XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.     

Effects of food availability on serum insulin and lipid concentrations in free-ranging baboons

The relationship between food availability and metabolic physiology was studied in groups of free-ranging baboons (Papio spp.) living in the Amboseli National Park and the Masai Mara National Reserve of Kenya. Three groups subsisted entirely on natural forage, while two other groups lived near tourist facilities and often consumed food wastes from these lodges. The refuse provided a very accessible food source with relatively high caloric density. Consumption of the refuse was associated with reduced locomotion. Sexually mature individuals from all five groups were sedated surreptitiously in the early morning and blood samples were collected. Compared to animals foraging exclusively in the wild, animals that supplemented their diet with the refuse items had two- to threefold elevations in serum insulin concentrations, as well as increased total cholesterol (C), HDL-C, and VLDL+LDL-C levels. No sex differences in physiological measures were observed except in body mass. Elevated serum insulin, and cholesterol and lipoprotein concentrations influence the development of cardiovascular disease and have been shown to be subject to dietary manipulation and exercise under controlled conditions. The present results suggest potentially deleterious effects of a highly accessible, calorically dense food source, and associated reduction of physical activity for baboons living in an otherwise natural environment.     

PARP inhibition during alkylation-induced genotoxic stress signals a cell cycle checkpoint response mediated by ATM

By limiting cell cycle progression following detection of DNA damage, checkpoints are critical for cell survival and genome stability. Methylated DNA damage, when combined with inhibition of PARP activity, results in an ATR-dependent S phase delay of the cell cycle. Here, we demonstrate that another checkpoint kinase, ATM, also is involved in the DNA damage response following treatment with a sub-lethal concentration of MMS combined with the PARP inhibitor 4-AN. Both ATM and PARP activities are important for moderating cellular sensitivity to MMS. Loss of ATM activity, or that of its downstream effector Chk2, limited the duration of the S phase delay. The combination of MMS and 4-AN resulted in ATM and Chk2 phosphorylation and the time course of phosphorylation for both kinases correlated with the S phase delay. Chk2 phosphorylation was reduced in the absence of ATM activity. The Chk2 phosphorylation that remained in the absence of ATM appeared to be dependent on ATR and DNA-PK. The results demonstrate that, following initiation of base excision repair and inhibition of PARP activity, ATM activation is critical for preventing the cell from progressing through S phase, and for protection against MMS-induced cytotoxicity.     

Hyperactivation of PARP Triggers Nonhomologous End-Joining in Repair-Deficient Mouse Fibroblasts

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.     

Tyrosine phosphorylation of myosin heavy chain during skeletal muscle differentiation: an integrated bioinformatics approach

Previously it has been shown that insulin-mediated tyrosine phosphorylation of myosin heavy chain is concomitant with enhanced association of C-terminal SRC kinase during skeletal muscle differentiation. We sought to identify putative site(s) for this phosphorylation event. A combined bioinformatics approach of motif prediction and evolutionary and structural analyses identified tyrosines163 and 1856 of the skeletal muscle heavy chain as the leading candidate for the sites of insulin-mediated tyrosine phosphorylation. Our work is suggestive that tyrosine phosphorylation of myosin heavy chain, whether in skeletal muscle or in platelets, is a significant event that may initiate cytoskeletal reorganization of muscle cells and platelets. Our studies provide a good starting point for further functional analysis of MHC phosphor-signalling events within different cells.     

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12–C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1^?/? mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12–C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.     

Duration of Adulthood Overweight,Obesity, and Cancer Risk in the Women’s Health Initiative: A Longitudinal Study from the United States

BackgroundHigh body mass index (BMI) has become the leading risk factor of disease burden in high-income countries. While recent studies have suggested that the risk of cancer related to obesity is mediated by time, insights into the dose-response relationship and the cumulative impact of overweight and obesity during the life course on cancer risk remain scarce. To our knowledge, this study is the first to assess the impact of adulthood overweight and obesity duration on the risk of cancer in a large cohort of postmenopausal women.ConclusionsIn summary, this study showed that a longer duration of overweight and obesity is associated with an increased risk of developing several forms of cancer. Furthermore, the degree of overweight experienced during adulthood seemed to play an important role in the risk of developing cancer, especially for endometrial cancer. Although the observational nature of our study precludes inferring causality or making clinical recommendations, our findings suggest that reducing overweight duration in adulthood could reduce cancer risk and that obesity prevention is important from early onset. If this is true, health care teams should recognize the potential of obesity management in cancer prevention and that excess body weight in women is important to manage regardless of the age of the patient.