The c-Abl protein tyrosine kinase is activated by ionizing radiation (IR) and certain other DNA-damaging agents. The present studies demonstrate that c-Abl associates constitutively with protein kinase C d (PKCd). The results show that the SH3 domain of c-Abl interacts directly with PKCd. c-Abl phosphorylates and activates PKCd in vitro. We also show that IR treatment of cells is associated with c-Abl-dependent phosphorylation of PKCd and translocation of PKCd to the nucleus. These ®ndings support a functional interaction between c-Abl and PKCd in the cellular response to genotoxic stress.

The c-Abl nonreceptor tyrosine kinase is activated in cells exposed to ionizing radiation (IR) 1 and certain other DNAdamaging agents (1-4). IR induces DNA double-strand breaks (5) and thereby activates the DNA-dependent protein kinase (DNA-PK) (6 -8). Recent work has shown that DNA-PK phosphorylates and activates c-Abl (9). Other studies have demonstrated that c-Abl interacts with the ataxia telangiectasia mutated (ATM) gene product and that ATM may activate c-Abl in the response to genotoxic stress (10, 11). Whereas cells deficient in DNA-PK or ATM are hypersensitive to killing by IR (12, 13), c-Abl-deficient cells are resistant to IR-induced apoptosis (14). Activation of c-Abl by genotoxic stress is associated with interaction of c-Abl with the p53 tumor suppressor in the G 1 arrest response (15,16). Other signals dependent on c-Abl activation include induction of the stress-activated protein kinase and p38 mitogen-activated protein kinase by genotoxic agents (1,2,17). The findings that c-Abl contributes to the regulation of p53 and certain stress-induced kinases associated with apoptosis have provided support for the activation of c-Abl as a pro-apoptotic signal (14). In this context, expression of c-Abl is associated with G 1 phase growth arrest and induction of apoptosis (14,18,19).Recombination plays a fundamental role in the repair of DNA damage. In Escherichia coli, the RecA protein mediates repair of double-strand breaks by initiating pairing and strand exchange between homologous DNAs (20). Identification of structural homologs of RecA in yeast, Xenopus laevis, mouse, and human cells has supported conservation of similar repair functions throughout evolution (21-25). ScRad51, the RecA homolog in Saccharomyces cerevisiae, is required for DNA damage-induced mitotic recombination (21). ScRad51 converts DNA double-strand breaks to recombinational intermediates, and rad51 mutants accumulate these breaks during meiosis (21). The finding that human Rad51 (HsRad51) promotes homologous pairing and strand exchange reactions in vitro has suggested that Rad51 may also play a role in recombinational repair in man (26). Whereas yeast deficient in Rad51 are viable (21), targeted disruption of the rad51 gene in mice results in an embryonic lethal phenotype (27,28). These findings in rad51 Ϫ/Ϫ mice have suggested that mammalian Rad51 has an essential role in cell proliferation and/or maintenance of genomic stability.The present studies demonstrate that c-Abl associates with Rad51. We show that c-Abl phosphorylates Rad51 on Tyr-54 in vitro and in irradiated cells. Importantly, phosphorylation of Rad51 by c-Abl inhibits Rad51 function in DNA strand exchange assays. MATERIALS AND METHODSCell Culture-U-937 cells, HeLa cells, 293 embryonal kidney cells, and mouse embryo fibroblasts (Abl Ϫ/Ϫ , Abl ϩ ) (29) were grown as described (1). Irradiation was performed using a Gammacell 1000 (Atomic Energy of Canada) with a 137 Cs source emitting at a fixed dose of 0.21 gray min Ϫ1 as determined by dosimetry. Immunoprecipitations ...

The nuclear p300/CBP proteins function as coactivators of gene transcription. Here, using cells deficient in p300 or CBP, we show that p300, and not CBP, is essential for ionizing radiation-induced accumulation of the p53 tumor suppressor and thereby p53-mediated growth arrest. The results demonstrate that deficiency of p300 results in increased degradation of p53. Our findings suggest that p300 contributes to the stabilization and transactivation function of p53 in the cellular response to DNA damage.In the exposure of cells to ionizing radiation (IR), 1 the formation of DNA double-strand breaks is associated with increases in p53 levels and the transactivation function of p53 (1-3). Activation of p53 in the response to IR induces transcription of the p21 (WAF1, Cip-1) gene (4). Thus, the growth arrest function of p53 is regulated at least in part by p21-mediated inhibition of cyclin-Cdk complexes and the proliferating cell nuclear antigen (PCNA) (1). In addition, p53-dependent induction of the bax gene contributes to the apoptotic response to DNA damage (5). Other genes implicated in p53-induced growth arrest and apoptosis include GADD45 (3), mdm2 (6, 7), cyclin G (8), and IGF-BP3 (9).Recent work has demonstrated that the DNA-dependent protein kinase (DNA-PK) is necessary but not sufficient for activation of p53 sequence-specific DNA binding (10). Phosphorylation of the p53 N-terminal region by DNA-PK may contribute to the transactivation function and stability of p53 (11,12). Other studies have shown that the ataxia telangiectasia-mutated (ATM) protein phosphorylates p53 on serine 15 in vitro (13,14). The findings that the p53 serine 15 site is phosphorylated in IR-treated cells (15,16) and that this effect is diminished in AT cells (16) have supported a role for ATM in the regulation of p53. The p300/CBP proteins (17-20) have also been implicated as coactivators of the p53 transactivation function (21,22). The N-terminal domain of p53 interacts with the C-terminal region of p300/CBP. Acetylation of the p53 C-terminal domain by p300/CBP stimulates the DNA binding activity of p53 (23). A dominant negative form of p300/CBP has also been found to inhibit p53-mediated transactivation and the G 1 arrest and apoptotic responses (24).Cells derived from p300-deficient embryos exhibit severe defects in proliferation (25). Consequently, in the present work, we have established cells expressing ribozymes specific for p300 or CBP such that the transfectants are selectively deficient in either protein. Our results demonstrate that p300, and not CBP, is essential for IR-induced increases in both p53 levels and the p53 transactivation function. MATERIALS AND METHODSCell Culture-MCF-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated bovine serum, 2 mM L-glutamine, 10 units/ml penicillin, and 10 g/ml streptomycin. The active p300 (p300-R), inactive p300 (p300-RI), active CBP (CBP-R), or inactive CBP (CBP-RI) ribozymes (26) were stably introduced into cells by LipofectAMINE (Life Tech...

We report here that the Rad51 recombinase is cleaved in mammalian cells during the induction of apoptosis by ionizing radiation (IR) exposure. The results demonstrate that IR induces Rad51 cleavage by a caspase-dependent mechanism. Further support for involvement of caspases is provided by the finding that IR-induced proteolysis of Rad51 is inhibited by Ac-DEVD-CHO. In vitro studies show that Rad51 is cleaved by caspase 3 at a DVLD/N site. Stable expression of a Rad51 mutant in which the aspartic acid residues were mutated to alanines (AVLA/N) confirmed that the DVLD/N site is responsible for the cleavage of Rad51 in IR-induced apoptosis. The functional significance of Rad51 proteolysis is supported by the finding that, unlike intact Rad51, the N- and C-terminal cleavage products fail to exhibit recombinase activity. In cells, overexpression of the Rad51(D-A) mutant had no effect on activation of caspase 3 but did abrogate in part the apoptotic response to IR exposure. We conclude that proteolytic inactivation of Rad51 by a caspase-mediated mechanism contributes to the cell death response induced by DNA damage.

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The cellular response to ionizing radiation (IR) includes the induction of apoptosis. The p300/CBP proteins possess histone acetyltransferase activity and function as transcriptional coactivators of p53. We have prepared cells de®cient in p300 or CBP to de®ne the roles of these proteins in the cellular response to DNA damage. The present results demonstrate that p300, but not CBP, contributes to IR sensitivity of cells. The results also demonstrate that IR-induced apoptosis is impaired in the p300-, but not CBP-, de®cient cells. These ®ndings indicate that p300 functions in the apoptotic response to DNA damage.

Activation of phosphatidylinositol (PI) 3-kinase by growth factors results in phosphorylation of phosphatidylinositol lipids at the D3 position. Although PI 3-kinase is essential to cell survival, little is known about mechanisms that negatively regulate this activity. Here we show that the c-Abl tyrosine kinase interacts directly with the p85 subunit of PI 3-kinase. Activation of c-Abl by ionizing radiation exposure is associated with c-Abl-dependent phosphorylation of PI 3-kinase. We also show that phosphorylation of p85 by c-Abl inhibits PI 3-kinase activity in vitro and in irradiated cells. These findings indicate that c-Abl negatively regulates PI 3-kinase in the stress response to DNA damage.

Differentiation induction by 12-o-tetradecanoyl 13-acetate (TPA) results in the growth arrest of HL60 cells in the G1 phase. However, little is known about the changes of cell cycle-regulating genes during this differentiation process. We investigated the changes of mRNA for various cyclins (A, C, D1, D2, D3 and E) and cdk2. Synchronized HL60 cells began to proliferate immediately after release from cell cycle block and cell cycle synchrony was obvious until the second S phase. TPA-treated cells accumulated in G1 phase within 24 h and most of the cells were arrested in this phase at 36 h. The expression of cyclins and cdk2 was studied by Northern blot hybridization of the reverse-transcription polymerase chain reaction (RT-PCR). TPA treatment altered the expression of all genes studied. The expression of cdk2 and cyclin A mRNA was markedly down-regulated. Cyclin E mRNA expression was also prominently down-regulated from 12 h to 36 h, at which time a second increase of its expression was observed in control cells. In contrast, the expression of cyclin D1 mRNA was induced by TPA, while its expression in control cells was undetectable by Northern blot hybridization throughout the cell cycle. Cyclin C expression was faint and fluctuated irrelevant of cell cycle, but its expression in both control and TPA-treated cells was higher than at baseline. Cyclin D2 expression remained stable in control cells and TPA treatment resulted in slight down-regulation at 12 h, but no difference was observed after 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)