PubMed · 9149529
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L Holm, C Sander. 1997. Enzyme HIT.. https://doi.org/10.1016/s0968-0004(97)01021-9
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The Atm protein kinase and Mre11-Rad50-nibrin (MRN) complex play an integral role in the cellular response to DNA double-strand breaks. Mutations in Mre11 and nibrin result in the radiosensitivity disorders ataxia-telangiectasia-like disorder (ATLD) and Nijmegen breakage syndrome (NBS), respectively. Cells from ATLD and NBS patients are deficient in activation of the Atm protein kinase and phosphorylation of downstream Atm targets following irradiation. However, the roles of individual MRN complex proteins in Atm function are not clear, because the mutations in NBS and ATLD cells result in global effects on the MRN complex. Previously we showed that the C-terminal 100 amino acids of nibrin were necessary and sufficient to translocate the MRN complex to the nucleus. Here we have taken advantage of this feature of nibrin to create isogenic cell lines lacking either nibrin or Mre11-Rad50 in the nucleus. We found that nuclear expression of Mre11-Rad50, but not nibrin, stimulated Atm activation at early times after low doses of radiation. At later times or higher doses of irradiation, Atm activation was independent of Mre11-Rad50 or nibrin. The requirement of MRN complex proteins for downstream Atm phosphorylation events following irradiation was more complex. Phosphorylation of nibrin and Chk2 by Atm required Mre11-Rad50 expression in the nucleus at early times after irradiation, reflecting the stimulation of Atm activation by Mre11-Rad50. By contrast, autophosphorylation of Chk2 and phosphorylation of Smc1 at Ser-957 was dependent on the MRN complex 60 min after irradiation, even though Atm was activated at that time point. These results indicate an independent role for Mre11-Rad50 in the activation of Atm and suggest nibrin and/or Mre11-Rad50 also act as adaptors for some downstream Atm phosphorylation events.
Fragile histidine triad (FHIT) gene plays an important role in the pathogenesis of lung cancer. However, the clinicopathological significance of CpG island hypermethylation of FHIT gene in non-small cell lung cancer (NSCLC) remains to be elucidated. We studied FHIT methylation in 254 NSCLCs in order to further understand the clinicopathological and prognostic significance of FHIT methylation in NSCLC. Methylation status of the FHIT gene was examined using Methylation-Specific PCR. All statistical analyses were two-sided, with a 5% type I error rate. Hypermethylation of the FHIT gene occurred more frequently in squamous cell carcinoma than adenocarcinoma. For 93 adenocarcinomas there was no statistically significant association between FHIT methylation and age, gender, smoking history, pathologic stage and p16 methylation. However, FHIT methylation in 125 squamous cell carcinomas was associated with exposure to tobacco smoke and p16 methylation, but not with age, gender and pathologic stage. Hypermethylation of FHIT in squamous cell carcinomas occurred more frequently in current smokers (45%) than in never-smokers (13%). FHIT methylation was significantly associated with p16 methylation in current- and ex-smokers (P = 0.02 and P = 0.01, respectively) with squamous cell carcinoma and in patients with pathologic stage I squamous cell carcinoma (P = 0.001). Patients with p16 methylation were 3.74 times [95% confidence interval (CI) = 1.62 - 7.95; P = 0.001] more likely to have FHIT methylation in squamous cell carcinoma. FHIT methylation in squamous cell carcinoma occurred at a 4.62 times (95% CI = 1.26 - 34.97; P = 0.02) higher prevalence in current smokers than in never-smokers. No prognostic effect of FHIT methylation was observed in stage I and stage II NSCLCs. In conclusion, hypermethylation of the FHIT gene did not have a prognostic significance in early stage NSCLCs. The FHIT methylation is associated with the p16 methylation and smoking in squamous cell carcinoma, suggesting that FHIT may cooperate with p16 for the development of squamous cell carcinoma of lung in individuals exposed to tobacco smoke.
Acylphosphatases catalyse the hydrolysis of the carboxyl phosphate bond in metabolites such as acetyl phosphate, 1,3-bisphosphoglycerate, succinoyl phosphate and carbamoyl phosphate. In this study, acylphosphatase (91 residues) from the hyperthermophilic archaeon Pyrococcus horikoshii has been cloned, overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method using sodium formate as a precipitant at 289 K. The crystals belong to space group P3(2)21, with unit-cell parameters a = b = 85.65, c = 75.51 A. The asymmetric unit contains two molecules of acylphosphatase, with a corresponding crystal volume per protein weight of 3.9 A Da(-1) and a solvent content of 68.6%. A data set diffracting to 1.6 A resolution was collected from a single crystal at 100 K.