicate that low concentrations of IKKβ inhibitor can apoptosis HTLV-1 cells much more efficiently as compared to uninfected cells.

We subsequently asked if IκB or p65 levels could be altered in drug treated infected and uninfected cells. We therefore Western blotted our drug treated cells with antibodies against IκB, phospho IκB (ser 32), p65, phospho p65 (ser 536), p50, p52, Tax and actin. Both ser 32 of IκB and ser 536 of p65 are phosphorylated by IKKβ in vivo. Results of such an experiment are shown in Figure 3 where IκB levels essentially stayed the same in all three cell lines except for a drop in C8166 cells at 5.0 μM. We have previously observed that cells, irrespective of infection, treated with BMS-345541 at higher does (i.e., 10.0 μM) are toxic and show non-specific activation of apoptotic machinery (data not shown). There was also no change in levels of p65 although a slight increase in C8166 cells was observed at higher concentrations. A more interesting set of results were observed with phosphor-IκB and phosphor-p65 blots. MT-2 cells treated with BMS-345541 showed a reduction of both phosphor-IκB and phosphor-p65 levels at 0.5 μM. Similar results were also seen in C8166 cells. Very little phosphor-IκB and phosphor-p65 were observed in CEM cells (or other control Jurkat cells, data not shown). P50, p52 levels were unchanged with various drug concentrations and Tax levels were not decreased at 0.5 or 1.0 μM concentration of the drug. No changes were seen in the actin levels in any of the treated cells. Collectively, these results indicate that inhibition of IKKβ in HTLV-1 infected cells by BMS-345541 affects phosphorylation of both IκB and p65 molecules, both of which may be the hallmarks of NF-κB activation in HTLV-1 infected cells.

We have previously shown that cyclin E/CDK2 kinase activity is de-regulated in HTLV-1 infected cells and these cells are especially susceptible to Purvalanol A treatment 62. Moreover, Purvalanol A, which is a purine analog that competes with the ATP binding site in CDKs, has been shown to inhibit cyclin E/CDK2 and cyclin A/CDK2 kinase activities with an IC₅₀ of 0.035 and 0.07 μM, respectively 74757677. We therefore treated both infected and uninfected cells for 48 hours with Purvalanol A and Western blotted for caspase-3 and PARP molecules. Results in Figure 4A show that the caspase-3 p17 molecule was present in infected cells treated with 0.1 and 0.5 μM of Purvalanol A (lanes 3 and 7). This was important since Purvalanol A did not significantly activate caspase-3 in CEM (lanes 11–15) or Jurkat cells (data not shown). There were no changes in actin (bottom panel), cyclin E, or cyclin A expression levels when treated with Purvalanol A (Panel B). Therefore Purvalanol A treatment induces caspase-3 activation in HTLV-1 infected, and not in uninfected cells. This is consistent with our previous results where Purvalanol A treatment of infected cells inhibited cyclin E/CDK2 complex activity in HTLV-1 infected cells, inhibited transcription of the LTR promoter and promoted apoptosis 62. Along these lines, we also assayed for changes in cell cycle progression and apoptosis in these cells using FACS analysis. Results in Figure 5 show the titration of Purvalanol A for all three cell types. Interestingly, significant apoptosis appeared in infected cells treated at 1.0 and 5.0 μM concentrations.

We next decided to use both drugs in a viral replication assay in MT-2 cells. MT-2 cells normally produce low levels of infectious HTLV-1 virions that could be detected in the supernatant using p19 gag ELISA. However, treatment of these cells with TNF can produce at least 1–2 log more virus that is shed into the supernatant. We therefore treated MT-2 cells with TNF for 2 hours and subsequently treated them with BMS-345541 alone (0.1 μM), Purvalanol A alone (0.5 μM), or a combination of both drugs. Results in Figure 6A show that, as compared to untreated cells, TNF treatment induced high amounts of p19 gag in the supernatant (lanes 1 and 2). Both drugs alone reduced p19 levels to some degree however; the best inhibition was seen with the combination of both drugs where NF-κB and CDK pathways were targeted in these cells. Similar results were also obtained in 293 cells transfected with ACH full-length infectious clone, where a combination of both drugs inhibited p19 expression as compared to when treated with one drug alone (Panel B). Collectively, these results imply that low concentrations of NF-κB and CDK inhibitors that normally do not cause cell death in uninfected cells are effective inhibitors against HTLV-1 infected cells.

MT-2, MT-4, C8166, and C10/MJ were all obtained from NIH AIDS Research & Reference Reagent Program. They are all HTLV-1 infected cell lines and some including C8166 contain defective viruses but still express Tax. MT-2 cells carry multiple copies of the HTLV-1 cosmopolitan subtype and normally produce some full length infectious HTLV-1 particles in the absence of any inducer [157]. MT-4 cells are established from the human T cells isolated from a patient with adult T-cell leukemia. CEM and Jurkat cells are the uninfected control T lymphocyte cell lines. All cell lines were cultured at 37°C up to 1 × 10⁵ cells per ml in RPMI 1640 medium containing fetal bovine serum (10%), streptomycin, penicillin antibiotics (1%) and L-Glutamine (1%) (Gibco/BRL). The CDK inhibitors used were: Aloisine A (270-385-M001), Alsterpaullone (270-275-M001), Bohemine (270-390-M001), CGP74514A (270-391-M001), Compound 52 (270-248-M001), 9-cyanopaullone (270-282-M001), 6-dimethylaminopurine (480-050-M100), indirubin-3'-monoxime (270-271-M001), 5-iodo-indirubin-3'-monoxime (270-424-M001), N-6-(Δ2-Isopentenyl)-adenine (350-034-M100), Kenpaullone (270-274-M001), Olomoucine (350-013-M005), N9-isopropylolomoucine (270-397-M001), Purvalanol A (270-246-M001), (R)-Roscovitine (350-251-M001), (S)-Roscovitine (350-293-M001) were purchased from Alexis Inc. and 6-benzyloxypurine (387606), 2,6-diaminopurine (247847), 2,6-dichloropurine (D73103), Flavone (F2003) were purchase from Sigma-aldrich Inc. Indirubin-3'-monoxime-5-sulfonic acid (402088), iso-olomoucine (495622), WHI-P180 (681500) were purchased from Calbiochem Inc. The CDK inhibitor, flavopiridol was a kind gift from Dr. Ajit Kumar at the GWUMC. The NF-κB inhibitors included BMS-345541 (401480), SC-514 (401479) were purchased from Calbiochem Inc. and 5-Aminosalicylic acid (430-110-G005), BAY 11-7082 (270-219-M010), BAY 11-7085 (270-220-M010), caffeic acid phenylethyl ester (270-244-M010), diethylmaleate (280-017-G005), Parthenolide (350-258-M025), pyrrolidinedithiocarbamic acid (400-002-G005) were purchased from Alexis Inc. and QNZ (6-amino-4-(4-phenoxyphenylethylamino)quinazoline (EI-352), Wedelolactone (EI-316) were purchased from Biomol Inc. All inhibitors were prepared in 10 mM stock solution. 2,6-Dichloropurine and diethylmaleate were dissolved in ethanol; Flavone was dissolved in acetone; Flavopiridol and pyrrolidinedithiocarbamic acid were dissolved in water; 5-aminosalicylic acid was dissolved in hydrochloric acid. The other twenty-nine inhibitors were all dissolved in DMSO.

HTLV-1 infected cells and uninfected cells were treated with thirty-five inhibitors at four concentrations including 0.01, 0.1, 1, and 10 μM. Forty-eight hours after treatment, cytotoxicity was primarily determined by the color of media and cell viability by trypan blue exclusion. Cells were counted for the number of living cells every 24–48 hrs. Subsequent focusing experiments used flow data to check for viability and apoptosis.

Cytoplasmic extracts were prepared according to the following procedure. Briefly, cells were collected and washed with PBS once and then once with 80 μl of ice-cold buffer A (Tris-HCl (pH 7.4, 10 mM), MgCl₂ (1.5 mM), KCl (10 mM), DTT (1 mM), 0.4% NP-40, phenylmethylsulfonyl fluoride (1 mM), aprotinin (10 μg/ml), pepstatin (1 μM), NaF (50 mM), and Na₃ VO₄ (1 mM)). Cells were lysed in 80 μl of buffer A by gently passing the cell suspension through a 28-gauge needle. The cytoplasmic extracts were collected by pelleting for 8 sec in an Eppendorf microcentrifuge and the supernatant was collected. The protein concentration for each preparation was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA).

Reaction mixtures (24 μl) contained (final concentrations) 40 mM β-glycerophosphate, pH 7.4, 7.5 mM MgCl2, 7.5 mM EGTA, 5% glycerol, [γ³²-P]ATP (0.2 mM, 1 μCi), 50 mM NaF, 1 mM orthovanadate, and 0.1% (v/v) β-mercaptoethanol. Phosphorylation reactions were performed with 2 mg of cytoplasmic extract immunoprecipitated with appropriate antibody and washed in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM EDTA, 50 mM NaF, 0.2 mM Na₃ VO₄ , 1 mM DTT, 0.5% NP-40 and protease inhibitors (Protease inhibitor cocktail tablets, Boehringer Mannheim, one tablet per 50 ml) or with 1 μg of purified recombinant GST-IκBα at 37°C for 1 hour. Reactions were stopped by adding 1 volume of Laemmli sample buffer containing 5% β-mercaptoethanol and ran on a 4–20% SDS/PAGE. Gels were autoradiographed and bands were counted using a Molecular Dynamics PhosphorImager software.

Total cellular extracts (20 μg) were separated by a 4–20% Tris-glycine gel then transferred to a PVDF membrane (Immobilon-P transfer membranes; Millipore Corp.) Following the transfer, the blots were blocked with 5% non-fat dry milk in PBS + 0.1% Tween-20 for 2 hr and washed three times with PBS + 0.1% Tween-20 at 4°C. The blots were then probed with 1:200 dilution of primary antibody against caspase-3 (H-277; Santa Cruz Biotechnology, sc-7148), PARP (H-250; Santa Cruz Biotechnology, sc-7150), CDK2 (M2; Santa Cruz Biotechnology, sc-163), cyclin A (H-432; Santa Cruz Biotechnology, sc-751), cyclin E (C-19; Santa Cruz Biotechnology, sc-198), and actin (c-11; Santa Cruz Biotechnology, sc-1615). The blots were then probed with a 1:750 dilution of secondary antibodies for 1 h at 4°C, followed by washes in PBS + 0.1% Tween-20 and detected using SuperSignal West Dura Extended Duration Substrate Kit (Pierce, Rockford, IL, USA).

MT-2 cells (HTLV-1 infected) were treated with TNF-α (10 ng/ml) for 2 h, washed, and subsequently treated with a specific NF-kB or CDK inhibitor. Media from MT-2 infected cells were centrifuged to pellet the cells, and supernatants were collected and diluted to 1:100 to 1:1,000 in RPMI 1640 prior to ELISA. Seven days later samples were collected and used for p19 gag ELISA. The HTLV-1 p19 core antigen ELISA kit was from Retro-Tek (Cellular Products) and RT/PCR using HTLV-1 specific Tax primers (data not shown).

Log phase 293 cells were transfected with 20 μg of ACH.pcTax (wild type HTLV-1 clone) using electroporation method. After transfection, the cells were cultured in complete medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 μg of penicillin/ml, and 50 U of streptomycin/ml. Cell culture supernatants were collected at 4 days post-transfection, and virus particle production was monitored by p19 ELISA as described above. Drug treatment was 6 hrs after transfection of the 293 cells for a total of 150 hrs.

For cell cycle analysis, cells treated with or without drugs were collected by low speed centrifugation and washed with PBS without Ca²⁺ and Mg²⁺ and then fixed with 70% ethanol. For fluorescence-activated cell sorting (FACS) analysis, cells were stained with a mixture of propidium iodide buffer (PBS with Ca²⁺ and Mg²⁺, 10 μg/ml RNase A, 0.1% Nonidet P-40, and 50 μg/ml propidium iodide) followed by cell sorting analysis. The acquired FACS data were analyzed by ModFit LT software (Verity Software House, Inc.). Cells were washed twice with cold PBS without Ca²⁺ and Mg²⁺, resuspended in 1× binding buffer (10 mM HEPES-NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂ ) and 5 μl of propidium iodide/10⁵ cells, and incubated at room temperature for 15 min. Cells were acquired and analyzed using CELLQuest software (BD Biosciences).

Detection of apoptosis through annexin V and PI staining was done according to the manufacturers protocol (BD Pharmingen, San Jose, CA). In brief, cells were washed three times in PBS and re-suspended in binding buffer at 1 × 106 cells/ml. An aliquot of 1 × 105 cells was stained with annexin V-FITC and PI for 15 minutes at room temperature. Analysis was performed on a BD FacsCalibur flow cytometer. Cells were considered to be early apoptotic if they exhibited staining for annexin V, but not PI. The double positive population was considered to be in the late stage of apoptosis.