Inducible expression of Tax in JPX9 and SS8tet Tax T cells. Expression of Tax was assessed by Western blotting with a monoclonal antibody. (A) Tax was induced in JPX9 with zinc (+). −, Mock-treated cells. (B) Expression of Tax was repressed in SS8tet Tax cells with 1 μg of doxycycline (Dox) added to the culture medium (+). −, Mock-treated cells.

PCR amplification of the p15^INK4b gene from JPX9 cells. Cellular DNAs were prepared from various cell lines: U2OS (lanes 1, 5, and 9), JPX9 (lanes 2, 6, and 10), and MT4 (lanes 3, 7, and 11). p15^INK4b-specific signal was assayed by amplification with two pairs of primers expected to produce a fragment of 130 bp in the first exon (lanes 1 to 4) and a fragment of 231 bp in the second exon (lanes 5 to 8) of p15^INK4b, respectively. A set of primers amplifying a fragment of 445 bp (lanes 9 to 12) in the actin gene was used as a positive control.

Effect of Tax on cyclin D-cdk4 and cyclin D-cdk6. (A) JPX9 cells were synchronized by serum starvation and then cultured with (+; lane 2) or without (−; lane 1) zinc for 8 h. Cell extracts were prepared and assayed by Western blotting for the expression of cyclin D3, cdk4, cdk6, or actin. Note that upon zinc induction, cyclin D3 appears as a doublet with a mass predominance in the slower-migrating form. (B) Sensitivity of the slower migrating form of cyclin D3 in JPX9 cells to phosphatase. Cell extracts from JPX9 cells were treated for 8 h with zinc (lanes 1 and 2); one extract was further incubated for 1 h at 0°C with 40 U of CIP (lane 2). Extracts were analyzed by Western blotting with anti-cyclin D3 antibody. The sample in lane 2 showed a distinct change in relative migration upon CIP treatment.

Binding of p16^INK4a but not p19^INK4d to Tax. Amylose-bound MBP-Tax fusion protein (lanes 1, 2, 5, and 6) and MBP protein (lane 3, 4, 5, and 6) were equilibrated for 3 h with 5 μl of extracts containing either GST-p16 (lanes 1 to 4) or GST-p19 (lanes 5 to 8). The resins were packed into columns, and flow-through fractions (ft; lanes 1, 3, 5, and 7) were collected. The resins were then extensively washed with buffer containing 0.1 M KCl. The final wash fraction did not contain any detectable GST-p16 or GST-p19 signal (data not shown). Beads were then eluted with buffer containing 0.25 M KCl (lanes 2, 4, 6, and 8). The presence of p16^INK4a or p19^INK4d in the elutions was assessed by Western blotting with anti-p16 or anti-p19 antibody.

Tax induces hyperphosphorylation of Rb. (A) The status of Rb phosphorylation was determined by Western blotting with anti-Rb in JPX9 cells synchronized first by serum starvation and then cultured with (+; lane 2) or without (−; lane 1) zinc. Cells were harvested 8 h after addition of zinc. pRb, Hypophosphorylated Rb; ppRb, hyperphosphorylated Rb. (B) Rb was detected by Western blotting in SS8tet Tax cells expressing Tax (+) and in SS8tet Tax cells not expressing Tax (−) (lanes 1 and 2) with anti-Rb. ppRb is the hyperphosphorylated form of Rb; pRb is the hypophosphorylated form. (C) Increased expression of E2F2 upon Tax induction. E2F2 was examined in extracts from SS8tet Tax cultured in the absence (+; lanes 2 and 4) or presence (−; lanes 1 and 3) of doxycycline by Western blotting by using an E2F2-specific antibody (left panel) or E2F2-specific antibody competed by mixing with a 2-μg/ml solution of the immunizing E2F2 peptide (right panel). The immunizing peptide competed specifically with a protein of an apparent size of 53 kDa but not with nonspecific lower-molecular-mass bands.

Tax induces the progression of cells from G₀/G₁ into S phase. The effect of Tax on the cell cycle was examined in either JPX9 or SS8tet Tax cells by FACS. (A) The DNA contents of synchronized cadmium-treated JPX9 cells expressing Tax in the presence (+serum+Cd) or absence (+Cd) of serum were analyzed by propidium iodide staining and compared to the identically treated parental Jurkat cell line with (+serum+Cd) or without (+Cd) serum. The DNA contents of JPX9 and Jurkat cells without cadmium and without serum are shown. Open line reflects actual values. Filled areas are the values interpreted from the MODFIT program. The short peaks to the left in the bottom panels are indicative of apoptotic cells. (B) The DNA contents of SS8tet Tax cells expressing (−Dox) or not expressing (+Dox) Tax were analyzed by propidium iodide staining. The cells were serum starved for 20 h and then stained with propidium iodide and analyzed by flow cytometry. In these profiles, the gating was such that apoptotic cells were not graphed. However, a significantly higher number of apoptotic cells was seen with cells expressing Tax. (C) Control DNA profiles of the parental cell line for SS8tet Tax (SS8 BPT) were examined after serum starvation for 20 h and treatment with (+Dox) or without (−Dox) doxycycline.

Tax coimmunoprecipitates with cyclin D1 and/or cyclin D3. (A) Intracellular complex of Tax with cyclin D3 or D1. In the left panel, each lane represents JEG-3 cells transiently transfected with 5 μg of pTM3-TaxH6 (lanes 3, 4, 7, and 8) and/or 5 μg of pCMV-cyclin D3 (cyc D3; lanes 2, 4, 6, and 8), infected with 2 × 10⁶ PFU of vTF7-3 vaccinia strain WR for 18 h, and subsequently labeled with [³⁵S]methionine. Protein complexes were identified by immunoprecipitation under native conditions with polyclonal antisera raised against either cyclin D3 (αcyclin D3) or Tax (αTax). Results are representative of two different experiments. In the right panel, JEG-3 cells were transiently transfected with 5 μg of pTM3-TaxH6 (lanes 2, 4, 6, and 8) and/or 5 μg of pCMV-cyclin D1 (cyc D1) (lanes 3, 4, 7, and 8), infected with 2 × 10⁶ PFU of vTF7-3 vaccinia strain WR for 18 h and subsequently labeled with [³⁵S]methionine. Protein complexes were identified by immunoprecipitation under native conditions with a polyclonal antisera raised against either cyclin D1 (αD1) or Tax (αTax). Results are representative of three different experiments. (B) In the top panel, detection of Tax coimmunoprecipitated with cyclin D1 or cyclin D3 by Western blotting is shown. Each lane represents 5 × 10⁶ 293T cells transiently transfected for 48 h with 20 μg of pCMV-Tax (lanes 1 to 6) and 20 μg of either pCMV-cyclin D1 (lanes 1 to 4) or pCMV-cyclin D3 (lanes 5 and 6). Protein complexes were immunoprecipitated under native conditions with polyclonal antisera raised against either Tax (αTax; lanes 1 and 2), cyclin D1 (αcyc D1; lanes 3 and 4) or cyclin D3 (αcyc D3; lanes 5 and 6), and the presence of Tax was detected by Western blotting with polyclonal antisera against Tax (αTax). Results are representative of two different experiments. In the middle and bottom panels, each lane represents 5 × 10⁶ 293T cells transiently transfected with 20 μg of pCMV-Tax (lanes 1 to 4) and 20 μg of either pCMV-cyclin D1 (middle panel) or pCMV-cyclin D3 (bottom panel) for 48 h. Protein complexes were immunoprecipitated under native conditions with polyclonal antisera raised against either cyclin D1 (αcyc D1; middle panel, lanes 1 and 2), Tax (αTax; middle panel, lanes 3 and 4; bottom panel, lanes 3 and 4), or cyclin D3 (αcyc D3; bottom panel, lanes 1 and 2). The presence of either cyclin D1 (middle panel) or cyclin D3 (bottom panel) was detected by Western blotting with polyclonal antisera raised against cyclin D1 (middle panel) or against cyclin D3 (bottom panel). Results are representative of two different experiments.

Activation of cyclin D-cdk4 and cyclin D-cdk6 kinases by Tax. (A) Jurkat or JPX9 cells were synchronized by serum starvation for 20 h. Cells propagated in the presence (+; lanes 3, 6, 10, 13, 15, and 17) or absence (−; lanes 1, 2, 4, 5, 8, 9, 11, 12, 14, and 16) of zinc were harvested at the indicated time (in hours). Cell extracts were immunoprecipitated with anti-cdk4 (lanes 1 to 6), anti-cyclin D3 (lanes 8 to 13), and anti-cdk6 (lanes 14 to 17), or a control irrelevant (lanes 7 and 18) antibody. The immunoprecipitates were tested for kinase activity with GST-Rb as substrate. In the top left panel, the cdk4 activities are shown; in the bottom left panel, the cyclin D3-associated kinase activities are shown. In the right panel (lanes 14 to 17) extracts were harvested after 8 h of induction with zinc, and the cdk6 kinase activities were assayed. (B) In vitro kinase assays with GST-Rb as substrate with anti-cdk4 immunoprecipitates from exponentially growing SS8tet Tax cells expressing (−Dox; lane 2) or not expressing (+Dox; lane 1) Tax. Quantitations of each series of activities are shown in bar graphs.

Tax induces cyclin D1-cdk4 kinase activity in C2C12 myoblasts and inhibits the differentiation of myoblasts into myotubes. (A) C2C12 cells stably expressing Tax were engineered by cotransfection of a vector encoding Tax under the control of the HTLV-1 LTR and a vector expressing neomycin resistance. The control cells were transfected with neomycin resistance vector alone. Stable expression of Tax (lanes 2 and 3) in two illustrative cell lines (C2C12 cl3 and C2C12 cl6) is shown by Western blotting with Tax-specific serum. (B) C2C12 cl6 and the C2C12 vector-selected control were cultured to 95% confluence, harvested, and probed for Rb expression (lanes 1 and 2) by Western blotting. Compared to C2C12, the hyperphosphorylated form predominates in C2C12 cl6 (upper panel). The lower panels show Rb-kinase assays performed with immunoprecipitates of normalized cell extracts with anti-cdk4 (lanes 3 and 4) or anti-cyclin D1 (lanes 5 and 6) antibody. (C) C2C12 vector-selected (top; non-Tax-expressing) and C2C12 cl6 (bottom; Tax-expressing) cells were grown in DMEM 2% horse serum (differentiating medium). After 6 days cells were fixed in staining solution. Long dark tubular morphology indicates differentiation of cells into myotubes. (D) Expression of Tax in C2C12 myoblasts decreases the differentiation specific marker, myogenin. C2C12 cells were transiently lipofected either with 2 μg of Tax expression vector (+) or with 2 μg of a control vector (−). At 15 h after lipofection, cells were cultured in differentiating medium (DMEM–2% horse serum) for an additional 48 h. Cell extracts were normalized and prepared as described in Materials and Methods, resolved by SDS-PAGE, and analyzed by immunoblotting with a monoclonal Tax antibody (upper panel) or a monoclonal myogenin antibody (lower panel).

A Tax point mutant neither coimmunoprecipitates with cyclin D3 nor induces increased cyclin D3-cdk6 kinase activity. (A) Inducible expression of Tax in JPX9 and JPX9m cell lines. Expression of Tax wild-type and Tax mutant was assessed by Western blotting with a monoclonal antibody to Tax. +, Tax wild type and mutant induced in JPX9 and JPX9m strains with zinc; −, control mock-treated cells. JPX9m expresses a transcriptionally inactive Tax mutant that contains a single amino acid (arginine) inserted at position 62. (B) JPX9 or JPX9m cells were treated (lanes 2 and 4) or mock treated with zinc (lanes 1 and 3) for 16 h. After solubilization in lysis buffer, complexes were immunoprecipitated under native conditions with polyclonal antisera against cyclin D3. The presence of Tax in complexes was assessed by Western blotting with a monoclonal antibody to Tax. (C) Tax mutant protein in JPX9m does not compel G₁-to-S progression as measured by [³H]thymidine incorporation. JPX9 and JPX9m cells were synchronized by serum starvation and then treated with or without zinc for 16 h. Cells were then plated into a 96-well plate and labeled with [³H]thymidine. Cell cycle progression from G₁ to S phase was measured by [³H]thymidine incorporation. The relative values for JPX9 and JPX9m cells were normalized against parallel values obtained in similarly treated Jurkat cells, with thymidine incorporation for JPX9 set at 100%. (D) Comparison of kinase activity of cyclin D3-cdk6 in cells expressing Tax mutant or Tax wild type. In vitro kinase assays with GST-Rb as substrate were performed. JPX9 or JPX9m cells were synchronized by serum starvation for 20 h. Cells grown in the presence (+; lanes 6, 8, 10, and 12) or absence (−; lanes 5, 7, 9, and 11) of zinc for 8 h were harvested, and cell extracts were immunoprecipitated with either anti-cdk6 (lanes 5 to 8) or anti-cyclin D3 (lanes 9 to 10) antibody. The immunoprecipitates were tested for kinase activity with GST-Rb as the substrate.