INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (37, 40). Due to the limited coding capacity of the viral genome, viral replication and transformation are largely dependent on modification of cellular regulatory protein function. HTLV-1 activates and immortalizes human T lymphocytes in vitro, resulting in polyclonal proliferation of the infected cells, followed by oligoclonal or monoclonal growth. The mechanism of HTLV-1 transformation appears to be distinct from that of chronic or acute leukemia viruses and is related to the viral activator Tax. Tax₁ transcriptionally activates viral mRNA synthesis, leading to an initial increase in the viral regulatory transcripts and ultimately to transformation (13, 14, 16).

Tax₁ is a 40-kDa (353-amino-acid) phosphoprotein, predominantly localized in the nucleus of the host cell, which functions to transactivate both viral and cellular promoters. Tax₁ has not been shown to bind directly to Tax₁-responsive sequences (TREs), suggesting that Tax₁ transactivation occurs through indirect effects of Tax₁ on transcription factors which bind to the TREs (6). Likely mechanisms for Tax₁ transactivation include (i) transcriptional induction of TRE-binding transcription factors, (ii) posttranslational modification of TRE-binding factors, and (iii) complex formation with transcription factors allowing indirect binding of Tax₁ to the TRE(s).

It seems likely that HTLV-1, through expression of the viral regulatory proteins Tax₁ and Rex₁, provides some initial alteration in cell metabolism predisposing to the development of ATL. Subsequently, the rearrangement or altered expression of a cellular oncogene(s) may provide the "second hit," leading to development of ATL. In fact, there have been reports that Tax₁ triggers DNA damage and inactivates p53 function. Diverse cytogenetic abnormalities have been observed in ATL patient peripheral blood lymphocytes. Although several karyotypic abnormalities, including trisomies 3 and 7 and rearrangements in the long arm of chromosome 6, have been found, no single chromosomal defect is pathognomonic for ATL (38).

Recently it has been shown that HTLV-1- and/or Tax₁-expressing cells have altered expression of some cell cycle-associated genes. Among these changes, high levels of inactive p53, cyclin-dependent kinase (cdk) inhibitor p21, and cyclin D2 and lower levels of cyclin D3 and the cdk inhibitor p16 have been observed (1). In vitro binding assays also indicate that Tax binds p16^INK4a (cdk/cyclin D inhibitor), but not p21^waf1 or p27^kip1, and forms complexes with p16^INK4a in vivo (31, 44). However, no careful analyses of Tax₁- or HTLV-1-infected cells have been performed to address the functional consequence of these seemingly dramatic changes at the cell cycle level. Of particular interest to us is the notion of very early events postmitosis that Tax₁ and/or HTLV-1 induce in the host cell cycle machinery. One such early event postmitosis is the activation of cyclin D family members.

Cyclins are the regulatory subunits of cdc2-related protein kinase complexes in the eukaryotic cell cycle. Cyclins C, D (D1, D2, and D3), E1, E2, and G are believed to be G₁ cyclins (28, 47). Cyclin A is an S-phase cyclin, and cyclin B (B1 and B2) are mitotic cyclins. Cyclin K and H are involved in phosphorylation of RNA polymerase II, and cyclins G1, G2, and I are involved in DNA damage response. The initial studies of G₁ cyclins were performed in budding yeast, which has three CLN-type cyclins (CLN1, CLN2, and CLN3) required for passage through Start, the G₁ restriction (R) point, and transition at G₁/S. Three novel types of putative mammalian G₁ cyclins were isolated by using human cDNA libraries to complement CLN-deficient yeast and designated cyclins C, D, and E (29). PRAD-1 was cloned as a gene rearranged in a parathyroid tumor and is identical to the human cyclin D1 gene (35). A murine homologue of cyclin D1 was independently isolated from a cDNA library prepared from murine macrophages synchronously progressing through G₁ in response to colony-stimulating factor 1. The murine cyclin D1 cDNA probe was used to identify two related genes, encoding murine cyclin D2 and D3. Unlike other types of cyclins, cyclins D1, D2, and D3 have unique cell- and tissue-specific patterns of expression, suggesting that each D-type cyclin may have a distinct mechanism for transcriptional regulation. Overexpression of any of the D-type cyclins can accelerate the timing of Start and shorten the G₁ interval (11).

In a quest to define models and events related to T-cell transformation, we have analyzed the G₁ cyclins in HTLV-1-transformed cells. We find that cyclin D2 is transcriptionally up-regulated in these cells and that the overexpression of this cyclin is associated with acquisition of two new cdk partners, cdk2 and cdk4, in infected T cells. The functional significance of this association is shortening of the G₁ phase of the cell cycle as shown by rapid phosphorylation of markers such as the Rb protein. Therefore, HTLV-1 infection and changes associated with the G₁ phase, as noted by changes in cyclins, may prove to be an ideal model system for study of T-cell transformation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Tax and CREB expression vectors and protein purification. Wild-type and mutant (M47) Tax proteins were overexpressed in Escherichia coli and purified as described previously (30). Proteins were purified by nickel affinity chromatography (Qiagen) followed by cation-exchange fast protein liquid chromatography (HiTrap SP; Amersham Pharmacia Biotech) (23). For protein electroporation assays, E. coli-expressed recombinant, purified Tax was electroporated as described previously (26).

Protein transfection. Lymphocyte (CEM [12D7]) cells were grown to the mid-log phase of growth and processed for protein electroporation as described previously (26), with the modification that cells were electroporated at 230 V and plated in 10 ml of complete RPMI 1640 medium for 18 h prior to harvest.

Detection and quantification of cyclin mRNA species. For the multiprobe RNase protection assay (RPA) system, we mixed 1 µl of RNasin, 1 µl of GACU pool, 2 µl of dithiothreitol (DTT), 4 µl of 5× transcription buffer, 1 µl of human cyclin 1 (RPA for human cell cycle regulator multiprobe template set; Pharmingen catalog no. 45352P), 10 µl of [-³²P]UTP, and 1 µl of T7 RNA polymerase. Samples were mixed gently and incubated at 37°C for 1 h, and reactions were terminated by adding 2 µl of DNase and further incubation at 37°C for 15 min. Following phenol-chloroform extraction, probes were incubated with 10 µg of total cellular RNA (using RNAzol; Pharmacia, Inc.), 8 µl of hybridization buffer, and 50 µl of mineral oil for each sample. Samples were placed in a 90°C heat block, and the temperature was reduced to 56°C over a 12- to 16-h period. The next day, a mixture of RNase A and RNase T₁ was added, and the mixture incubated for 45 min at 30°C. Following the incubation, 390 µl of proteinase K buffer, 30 µl of proteinase K, 30 µl of yeast RNA, 120 µl of 4 M ammonium acetate, and 650 µl of ice-cold 100% ethanol were added to each sample. Samples were trichloroacetic acid (TCA) precipitated, loaded on a 6% Tris-borate-EDTA-urea gel (Novex, Inc.), and run at a constant current of 180 V for 50 min. Gels were subsequently dried and placed on a PhosphorImager cassette for overnight exposure.

Microscale preparation of nuclear extracts. To prepare nuclear extracts, cells were collected and washed with phosphate-buffered saline (PBS) once 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 is done with the tube containing the cells submerged in ice. The nuclei were collected by pelleting for 8 s in an Eppendorf microcentrifuge, and the supernatant was discarded. 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 phenylmethylsulfonyl fluoride [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 spun in an Eppendorf microcentrifuge 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 a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, Calif.).

Immunoprecipitation and immunoblotting. Cells grown in culture were spun at 10,000 × g for 15 min. The supernatants were discarded, and the pellets were washed twice with 25 ml of PBS without calcium or magnesium. The pelleted cells were lysed with 1 ml of lysis buffer containing 50 mM Tris-Cl (pH 7.4), 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF (phosphotyrosine phosphatase inhibitor), 1 mM DTT, and 1 mM PMSF. The cells were incubated on ice for 15 min and mixed gently every 5 min. Cells were transferred to an Eppendorf tube and microcentrifuged at 4°C for 10 min. Protein concentrations in the lysates were determined by using a bicinchoninic acid BCA protein assay kit (Bio-Rad). A total of 2 mg of cellular proteins with 50 µl of rabbit anti-human cyclin D2 antibody C-17 (Santa Cruz Biotechnology catalog no. sc-181) was used for immunoprecipitation. The proteins and antibody were mixed for 12 to 14 h at 4°C, and the next day 150 µl of 30% protein G PLUS/protein A (protein G+A)-agarose beads (Oncogene Research Products/Calbiochem catalog IP05) was added to TNE 50-0.1% NP-40 buffer and mixed at 4°C for 3 h. The samples were microcentrifuged for 10 min at 4°C, and the supernatants were discarded. Agarose beads were washed three times with TNE 50-0.1% NP-40, gently vortexed, and pelleted. To the pellets, 20 µl of 2× Tris-glycine sodium dodecyl sulfate (SDS) sample buffer was added; the samples were heated at 95°C for 5 min and separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 4 to 20% polyacrylamide gel (Novex) at 200 V for 60 min. The proteins were then transferred to nylon-reinforced nitrocellulose membranes (Immobilon-P transfer membranes; Millipore Corp.), and transferred overnight at 0.08 A. Following the transfer, the blots were blocked with 5% nonfat dry milk in 50 ml of TNE 50-0.1% NP-40 for 30 min and washed twice with 25 ml of TNE 50-0.1% NP-40 at 4°C. After discarding of the wash, the blots were probed with 1:1,000 dilution of rabbit anti-human cdk2 (H-298; Santa Cruz Biotechnology catalog no. sc-748), rabbit anti-human cdk4 (H-303; Santa Cruz Biotechnology catalog no. sc-749), or rabbit anti-human cdk6 (H-96; Santa Cruz Biotechnology catalog no. sc-7180). The blots were probed for a period of 12 to 14 h in the cold, washed twice with 25 ml of TNE 50-0.1% NP-40, and then treated with 10 ml of ¹²⁵I-protein G (Amersham catalog no. IM.244; 50 µl) in TNE 50-0.1% NP-40 for 2 h at 4°C. Finally, the blots were washed twice in 25 ml of TNE 50-0.1% NP-40 and placed on a PhosphorImager cassette for further analysis. For direct Western blotting, a total of 25 to 50 µg of cellular proteins was separated by SDS-PAGE on a 4 to 20% gel transferred, and blotted with a 1:1,000 dilution of cyclin D2 antibody or, in some cases, TATA-binding protein (TBP) antibody.

Cell culture. MT-2 (34) and C81 (43) are HTLV-1-infected T-cell lines; Jurkat and CEM (8) are uninfected human T-cell lymphocyte lines established from patients with T-cell leukemia. These and other cell lines were cultured at 37°C at a density of up to 10⁵ cells per ml in RPMI 1640 medium containing 10% fetal bovine serum (FBS) treated with a mixture of 1% streptomycin, penicillin antibiotics, and 1% L-glutamine (Gibco/BRL).

cdk assays. cdk4 and cdk6 activities were determined by a modification of the method described by Matsushime et al. (33). Twenty million T cells were cultured to the mid-log phase of growth and lysed in a buffer containing 150 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Tween 20, 100 µM Na₃VO₄, 1 mM NaF, 30 nM aprotinin, 500 nM leupeptin, 100 µM PMSF, 10 mM -glycerophosphate, and 1 mM sodium pyrophosphate. Kinase activities of the immunoprecipitated anti-cyclin D2 complexes were assessed by transfer of phosphate from [-³²P]ATP to truncated recombinant glutathione S-transferase (GST)-Rb protein in a reaction buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 2.5 mM EGTA, 10 mM -glycerophosphate, 100 µM Na₃VO₄, 1 mM NaF, 20 µM ATP, 200 ng of the substrate GST-Rb protein (eluted from glutathione beads), and 10 µCi of [-³²P]ATP (specific activity, 11 Ci/mmol; ICN Biochemical). The reactions were performed for 30 min at 30°C and stopped by addition of SDS sample buffer. The samples were boiled for 5 min at 65°C, and the proteins were separated by SDS-PAGE on 4 to 20% gels. The gels were autoradiographed, and bands were counted on a Molecular Dynamics PhosphorImager plate.

Northern blot. Total cellular RNA was extracted by using the Trizol reagent (Gibco/BRL). Total RNA (5 µg) was spotted onto a 0.2-µm-pore-size nitrocellulose (Millipore), UV cross-linked, and hybridized overnight at 42°C with various 40-mer ³²P-end-labeled, cyclin D2, cyclin D3, cyclin E, HTLV-1 long terminal repeat (LTR; R region, +1 to +260) and actin probes (11, 47). The next day, they were washed two times (10 ml; 15 min each time) with 0.2% SDS-2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 37°C, exposed, and counted on a PhosphorImager cassette (Molecular Dynamics).

Cell cycle block and analysis. Cells for transfection experiments were grown to mid-log phase, washed, and kept in complete medium with 1% FBS and 100 ng of nocodazole per ml for 24 h. For fluorescence-activated cell sorting (FACS) analysis, cells were removed from the medium at each time point, washed with Mg²⁺/Ca²⁺-free PBS fixed with 70% ethanol, and stained with a cocktail of PI buffer (PBS with Ca²⁺ and Mg²⁺, RNase A [10 µg/ml], NP-40 [0.1%], and propidium iodide [50 µg/ml]) followed by FACS analysis on a Coulter Epic model (Department of Pediatrics, UMDNJ-New Jersey Medical School).

Processing of patient samples. Informed consent was obtained from all patients. Briefly, heparinized blood was obtained from four HTLV-1-positive (two ATL and two HAM/TSP) patients. Peripheral blood mononuclear cells were separated, put in culture, and maintained in a humidified 5% CO₂ atmosphere with biweekly changes of RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 10% interleukin-2 (IL-2), 1% L-glutamine, and 1% penicillin-streptomycin. During the first 3 days, the cells were stimulated with phytohemagglutinin at 2 µg/10⁶ cells. After 3 months of culture, continuous IL-2-dependent cell lines were obtained, lysed, and Western blotted for cyclin D2. Then 10⁶ cells were lysed in TNN buffer (50 mM Tris-HCl [pH 7.4], 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na₃VO₄, 1 mM DTT, 1 mM PMSF, 20 µg of aprotinin per ml), and centrifuged at 12,000 rpm for 10 min; 40 µg of total cellular protein was loaded onto an SDS-4 to 20% polyacrylamide gel and Western blotted with either rat monoclonal or rabbit polyclonal anti-cyclin D2 antibody.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Cyclin D2 expression in HTLV-1 infected cells. To determine whether any of the cyclins are deregulated in HTLV-1-infected cells, we used RPA with total cellular RNA from both infected and uninfected cells. The transcriptional regulation of these cyclins was scored by using a sensitive RPA which relies on gene expression from bona fide endogenous cyclin promoters with their chromatin structures. The transcriptional regulation in these assays can be quantitated in comparison to endogenous cytoplasmic (L32) and nuclear (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) positive control RNAs. We initially used two cell lines in RPAs: MT-2, which expresses wild-type HTLV-1 particles, and a related uninfected T-lymphocyte line, CEM (12D7). As shown in Fig. 1A, control uninfected cells showed normal transcription levels of cyclins A, B, and D3. However, an inverse effect was seen in the transcriptional regulation of cyclins D2 and D3, which were dramatically changed in the HTLV-1-infected cells. Cyclin D2 levels were up-regulated (12-fold) and cyclin D3 levels were down-regulated (3-fold) in infected cells. Similar results were obtained for cyclin D2 and D3 primers in reverse transcription-PCR assays (data not shown). To determine whether any other known human cyclins are affected at the level of transcription, we performed a series of similar RNase protection and Western blot assays of all known cyclins (cyclins A1, A2, B1, B2, C, D1, D2, D3, E1, E2, F, G1, G2, H, I, and K) in infected and uninfected cells. Only one other cyclin, cyclin G1, was transcriptionally up-regulated in HTLV-1-infected (MT-2 and C81) cells. However, Western blot analysis of infected and uninfected cells revealed no difference of cyclin G1 protein levels between infected and uninfected cells (data not shown). Collectively, these results indicated that HTLV-1 infection affects G₁ cyclins by regulating the cyclin D family members.

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Transcriptional activation of cyclin D2 in HTLV-1-infected cells. (A) Ten micrograms of RNA was used for hybridization with probes specific for cyclins A, B, C, D1, D2, D3, and A1. The human probe set used was human cyclin 1 from PharMingen. Following RNA preparation, hybridization, and digestion with RNases A and T1 as recommended by the manufacturer, protected fragments were separated on a 6% urea-polyacrylamide gel (Novex), dried, and exposed to a PhosphorImager cassette. Lane 1, 1/10 of the probe used for protection; lane 2, negative control sample hybridized with yeast tRNA; lanes 3 and 4, hybridization of uninfected (CEM) and HTLV-1-infected (MT-2) cells with the cyclin probes. Both L32 (cytoplasmic) and GAPDH (nuclear) RNA protections serve as internal controls in each lane. (B) Twenty-five micrograms of total cellular protein from uninfected (CEM and Jurkat) and infected (MT-2 and C8166) cells was prepared, separated by SDS-PAGE on a 4 to 20% gel, and blotted with anti-cyclin D2 polyclonal antibody or anti-TBP monoclonal antibody (generous gift from Nancy Thompson) (bottom). The antigen-antibody complex was further detected with 125I-protein G. The marker is a 14C-labeled Rainbow (high-molecular-weight) marker from Amersham; positions are indicated in kilodaltons. Cyclin D2 protein was seen at higher levels in HTLV-1-infected cells, as evident in lanes 2 and 3. Similar results have been obtained with two other cyclin D2 monoclonal antibodies, DCS-3 and DCS-5 (Neomarkers, Union City, Calif.). NS, nonspecific cross-reaction with cellular proteins. (C) Two hundred microgram of nuclear Jurkat or CEM extracts was treated with 100 U of CIP (Gibco/BRL catalog no. 18009-019), TCA precipitated, and run on a 6% Tricine-polyacrylamide (Novex) (lanes 3 and 4). Lanes 1, 2, 5, and 6 serve as controls (10 µg in each lane) for both phosphorylated and unphosphorylated cyclin D2. (D) Cellular extracts from four HTLV-1-infected patients, two with HAM/TSP and two with ATL, were processed and Western blotted with rabbit polyclonal anti-cyclin D2 antibody. All four samples were kept in culture for 4 to 5 months in the presence of exogenously added IL-2 (recombinant human IL-2; 200 U/ml; Boehringer Mannheim). A control TBP Western blot of the samples is shown at the bottom. The cells from ATL and HAM/TSP patients were not able to grow in the absence of IL-2, indicating that they are not fully transformed.

View larger version (200K): [in this window] [in a new window]   FIG. 2.   Activity of wild-type and mutant Tax protein on the endogenous cyclin D2 promoter. (A) Four hundred micrograms of purified and dialyzed wild-type (WT) and M47 Tax were run on an SDS-4 to 20% polyacrylamide gel and stained with Coomassie blue. MW, molecular weight markers (positions are indicated in kilodaltons on the right). (B) Two micrograms of each Tax protein and 3 µg of HTLV-1 reporter plasmid were transfected into CEM cells, and the cells were processed for CAT assay the next day (26). (C) As for panel B except that the reporter was HIV LTR-CAT and 200 ng of purified E. coli Tat was used as a control activator for this construct (lane 2). (D) Two micrograms of each Tax protein was transfected into 20 million CEM cells and processed 24 h later for Western blotting. Samples were lysed, and nuclear extracts were made as described in Materials and Methods, TCA precipitated, run on an SDS-4 to 20% polyacrylamide gel, and Western blotted with cyclin D2 antibody. In the IPed Tax (WT) lane (control), the wild-type Tax protein was immunoprecipitated with a cocktail of Tax monoclonal antibodies (Tab169, Tab170, Tab171, and Tab172) and pelleted in the presence of protein A+G-agarose, and the supernatant was used for transfection of CEM cells. NS, nonspecific reaction. (E) Recovery of Tax protein from the transfected cells. Details are as for panel D except that Western blotting was done with a cocktail of four anti-Tax monoclonal antibodies (1:500) and the antigen-antibody complex was detected with 125I-protein G (1:100; Amersham). Lane 1 and 2, controls where Tax was immunodepleted prior to transfection; lanes 3 and 4, nuclear extracts from transfected cells; lanes 5 and 6, 1/20 of the initial material used for transfection.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Effect of Tax on cyclin D2 expression. Mouse CTTL-2 (IL-2 dependent) cells were transfected with either wild-type or M47 Tax and selected for the ability to become IL-2 independent. Both cell types (described elsewhere [20]) were grown to mid-log phase of growth, and nuclear extracts were processed, run on an SDS-4 to 20% polyacrylamide gel, and Western blotted for either Tax (Tab172) or cyclin D2. (A) Wild-type (WT-14) and mutant (703-3) Tax Western blot analysis using 50 µg of extract. (B) Western blot analyses for mouse cyclin D2, using human antibody (top) and for both mouse and human TBP (hTBP), using polyclonal antibody (Santa Cruz) (bottom). Human and mouse cyclin D2 are more than 90% identical in primary sequence, and the human antibody cross-reacts with the mouse protein. CTLL (703-3) cells, which contain mutations at amino acids 319 and 320, show more than 80% reduction (wild type, 522,789 counts; 703-3, 6,325 counts) when quantitated on a PhosphorImager (Molecular Dynamics). MW lanes are as in Fig. 2A.

View larger version (25K): [in this window] [in a new window]   FIG. 4.   Endogenous promoter activities of HTLV-1 and early cyclin genes. MT-2 and CEM cells were blocked in low serum and nocodazole (Noco), washed the next day, and released. Samples were collected at time zero or at 2 h postrelease for RNA analysis. (A) Diagram of the experiment. (B) FACS analysis of both cell types, using propidium iodide DNA staining (FAST systems; Gaithersburg, Md.); (C) hybridization of 10 µg of total RNA, using HTLV-1 (nick translated sequence of HTLV-1 LTR, R region, +1 to +260) and cyclin D2, cyclin D3, cyclin E, and actin probes (1).

Physical and functional significance of cyclin D2 overexpression. Since the cyclin D2 protein levels were up-regulated in HTLV-1-infected cells, we wished to examine whether this cyclin could partner up with any of the known cdks. To date, cyclin D2 has been shown to partner up with either cdk2, cdk4, cdk5, or cdk6 in various cell lines (4, 17, 45). We therefore used anti-cyclin D2 antibody for immunoprecipitations followed by Western blotting to detect the presence of various cdks. As shown in Fig. 5, the anti-cyclin D2 immunoprecipitate contained only cdk6 in uninfected CEM and Jurkat cells. However, a more interesting pattern emerged from HTLV-1-infected cells: cdk2, cdk4, and cdk6 were all present in the cyclin D2 immunoprecipitated complex (Fig. 5A). This pattern was also evident in immunoprecipitations using only one-fourth of the original infected extracts. As seen in Fig. 6B, when the cyclin D2 levels were normalized between MT-2 and CEM cells, all three cdks still were complexed with cyclin D2. As controls, a number of other cdk antibodies (cdk5, cdk7, cdk9, and cdc2) which were absent in the infected cyclin D2 immunoprecipitates were used in Western blot (data not shown). Similar results were obtained for monoclonal antibodies DCS-3 and DCS-5, against cyclin D2 protein (32) (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 5.   Various cdk partners of cyclin D2 in HTLV-1 infected cells. (A) Extracts from uninfected (Jurkat and CEM) and HTLV-1-infected (MT-2 and C8166) cells were used for immunoprecipitation with anti-cyclin D2 antibody and subsequently Western blotted with anti-cdk2, -4, and -6. Only cyclin D2 from HTLV-1-infected cells showed the presence of all three cdks in the complex. A number of antibodies specific to other cdks (cdk5, cdk7, cdk9, and cdc2) were used in cyclin D2 immunoprecipitation-Western blot assays and were found to be negative in HTLV-1-infected cells (data not shown). (B) 1/10 of the input cellular lysates used in immunoprecipitations.

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Normalized concentrations of cyclin D2-associated complexes from infected and uninfected cells. A total of 500 µg of cellular proteins (MT-2 and CEM) was mixed with 50 µl of rabbit anti-human cyclin D2 antibody C-17 (Santa Cruz Biotechnology catalog no. sc-181) for immunoprecipitation and mixed for 12 to 14 h at 4°C; the next day, 150 µl of 30% protein G+A-agarose beads was added for 2 h, and the samples were pelleted, washed, and processed as in experiments represented in Fig. 5. (A) Western blot with anti-cyclin D2 antibody; (B) immunoprecipitation with anti-cyclin D2 antibody followed by Western blotting with anti-cdk2, -4, and -6 antibodies. Similar results were observed at higher concentrations of input (up to 10 mg) of MT-2 or CEM extract (data not shown).

Substrate specificity of cyclin D2-associated complexes from HTLV-1-infected cells. We next examined whether cyclin D2-associated complexes were functional and could phosphorylate substrates such as the Rb and/or histone H1 proteins. The Rb protein, by means of phosphorylation, has been shown to be the protein at the R point which is involved in preparing cells to enter S phase. Rb is normally phosphorylated by cdk4 and cdk6 but not cdk2. The cdk2-cyclin complex can, however, phosphorylate other substrate such as histone H1 protein. The cyclin D2 immunoprecipitates from both infected and uninfected cells were used in Rb and H1 kinase assays. Cellular extracts of both infected and uninfected cells from various stages of G₁ phase were obtained and used for immunoprecipitations followed by a kinase assay. As shown in Fig. 7, Rb is phosphorylated within the first 2 h of nocodazole release. The level of phosphorylation before the R time point was much more pronounced in infected cells (10-fold) than in uninfected cells (2.6-fold). More importantly, the cyclin D2 immunoprecipitate from HTLV-1-infected cells was able to phosphorylate histone H1, a substrate for cdk2-associated complexes. Taken together, these results suggest that the cyclin D2-cdk2, cyclin D2-cdk4, and cyclin D2-cdk6 complexes physically associate and are functionally active in HTLV-1-infected cells.

View larger version (25K): [in this window] [in a new window]   FIG. 7.   Functional effects of cyclin D2-cdk partners from HTLV-1-infected cells. (A) Diagram of immunoprecipitations using anti-cyclin D2 antibody from both infected and uninfected cells treated with an M-phase blocker (nocodazole) and released. Following release, samples at various time points were processed and used for immunoprecipitations with cyclin D2 antibody. (B) FACS analysis of cells depicted in panel A following block and release with nocodazole. (C) Cyclin D2-immunoprecipitated complexes from infected and uninfected cells were washed and used in kinase assays with histone H1 and recombinant Rb proteins. Both cells traversed into the G1 phase following release, with higher kinase activity present in HTLV-1-infected cells when using Rb as a substrate (compare 2 to 4 h postrelease in MT-2 and CEM cells). However, only histone H1 (H1) was phosphorylated from HTLV-1-infected immunoprecipitates, implying that cdk2, which preferentially phosphorylated H1, is active in these cells (compare lanes 4 to 8).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The functional significance of cyclin D2 in vivo has been demonstrated in knockout animal models. Cyclin D2-deficient females were sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone, whereas mutant males display hypoplastic testes. In ovarian granulosa cells, this hormone specifically induced cyclin D2 via a cyclic AMP (cAMP)-dependent pathway, indicating that expression of the various D-type cyclins is under control of cAMP response element (CRE) signaling pathways (41).

The human cyclin D2 gene (CCND2) has been mapped to chromosome 12p13 and trisomy 12, which is the most common chromosomal change in lymphomas of B-CLL and immunocytomas. Previously, cyclin D2 mRNA was found to be overexpressed in 29 of 34 B-CLL cases and in all cases of LPL; the level of cyclin D2 expression in these disorders was, on average, 5- to 10-fold higher than in normal resting B lymphocytes (11). Cyclin D3 was not detected in any sample from B-cell chronic lymphocytic leukemia or lymphoplasmacytic lymphoma (LPL) patients, whereas cyclin D1 was expressed in only three cases (one LPL and two mantle cell lymphoma) associated with a t(11;14) translocation. Other interesting observations on the cyclin D2 gene have been noted when retroviral sequences were found adjacent to the cyclin D2 open reading frame. The vin-1 gene, first identified as the common site of provirus integration in retrovirus-induced rodent T-cell leukemia, was shown to be identical to the cyclin D2 gene (46). The possible role of the vin-1/cyclin D2 gene regulation in rodent oncogenesis is suggested by the overexpression of cyclin D2 that results from adjacent provirus integration.

Human DNA viruses have also been shown to either regulate cyclin D2 or acquire a gene homologous to the human counterpart. For instance, Epstein-Barr virus (EBV)-infected cells have shown an up-regulation of the cyclin D2 promoter in their infected hosts. The presence of either wild-type EBV or its transforming latent membrane protein 1 was found to induce the expression of cyclin D2; in control normal B cells or EBV-negative Burkitt's lymphoma cells, there is no expression of D-type cyclins. Up-regulation of latent membrane protein 1 can lead to Rb hyperphosphorylation and uncontrolled cell proliferation (2). Human herpesvirus 8, another herpesvirus family member, contains a gene, v-cyclin D, that is a homologue of the cellular cyclin D2 gene and encodes a protein that promotes passage through the G₁ phase of the cell cycle. Spindle cells of Kaposi's sarcoma, which have been regarded as the tumor cells of this cancer, contain v-cyclin D mRNA. Expression of v-cyclin D protein may be involved in the pathogenesis of Kaposi's sarcoma by promoting cell proliferation (10).

Schmitt and colleagues recently demonstrated that upon transduction of primary human cord blood T cells, Tax suppression stopped lymphocyte growth and caused cell cycle arrest in the G₁ phase (39). Upon reinduction of Tax expression, the arrested cells entered the S phase. These authors have suggested that Tax has mitogenic activity, which is required for stimulating the G₁- to S-phase transition of immortalized lymphocytes. Along the same lines, others have suggested that Tax affects cell phase transition by forming a direct protein-protein complex with p16^INK4a, thereby inactivating an inhibitor of G₁-to-S-phase progression. Tax formed a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein (36).

We have observed that the activation of the endogenous cyclin D2 mRNA by Tax, at the G₁ phase of the cell cycle, is evident in not only human but also mouse cells transfected with Tax. This was seen in RPAs using bona fide endogenous promoters that carry all necessary elements, including proper DNA structure, copy number, and chromatin structure. This phenomenon seems to be general to HTLV-1-infected cell lines (IL-2 independent), Tax-transfected mouse cells (CTLL), and ATL and HAM/TSP patient samples (IL-2 dependent). Interestingly, all uninfected lines tested, including CEM, Jurkat, Molt, and H9, and normal peripheral blood mononuclear cells show an up-regulation of the cyclin D3 promoter and not the cyclin D2 promoter. This intriguing observation implies that cyclin D family members are the first targets of HTLV-1 regulation when the host enters the cell cycle.

The cyclin D2 promoter contains a number of visible DNA-binding elements. The general structure of the cyclin D2 promoter contains no TATA box but does contain putative DNA-binding sites for Sp1, CREB, C/EBP, PEA3, NF-B, SIF, E2F, GCF, and AP1. The CAP site in the promoter was shown to be a loosely conserved sequence where a number of transcription sites have been observed (7, 22). We have shown that the proximal CRE in the promoter is partially responsible for the activation seen by Tax (38a). As expected, the activation was enhanced by CBP, a general coactivator of the cAMP pathway. It remains to be seen if other sites such as NF-B and/or AP2 contribute to overall activity of the activated transcription by Tax. High levels of NF-B and AP2 have previously been found in HTLV-1-infected cells (3). We are currently using 5' deletion constructs of the cyclin D2 promoter, in transfections as well as in in vitro transcription reactions, to define the contribution of various DNA-binding elements as well as coactivator p300/CBP within the cyclin D2 promoter.

A number of cdks, including cdk2, -4, -5, and -6, have been reported to interact with cyclin D2. In a two-hybrid system, cyclin D2 interacted with cdk5, a serine/threonine kinase that displays neuron-specific activity. Sweeney and colleagues (45) have also shown that the D-type cyclins are not necessarily redundant in their function. For instance, the cyclin D2-associated kinase activity could phosphorylate histone H1, a substrate for cdk2 but not for cdk4 and cdk6, and was largely inhibited by cdk2-specific inhibitors. Consistent with the hypothesis that cyclin D2 can bind to other cdk partners, we have shown that cyclin D2 can pair up with kinases such as cdk4 and cdk6, which can phosphorylate the R checkpoint protein Rb, as well as cdk2-phosphorylating histone H1, a general protein marker for chromatin remodeling and gene expression (12, 15). The interactions of cyclin D2 and cdk2, -4, and -6 are independent of Tax, as we have not observed the presence of Tax protein in the cyclin D2 immunoprecipitates (data not shown). Therefore, the activation pathway of cyclin D2 and its cdk partners does not directly involve the physical interaction with the Tax protein, as observed in the case of p16 inhibitor and Tax.

It remains to be seen what substrates other than Rb are regulated by the cyclin D2-associated kinases which result in accelerated transition from G₁ to S phase. For instance, we have recently observed that p53, a major checkpoint protein in HTLV-1-infected cells, can be phosphorylated by the cyclin D2-cdk complex in vitro (23a), reinforcing the notion that proteins downstream of the R checkpoint may be the target of cyclin D2-associated kinases, thereby inactivating G₁/S checkpoint controls. Further experiments will shed light on the effect of this complex and its associated polypeptides at early G₁ phase.