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 ...

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. To study effects of RPS19 deficiency in hematopoiesis we transduced CD34 ؉ umbilical cord blood (CB) and bone marrow (

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 performed post-marketing surveillance to evaluate the safety and efficacy of cell-free and concentrated ascites reinfusion therapy (CART). In total, 356 CART sessions in 147 patients at 22 centers were performed. The most common primary disease was cancer (128 cases, 300 sessions). Mean amount of ascites collected was 3.7 L, and mean concentration ratio was 9.2. Mean amount of reinfused protein was 67.8 g (recovery rate, 72.0%). Performance status, dietary intake, urine volume, body weight and abdominal circumference were significantly improved after CART. Body temperature increased significantly, by 0.3°C on average. Concomitant steroids and/or NSAIDs use before reinfusion was significantly and negatively associated with increases in body temperature. Most adverse events were fever and chills. This study examined a large number of patients compared with previous studies, and showed that CART is an effective and relatively safe treatment for refractory ascites, such as malignant ascites.

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.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. No models exist for RPS19-deficient DBA and the molecular pathogenesis is unknown. To establish an in vitro inducible model for DBA, human erythroid leukemic cell lines, TF-1 and UT-7 cells, were cotransduced with a lentiviral vector expressing the green fluorescent protein (GFP) gene and small interfering RNA (siRNA) against RPS19 controlled by a tet operator regulatory element and another transactivator vector containing the red fluorescent protein (RFP) gene and the cDNA encoding a tetracycline-controllable transcriptional repressor. Following transduction, the RFP-positive and GFP-negative cell population was sorted by flow cytometry. Upon incubation with doxycycline (0.5 mug/ml), more than 98% of cells expressed GFP and the siRNA. Significant suppression of erythroid differentiation, cell growth, and colony formation was observed in cells treated with siRNA against RPS19 but not in cells treated with a control vector. These findings show that RPS19 plays an important role in the regulation of hematopoietic cell proliferation and erythroid differentiation. These novel cell lines represent models for RPS19-deficient DBA and can be used to identify the molecular mechanisms in RPS19-deficient DBA.

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