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Kevin D Brown

Publications and source records attributed to Kevin D Brown.

23 records · Page 2Linked to original sources
PubMed2002

ATM is activated in response to N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA alkylation.

p53 plays an important role in response to ionizing radiation by regulating cell cycle progression and triggering apoptosis. These activities are controlled, in part, by the phosphorylation of p53 by the protein kinase ATM. Recent evidence indicates that the monofunctional DNA alkylating agent N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) also triggers up-regulation and phosphorylation of p53; however, the mechanism(s) responsible for this are unknown. We observed that in MNNG-treated normal human fibroblasts, up-regulation and phosphorylation of p53 was sensitive to the ATM kinase inhibitor wortmannin. ATM-deficient fibroblasts exhibited a delay in p53 up-regulation indicating a role for ATM in triggering the MNNG-induced response. Likewise, a mismatch repair (MMR)-deficient colorectal tumor line failed to show rapid up-regulation of p53. However, unlike ATM-deficient cells, these MMR-deficient cells displayed rapid phosphorylation of the p53 residue serine 15 after MNNG. In vitro kinase assays indicate that ATM is rapidly activated in both normal and MMR-deficient cells in response to MNNG. Using a number of morphological and biochemical approaches, we failed to observe MNNG-induced apoptosis in normal human fibroblasts, suggesting that apoptosis-induced DNA strand breaks are not required for the activation of ATM in response to MNNG. Comet assays indicated that strand breaks accumulated, and p53 up-regulation/phosphorylation occurred quite rapidly (within 30 min) after MNNG treatment, suggesting that DNA strand breaks that arise during the repair process activate ATM. These findings indicate that ATM activation is not limited to the ionizing radiation-induced response and potentially plays an important role in response to DNA alkylation.

Alkylation↗
PubMed2002

Aberrant methylation of the ATM promoter correlates with increased radiosensitivity in a human colorectal tumor cell line.

Recent findings suggest that DNA alkylating agents trigger cellular responses that overlap those activated after ionizing radiation. Moreover, activation of these responses is dependent upon a functional mismatch repair (MMR) system. These developments led us to test if MMR-deficient cells may be compromised in their ability to activate appropriate cellular signaling pathways after ionizing radiation. An initial experiment to address this notion was to determine the level of radiosensitivity of several MMR-deficient cell lines derived from patients with Hereditary Non-Polyposis Colorectal Cancer (HNPCC). While two of the three HNPCC lines investigated show levels of radiosensitivity consistent with that displayed by normal human fibroblasts, HCT-116 cells display moderate radiosensitivity compared to the other MMR-deficient lines. This increased sensitivity to ionizing radiation correlates with lowered levels of ATM expression in HCT-116. Analysis of genomic DNA from HCT-116 cells determined that these cells possess aberrant methylation of multiple CpG dinucleotides within the proximal promoter region of the ATM gene. The significance of this finding is underscored by our observations that co-culturing HCT-116 cells with the DNA demethylating agent 5-azacytidine reverses promoter methylation, promotes normal levels of ATM expression, and restores normal radiosensitivity. The proximal ATM promoter is a approximately 520 bp region shared with the NPAT gene, and current evidence suggests that this region functions as a bi-directional promoter. We found that, unlike ATM, the methylation status of this intergenic region does not effect the expression of the NPAT gene. In sum, these observations indicate that the ATM gene is a novel target for epigentic silencing through inappropriate methylation of its proximal promoter region.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2002

Regulation of TRAF2 signaling by self-induced degradation.

Receptors belonging to the tumor necrosis factor receptor (TNF-R) family utilize cytoplasmic adapter proteins called TNF-R-associated factors (TRAFs) as key elements in their signaling pathways. However, it is not yet clear how individual TRAFs regulate signaling by this large and growing receptor family. Signaling via the TNF-R family member CD40 has recently been shown to result in recruitment of TRAF2 to plasma membrane detergent-resistant microdomains (lipid rafts) as well as to subsequently initiate TRAF2 degradation. As TRAF2 associates with most members of the TNF-R family, we wished to determine how this degradation occurs. We show here that CD40-mediated TRAF2 degradation requires the zinc-binding RING domain of TRAF2 and is preceded by TRAF2 ubiquitination, suggesting that the TRAF2 RING may promote ubiquitination although the RING itself is not a target of ubiquitination. Several approaches show that ubiquitination and proteasomal activity are integral to TRAF2 degradation, and inhibition of this process potentiates CD40 signaling.

Animals↗
PubMed2002

Mechanisms of TNF receptor-associated factor (TRAF) regulation in B lymphocytes.

A key component of signaling by members of the tumor necrosis factor receptor (TNF-R) family is interaction with the cytoplasmic adapter proteins known as TRAFs. Several proteins encoded by microbes also interact with TRAFs. A notable example is the CD40 receptor, expressed on antigen presenting cells and providing key activation signals in T cell-dependent B cell activation. CD40 signals to B cells are mimicked by a constitutively active viral protein produced by the Epstein-Barr virus. For each of these receptors, multiple mechanisms of TRAF regulation contribute to signal transduction and the ultimate effect on the B cell. Recent findings concerning these regulatory mechanisms are summarized in this overview.

Animals↗