Activation of protein kinase C delta by the c-Abl tyrosine kinase in response to ionizing radiation

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 delta (PKCdelta). The results show that the SH3 domain of c-Abl interacts directly with PKCdelta. c-Abl phosphorylates and activates PKCdelta in vitro. We also show that IR treatment of cells is associated with c-Abl-dependent phosphorylation of PKCdelta and translocation of PKCdelta to the nucleus. These findings support a functional interaction between c-Abl and PKCdelta in the cellular response to genotoxic stress.     

The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation

p53 accumulates after DNA damage and arrests cellular growth. These findings suggest a possible role for p53 in the cellular response to DNA damage. We have previously shown that the C terminus of p53 binds DNA nonspecifically and assembles stable tetramers. In this study, we have utilized purified segments of human and murine p53s to determine which p53 domains may participate in a DNA damage response pathway. We find that the C-terminal 75 amino acids of human or murine p53 are necessary and sufficient for the DNA annealing and strand-transfer activities of p53. In addition, both full-length wild-type p53 and the C-terminal 75 amino acids display an increased binding affinity for DNA damaged by restriction digestion, DNase I treatment, or ionizing radiation. In contrast, the central site-specific DNA-binding domain together with the tetramerization domain does not have these activities. We propose that interactions of the C terminus of p53 with damaged DNA may play a role in the activation of p53 in response to DNA damage.     

p53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation

BACKGROUND: The complications associated with anterior craniofacial resections for benign and malignant tumors were reviewed in 104 patients treated between January 1981 and June 1996. METHODS: Information regarding patient characteristics, histologic type, history of prior therapy, extent of the disease, extent of surgical procedure, and type of reconstruction were entered in a microcomputer database. To better understand and stage postoperative complications, we divided them into early (<14 days) and late (>14 days) according to the time of presentation, into major and minor depending on the morbidity potential of complication, and into local and systemic ones. Comparison between risk factors associated with complications was made using chi-square analysis with Yates' correction for continuity. Survival analysis was performed using the Kaplan-Meier product limit method. RESULTS: There were 8 (7.6%) postoperative deaths, with only 1 occurring from systemic complications. Complications occurred in 53 (48.6%) patients. Local major complications occurred in 49 (45%) patients, local minor in 29 (26.6%), and systemic in 11 (10%). Early complications occurred in 40 (38.5%) patients and late complications in 13 (12.5%) patients. These complications developed during a period ranging from 1 day to 5 months. More than one complication occurred in a number of patients. Bacterial contamination leading to local septic complications was the principal cause of morbidity, accounting for 54.7% (29/53) of complications. Major complications included meningitis in 8 patients associated with cerebrospinal fluid leak in 7, cerebral abscess in 2, sepsis in 1, and subdural hemorrhage in 1, all of which resulted in death except for one case. The extent of the craniofacial resection (p = .011) was the most important factor associated with major complications. Invasion of the dura and the type of reconstruction of the anterior skull base were the most important factors related to cerebrospinal fluid leakage (p = .048 and p = .032) and meningitis (p = .011). CONCLUSION: Contemporary surgical approaches and methods of reconstruction have enabled skull base surgeons to extend their cranial base resections and increase the 5-year survival rates of patients. Nevertheless, significant complications persist. Knowledge and high index of suspicion together with early recognition of these complications are essential for effective management of patients undergoing craniofacial resection. The factors related to major complications found in this study stressed the need to develop more effective methods to prevent contamination of intracranial structures.     

Stabilization and activation of p53 are regulated independently by different phosphorylation events

Rheumatoid factors (RF) recognize conformational determinants located within the Fc portion of IgG. By analyzing a panel of monoclonal rheumatoid arthritis (RA)-derived RFs, we previously demonstrated that the somatically generated light chain complementarity-determining region 3 (CDR3) contributes to RF specificity. We have now generated a panel of heavy chain mutants of the B'20 Ab, a high affinity RA-derived IgM RF. B'20 also binds avidly to protein A and weakly to ssDNA and tetanus toxoid. B9601, a RF negative Ab that is highly homologous to B'20 but does not bind any of the Ags tested, and RC1, a low affinity polyreactive RF, were used to generate heavy chain mutants with framework (FR) and CDR switches. The mutated heavy chains were cotransfected into a myeloma cell line with the germline counterpart of the B'20 light chain, and the expressed Ig tested for antigenic specificity. We show that both RF specificity and polyreactivity of B'20 is dependent on its unique heavy chain CDR3 region. Replacement with a B9601 CDR3 shortened to the same length as the B'20 CDR3, and with only 5 amino acid differences, did not restore Fc binding. Conversely, absence of protein A binding of B9601 is due to the presence of a serine residue at position 82a in the B9601 heavy chain FR3 region. Together, our data suggest that Ig gene recombination events can generate B cells with autoantibody specificities in the preimmune repertoire. Abnormal release, activation, expansion, or mutation of such cells might all contribute to the generation of a high titer RF response in patients with RA.     

Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms

The p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that are induced in response to lipopolysaccharide, hyperosmolarity, and interleukin 1. p38 MAP kinase appears to play a role in regulating inflammatory responses, including cytokine secretion and apoptosis. Here we show that diverse classes of DNA-damaging agents such as cisplatinum, 1-beta-D-arabinofuranosylcytosine, UV light, ionizing radiation, and methyl methanesulfonate activate p38 MAP kinase. We also demonstrate that cells deficient in c-Abl fail to activate p38 MAP kinase after treatment with cisplatinum and 1-beta-D-arabinofuranosylcytosine but not after exposure to UV and methyl methanesulfonate. Reconstitution of c-Abl in the Abl-/- cells restores that response. Similar results were obtained for induction of the Jun-NH2-kinase/stress-activated protein kinase. These findings indicate that p38 MAP and Jun-NH2-kinase/stress-activated protein kinases are differentially regulated in response to different classes of DNA-damaging agents.     

Ultraviolet radiation, but not gamma radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389

Polyclonal antibodies were produced and purified that selectively react with a p53 epitope containing the murine phosphoserine-389 or the human phosphoserine-392 residue, but not the unphosphorylated epitope. These antibodies, termed alpha-392, were employed to demonstrate that the phosphorylation of this serine-389 residue in the p53 protein occurs in vivo in response to ultraviolet radiation of cells containing the p53 protein. After ultraviolet radiation of cells in culture, p53 levels increase and concomitantly serine-389 is phosphorylated in these cells. By contrast, the serine-389 phosphorylation of the p53 protein was not detected by these antibodies in the increased levels of p53 protein made in response to gamma radiation or the treatment of cells with etoposide. These results demonstrate an ultraviolet responsive and specific phosphorylation site at serine-389 of the mouse or serine-392 of the human p53 protein. Previous studies have demonstrated that this phosphorylation of p53 activates the protein for specific DNA binding. This study demonstrates in vivo a unique phosphorylation site in the p53 protein that responds to a specific type of DNA damage.     

Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis

The response of eukaryotic cells to ionizing radiation (IR) includes induction of apoptosis. However, the signals that regulate this response are unknown. The present studies demonstrate that IR treatment of U-937 cells is associated with: (i) internucleosomal DNA fragmentation; (ii) cleavage of poly(ADP-ribose) polymerase; (iii) cleavage of protein kinase C delta; and (iv) induction of an Ac-DEVD-p-nitroanilide cleaving activity. Overexpression of the cowpox protein CrmA blocked tumor necrosis factor (TNF)-induced apoptosis but had no effect on IR-induced DNA fragmentation or cleavage of poly(ADP-ribose) polymerase and protein kinase C delta. By contrast, overexpression of the baculovirus p35 protein blocked both IR- and TNF-induced apoptosis. The results further demonstrate that the IR-induced proteolytic activity is directly inhibited by the addition of purified recombinant p35, but not by CrmA. We show that the CPP32 protease is sensitive to p35 and not CrmA. We also show that IR induces activation of CPP32 and that this event, like induction of apoptosis, is sensitive to overexpression of p35 and not CrmA. These findings indicate that IR-induced apoptosis involves activation of CPP32 and that this CrmA-insensitive apoptotic pathway is distinct from those induced by TNF and certain other stimuli.     

Activation of microtubule-associated protein kinase (Erk) and p70 S6 kinase by D2 dopamine receptors

The ability of human and rat D2(short) and D2(long) dopamine receptors to activate microtubule-associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose-dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D2 dopamine receptor. Activation of Erk and of p70 S6 kinase via the D2 dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the Gi or Go family. Inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3-kinase-dependent and -independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3-kinase.     

Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase

Blastic transformation of chronic myelogenous leukemia (CML) is characterized by the presence of nonrandom, secondary genetic abnormalities in the majority of Philadelphia1 clones, and loss of p53 tumor suppressor gene function is a consistent finding in 25-30% of CML blast crisis patients. To test whether the functional loss of p53 plays a direct role in the transition of chronic phase to blast crisis, bone marrow cells from p53+/+ or p53-/- mice were infected with a retrovirus carrying either the wild-type BCR/ABL or the inactive kinase-deficient mutant, and were assessed for colony-forming ability. Infection of p53-/- marrow cells with wild-type BCR/ABL, but not with the kinase-deficient mutant, enhanced formation of hematopoietic colonies and induced growth factor independence at high frequency, as compared with p53+/+ marrow cells. These effects were suppressed when p53-/- marrow cells were coinfected with BCR/ ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow cells had a proliferative advantage, as reflected by an increase in the fraction of S+G2 phase cells and a decrease in the number of apoptotic cells. Immunophenotyping and morphological analysis revealed that BCR/ABL-positive p53-/- cells were much less differentiated than their BCR/ABL-positive p53+/+ counterparts. Injection of immunodeficient mice with BCR/ABL-positive p53-/- cells produced a transplantable, highly aggressive, poorly differentiated acute myelogenous leukemia. In marked contrast, the disease process in mice injected with BCR/ABL-positive p53+/+ marrow cells was characterized by cell infiltrates with a more differentiated phenotype and was significantly retarded, as indicated by a much longer survival of leukemic mice. Together, these findings directly demonstrate that loss of p53 function plays an important role in blast transformation in CML.     

Power lines and ionizing radiation

The magnetic field and ionizing radiation dose rate were measured near ground level in the vicinity of a utility power line. The dose rate was monitored using thermoluminescence detectors. Measurements were made at distances up to about 100 m from the main transmission line from the Kewaunee Nuclear Power Plant while the plant was in operation and while the plant was shut down for refueling. No evidence was found that the dose rate is significantly different in the presence of the magnetic field from the power line than in its absence.