Article Figures & Data
Figures
- Fig. 1.
Generation of Mv1Lu cell lines with inducible c-Myc expression. (A) Clonal tet-Myc cell lines (TM1 to TM3) were maintained in medium containing 1 μg of tetracycline per ml and then grown in the absence of tetracycline for 18 h before being harvested and analyzed by anti-human c-Myc Western immunoblotting. Parental Mv1Lu cells were also analyzed in the presence and absence of tetracycline with the anti-human c-Myc antibody. (B) Comparison of the levels of induced human c-Myc expressed in tet-Myc clones with other human cell lines by anti-human c-Myc Western immunoblotting. The same amount of cell lysate protein was loaded in each lane. (C) The human c-Myc protein levels visualized in panel B were quantitated by densitometry of the signal and plotted as relative levels of c-Myc protein in arbitrary units.
- Fig. 2.
Analysis of cell cycle progression and G1regulators upon treatment of tet-Myc cells with TGF-β. (A) Parental Mv1Lu cells were maintained in growth medium, and TM2 cells grown in the absence of tetracycline (Myc on) or in the presence of 1 μg of tetracycline per ml (Myc off) for 18 h before the addition of 200 pM TGF-β. Cells were harvested for flow-cytometric analysis of DNA content after 20 h in the presence or absence of TGF-β. The percentage of cells in the G1 phase at this time is indicated. (B) TM2 cells were harvested and analyzed by anti-pRB Western immunoblotting at the indicated times after TGF-β addition. (C) TM2 cells were analyzed for the presence of p27-cdk complexes following 20 h of TGF-β treatment. Immunoprecipitations were performed on cell lysates with anti-p27, and Western immunoblotting was used to determine total p27 or p27-associated cdk4 and cdk2 levels. cdk2 was also immunoprecipitated from these lysates, and the ability of these complexes to phosphorylate histone H1 in vitro was determined by kinase assays.
- Fig. 3.
Effect of exogenous c-Myc expression on G1cdk components. TM2 cells were induced to express exogenous Myc by the removal of tetracycline and harvested at the indicated times. Immunoblots were probed for exogenous human c-Myc and endogenous mink cdk2, p27, cdk4, cdk6, cyclin D1, and cyclin D2 proteins.
- Fig. 4.
Induction of p15Ink4b in tet-Myc clones. (A and B) Three tet-Myc clones (TM1 to TM3) were induced to express c-Myc by the absence of tetracycline for 18 h and then treated with 200 pM TGF-β. At 6 h after TGF-β addition, the cells were harvested and their RNA was analyzed by Northern blotting for p15Ink4b expression. A representative blot with the TM1 cell lines is shown (A). The same blot was probed for GAPDH expression as a loading control. Quantitation of such p15 blots for all three TM clones was carried out and is shown as the fold induction of this RNA by addition of TGF-β, normalized to GAPDH values (B). (C) p15Ink4b induction in parental Mv1Lu cells and TM1 cells after 20 h without tetracycline followed by TGF-β addition for the indicated times. The same Northern blot was probed forPAI-1 as another representative TGF-β-responsive gene and GAPDH as a loading control. (D) TM1 cells were transfected with the p15p113-Luc reporter. Transfections were carried out without or with TGFβ in the absence or presence of tetracycline for 24 h prior to analysis of luciferase activity. Results are means ± standard deviations of triplicate transfections. HaCaT cells were transfected with p15 p113-Luc reporter, without or with a human c-Myc expression vector, and analyzed as described above.
- Fig. 5.
Analysis of cdk4 complexes by gel filtration. (A) Fractions from Superdex 200 gel filtration of lysates from parental Mv1Lu cells were analyzed by immunoblotting and immunoprecipitation for the composition of endogenous cdk4 complexes. Fractions 20 to 36 are shown, with the positions of protein molecular weight markers indicated at the top. Fractions were subjected directly to anti-cdk4 immunoblotting in both proliferating cells (−TGFβ), cells treated with 200 pM TGF-β for 20 h (+TGFβ), and cells allowed to grow to confluence and maintained in this arrested state for three days in the presence of growth media (contact arrested). Western immunoblotting analysis of p27 and hsp90 was also carried out on fractions from proliferating Mv1Lu cells. Immunoprecipitations of endogenous cyclin D1 and p27 from fractions were subjected to anti-cdk4 Western immunoblotting to determine the levels of cyclin D1-bound cdk4 (D1-K4) and p27-bound cdk4 (p27-K4) respectively. (B) Fractions from Superdex 200 gel filtration of lysates from tet-K4 cells grown in the absence of tetracycline were collected. Fractions 22 to 24 (lane 1), 27 to 29 (lane 2), and 32 to 33 (lane 3) were pooled and immunoprecipitated with anti-cdk4 antibodies, followed by cdk4-associated Rb kinase assays (top) or subjected directly to anti-cdk4 immunoblotting analysis (middle). The Rb kinase specific activity for each pool was plotted as the value of Rb kinase activity over protein units (bottom). Kinase activity and protein level are plotted as arbitrary units determined by PhosphorImager quantitation and densitometry, respectively. (C) The level of cdk4 in cells under each of the conditions in panel A was determined by Western immunoblotting of the lysates loaded on the column. The percentage of cells in G1 phase under each of these conditions before lysis, as determined by flow cytometry, is indicated.
- Fig. 6.
Effect of overexpression of cell cycle regulators on cdk4 complexes in Mv1Lu cells. (A to C) Mv1Lu cells engineered to conditionally overexpress p15 (tet-p15) (A), p27 (tet-p27) (B), or cyclin D1 (tet-D1) (C) in the absence of tetracycline were analyzed by gel filtration for cdk4 complexes. Cell lines were either maintained in the presence of tetracycline (exogenous gene off) or removed from tetracycline for 18 h (exogenous gene on) before being harvested for analysis. Exogenous p15 in tet-p15 cells (p15 on) was visualized by Western immunoblotting with an antibody specific to human p15 (A). (D and E) tet-D1 cells were removed from tetracycline for 18 h and treated with 200 pM TGF-β (+TGF-β) for 20 h before being lysed. The proportion of cells in the G1 phase and the level of cdk4 under each of these conditions are shown. The expression of the exogenous genes in tet-p15 and tet-p27 cells has been described previously (47, 48). The expression of exogenous cyclin D1 in tet-D1 cells was analyzed by Western immunoblotting with an antibody specific for human cyclin D1 and is also shown in panel E. (F) Comparison of cyclin D1 (top) and cyclin D2 (bottom) protein levels in tet-D1, TM2, and parental Mv1Lu cells in the absence and presence of tetracycline.
- Fig. 7.
c-Myc overexpression and cdk4 complexes in Mv1Lu cells. (A) TM2 cells grown in the presence of tetracycline (Myc off) or grown for 18 h in the absence of tetracycline (Myc on) were harvested for gel filtration analysis either directly (−TGF-β) or after incubation with TGF-β for 20 h (+TGF-β). Following gel filtration of these cell lysates, fractions were subjected to anti-cdk4 Western immunoblotting either directly (cdk4) or following anti-p27 immunoprecipitation (p27-K4). (B) Lysates (3 mg of total protein) from proliferating Mv1Lu cells and TM2 cells induced to express c-Myc for 18 h in the absence of tetracycline (tet-Myc on) were incubated with 80 μg of recombinant human p15 (+rhp15) at 37°C for 30 min and then subjected to gel filtration and anti-cdk4 immunoblotting. (C) The proportion of cells in G1 phase and the level of cdk4 under each of these conditions.
- Fig. 8.
Schematic representation of cdk4 complexes and their control by TGF-β, p15, and c-Myc in Mv1Lu cells. In proliferating epithelial cells, endogenous cdk4 is distributed among three populations: an abundant high-molecular-weight pool of latent cdk4, a low-abundance pool containing active cyclin D-cdk4 and p27-cyclin D-cdk4 complexes, and an inactive population of monomeric and Ink4b-bound cdk4. The latent pool serves as a source of cdk4 for cyclin D, yielding active cyclin D-cdk4 complexes. TGF-β and p15 target both the latent and active forms of cdk4, converting these to the inactive cdk4 population. TGF-β causes rapid downregulation of c-myc and upregulation of p15. cdk4 targeting by TGF-β is prevented by the enforced expression of normal levels of c-Myc, suggesting that c-Myc downregulation by TGF-β is required for activation of the p15 G1 arrest pathway.
