The Role of γH2AX in Replication Stress-induced Carcinogenesis: Possible Links and Recent Developments.

Cancer is a condition characterized by genomic instability and gross chromosomal aberrations. The inability of the cell to timely and efficiently complete its replication cycle before entering mitosis is one of the most common causes of DNA damage and carcinogenesis. Phosphorylation of histone 2AX (H2AX) on S139 (γH2AX) is an indispensable step in the response to DNA damage, as it is required for the assembly of repair factors at the sites of damage. γH2AX is also a marker of DNA replication stress, mainly due to fork collapse that often follows prolonged replication stalling or repair of arrested forks, which involves the generation of DNA breaks. Although the role of γH2AX in the repair of DNA breaks has been well defined, the function of γH2AX in replicative stress remains unclear. In this review, we present the recent advances in the field of replication stress, and highlight a novel function for γH2AX that is independent of its role in the response to DNA damage. We discuss studies that support a role for γΗ2ΑΧ early in the response to replicative stress, which does not involve the repair of DNA breaks. We also highlight recent data proposing that γH2AX acts as a chromatin remodeling component, implicated in the efficient resolution of stalled replication forks. Understanding the mechanism by which γH2AX enables cellular recovery after replication stress will allow identification of novel cancer biomarkers, as well as new targets for cancer therapies.

Post-transcriptional modification of spliceosomal RNAs is normal in SMN-deficient cells.

The survival of motor neuron (SMN) protein plays an important role in the biogenesis of spliceosomal snRNPs and is one factor required for the integrity of nuclear Cajal bodies (CBs). CBs are enriched in small CB-specific (sca) RNAs, which guide the formation of pseudouridylated and 2'-O-methylated residues in the snRNAs. Because SMN-deficient cells lack typical CBs, we asked whether the modification of internal residues of major and minor snRNAs is defective in these cells. We mapped modified nucleotides in the major U2 and the minor U4atac and U12 snRNAs. Using both radioactive and fluorescent primer extension approaches, we found that modification of major and minor spliceosomal snRNAs is normal in SMN-deficient cells. Our experiments also revealed a previously undetected pseudouridine at position 60 in human U2 and 2'-O-methylation of A1, A2, and G19 in human U4atac. These results confirm, and extend to minor snRNAs, previous experiments showing that scaRNPs can function in the absence of typical CBs. Furthermore, they show that the differential splicing defects in SMN-deficient cells are not due to failure of post-transcriptional modification of either major or minor snRNAs.

Impaired minor tri-snRNP assembly generates differential splicing defects of U12-type introns in lymphoblasts derived from a type I SMA patient.

The survival of motor neuron (SMN) protein is essential for cytoplasmic assembly of spliceosomal snRNPs. Although the normal proportion of endogenous snRNAs is unevenly altered in spinal muscular atrophy (SMA) tissues, the biogenesis of individual snRNPs is not dramatically affected in SMN-deficient cells. The SMN protein is also required for normal Cajal body (CB) formation, but the functional consequences of CB disruption upon SMN deficiency have not yet been analyzed at the level of macromolecular snRNPs assembly. Here, we show that the SMN protein is required for tri-snRNPs formation and that the level of the minor U4atac/U6atac/U5 tri-snRNPs is dramatically decreased in lymphoblasts derived from a patient suffering from a severe form of SMA. We found also that splicing of some, but not all, minor introns is inhibited in these cells, demonstrating links between SMN deficiency and differential alterations of splicing events mediated by the minor spliceosome. Our results suggest that SMA might result from the inefficient splicing of one or only a few pre-mRNAs carrying minor introns and coding for proteins required for motor neurons function and/or organization.

Consistent absence of BRAF mutations in cervical and endometrial cancer despite KRAS mutation status.

BACKGROUND: Mutational activation of KRAS and BRAF proto-oncogenes contributes to the development of many human cancers. Current research on gynecological cancer and specifically in cervical and endometrial cancer is focused on the mechanisms of their mutational activation. OBJECTIVES: In view of the paucity of data on their mutation frequency and the status of BRAF in these two types of gynecological cancer, we performed a systematic molecular study in 114 clinically and histologically well-defined malignant tumors of uterine cervix and endometrium and correlated the mutation status of KRAS and BRAF with the age at diagnosis and with tumor grade, stage or histological type. METHODS: Direct sequence analysis of the PCR products of KRAS and BRAF genes was used to screen for known activating mutations. RESULTS: In 67 cases of endometrial cancer, six KRAS mutations (8.9%) were found, four at codon 12 (5.9%) and two at codon 13 (2.9%), while no mutation was detected at codon 61. Most of the mutations occurred in surgical stage I and in the endometrioid adenocarcinoma subtype. We also detected three KRAS point mutations (6.3%) in the 47 cervical cancer samples, two at codon 12 (4.2%) and one at codon 13 (2.1%), while there was no mutation at codon 61. On the contrary, no mutation was identified in BRAF exon 15 for either endometrial or cervical cancer samples at position V600, which represents the most frequently mutated site of BRAF in human cancer. There was no association between KRAS mutations with either histological type, tumor grade or stage. Interestingly, however, KRAS mutation status in endometrial cancer was strongly associated with increased age at diagnosis (P < 0.001). CONCLUSIONS: Our data document (a) the absence of BRAF mutations in cervical and endometrial cancer, despite the mutation status of KRAS, (b) suggest that KRAS mutations reflect an early event in endometrial carcinogenesis and (c) imply that BRAF activation is involving alternative pathways in these two types of cancer.