Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition

Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.     

Involvement of poly(ADP-ribose) polymerase activity in regulating Chk1-dependent apoptotic cell death

The activity of poly(ADP-ribose) polymerase (PARP) is highly stimulated following DNA damage resulting in formation of DNA nicks and strand breaks. This leads to modification of numerous proteins, including itself, using NAD(+) as substrate and to exhaustion of intracellular ATP. A highly cytotoxic concentration of the DNA methylating agent methyl methanesulfonate (MMS) results in cellular ATP depletion and cell death primarily by necrosis in both wild-type and DNA polymerase beta null mouse fibroblasts. The loss of ATP can be prevented by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN), and now cells die by an energy-dependent apoptotic pathway. We find that inhibition of PARP activity transforms a sub-lethal exposure to MMS into a highly cytotoxic event. Under this condition, ATP is not depleted and cell death is by apoptosis. The caspase inhibitor, Z-VAD, shifts the mechanism of cell death to necrosis indicating a caspase-dependent component of the apoptotic cell death. Co-exposure to the Chk1 inhibitor UCN-01 also produces a decrease in apoptotic cell death, but now there is an increase in viable cells and an enhancement in long-term survival. Taken together, our results suggest that inhibition of PARP activity, induced as a result of low dose MMS exposure, signals via a Chk1-dependent pathway for cell death by apoptosis.     

Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition

Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773-15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.     

Men Gain Additional Psychological Benefits by Adding Exercise to a Weight‐Loss Program

Objective: Adding exercise to a comprehensive weight‐loss program might not only attenuate any psychological distress associated with weight‐loss attempts but also may provide psychological benefits. This study examined whether a diet‐plus‐exercise weight‐loss program improved psychological outcomes more than a diet‐only weight‐loss program or an assessment‐only control group. Research Methods and Procedures: This study was part of a larger 1‐year randomized weight‐loss trial examining the effects of diet and exercise on cardiovascular disease risk factors in 264 overweight adults. Psychological measures specific to weight control (e.g., cognitive restraint, disinhibition, hunger, and body dissatisfaction) as well as traditional measures of psychological distress (e.g., symptoms of depression, anxiety, and stress) were obtained at baseline and 1 year. Results: Men and women in either weight‐loss program reported greater restraint, less disinhibition, and less hunger at 1 year than those in no program. Men in the diet‐plus‐exercise program experienced additional increases in restraint and decreases in hunger than did men in the diet‐only program. Women in the diet‐plus‐exercise program did not experience additional psychological benefits specific to weight control than those in the diet‐only program, despite increases in aerobic capacity. Discussion: The pattern seen for overweight men in the diet‐plus‐exercise program at 1 year—greater restraint, less disinhibition, and less hunger—is similar to the pattern seen in successful weight maintainers. These results underscore the need for innovative strategies that will enhance and sustain the pattern of psychological benefits specific to weight control associated with successful weight loss, especially for overweight women.     

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of the cytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNA COI-II) were deduced to consist of multiple haplotypes in three Sitobion species. We investigated the molecular basis of these observations. PCR products were cloned, and six clones from one individual per species were sequenced. In each individual, one sequence was found commonly, but also two or three divergent sequences were seen. The divergent sequences were shown to be nonmitochondrial by sequencing from purified mtDNA and Southern blotting experiments. All seven nonmitochondrial clones sequenced to completion were unique. Nonmitochondrial sequences have a high proportion of unique sites, and very few characters are shared between nonmitochondrial clones to the exclusion of mtDNA. From these data, we infer that fragments of mtDNA have been transposed separately (probably into aphid chromosomes), at a frequency only known to be equalled in humans. The transposition phenomenon appears to occur infrequently or not at all in closely related genera and other aphids investigated. Patterns of nucleotide substitution in mtDNA inferred over a parsimony tree are very different from those in transposed sequences. Compared with mtDNA, nonmitochondrial sequences have less codon position bias, more even exchanges between A, G, C and T, and a higher proportion of nonsynonymous replacements. Although these data are consistent with the transposed sequences being under less constraint than mtDNA, changes in the nonmitochondrial sequences are not random: there remains significant position bias, and probable excesses of synonymous replacements and of conservative inferred amino acid replacements. We conclude that a proportion of the inferred change in the nonmitochondrial sequences occurred before transposition. We believe that Sitobion aphids (and other species exhibiting mtDNA transposition) may be important for studying the molecular evolution of mtDNA and pseudogenes. However, our data highlight the need to establish the true evolutionary relationships between sequences in comparative investigations.      

Metabolic Syndrome and Changes in Body Fat From a Low‐fat Diet and/or Exercise Randomized Controlled Trial

It is difficult to identify the successful component(s) related to changes in metabolic syndrome (MetS) from lifestyle interventions: the weight loss, the behavior change, or the combination. The purpose of this study is to determine the effects of a weight‐stable randomized controlled trial of low‐fat diet and exercise, alone and in combination, on MetS. Men (n = 179) and postmenopausal women (n = 149) with elevated low‐density lipoprotein cholesterol (LDL‐C) and low high‐density lipoprotein cholesterol (HDL‐C) were randomized into a 1‐year, weight‐stable trial with four treatment groups: control (C), diet (D), exercise (E), or diet plus exercise (D+E). MetS was defined using a continuous score. Changes in MetS score (ΔMetS) were compared between groups using analysis of covariance, stratified by gender and using two models, with and without baseline and change in percent body fat (ΔBF) as a covariate. In men, ΔMetS was higher for D vs. C (P = 0.04), D+E vs. C (P = 0.0002), and D+E vs. E (P = 0.02). For women, ΔMetS was greater for D vs. C (P = 0.045), E vs. C (P = 0.02), and D+E vs. C (P = 0.004). After adjusting for ΔBF, all differences between groups were attenuated and no longer significant. ΔMetS were associated with ΔBF for both men (P < 0.0001) and women (P = 0.004). After adjustment for ΔBF, low‐fat diet alone and in combination with exercise had no effect on MetS. The key component for MetS from low‐fat diet and/or increased physical activity appears to be body fat loss.     

Hormones and sexual behavior in relationship to aging in male rats.

To determine if the age-related decline in male sex behavior is correlated with hormonal factors, a longitudinal study was conducted. Sexually experienced males were given mating tests every 2 months from 7 through 27 months of age. To study possible relationships between changes in behavior and alterations in hormone levels, blood samples were taken before and after these bimonthly tests. At 23 months, cross-sectional studies were also conducted comparing results to those obtained in 5-month-old males. Significant changes in mating behavior first appeared at 11 months; mount latency, intromission latency, ejaculation latency, postejaculatory interval, and intercopulatory interval were increased. Similarly, detectable decreases in testosterone (T) also occurred at this age. A significant decline in luteinizing hormone (LH) was not seen until 19 months. Correlational analyses revealed small (r less than or equal to -0.29) but significant negative correlations between T and parameters of mating behavior with age. When each age was examined separately, no significant correlations appeared. Plasma T was not predictive of behavioral performance. At 23 months, cross-sectional studies revealed deficits in mounting and penile reflex behavior but ejaculatory reflex capacity was unimpaired. At 28 months, males were decapitated. Only T levels showed a significant effect of age; estradiol, prolactin, and LH were unaffected when compared to 5-month-old males. The data suggest that although there are small and significant negative correlations between circulating testosterone and parameters of mating behavior with advancing age, it is unlikely that the observed decline in testosterone is the primary cause of the age-induced behavioral deficits. It is likely that the major causal factor(s) involves non-hormone-dependent changes within the CNS.     

ATR signaling mediates an S-phase checkpoint after inhibition of poly(ADP-ribose) polymerase activity

Human fibroblasts, capable of expressing a kinase-dead form of ATR (ATRkd), can be sensitized to the cytotoxic effects of methyl methanesulfonate (MMS) by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN). The combination of MMS+4-AN results in accumulation of cells in S-phase of the cell cycle and activation of Chk1. Inhibition of ATR activity by expression of ATRkd suppresses the S-phase accumulation and partially reverses the Chk1 phosphorylation. The results confirm involvement of an ATR-mediated damage response pathway in the MMS+4-AN-induced S-phase cell cycle checkpoint in human fibroblasts. Consistent with this hypothesis, the inhibitors caffeine and UCN-01 also abrogate the ATR- and Chk1-mediated delay in progression through S-phase. In the absence of ATR-mediated signaling, MMS+4-AN exposure results in a G(2)/M arrest, rather than an S-phase checkpoint. Thus, whereas ATR mediates the S-phase response, it is not critical for arrest of cells in G(2)/M.