Article Figures & Data
Figures
- Fig. 1.
Generation of MCF-7 cell lines with Zn-inducible c-Myc or cyclin D1. MCF-7.7 cell lines stably transfected with the Zn-inducible pΔMT vector containing c-Myc cDNA (myc), cyclin D1 cDNA (D1), or no cDNA (mt) were growth arrested for 48 h with 10 nM antiestrogen ICI 182780. (A) Cells were treated at time zero with either 50 μM Zn (+) or vehicle (−). After 6 to 8 h, cell lysates were prepared and immunoblotted with antibodies against c-Myc and cyclin D1. (B) Cells were treated at time zero with the indicated concentration (micromolar) of Zn. Cells were harvested (18 h for myc cells; 21 h for D1 and mt cells) and stained for DNA content, and the proportion of cells in S phase was determined by flow cytometry. (C) Cells were treated at time zero with either the indicated concentration (micromolar) of Zn, 100 nM 17β-estradiol (E2) or vehicle (ethanol [EtOH]). At intervals thereafter, cells were harvested and stained for DNA content, and the proportion of cells in S phase was determined by flow cytometry.
- Fig. 2.
c-Myc and cyclin D1 protein expression following E2 treatment or Zn induction of c-Myc or cyclin D1. Three of the clonal MCF-7.7 cell lines used for Fig. 1 (myc.3, D1.13, and mt.4) were growth arrested with 10 nM ICI 182780 for 48 h. (A) Cells were treated at time zero with 50 μM Zn or 100 nM E2 (+) or with vehicle (−). Whole-cell lysates were prepared at intervals thereafter (shown in hours). Lysates were immunoblotted with antibodies against c-Myc and cyclin D1. (B) Cells were treated at time zero with either the indicated concentration (micromolar) of Zn, 100 nM E2, or vehicle (Con). At intervals thereafter, cell lysates were prepared and immunoblotted with antibodies against c-Myc or cyclin D1. Autoradiographs were quantitated by densitometry and expressed relative to time-matched controls. After 18 h (myc.3) or 21 h (D1.13), cells were harvested and stained for DNA content, and the proportion of cells in S phase was determined by flow cytometry. Data for protein and S phase are from the same experiment.
- Fig. 3.
Cdk4 activity following E2 treatment or Zn induction of c-Myc or cyclin D1. The experimental design was as described for Fig. 2A. (A) Cdk4 immunoprecipitates were assayed for kinase activity toward a GST-pRB773-928 substrate. Autoradiographs were quantitated by densitometry and expressed relative to time-matched controls. E2 treatment of all cell lines resulted in similar levels of Cdk4 activity and is represented by results from myc.3 cells. Points shown represent the means of two independent experiments. (B) Total cell lysates were immunoblotted with antibodies against a pRB-derived phosphopeptide that contains a Cdk4-specific target (phospho-Ser 780). The immunoreactive band labeled with an asterisk is nonspecific since it was not detected in pRB immunoprecipitates (data not shown).
- Fig. 4.
Cyclin E-Cdk2 activation and hyperphosphorylation of pocket proteins following E2 or Zn treatment. The experimental design was as described for Fig. 2A. Control lanes (C) represent results from cells treated with vehicle. (A) Cyclin E immunoprecipitates were assayed for kinase activity toward histone H1 substrate. A 1.5-h time point is included for D1.13 and mt.4 cells. For each cell line, the results shown are from the same autoradiograph. Autoradiographs were quantitated by densitometry, and results are expressed relative to those for time-matched controls. E2treatment of all cell lines resulted in similar levels of cyclin E-associated kinase activity and is represented by results for D1.13 cells. Points shown for 3 to 16 h represent the means of two independent experiments. (B) Cell lysates were immunoblotted with either pRB or p130 antibodies. Three distinct phosphorylated species of p130 are indicated. For each cell line and antibody the results shown are from the same autoradiograph.
- Fig. 5.
Inhibition of E2-, c-Myc-, or cyclin D1-induced G1-S-phase progression by the Cdk2-specific inhibitor roscovitine. The experimental design was as described for Fig. 2A except that cells were pretreated with the indicated concentration of roscovitine 30 min prior to treatment with Zn, E2, or vehicle. After 18 h (myc.3) or 21 h (D1.13), cells were harvested and stained for DNA content, and the proportion of cells in S phase was determined by flow cytometry. For each concentration of roscovitine the increase in S phase with either Zn or E2 (above the vehicle-treated control level) was expressed as a percentage of the increase in S phase with 0 μM roscovitine. Points represent the means of three (myc.3) or four to five separate experiments (D1.13), and error bars indicate the standard errors of the means.
- Fig. 6.
Mechanism of activation of cyclin E-Cdk2 by E2 treatment or Zn induction of c-Myc or cyclin D1. The experimental design was as described for Fig. 2A. Lysates from myc.3 and D1.13 cells were prepared 10 h after treatment with Zn and fractionated on a HiLoad 16/60 Superdex 200 gel filtration column. Cyclin E complexes were immunoprecipitated from 3-ml fractions and then either assayed for histone (H1) kinase activity (as described for Fig.3A) or resuspended in 20 μl of sample buffer, separated by SDS-PAGE, transferred to a nitrocellulose filter, and sequentially blotted with the indicated antibodies. Fraction 5 (which contained high levels of cyclin E) was loaded in variable amounts in order to permit comparison of the relative levels of coimmunoprecipitating proteins with those in fractions 1 and 2 (combined). Lanes containing similar levels of cyclin E protein are indicated with either an asterisk (Zn treated) or an arrowhead (vehicle treated). The elution volumes for marker proteins of known molecular weight are indicated at the top.
- Fig. 7.
Increased association of cyclin E with p130 follows E2 treatment or Zn induction of c-Myc or cyclin D1. The experimental design was as described for Fig. 2A. Lysates were prepared 10 h after treatment. (A) Cyclin E or p130 immunoprecipitates (IP) from total cell lysates were immunoblotted with the indicated antibodies. (B) p130 was immunoprecipitated from lysates with p130 antibodies in the presence (+) or absence (−) of immunizing peptide. The supernatant was fractionated on a gel filtration column as described for Fig. 6. Representative cyclin E protein immunoblots and cyclin E histone (H1) kinase assays are shown following E2and Zn treatment of myc.3 cells and Zn treatment of D1.13 cells. The elution volumes for marker proteins of known molecular weight are indicated.
- Fig. 8.
Effects of E2 treatment or Zn induction of c-Myc or cyclin D1 on the binding of p21 to recombinant cyclin E-Cdk2. The experimental design was as described for Fig. 2A. (A) Lysates prepared 10 h after treatment were incubated with GST-cyclin E-Cdk2 complexes or GST. GST proteins were recovered on glutathione-agarose beads and then immunoblotted for p21. (B) Cyclin D1 immunoprecipitates were immunoblotted for p21. Immunoblots of control nonimmune rabbit antiserum immunoprecipitates failed to detect p21. Control lanes (C) represent results from cells treated with vehicle. For each cell line the results shown are from the same radiograph.
