p21 functions in a post-mitotic block checkpoint in the apoptotic response to vinblastine

Abstract

We have shown previously that in KB-3 (HeLa) cells vinblastine causes downregulation of the CDK inhibitor p21 through a c-Jun regulated pathway. To test the hypothesis that p21 downregulation is necessary to alleviate a protective function, we transfected p21 in KB-3 cells and examined the apoptotic response to vinblastine. The results showed that cells overexpressing p21 were apoptosis-resistant, not through an ability of p21 to cause cell cycle arrest prior to mitotic arrest, but through altering the fate of mitotically arrested cells after drug treatment. Moreover, p21 null HCT116 cells were more prone to vinblastine-induced apoptosis relative to wild-type cells. The results provide support for a model whereby p21 downregulation promotes vinblastine-induced apoptosis by alleviating its protective function following mitotic arrest.

Fig. 1

Overexpression of p21 in KB-3 cells. (A) KB-3 cells were cotransfected with plasmids encoding GFP and p21/WAF1 as described in the text. Images were taken 24 h post-transfection and are representative of several fields and several independent experiments. (a) phase contrast; (b) green fluorescence. (B) KB-3 cells were untransfected or transfected with empty vector (EV) or p21/WAF1 expression plasmid. At 24, 48 and 72 h post-transfection, cell extracts were prepared and subjected to immunoblotting for p21 and GAPDH as a loading control. (C) 24 h after transfection, cells were washed, fixed and incubated with FITC-conjugated p21 monoclonal antibody. Samples were analyzed by flow cytometry using FACS Aria. (a) no p21 antibody as negative control; (b) untransfected KB-3 cells; (c) p21 transfected cells. Percentages of p21 negative cells (P2 fraction) and p21 positive cells (P3 fraction) are given and are representative of data from two independent experiments.

Fig. 1

Overexpression of p21 in KB-3 cells. (A) KB-3 cells were cotransfected with plasmids encoding GFP and p21/WAF1 as described in the text. Images were taken 24 h post-transfection and are representative of several fields and several independent experiments. (a) phase contrast; (b) green fluorescence. (B) KB-3 cells were untransfected or transfected with empty vector (EV) or p21/WAF1 expression plasmid. At 24, 48 and 72 h post-transfection, cell extracts were prepared and subjected to immunoblotting for p21 and GAPDH as a loading control. (C) 24 h after transfection, cells were washed, fixed and incubated with FITC-conjugated p21 monoclonal antibody. Samples were analyzed by flow cytometry using FACS Aria. (a) no p21 antibody as negative control; (b) untransfected KB-3 cells; (c) p21 transfected cells. Percentages of p21 negative cells (P2 fraction) and p21 positive cells (P3 fraction) are given and are representative of data from two independent experiments.

Fig. 2

Cell cycle distribution is unchanged in p21 expressing cells. KB-3 cells were transfected with empty vector (EV) (A) or p21/WAF1 plasmid (B) using established transfection conditions. 24 hours after transfection, cells were washed, fixed and incubated with propidium iodide/RNase for 20 min. DNA content was analyzed by flow cytometry. 10,000 events were analyzed for each sample. Data presented in the table (panel C) represent averages from three independent experiments (mean + S.D.). p-values > 0.2, no significant differences.

Fig. 3

p21-expressing cells are more resistant to vinblastine-induced apoptosis. (A) KB-3 cells were transfected with empty vector (EV) or p21 expression plasmid and after 20 h were treated with vehicle (1% DMSO) or 30 nM vinblastine for 36 h. Cells were washed, fixed and incubated with PE-conjugated anti-active caspase-3 specific antibody using the manufacturer’s instructions (BD Biosciences) and analyzed by flow cytometry using FACS Aria. Percentages of cells negative for active caspase-3 (P2) or positive for active caspase-3 (P3) are indicated and are representative of two independent experiments. (B) KB-3 cells were transfected as in (A) and treated with 30 nM vinblastine for the times indicated. Whole cell extracts were immunoblotted for PARP, with uncleaved (110 kDa) and cleaved (85 kDa) species indicated. GAPDH was used as a loading control.

Fig. 3

p21-expressing cells are more resistant to vinblastine-induced apoptosis. (A) KB-3 cells were transfected with empty vector (EV) or p21 expression plasmid and after 20 h were treated with vehicle (1% DMSO) or 30 nM vinblastine for 36 h. Cells were washed, fixed and incubated with PE-conjugated anti-active caspase-3 specific antibody using the manufacturer’s instructions (BD Biosciences) and analyzed by flow cytometry using FACS Aria. Percentages of cells negative for active caspase-3 (P2) or positive for active caspase-3 (P3) are indicated and are representative of two independent experiments. (B) KB-3 cells were transfected as in (A) and treated with 30 nM vinblastine for the times indicated. Whole cell extracts were immunoblotted for PARP, with uncleaved (110 kDa) and cleaved (85 kDa) species indicated. GAPDH was used as a loading control.

Fig. 4

p21 expression promotes DNA endoreduplication. KB-3 cells were transfected with empty vector (EV) or p21/WAF1 plasmid using established transfection conditions. Twenty-four post-transfection, cells were left untreated or treated with vinblastine for 16, 24 or 36 h, then stained with PI and analyzed by flow cytometry. At least 10,000 events were counted for each sample. A. DNA content profiles determined by flow cytometry. B. Tabulation of data of panel A.

Fig. 4

p21 expression promotes DNA endoreduplication. KB-3 cells were transfected with empty vector (EV) or p21/WAF1 plasmid using established transfection conditions. Twenty-four post-transfection, cells were left untreated or treated with vinblastine for 16, 24 or 36 h, then stained with PI and analyzed by flow cytometry. At least 10,000 events were counted for each sample. A. DNA content profiles determined by flow cytometry. B. Tabulation of data of panel A.

Fig. 5

p21 null cells are more sensitive to vinblastine-induced apoptosis. (A) Wild-type and p21 null HCT116 human colon carcinoma cells were treated with vehicle (1% DMSO) or 100 nM vinblastine for 24 or 48 h. After cell lysis and centrifugation, nucleosomes were detected in the soluble fraction using the Cell Death ELISA kit (Roche) following manufacturer’s instructions. Data represent mean ±S.D. (n = 3). (B) Wild-type and p21 null HCT116 cells were untreated or treated with the DNA damaging agent VP-16 (15 μM, 16 h) and cell extracts were subjected to immunoblotting for p21 and GAPDH as a loading control.