Fig. 9

Abl1 stimulates p53-mediated apoptosis. Panel A: Human MCF7 cells were transiently transfected with a consensus p53 reporter vector (PG13) along with p53 or/and Abl1 expression vectors. Additionally, cell cultures were treated with 1.5 µM doxorucibin for 16 h. Results are expressed as average relative light units (RLU), normalized to the Renilla control. Abl1 overexpression induced p53-dependent activity on the PG13 reporter. The combined expression of Abl1 and p53 caused a greater than additive induced activation, therefore demonstrating cofactor activity of Abl1 on p53 dependent transcriptional responses. Using the same experimental approach treatment with doxorubicin caused a further significant increase in the reporter assay. Bars plots are averages with standard deviations for 3 independent biological replicates. * = p < 0.01 Student t-test. Panels B-C: MCF7 vector and short hairpin p53 silenced cells were transiently transfected with an empty vector or an Abl1 expression vector. Forty-eight hours after treatment cells were stained both with FITC-Annexin V and TO-PRO-3 iodide to detect apoptosis (represented by the population of Annexin V positive cells). The involvement of p53 was calculated by subtracting the apoptotic rate obtained with the p53 proficient cell line (MCF7 vector, panel B) from the one stably silenced for p53 (MCF7 shp53, panel C). Doxorubicin treatment alone induced apoptosis that was superseded by Abl1 (panel B). The effects of Abl1 on the apoptosis rate is even more apparent in MCF7 cells silenced for p53 (panel C). Shown are bar graphs of 3 independent biological replicates. * = p < 0.05 and ** = p < 0.01 Student’s t-test. Panels D-F) Examples of the Western blots in MCF7 cells demonstrating the stabilization of p53 protein and the activation of p53 targets (p21 and MDM2) in response to doxorubicin treatment (D); overexpression of Abl1 protein following doxorubicin treatment (E) and cleavage of PARP-1 by caspases to hallmark apoptosis. Protein molecular weights are indicated according to the ladder. GAPDH was used as housekeeping protein. The blots were quantified with the Image J64 software, and the data are arbitrary units (AU) of the normalized (with GAPDH) signals. In case of PARP-1 (F), the histograms represent the ratio of normalized signal for cleaved PARP to total PARP protein. * = p < 0.05 and ** = p < 0.01 Student’s t-test