Higher order chromatin structure presents a barrier to the recognition and repair of DNA damage. Double-strand breaks (DSBs) induce histone H2AX phosphorylation, which is associated with the recruitment of repair factors to damaged DNA. To help clarify the physiological role of H2AX, we targeted H2AX in mice. Although H2AX is not essential for irradiation-induced cell-cycle checkpoints, H2AX −/− mice were radiation sensitive, growth retarded, and immune deficient, and mutant males were infertile. These pleiotropic phenotypes were associated with chromosomal instability, repair defects, and impaired recruitment of Nbs1, 53bp1, and Brca1, but not Rad51, to irradiation-induced foci. Thus, H2AX is critical for facilitating the assembly of specific DNArepair complexes on damaged DNA.The first 120 amino acids of the H2AX and the H2A1/2 bulk isoprotein species exhibit a high degree of similarity, but H2AX carries a unique COOH-terminal tail that contains the * To whom correspondence should be addressed. andre_nussenzweig@nih.gov. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript consensus phosphatidyl inositol 3-kinase (PI-3 kinase) motif that is activated by DSBs (1, 2). Phosphorylation of H2AX (γ-H2AX) is induced by external genotoxic agents (2, 3) and is activated at physiological sites of recombination in lymphocytes (4, 5) and germ cells (6). Several essential DNA-repair factors implicated in homologous recombination (HR) (e.g., Brca1, Brca2, and Rad51) or that participate in both HR and nonhomologous end-joining (NHEJ) (e.g., Rad50, Mre11, Nbs1) form immunofluorescent foci that colocalize with γ-H2AX (7). However, the precise relation between focus formation and DNA repair is not understood.To determine the physiological role of H2AX in mammalian cells, we produced a targeted disruption of mouse H2AX (Web fig. 1A) (5,8). H2AX −/− mice were born at the expected frequency, and absence of H2AX protein was confirmed by two-dimensional gel electrophoresis and Western blotting (Web fig. 1, B to E) (8). Despite the loss of H2AX, treatment with γ-irradiation resulted in normal phosphorylation of Nbs1 (Web fig. 1E) (8).We conclude that H2AX is not essential for survival, or for irradiation-induced phosphorylation of Nbs1.H2AX −/− mice were growth retarded (Web fig. 2) (8), and H2AX −/− mouse embryo fibroblasts (MEFs) proliferated poorly in vitro (Fig. 1A). The difference in the growth of MEFs was partly due to a decrease in the number of dividing cells in H2AX −/− cultures as determined by incorporation of bromodeoxyuridine (BrdU) into DNA. During a 24-hour labeling period, only 44% of passage 1 H2AX −/− MEFs were actively cycling, compared with 72% for the controls, and the mitotic index of H2AX −/− MEFs was at least 50% lower than in wild-type cultures (see below; Fig. 1, D and F). By passage 4, H2AX −/− MEFS accumulated nondividing giant cells, suggesting premature entry into senescence. With continual passage, both H2AX −/− and wild-type MEFs went through crisis, after wh...

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for “classic” nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

Cholangiocarcinomas (CCA) are aggressive cancers, with a high mortality and poor survival rate. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of the late diagnosis secondary to relatively poor accuracy diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on 5 primary CCAs and 5 normal bile duct specimens (NBD). Several miRs were dysregulated, and miR-21 was overexpressed, in CCAs. miR-21 differential expression in these 10 specimens was verified with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). To validate these findings, qRT-PCR for miR-21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR-21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the Receiver Operating Characteristic (ROC) curve of 0.995. Inhibitors of miR-21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA (mRNA) levels of TIMP3 were significantly lower in CCAs than in normals. Conclusions MiR-21 is overexpressed in human CCAs. Furthermore, miR-21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree.

Background & Aims-Barrett's esophagus (BE) is a highly premalignant disease that predisposes to the development of esophageal adenocarcinoma (EAC); however, the involvement of microRNAs (miRs) in BE-EAC carcinogenic progression is not known.