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Journal of Virology, August 2000, p. 7270-7283, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Overexpression of p21waf1 in Human T-Cell Lymphotropic Virus Type 1-Infected Cells and Its Association with Cyclin A/cdk2

Cynthia de la Fuente,1 Francisco Santiago,1 Siew Yen Chong,1 Longwen Deng,1 Todd Mayhood,1 Peng Fu,1 Dana Stein,2 Thomas Denny,2 Frederick Coffman,3 Nazli Azimi,4 Renaud Mahieux,5 and Fatah Kashanchi1,*

Department of Biochemistry and Molecular Biology,1 Department of Pediatrics,2 and Department of Pathology,3 University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103; National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208744; and Unite d'Oncologie Virale, Department SIDA-Retrovirus, Institut Pasteur, 75724 Paris, France5

Received 30 November 1999/Accepted 19 May 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G1, S, or G2/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G1 phase prior to gamma-irradiation, arrested in G1 and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G1/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+ T cells are the main target of infection and transformation by the HTLV-1 virion. T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax, a 40-kDa protein (353 amino acids), functions to transactivate viral and cellular promoters, causing uncontrolled cellular proliferation. Tax interacts with multiple transcriptional factors, such as cyclic AMP responsive element (CREB), CREB-binding protein, NF-kappa B family members, TATA-binding protein (TBP), and TFIIA. Tax also stimulates the transcription of many cellular genes, including those encoding interleukin 2 (IL-2), IL-2Ralpha , PCNA, and PTHrP as well as c-fos and the c-sis proto-oncogene (11).

Cell cycle regulation is accomplished by modulating the activity of cyclin-dependent kinases (cdk's) and their catalytic subunit, cyclins. This is usually achieved by the phosphorylation and dephosphorylation of the enzyme complex, by the reduction of cyclin levels (either transcriptionally or by proteolytic degradation), and by binding to cdk inhibitors (CKIs) (7). One such CKI, p21/waf1/cip1/sdi1, has been the source of concentrated study since its discovery in 1992 as part of the cyclin D1/cdk4/PCNA complex (4). p21/waf1 has been characterized as a p53-transactivated gene (waf1), as a cdk-interacting protein (CIP1), and as a DNA inhibitor in senescent human fibroblasts (Sdi1) (29). p21/waf1 overexpression has been seen to inhibit two critical checkpoints in the cell cycle, namely G1 and G2, through both p53-dependent and -independent pathways (17).

While p21/waf1 can effectively inhibit cyclin/cdk's involved in the G1 and S phases of the cell cycle, it is able to bind to a wide variety of these holoenzymes (4). The major targets of p21/waf1 include cyclin D/cdk4/PCNA, cyclin B1/cdc2/PCNA, cyclin E/cdk2/PCNA, and cyclin A/cdk2/PCNA (4, 7, 16, 28, 29, 35). The effect of p21/waf1 on various in vitro-purified cdk's has also been explored. p21/waf1 effectively inhibits cdk2, cdk3, cdk4, and cdk6 kinases (Ki, 0.5 to 15 nM) but is much less effective toward cdc2/cyclin B (Ki, approximately 400 nM) and cdk5/p35 (Ki, >2 µM) and does not associate with cdk7/cyclin H (9). Thus, p21/waf1 is not a universal inhibitor of cdk's but displays selectivity for G1/S cyclin/cdk complexes. Association of p21/waf1 with cdk's is greatly enhanced by cyclin binding. Reconstruction experiments using purified components indicate that multiple molecules of p21/waf1 can associate with cdk/cyclin complexes, and inactive complexes containing more than one molecule of p21/waf1 per cyclin/cdk holoenzyme have been described (9, 35). In general agreement with its inhibitory role, mice lacking p21/waf1 (p21-/- embryonic fibroblasts) are significantly deficient in their ability to arrest in G1 in response to DNA damage. p21-/- cells also exhibit a significant growth alteration in vitro, achieving a saturation density as high as that observed in p53-/- cells (6).

While p21/waf1 has been seen as a cell cycle inhibitor, it has also been proposed to play a role as an assembly factor. LaBaer et al. (15), like the authors of other reports (12, 18), found that cyclin D-cdk4 complexes are not efficiently assembled in cells or in vitro. However, in the presence of p21/waf1, the amount of complexed cyclin D/cdk4 increases proportionately to p21/waf1 levels. By using a purified system, this effect can be shown to be through a direct interaction of p21/waf1 with the cyclin/cdk and to require both the N-terminal cyclin and cdk-binding sites on p21/waf1. Although p21/waf1 increased the rate of cyclin D-cdk4 association, the primary effect seemed to be stabilizing the interaction and preventing rapid dissociation of the holoenzyme. Interestingly, the authors reported that p21/waf1, but not other members of the p21 family, can stimulate cyclin D1-cdk4 activity when present at low concentrations. Thus, in agreement with previous results (35), the study suggested that p21/waf1 can be both an activator and an inhibitor of cyclin D1-cdk4 activity, depending on its relative abundance.

A second, perhaps more provocative, observation was made when the cellular localization of transfected complexes was monitored (15). Evidence was found that after promoting cyclin D1/cdk4 assembly, p21/waf1 targeted the complex to the nucleus. This led to the suggestion that p21/waf1, and other members of the p21 family, may direct cyclin D1-cdk4 complexes to different targets, e.g., different nuclear structures or different substrates, and that these could be determined by the divergent C-terminal domains of p21, p27, and p57 proteins. This would add another cyclin/cdk regulatory function to the p21/waf1 arsenal (4).

Paradoxically, HTLV-1-infected T cells show high levels of tumor suppressor protein p53 (5, 19, 24, 26) as well as p21/waf1 protein (2, 5). It is speculated that the high levels of p21/waf1 are related to p53 levels. In agreement with others (2, 5), we find here that p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as patient samples. The p21/waf1 protein is associated with cyclin A/cdk2 and not with other known G1, S, or G2/M cyclins. Functionally, the association of p21/waf1 with cyclin A/cdk2 decreases the histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affects the phosphorylation of other substrates such as the C terminus of Rb protein. Down regulation of Rb function is most prominent at the C-terminal domain of Rb, where E2F binding has been observed. To elucidate the in vivo function of the p21/cyclin A/cdk2 complex, we used elutriated purified cell cycle fractions and a stress signal, such as gamma-irradiation, and found that the complex is functionally important for stopping the infected host cell from entering the next phase of the cell cycle. This may be an important mechanism for a cancer-causing virus, such as HTLV-1, to ensure host survival upon DNA damage.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cell culture. C81 is an HTLV-1-infected T-cell line, and CEM (12D7) is an uninfected human T-cell line established from patients with T-cell leukemia (28). Chronic T-lymphocytic leukemia (CTLL) is a mouse T-cell line that is IL-2 dependent; however, upon transfection and selection of the Tax gene, these cells became IL-2 independent (10). Here they are designated as CTLL (WT), and CTLL cells transfected with the M47 Tax mutant are designated CTLL (703). The M47 Tax mutant has two amino acid substitutions, at positions 319 and 320 of the Tax protein (10). All cultures were grown in RPMI 1640 containing 10% fetal bovine serum (FBS), 1% streptomycin, penicillin antibiotics, and 1% L-glutamine (Quality Biological) and were incubated in a 5% CO2 incubator at 37°C.

Cell extract preparations and immunoprecipitation. Cells were initially centrifuged at 4°C for 15 min at 3,000 rpm in a Sorvall RT 6,000 centrifuge. Pelleted cells were washed twice with 25 ml of Dulbecco's phosphate-buffered saline without calcium or magnesium (D-PBS without Ca2+/Mg2+; Quality Biological) and were centrifuged again. Cell pellets were resuspended in lysis buffer containing 50 mM Tris-Cl, pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40 (NP-40), 50 mM NaF, 0.2 mM Na3VO4 (phosphotyrosine phosphatase inhibitor), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT). Cell lysates were incubated on ice for 15 min with occasional mixing. Cell lysates were transferred to 1.5-ml Eppendorf tubes and were centrifuged in an Eppendorf microcentrifuge at 4°C and 12,000 rpm for 10 min. Supernatants were extracted, and protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad, Hercules, Calif.).

To prepare nuclear extracts, cells were collected and washed once with phosphate-buffered saline (PBS) without Ca2+/Mg2+ and once with 200 µl of ice-cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT). Cells were lysed in 200 µl of buffer A by gently passing the cell suspension through a 28-gauge needle. This procedure was carried out with the tube containing the cells submerged in ice. Nuclei were collected by pelleting for 30 s in an Eppendorf microcentrifuge, and the supernatant was removed and kept for further analysis. Crude nuclei were extracted with ice-cold buffer C (20 mM HEPES [pH 7.9], 25% [vol/vol] glycerol, 420 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF), 60 µl per 100 µl of cell pellet, for at least 15 min on ice. An equal volume of buffer D (20 mM HEPES [pH 7.9], 20% [vol/vol] glycerol, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) was added, and the mixture was centrifuged for at least 10 min at 4°C. Supernatants were collected, and their volumes were measured. The protein concentration for each preparation was determined by using the Bio-Rad protein assay kit.

For immunoprecipitations, 1.5 mg of proteins from cell extracts was mixed with lysis buffer to bring the volume up to 1 ml. Fifty microliters of the appropriate antibody (Ab) (200 µg/ml) was added, and the extract was incubated overnight at 4°C on a rotator. One hundred microliters of 30% slurry (protein G- and protein A-agarose beads in TNE 50 + 0.1% NP-40 [100 mM Tris, pH 8.0; 50 mM NaCl; 1 mM EDTA, 0.1% Nonidet P-40]) was added to the mixture and incubated for 3 h at 4°C. Immune complexes bound to beads were pelleted by centrifugation at 12,000 rpm in an Eppendorf microcentrifuge for 5 min at 4°C, and the beads were washed three times with TNE 150 + 1% NP-40 (100 mM Tris [pH 8.0] 150 mM NaCl, 1 mM EDTA, and 1% NP-40). Samples were treated with 2× Tris-glycine-sodium dodecyl sulfate (SDS) sample buffer, vortexed, heated at 95°C for 5 min, placed on ice for 1 min, and further centrifuged at 14,000 rpm for 2 min. Twenty microliters of supernatant was loaded onto a Tris-glycine-4 to 20% polyacrylamide gel (Novex), with 1 µl of Rainbow 14C-methylated protein molecular weight (MW) marker (Amersham). Lanes designated as "input" contained appropriate amounts of the starting cell extract, which served as a positive control for each Western blot.

Antibodies and Western blots. Anti-p21/waf1 (C-19) rabbit or goat polyclonal immunoglobulin G (IgG) Ab (Santa Cruz) were used for immunoprecipitations and Western blotting. These Abs were specific for the carboxy terminus of human p21/waf1 and were rat, mouse, and human reactive. The alpha -cyclin A (H-432) rabbit polyclonal IgG Ab (Santa Cruz) was used for Western blotting and immunoprecipitations. The alpha -cdk2 (H-298) rabbit polyclonal IgG Ab (Santa Cruz) was used in Western blotting. The alpha -TBP (N-12; Santa Cruz) was used as an indicator of the amount of protein in each lane. Normally, 50 ml of each antibody was used in 10 ml of TNE buffer for each Western blot.

Protein transfers were carried out overnight at 80 mA, at room temperature, onto a polyvinylidene difluoride (PVDF) membrane (Millipore). During the last 30 min of the transfer, the amperage was increased to 240 mA. Membranes were blocked with 5% milk solution (dry milk and TNE 50-0.1% NP-40) at 4°C for 3 h, with gentle rocking. Membranes were washed once with TNE 50-0.1% NP-40 and were incubated with primary Ab overnight at 4°C. The next day, membranes were washed once and protein G labeled with 125I (50 µl/10 ml of solution; Amersham) was placed on membranes for 2 h with gentle rocking. Membranes were finally washed three times with TNE 50-0.1% NP-40, were air dried, and were placed in a PhosphorImager cassette overnight and scanned the next day.

Gamma-irradiation. Cell cultures were serum starved (1% FBS) for 3 days. Gamma-irradiation was performed on the third day by using a J. L. Shepherd and Associates Mark I Irradiator machine (model 68A, utilizing a pair of 6,000-Ci 137Cs sources in type 6810 capsules). Cells were irradiated at 770 rad for a period of 1.04 min. For serum-starved cells, immediately after irradiation, FBS was added to each flask to 10%, and samples were cultured and processed at appropriate time points.

To prepare cells for flow cytometry analysis, samples were centrifuged in a Sorvall RT 6,000 centrifuge at 3,000 rpm at room temperature for 5 min. Cell pellets were washed twice with D-PBS without Ca2+/Mg2+ and were centrifuged. Cell pellets were then resuspended in 70% ethanol and kept at 4°C. Once all samples were collected from various time points, they were centrifuged at 3,500 rpm at 4°C for 6 min. Cell pellets were rehydrated on ice for 15 min with D-PBS without Ca2+/Mg2+. Cells were pelleted and resuspended in 1 ml of propidium iodide (PI) staining solution (50 µg of PI per ml, 10 µg of RNase per ml, 0.1% NP-40, D-PBS with Ca2+/Mg2+). Samples were then subjected to flow cytometry by using a Becton Dickinson FACSCaliber with an argon laser (488 nm). Acquisition was carried out by using CELLQuest software (Becton Dickinson), and analyses were performed with ModFit LT software (Verity Software House, Inc.).

Kinase assays. Immunoprecipitates (IPs) were allowed to incubate for 2 h with protein G- and protein A-agarose beads, as described above. IPs were then washed and centrifuged twice with lysis buffer and twice with kinase buffer (50 mM HEPES, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 1 mM PMSF, 50 µM NaF, 0.2 mM Na3VO4, leupeptin, aprotinin, and pepstatin [or one complete tablet of protease cocktail inhibitor/50 ml of buffer; Boehringer Mannheim]). Equal amounts of beads and complex were allocated for each kinase reaction. A kinase reaction mixture was made up containing 10 µM ATP, 2.5 µCi of [gamma -32P]ATP (Amersham) per 50 µl, 1 mg of the substrate per ml, and kinase buffer. Beaded immune complexes were incubated with 40 µl of kinase reaction mixture for 30 min at 37°C and were mixed every 5 min. Reactions were terminated by adding 10 µl of 2× SDS sample buffer, and reaction mixtures were heated at 95°C for 3 min and were centrifuged at 3,000 rpm for 3 min. Twenty microliters of supernatant was loaded and separated on an SDS-Tris-glycine-4 to 20% polyacrylamide gel. Gels were dried for 2 h and were exposed to a PhosphorImager cassette.

For the peptide kinase assays, the following procedure was performed. Whole-cell lysates were prepared from CEM and C81 cells in IP buffer. Lysates (2 mg) were treated with protein A-Sepharose CL-4B (Sigma, St. Louis, Mo.) to avoid nonspecific binding and were centrifuged. The supernatants were incubated with Abs against cyclin A and control rabbit IgG and then with protein A- and G-agarose beads. After centrifugation, the immunoprecipitates were washed five times with IP buffer. The immunopurified cyclins and substrates were incubated at 30°C for 30 min in R buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 4.5 mM 2-mercaptoethanol, 1 mM EGTA) that contained 50 µM ATP and 10 mCi of [gamma -32P]ATP (6,000 Ci/mmol; Amersham, Little Chalfont, Buckinghamshire, United Kingdom) in a final volume of 25 ml. The supernatants were separated by thin-layer chromatography on cellulose plates with solvent A (n-butanol-ethanol-25% ammonia-water-chloroform, 4:5:9:2 by volume) as a mobile phase. Phosphorylated peptides were detected with a Bio-Image Analyzer (BAS2000; Fuji, Tokyo, Japan). Alternatively, peptides were trapped on P81 papers (Whatman Co., Ltd., Maidstone, United Kingdom) which were washed six times with 75 mM NaH3PO4 and then monitored for radioactivity in a liquid scintillation counter.

Centrifugal elutriation. CEM and C81 cultures were grown up and harvested at log phase of growth (109 cells/ml). Cultures were washed once with D-PBS without Ca2+/Mg2+ and 3 mM EDTA, pH 7.5 (elutriation buffer), and were resuspended in the same buffer. A Beckman J6-MI elutriation rotor was washed with 70% ethanol followed by elutriation buffer; then the rotor was brought to 2,700 rpm and 18°C. Cells were loaded at 18 ml/min, and 150-ml fractions were collected at flow rates of 23, 27, 30, 38, 45, 50, and 70 ml/min. Fractions were washed once, centrifuged, resuspended with D-PBS with Ca2+/Mg2+, and divided equally for zero-time and gamma-irradiated 24-h sample collections. The zero-time-fraction aliquots were processed and placed in 70% ethanol for fluorescence-activated cell sorter (FACS) analysis. The gamma-irradiated 24-h samples were placed in complete medium, gamma-irradiated with 770 rads, and cultured for 24 h at 37°C. All samples were then processed (as described above) for FACS analysis by using PI staining.

Transfection and luciferase assay. Various 5'-deletion p21/waf1 constructs, generously donated by Wafik El-Deiry (30, 34), were used to transfect mid-log-phase Jurkat cells that had been passaged no more than 10 times. The transfection was performed with Superfect reagent (QIAGEN). Three micrograms of the reporter plasmid was mixed with various concentrations of pCTax construct (0, 0.5, 1, and 2 µg). Cells were harvested the next day, and luciferase assays were performed by using the Promega Dual luciferase kit according to the manufacturer's recommendations. A control plasmid, TK-RL reporter construct, was used to normalize counts to activity in these experiments. Titrations for each construct were done at least twice. The M47 mutant was also used in some experiments to check for a specific activation of the p21 promoter.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Overexpression of p21/waf1 protein in HTLV-1-infected cells. The hallmark of most cancers is uncontrolled cellular proliferation, an event that would, under normal circumstances, be controlled by cell cycle checkpoint proteins such as p53 and its downstream mediator, p21/waf1. HTLV-1-infected cells, however, show abnormally high levels of p53 (5, 19, 24, 26) and p21/waf1 proteins (2, 5). It has previously been shown that wild-type (WT) p53 is stabilized and transcriptionally inactive in HTLV-1-transformed cells, and Tax plays a role in both the stabilization and inactivation of p53 through a mechanism involving the phosphorylation of the first 52 amino acids of p53 (23). Although p53 is an important cell cycle regulatory protein, its downstream activator, p21/waf1, is considered primarily responsible for inhibiting cells from progressing through various phases of the cell cycle. However, to date there is no clear understanding of why p21/waf1 levels are upregulated in HTLV-1-infected cells and how this complex would regulate the infected host cell cycle machinery. In an attempt to further clarify this point, we investigated the role of p21/waf1 in HTLV-1-infected cells.

We first investigated the amount of p21/waf1 in HTLV-1-infected and uninfected T cells. The results of such an experiment are shown in Fig. 1A, where equal amounts of whole-cell lysates were loaded onto the Tris-glycine-4 to 20% polyacrylamide gel, transferred to a PVDF membrane, and Western blotted with anti-p21/waf1 rabbit polyclonal Ab. As shown in Fig. 1A, C81 (lanes 3), in contrast to CEM (lanes 4), demonstrated an increase in p21/waf1 protein levels. This observation is consistent with previous reports of increased p21/waf1 in HTLV-1-infected T cells (C81, MT-4, MT-2, HUT102, OCH, and our similar unpublished Western blot results) and in Tax1-immortalized T-cell lines (2, 5). Interestingly, when using mouse Tax+ clone CTLL (WT) and a CREB mutant Tax clone, CTLL (703), we observed an increase of p21/waf1 protein expression only in CTLL (WT) and not CTLL (703) cells (Fig. 1A, lanes 1 and 2). Similar levels of Tax were expressed in both C81 and CTLL (WT) cells (Fig. 1, Tax Western blot). Taken together, these data suggest that HTLV-1 and/or Tax protein may be responsible for the up regulation of the p21/waf1 protein.


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  		FIG. 1.   Overexpression of p21/waf1 protein in HTLV-1-infected cells. (A) Western blot of cell lines CEM, C81, CTLL (WT), and CTLL (703). All of these cell lines were IL-2 independent for their growth in vitro. Fifty-microgram quantities of whole-cell extracts were loaded onto a Tris-glycine-4 to 20% polyacrylamide gel (Novex), transferred to a PVDF membrane, and Western blotted with alpha -p21/waf1 rabbit polyclonal Ab. Lane 1 contains the CTLL (WT) Tax+ extract, and lane 2 contains CTLL (703), a Tax mutant (M47) extract. Lanes 3 and 4 contain extracts from C81 and CEM, respectively. After the first Western blot, the same blots were stripped and reprobed with alpha -TBP (N-12) rabbit polyclonal Ab to determine the amount of protein loaded in each lane. A Tax Western blot was also performed on all extracts (panel A, bottom) by using four monoclonal Tab (169, 170, 171, and 172) Abs. Extracts were run on a Tricine-10 to 20% polyacrylamide gel (Novex) prior to Western blot analysis. (B) Western blot of CEM, C81, PBMCs, and patient samples Bes, Boul, and Bak. Forty micrograms of total cell extract was loaded onto a 4% Tris-glycine gel, transferred to a PVDF membrane, and Western blotted with alpha -p21/waf1 (N-20) goat polyclonal Ab, using the enhanced chemiluminescence method of detection. Lanes 1 to 3 (CEM, C81, and PBMC) represent positive and negative controls for p21/waf1 Western blot. Lane 4 contains an ATL patient cell line, Bes, while lanes 5 and 6 contained HAM/TSP patient cell lines Boul and Bak. All patient samples were IL-2 dependent for their growth in vitro. Following the first procedure, blots were stripped and reprobed with alpha -TBP (N-12) rabbit polyclonal Ab.

We next examined the p21/waf1 levels in three French ATL and HAM/TSP patients infected with HTLV-1 (28). Figure 1B shows the results of such an experiment, where levels of p21/waf1 present in infected cells were consistently higher than in uninfected peripheral blood lymphocyte cells. Similar results have also been obtained with two other ATL cell samples from patients in Japan and with three ATL cell samples from patients in the Middle East (data not shown).

p21/waf1 promoter expression has been observed to be up regulated by the p53 protein. The p21/waf1 promoter contains five natural p53 binding sites, at positions -4001, -3764, -2311, -2276, and -1391 (start of transcription at +1; GenBank accession number U24170), where the p53 can bind and activate transcription of this promoter. We therefore examined whether Tax was able to transactivate the p21/waf1 promoter either from an endogenous promoter or using a series of WT and 5'-deletion constructs linked to a luciferase reporter cassette. We first examined the levels of endogenous p21/waf1 transcription by using an RNase protection assay. The assay relies on specific hybridization of various cellular RNA products with multiple probes in the same reaction test tube. Figure 2A shows the result of such an experiment, where p21/waf1 transcription was up regulated in infected cells (C81 and MT-2) and not in uninfected CEM control cells. Two cellular control RNAs were used in each test tube, namely L32, which scores for quality and amounts of cytoplasmic RNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which scores for nuclear RNA (PharMingen hSTRESS-1 set, custom designed for p21/waf1, L32, and GAPDH genes). Similar results were also obtained when transfecting WT Tax protein into CEM cells (data not shown). We next performed deletion construct transfection assays to pinpoint which promoter elements, especially p53-binding sites, were important in activated transcription by Tax. When using Jurkat cells, we found that all 11 of the 5'-deletion constructs up to position -49 could be activated by Tax. The results of such an experiment are shown in Fig. 2B, where the tax gene was able to up regulate the WT promoter by fourfold and the minimal promoter by 10-fold. The transactivation was specific to WT, but not to M47 mutant, Tax and not to a control luciferase plasmid (TK-RL) (data not shown). Similar results were also obtained when using a minimal human immunodeficiency virus type 1 promoter, where only a functional TATA box and no E2A binding sites were present (Fig. 2C, E1 and E2 sites represent E2A transcription factor binding sites). More importantly, the minimal p21/waf1 (-49) construct had no p53 binding sites in the promoter. A complete GCG search also confirmed the absence of any p53 binding site in this minimal promoter. Therefore, the effect of Tax on the p21/waf1 is p53 independent and may involve other regulatory elements that are Tax responsive in the minimal promoter. Interestingly, the minimal promoter contains two E2A (helix-loop-helix) binding sites, at positions -22 and -6, that have been shown to be critical for p21/waf1 activity (25).



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  		FIG. 2.   Tax transactivation of the endogenous and transfected p21/waf1 promoter. (A) Demonstration of RNase protection from CEM, C81, and MT-2 (HTLV-1-infected) cells. A custom-made kit from PharMingen (modification of hSTRESS-1 probe set) along with 2 mg of total cellular RNA was used for RNase protection analysis. Protected fragments for p21/waf1, L32, and GAPDH were 202, 113, and 96 bases, respectively. (B) Various p21/waf1 promoter 5'-deletion luciferase constructs. The 5'-deletion constructs ranged from -2326 (0-Luc) to -49 (11-Luc), which included the TATA box and the transcriptional start site. Three micrograms of the reporter plasmid alone or in the presence of 2 mg of pCTax construct was used to transfect Jurkat cells. A similar pattern of luciferase counts was obtained when using a pCTax construct titration of 0.1, 0.5, 1.0, 2.0, and 4.0 mg (data not shown). Results in the right panel depict the basal and Tax-mediated activation counts of various p21/waf1 luciferase constructs. (C) Transfection of the p21/waf1 minimal promoter either alone, with WT Tax, or with M47 Tax mutant. A minimal HIV-1 promoter, which did not contain E2A-binding sites (E1 and E2) but had a WT TATA box, was also used in transfection assays. A second control luciferase plasmid, TK-RL, was also used for each transfection in panel A (data not shown).

Identification of p21/waf1 partners in HTLV-1-infected T cells. The p21/waf1 protein is able to bind to a wide variety of cyclin/cdk's, depending on the cell line tested, including cyclin D/cdk4, cyclin B1/cdc2, cyclin E/cdk2, and cyclin A/cdk2 (4, 29, 35), and inhibit their enzymatic activity. We examined which of the cyclin/cdk partners were complexing with p21/waf1 in HTLV-1-infected and uninfected T cells. We initially performed a series of immunoprecipitations by using anti-p21/waf1 antibody and whole-cell extracts from unsynchronized CEM and C81 cells. After immunoprecipitation, we Western blotted for 15 various human cyclins and 12 different cdk's. Only one cyclin/cdk complex was reproducibly observed to be complexed with p21/waf1 in HTLV-1-infected cells. The results of such an experiment are shown in Fig. 3, where cyclin A (top panel) and cdk2 (bottom panel) associated with p21/waf1. The cyclin A/cdk2 complex was resistant to 150 mM salt during incubation and under wash conditions. None of the other cyclin/cdk complexes could withstand 150 mM salt wash conditions. A representation of the p21/waf1 immunoprecipitations followed by Western blotting for some of the cyclin/cdk proteins is shown in Fig. 3B. Similar results were obtained with other HTLV-1-infected cells, including HUT102, MT-2, and CTLL (WT) cells (data not shown). Collectively, these findings suggest that p21/waf1 complexes with cyclin A/cdk2 in HTLV-1-infected T cells.


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  		FIG. 3.   Detection of p21/waf1 partners in HTLV-1-infected T cells. (A) The p21/waf1 and Tax immunoprecipitates were used for Western blotting with anti-cyclin A and cdk2 Abs. Infected and uninfected cell extracts (1.5 mg) were treated with alpha -p21/waf1 rabbit polyclonal Ab and/or alpha -Tax mouse monoclonal Ab (Tabs 169, 170, 171, and 172) overnight at 4°C. Immune complexes were precipitated with protein A+G beads, were washed with 150 mM NaCl buffer, and were separated on a Tris-glycine-4 to 20% polyacrylamide gel and transferred onto a PVDF membrane. Lanes 1 and 2 are input lanes containing whole-cell extracts from C81 and CEM. Lanes 3 and 4 contain C81 and CEM, respectively, immunoprecipitated with anti-Tax Abs. Lanes 5 and 6 contain C81 and CEM, respectively, immunoprecipitated with anti-p21/waf1 Abs. alpha -Cyclin A rabbit polyclonal and alpha -CDK2 rabbit polyclonal antibodies were used for Western blots. Bottom panel lanes 5 and 6 represent immunoprecipitations with anti-p21/waf1 (C-19) goat polyclonal Ab. NS, nonspecific cross-reactivity. (B) Representation of some of the immunoprecipitations with alpha -p21/waf1 rabbit polyclonal Ab followed by Western blotting for cdc2, cdk6, cyclin E, and B1.

Activity of cyclin A/cdk2/p21/waf1 complex from HTLV-1-infected cells. In proliferating immortalized cell lines, many cyclin/cdk complexes can be isolated by immunoprecipitation procedures and found to be catalytically active in an in vitro kinase assay. It is only after induction of CKIs, such as p21/waf1 in response to stimuli such as DNA-damaging agents, that the cyclin/cdk complexes are found to be catalytically inactive (4). Normally, two substrates are used to score for cyclin A/cdk2 activity in vitro, namely histone H1 and pRB proteins (21, 33, 35). Both are relevant substrates, since histone H1 is involved in higher-order chromatin fiber formation and Rb is the restriction protein prior to commitment of cells to DNA replication. Therefore, we focused on the activity of p21/waf1-associated complexes in HTLV-1-infected and uninfected T cells under normal and stressed conditions. Figure 4 shows results of such an experiment where CEM, C81, CTLL (WT), and CTLL (703) cells were used for immunoprecipitation with anti-cyclin A antibody and subsequently assayed by using an in vitro kinase assay. The three sets represent unsynchronized cells, serum-starved (G0/G1, 0 h) cells, and serum-starved cells that had been gamma-irradiated and released with complete medium, respectively. The purpose of serum starvation was to synchronize cells at G0/G1 prior to gamma-irradiation. When immunoprecipitating with anti-cyclin A Ab, the unsynchronized group represented in Fig. 4 (lanes 1 to 4) showed the highest overall phosphorylation levels compared to other sets when using histone H1 as a substrate. In all sets, we consistently observed uninfected CEM cells, as well as Tax mutant CTLL (703) cells, to have higher kinase activity than C81 or WT cells. In serum-starved cells (0 h, lanes 5 to 8), there was an overall decrease of counts for all samples. This decrease was expected, since cyclin mRNAs and their corresponding proteins (e.g., cyclin A) don't start expressing until late G1 phase. Interestingly, and perhaps more importantly, C81 cells that had been serum starved and released for 16 h contained higher kinase activity than their gamma-irradiated counterparts (compare lanes 6, 10, and 14). Western blot analysis for cdk2, p21/waf1, and cyclin A from various extracts (Fig. 4B) and their corresponding FACS analyses (Fig. 4C) showed no dramatic differences between various samples (Fig. 4C). Perhaps the only notable difference was observed in FACS analysis, where there was an increase of apoptosis in C81 cell populations, from zero to 6.6%, after gamma-irradiation. However, this change is unlikely to contribute to the overall H1 phosphorylation activity in vitro, since similar levels of cdk2, p21/waf1, and cyclin A were present at 0 and 16 h in gamma-irradiated C81 cells. Collectively from these results, we deduced that the cyclin A-associated complex in C81 and WT cells were more inhibitory in their H1 kinase activity when placed under DNA-damaging stress conditions, such as gamma-irradiation.





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  		FIG. 4.   Activity of cyclin A/cdk2/p21/waf1 complex from HTLV-1-infected cells. (A) Cyclin A immunoprecipitates which were used for in vitro kinase reaction using histone H1 as the substrate. Whole-cell lysates were prepared from CEM, C81, WT, and 703. The first set (lanes 1 to 4) was from normally growing unsynchronized cells cultured in complete medium containing 10% FBS. The second set (lanes 5 to 8) was 3-day-old serum-starved G0/G1 cells (1% FBS; 0 h). The third and fourth sets (lanes 9 to 12 and 13 to 16, respectively) were 3-day-old serum-starved cells, either released with 10% FBS (lanes 9 to 12) or gamma-irradiated (7.7 Gy) and released with 10% FBS (lanes 9 to 12). Samples were harvested 16 h later, corresponding to populations of cells at the G1/S boundary. Kinase reactions were separated by SDS-polyacrylamide gel electrophoresis, dried, and exposed to a PhosphorImager cassette. (B) p21/waf1, cyclin A, and cdk2 Western blots of various extracts used in the kinase assay above. (C) FACS analysis of all cells used in panel A. Actual numbers of G0/G1, S, and G2/M cells are given at the upper right-hand corner of each histogram. Apop, cumulative number of cells that are in the process of apoptosis from all four stages of cell cycle; gamma , gamma-irradiation.

Cyclin A/cdk2/p21/waf1 complex and Rb phosphorylation in CEM and C81 cells. The tumor suppressor retinoblastoma protein assists in mediating the G1/S checkpoint, which is important and necessary in cell proliferation. The Rb protein (and its family members) has repressor activity, and its repressor activity is reversed by phosphorylation, which is catalyzed by cyclin/cdk complexes such as cyclin D/cdk4 and -6, cyclin E/cdk2, and cyclin A/cdk2 (8). We therefore considered the status of Rb phosphorylation for both HTLV-1-infected and uninfected cells. To utilize the phosphorylation sites within the Rb protein, we synthesized nine different peptides, corresponding to all of the known sites that have been reported to be phosphorylated by various cyclin/cdk's (8, 13, 14, 33). A general diagram of the human pRb protein and its corresponding peptide maps is depicted in Fig. 5A. We next performed kinase assays by using immunoprecipitations with anti-cyclin A Ab from infected and uninfected cells. The results of such an experiment are shown in Fig. 5B. Two carboxy-terminal peptides, peptides I and J, were drastically hypophosphorylated when using IPs from infected, as compared to uninfected, cells. A similar result was also obtained for MT-2 cells (data not shown). Interestingly, the peptides I and J correspond to a portion of the C domain of the Rb protein. The I peptide contains serines 807 and 811, which, when phosphorylated, block binding of Rb to c-Abl protein. Threonines 821 and 826 present in the J peptide regulate the interactions in the A/B pocket, disrupting binding of proteins such as HDAC1. However, a more dramatic change in phosphorylation pattern emerged when we immunoprecipitated cyclin A from gamma-irradiated C81 cells. The results shown in Fig. 5B (C81 + gamma ) show that virtually all the Rb peptides were hypophosphorylated after gamma-irradiation in C81 cells. This was in marked contrast to control CEM cells, where only the last two C-terminal peptides were affected by gamma-irradiation. Therefore, the results presented above collectively point to the possibility that the hypophosphorylation of histone H1 and Rb may contribute to the arrest of the cell cycle in Tax-containing cells after DNA damage.


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  		FIG. 5.   Rb peptide phosphorylation using cyclin A IPs from CEM and C81 cells. (A) A general diagram of the human pRb protein and the A, B, and C pocket domains. Black bars underneath represent areas where E2F and HDAC bind. (B) The results of the in vitro kinase assay when using various pRb peptides (A thru J). Cyclin A immunoprecipitates from untreated or gamma-irradiated CEM and C81 cells were incubated with various peptides, and the phosphorylated products were separated by thin-layer chromatography on cellulose plates. The peptides were detected with a Bio-Image Analyzer (BAS2000; Fuji) or were simply trapped on P81 papers (Whatman Co., Ltd.), washed, and monitored for incorporation of 32P in a liquid scintillation counter.

Functional effect of gamma-irradiation on Tax-expressing cells. Finally, to determine the in vivo function of the p21/waf1-associated complex, we examined the effects of gamma-irradiation on Tax-expressing cells. Initially, we used two sets of cell lines, namely CEM and C81, to determine if gamma-irradiation had any effect on Tax-expressing cells. The results of such an experiment are shown in Fig. 6A, where two very different phenomena were observed. First, even though both infected and uninfected C81 and CEM cells had similar FACS profiles at time zero, their cell cycle patterns had changed upon gamma-irradiation. After 48 h, CEM cells had a lower percentage of G0/G1 cells (22.55 versus 42.04%), a higher percentage of S-phase cells (52.65 versus 2.61%), and lower levels of G2/M cells (24.80 versus 48.29%) than C81 cells. A similar pattern of events was also seen in CTLL (WT) versus CTLL (703) cells, where CTLL (WT) cells had a higher percentage of G0/G1 and G2/M cells and a lower percentage of S-phase cells upon gamma-irradiation (data not shown). A second interesting observation was also made: C81 cells had more apoptosis following gamma-irradiation (1.3 versus 28.97%) than did CEM cells. A similar pattern of increased apoptosis was also observed in CTLL (WT) cells (data not shown). Because we were interested in the effect of the cyclin A/cdk2/p21/waf1 complex and its possible involvement in the G1/S boundary, we focused on studying and physically separating G1 cells, followed by gamma-irradiation. Therefore, we utilized the centrifugal elutriation technique to obtain cells at early G1, S, and G2/M phases of the cell cycle. Flow rates were calibrated to give definable G1 (early G1, 23 ml/min; mid-G1, 27 ml/min; and late G1, 30 ml/min), S, and G2/M phases. G1-phase cells were the smallest in size and were contained in the initial fractions, followed by S-phase and G2/M-phase cells, which had the largest mass.


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  		FIG. 6.   Effect of gamma-irradiation on Tax-expressing cells. (A) Cells that were grown to mid-log phase, serum starved for 3 days (1% FBS), and either harvested at 0 h or released in complete medium for 48 h following gamma-irradiation. FACS analyses were performed on 0-h samples (left panel) and 48-h samples (right panel). Uninfected T cells, CEM (12D7), and HTLV-1-infected T-cells, C81, were used in panel A. Panel B represents centrifugal elutriated CEM and C81 cells from the G0/G1 phase. The elutriated G0/G1 cell fractions were harvested, washed in PBS, and either directly analyzed by FACS at 0 h (left panels) or gamma-irradiated and kept in culture for 48 h prior to FACS analysis. Each panel depicts cell cycle histogram profiles and percentages of cell numbers at various stages of the cell cycle. Apoptotic cells represent a collection of cell populations that were either at G0/G1, S, G2, or M phase of the cell cycle.

Utilizing this method on CEM and C81 cells, we were able to take cell populations in G1, S, and G2/M phases and apply stress with gamma-irradiation. Figure 6B depicts the results of flow cytometry analysis of CEM and C81 cells followed by gamma-irradiation of G1 cells. C81 cells that had been at early or mid-G1 phase prior to gamma irradiation were stopped at the G1/S border, unlike CEM cells, which traversed into S phase. We observed a complete block of C81 cells at G0/G1 and no apparent apoptosis. However, there was an increase of apoptotic cells from the latter fractions of C81 (S or G2/M population), which may correspond to the apoptotic cells shown in Fig. 6A (data not shown). Taken together, these results imply that when Tax-expressing cells are at early G0/G1 and are introduced to stress, they will be blocked at the G1/S checkpoint, possibly by the action of cyclin A/cdk2/p21/waf1, and will not initiate apoptosis. On the other hand, if they have passed the G1/S checkpoint and are introduced to stress, they will quickly finish S phase (possibly assisted by the mitogenic action of Tax), lose the G2/M checkpoint, and eventually undergo apoptosis.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

cdk's are generally active at specific stages of the cell cycle when bound to specific cyclin partners. The cyclin/cdk complexes are subject to regulation by CKIs, which bind to and suppress the enzymatic functions of cyclin/cdk complexes, thereby stopping cells at specific checkpoints. The G1 phase of the cell cycle is regulated by two sets of inhibitors, the INK and KIP family members for early G1 and late G1 phase, respectively. The INK family members consist of p16 (INK4A), p15 (INK4B), p18 (INK4C), and p19 (INK4D), and they mainly inhibit early G1 kinases such as cyclin D1 to -3/cdk4 and -6. The CIP/KIP family members are p21/waf1/CIP1, p27 (KIP1), and p57 (KIP2), and they inhibit some early G1 kinases (e.g., p27 association with cyclin D1/cdk complex) but primarily inhibit the late G1/S checkpoint kinase, cyclin E/cdk2.

The p21/waf1 protein was seen in this study to be expressed at high levels in HTLV-1-infected T cells (C81), Tax+ mouse cell clones [CTLL (WT)], and peripheral blood mononuclear cell (PBMC) samples from ATL patients. This is consistent with previous reports that HTLV-1-infected cell lines and Tax1-immortalized T-cell lines both have increased amounts of mRNA and protein expression (2, 5). We also obtained similar mRNA results when using the hSTRESS-1 riboprobe set (PharMingen), which contains the p21/waf1 probe and scores for the activity of the real endogenous promoters containing the proper chromatin structure.

There appear to be two forms of p21/waf1 in cells, caused by either proteolytic cleavage or phosphorylation differences. A novel form of p21/waf1 has been observed both in 12-O-tetradecanoylphorbol-13-acetate-treated Calu-1 lung carcinoma cells (27) and in active and inactive cyclin A/cdk2/p21 complexes (35). In the case of the 12-O-tetradecanoylphorbol-13-acetate-induced levels of p21/waf1, the cause was attributed to proteolytic cleavage of the protein at the C terminus, resulting in doublet bands of p21/waf1 and linked to the G2/M arrest. This was evident when two Abs, one targeting the epitope at the N terminus (amino acids 2 to 21) and another targeting that at the C terminus (amino acids 146 to 164), were used in Western blotting. We also used both the N- and C-terminal Abs and observed no difference in the p21/waf1 reactivity between the CEM and C81 cells (data not shown). Therefore, we focused our attention on the phosphorylation status of p21/waf1 in the two cell types. We have seen that the different forms of p21/waf1 observed in CEM and C81 cells are due to phosphorylation differences. Alkaline phosphatase treatment of CEM extracts showed a faster migrating band corresponding to the same position as the dephosphorylated p21/waf1 in C81 cells (data not shown). The change in the p21/waf1 mobility shift has also been observed by others (35) and contributed to the dephosphorylation form of the protein at serines 98 and 130. We are, therefore, currently investigating whether the dephosphorylated form of p21/waf1 in Tax-expressing cells contributes to the G1/S block observed in HTLV-1-infected cells.

Since p21/waf1 protein expression was significantly higher in both mouse CTLL (WT) and C81 cells, we examined the effect of Tax on the p21/waf1 promoter. When using a series of 5'-deletion constructs, we found that there was a significant activation by Tax up to and including the -49 construct (Fig. 2, 11-Luc). We hypothesize that the Tax activation on this deletion construct may be due to the effects of Tax on the E2A transcription factor. The E2A transcription factor is part of the basic helix-loop-helix family of proteins, which contains a conserved basic region responsible for DNA binding and a helix-loop-helix domain for dimerization (20). From the E2A gene, there are two alternatively spliced products that are normally produced, E12 and E47. These two proteins differ in their basic helix-loop-helix domains and in their DNA-binding properties. Hetero- and homodimers can be formed, but it is the E47 homodimer that has a strong affinity for the E-box sequence (CANNTG). Overexpression of E2A has been shown to induce growth arrest before the G1-to-S transition (22, 25). Interestingly, the WT p21/waf1 promoter contains eight putative E-box consensus sequences, two of which lie between the TATA box and the transcription start site, E2 and E1 (Luc-11 construct, a minimal promoter in this study). The E1 sequence (GCAGCTG), which lies immediately upstream of the start site, belongs to the E-boxes (group I) that have a strong binding to E47 hetero- and homodimers. The E2 sequence (CCAGCTG) lies upstream from the E1 box, is part of the group III E-boxes, and has much less affinity for E47 (25). Therefore, we are currently investigating whether the E2A sites within the minimal p21/waf1 promoter are able to respond to Tax in in vitro transcription assays. Preliminary results indicate that Tax may aid in multimerization of the E2A-related proteins on the p21/waf1 promoter, much like the stimulation and enhancement of the bZIP proteins by Tax (31).

Cyclin A/cdk2 interactions with p21/waf1 had been explored in quaternary complexes (cyclin A/cdk2/PCNA/p21) in normal human fibroblasts (16) and in inactive and active complexes with varying levels of p21/waf1 protein (35). Based on the structure of a complex between another CKI, p27/kip2, and cyclin A/cdk2, one can reason that the N-terminal inhibitory domain of p21/waf1 interacts with a groove on the surface of cyclin A through the conserved LFG sequence near the N terminus of the inhibitory domain, allowing the C-terminal end of the inhibitory domain to displace the first beta  strand of the N-terminal lobe of cdk2, thereby disrupting the ATP-binding site (27, 29). A second cyclin-binding motif near the C terminus of p21 has been shown to independently inhibit cyclin/cdk activity toward certain substrates (3). It remains to be seen whether Tax-expressing cells contain free N- or C-terminal p21/waf1, which may be responsive to stress signals such as gamma-irradiation. Future experiments will address the stoichiometry of the p21/waf1-associated complex(s) and its partners in HTLV-1-infected cells before and after stress signals.

Ultimately, the functional consequence of the p21/waf1 protein in cells is its regulation of the Rb protein. The phosphorylation seems to be well regulated, in that sites are phosphorylated strongly by one or the other cyclin/cdk complex (13, 33). Several cyclin/cdk combinations, including D cyclins (D1, D2, and D3) with cdk4 or cdk6, cyclin E associated with cdk2, and cyclin A with cdc2 or cdk2, mediate the phosphorylative state of Rb. Cyclin D/cdk4 and -6 and cyclin E/cdk2 phosphorylation starts during G1 and continues into S phase with cyclin A/cdk2 (8). Continued phosphorylation of Rb is a requirement for the progression through the S phase and completion of DNA replication. While there are at least 16 consensus sequences for cdk phosphorylation, it is the C-terminal region of Rb (amino acids 729 to 928) that is the main target for inhibitory phosphorylation. The peptides I and J correspond to the C pocket containing serines 807 and 811 and threonines 821 and 826. Phosphorylation of serine 807/811 blocks binding of the c-Abl tyrosine kinase protein to Rb in the C pocket region (8). Free c-Abl protein binds and phosphorylates such proteins as p73, the homologue to the tumor suppressor p53, thereby stimulating p73-mediated transactivation and apoptosis (1, 32). Also, phosphorylation of threonine 821/826, in the C pocket domain, leads to the inhibition in the A/B pocket. It has been deduced that the cyclin A/cdk2 complex specifically phosphorylates the threonine 821 site, both blocking and disrupting the binding of the LXCXE protein to the A/B region. Proteins containing the consensus sequence LXCXE are blocked or their bindings are disrupted. HDAC1 and -2 contain an LXCXE-like sequence that connects to the LXCXE-binding site on Rb. These enzymes remove inhibitory acetyl groups from the amino-terminal regions of histone octamers, thereby promoting nucleosome assembly that blocks transcription factors from the promoter (8). Therefore, it is tempting to speculate that the decreased phosphorylation of the Rb protein (I and J peptides) from HTLV-1-infected T cells may help to acquire proteins such as HDAC (to block transcription) and c-Abl (to block apoptosis), thereby modulating either specific gene transcription and/or the apoptosis pathway. Perhaps a more significant finding related to Rb phosphorylation emerged when we examined the phosphorylation pattern of immunoprecipitated cyclin A from gamma-irradiated C81 cells. The results shown in Fig. 5B clearly indicate that virtually all the Rb peptides were hypophosphorylated after gamma-irradiation in C81 and not in control CEM cells. This dramatic inhibition in Rb phosphorylation may explain why purified C81 G0/G1 cells were blocked at G1/S after gamma-irradiation. Of notable interest, cells blocked at G1/S after DNA damage have a reversible block (72 to 96 h) and eventually traverse into S phase, indicating that DNA damage machinery prior to the G1/S checkpoint is intact in HTLV-1-infected cells. Therefore, the net functional effect of these interactions may be a block at the G1/S boundary and inhibition of apoptosis upon cell stress. Perhaps in this way, HTLV-1 virus would be able to prevent its host from inappropriately entering the S phase. This may be an advantage for a cancer-causing virus, such as HTLV-1, to ensure proper host cell survival and continue proliferation after cell stress.


    ACKNOWLEDGMENTS

We thank Wafik El-Deiry for supplying the p21/waf1 promoter constructs and Ebony Brooks for assistance in preparing the manuscript.

This work was supported in part by National Institutes of Health grants AI42524, AI43894, and 13969 and UMDNJ foundation funds to F.K.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, MSB E635, UMDNJ-New Jersey Medical School, Newark, NJ 07103. Phone: (973) 972-1089. Fax: (973) 972-5594. E-mail: Kashanfa{at}umdnj.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, August 2000, p. 7270-7283, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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