Regulation of DNA damage-induced apoptosis by the c-Abl tyrosine kinase

Abstract

Activation of the c-Abl protein tyrosine kinase by certain DNA-damaging agents contributes to downregulation of Cdk2 and G1 arrest by a p53-dependent mechanism. The present work investigates the potential role of c-Abl in apoptosis induced by DNA damage. Transient transfection studies with wild-type, but not kinase-inactive, c-Abl demonstrate induction of apoptosis. Cells that stably express inactive c-Abl exhibit resistance to ionizing radiation-induced loss of clonogenic survival and apoptosis. Cells null for c-abl are also impaired in the apoptotic response to ionizing radiation. We further show that cells deficient in p53 undergo apoptosis in response to expression of c-Abl and exhibit decreases in radiation-induced apoptosis when expressing inactive c-Abl. These findings suggest that c-Abl kinase regulates DNA damage-induced apoptosis.

Figure 1

Overexpression of c-Abl induces apoptosis. MCF-7 cells were transfected with 8 μg of control pSRαMSVtkNeo, c-Abl, or c-Abl(K-R) vector. (A) Cell lysates were subjected to immunoblot analysis with anti-Abl and anti-proliferating cell nuclear antigen (PCNA). (B) Cells were assayed for death by trypan blue exclusion at 48 h after transfection. Results are expressed as mean ± SEM of three independent transfections. Transfection efficiency as determined by cotransfection with a pSV-β-gal vector was 40–45%. Cell death for nontransfected MCF-7 cells was approximately 1%. (C) Cells were assayed for DNA content by flow cytometry at 48 h after transfection. Transfection with pSR, c-Abl, and c-Abl(K-R) resulted in 4.5% ± 0.9%, 29.4% ± 4.6%, and 4.1% ± 1.0% (mean ± SEM, three independent experiments) cells with sub-G₁ DNA content, respectively.

Figure 2

c-Abl(K-R) transfectants are resistant to ionizing radiation-induced apoptosis. (A) Wild-type MCF-7 (▪), MCF-7/pSR (•), and MCF-7/c-Abl(K-R) (clones a and b; □ and ○) cells (23) were exposed to the indicated doses of ionizing radiation (IR) and assayed for colony formation at 10 days (Left). MCF-7/pSR (•) and MCF-7/c-Abl(K-R) (□) cells were exposed to staurosporine (STSP) for 1 h, washed, and then assayed for colony formation at 10 days (Right). Results (mean ± SEM of three experiments) are expressed as the percentage clonogenic survival relative to untreated cells. (B) MCF-7/pSR and MCF-7/c-Abl(K-R) cells were treated with 5 Gy of ionizing radiation (IR). The cells were stained in TUNEL assays at 120 h after irradiation. (×400.) (C) DNA content was analyzed at 120 h after irradiation with 5 Gy.

Figure 3

c-Abl kinase is involved in radiation-induced apoptosis. (A) Wild-type (Abl^+/+; ○) and Abl^−/− (•) MEFs (15) were exposed to the indicated doses of ionizing radiation (IR) and assayed for colony formation after 10 days. (B) Abl^+/+ and Abl^−/− MEFs were exposed to 0 or 5 Gy and collected at 3 h. Cell lysates were immunoblotted with anti-p53 and anti-p21 antibodies. (C) Abl^+/+ and Abl^−/− MEFs were exposed to 0 or 10 Gy and collected at 120 h for analysis of DNA content. Irradiation of the Abl^+/+ and Abl^−/− cells resulted in 17.3% ± 4.3% and 8.9% ± 2.1% with sub-G₁ DNA, respectively.

Figure 4

c-Abl kinase regulates irradiation-induced apoptosis by p53-dependent and -independent mechanisms. (A) MCF-7/E6 cells were transfected with 8 μg of pSR, c-Abl, or c-Abl(K-R) vector. Cells were harvested at 48 h and assessed for DNA content. Transfection efficiency as determined by cotransfection with pSV-β-gal vector was 40–45%. (B) (Left) MCF-7/pSR (lanes 1 and 2, counting from the left) and MCF-7/E6 (lanes 3 and 4) were irradiated with 5 Gy (lanes 2 and 4). Lysates prepared at 3 h were subjected to immunoblotting with anti-p53 (Ab-6; Oncogene Science) or anti-p21 (Ab-1; Oncogene Science). (Right) MCF-7/pSR (□), MCF-7/E6 (•), and MCF-7/E6/c-Abl(K-R) (▪) cells were irradiated with the indicated doses. Colony formation was assessed at 10 days. (C) p53^−/− MEFs (33) were transfected with 8 μg of pSR, c-Abl, or c-Abl(K-R) vector. Transfection efficiency as determined by cotransfection of pSV-β-gal vector was 15–20%. Cells were harvested at 48 h and assessed for DNA content. (D) p53^−/− MEFs stably tranfected with pSR vector or c-Abl(K-R) were treated with 10 Gy of ionizing radiation and collected at 120 h for assessment of DNA content. The results (mean ± SEM of three experiments) are expressed as the percentage of apoptotic cells with sub-G₁ DNA.