Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region.

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

53BP1 Supports Immunoglobulin Class Switch Recombination Independently of Its DNA Double-Strand Break End Protection Function.

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector functions of antibodies. CSR occurs via the formation and non-homologous end joining (NHEJ) repair of programmed DNA double-strand breaks (DSBs) at the immunoglobulin heavy chain locus. The DNA repair factors 53BP1 and Rif1 promote NHEJ and CSR by protecting DSBs against resection. However, to what extent repression of DNA end resection contributes to CSR is unknown. Here, we show that B lymphocytes devoid of 53BP1-Rif1-dependent DSB end protection activity undergo robust CSR. Inactivation of specific sets of phospho-sites within 53BP1 N-terminal SQ/TQ motifs abrogates Rif1 recruitment and inhibition of resection but only mildly reduces CSR. Furthermore, mutations within 53BP1 oligomerization domain abolish CSR without substantially affecting DNA end processing. Thus, inhibition of DNA end resection does not correlate with CSR efficiency, indicating that regulation of DSB processing is not a key determinant step in CSR.

The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination.

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.

Alternative splicing variants of carbonic anhydrase IX in human non-small cell lung cancer.

In human cancers, carbonic anhydrase IX (CAIX) contributes to maintain intracellular and extracellular pH under hypoxic conditions, but also influences regulation of cell proliferation and tumor progression. CaIX was previously indicated as an independent prognostic marker in non-small cell lung carcinoma (NSCLC). Very recently a CAIX alternative splicing isoform, generating a transcript lacking of exons 8-9, was detected in cancer cells independently from the levels of hypoxia. This alternative splicing (AS) generates a truncated protein lacking the transmembrane region, the intracellular tail and the C-terminal of the catalytic domain and competes with the full-length (FL) isoform in the regulation of the extracellular pH, mainly in a mild hypoxic status. In the present study we measured the mRNA expression of FL and AS CAIX isoforms in 101 NSCLC and in paired not affected tissues. The two isoforms were coexpressed in all NSCLC and normal tissues but while AS mRNA was prevalent in normal tissues (66+/-3%), the FL isoform was higher in NSCLC (58+/-2%, p=0.001). FL mRNA, but not AS, was statistically increased in NSCLC (p=0.01) and showed a statistical association with lymphnode involvement (p=0.009) and tumor stage (p=0.04). Global survival analysis of cancer/related death showed that high levels of FL mRNA were predictive of unfavorable outcome (p<0.0001) and shorter disease-free survival (p<0.0001). Multivariate analysis indicated that FL is an independent prognostic factor for overall survival and higher levels of mRNA in NSCLC sensibly increase hazard ratio ( approximately sixfold). In conclusion, our results seems to indicate that, at least in NSCLC, FL CAIX is the most accurate surrogate of hypoxic stress and represents the only variant with a prognostic role. These data indicate the importance of a separate measurement of the two isoforms in cancer and the need of an accurate re-evaluation of most studies on the clinical role of CAIX in cancer diagnosis.

Functional polymorphisms of the microsomal epoxide hydrolase gene: a reappraisal on a early-onset lung cancer patients series.

Microsomal epoxide hydrolase gene (EPHX1) is polymorphic and encodes an enzyme involved in both the activation and detoxification of several tobacco carcinogens. Therefore, a contribution of EPHX1 enzymatic activity on lung cancer risk is possible. A genetic component of early-onset lung cancer has been suggested but variations in enzyme activity and polymorphisms in EPHX1 have seldom been studied in young patients with lung cancer. Primary lung cancer cases of both sexes and under age 45 at diagnosis were considered for this study. Controls fulfilled the following criteria: over 60 years old, smoking history of at least 40 years, no malignancies. Because of these criteria, they are referred to as super controls. The polymorphisms at exons 3 (Tyr113His) and 4 (His139Arg) as well as at the 5'-UTR-290T/G of the EPHX1 gene were genotyped by minisequencing. The association of these three polymorphisms with the development of early-onset lung cancer and the group of the super controls was evaluated by means of 2x2 tables using Yate's X(2) test or Fisher's exact test. Overall, data were obtained from 42 cases and 72 super controls. There was a significant association between early-onset lung cancer and the presence of the EPHX1 exon 4 variant (OR=3.33, 95% CI=1.50-7.41). This was confirmed at the phenotypic level when the data of both patients and super controls were stratified according to the predicted enzymatic activity (X(2) for linear trend=7.23, p=0.007). This analysis of lung cancer in subjects under age 45 supports the hypothesis that EPHX1 polymorphisms may have a role in cancer susceptibility in this age group.

Quantitative analysis of carbonic anhydrase IX mRNA in human non-small cell lung cancer.

Hypoxia is associated with malignant progression and poor outcome in human cancers. The effects of hypoxia are mediated by a series of genomic changes that enable tumor cells to survive or escape their oxygen deficient environment. Recent studies indicated that carbonic anhydrase IX (CA IX) is an intrinsic marker of hypoxia. In the present study we investigated with quantitative RT-PCR the expression of CA IX mRNA in 93 non-small cell lung carcinomas (NSCLC) and in their paired not affected tissues. CA IX mRNA was expressed in 100% NSCLC and in 76% of paired not affected tissues, even if tumoral CA IX expression was found constantly higher (p < 0.02) than that found in normal tissues. The increase of CA IX mRNA expression in cancer tissues was significantly correlated to the increase of corresponding protein, as determined with conventional immunoblotting (p = 0.027). In addition the expression of CA IX mRNA in NSCLC samples was significantly correlated to VEGF (p = 0.002) and MMP-9 (p = 0.002) mRNAs. Whereas CA IX mRNA expression was not associated to any clinical-pathological parameters in our patients, global survival analysis of cancer-related death revealed that high expression of CA IX mRNA predicted unfavorable outcome (p = 0.001) and shorter disease free survival interval (p = 0.004). A multivariate analysis showed that CA IX expression was the strongest prognostic parameter (p = 0.000) in comparison to other conventional predictive markers. In addition, differences emerged on the basis of clinical-pathological parameters: in fact separate Kaplan-Meyer analyses of patients indicated that whereas high levels of CA IX mRNA expression were not predictive of worse prognosis in early NSCLC (G1, T1, Stage 1 and pN- patients), this parameter appeared highly significant in advanced NSCLC (G2-G3, T2-T3, Stage 2-3 and pN+ patients). Finally we demonstrated that CA IX expression was not able to discriminate different survival probability in adenocarcinomas, whereas the same parameter was highly predictive in squamous (p = 0.03) and adenosquamous cell carcinomas (p = 0.001).

Simultaneous measurement of MMP9 and TIMP1 mRNA in human non small cell lung cancers by multiplex real time RT-PCR.

Extracellular matrix (ECM) homeostasis is strictly maintained by a coordinated balance between the expression of matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs). Our study was focused on the simultaneous measurement of the expression profile of MMP9 mRNA and its principal inhibitor, TIMP-1, in 100 non small cell lung cancers (NSCLC) and in corresponding adjacent non malignant tissues. The measurement was performed with a multiplex quantitative RT-PCR assay based on TaqMan assay, using two probes labelled with different fluorocromes. We found that both MMP9 and TIMP-1 mRNAs were significantly higher in NSCLC (P < 0.0001) in comparison to corresponding controls as well as the MMP9/TIMP-1 ratio (P = 0.014). MMP9 and TIMP-1 mRNA expression was highly correlated in cancer samples (r = 0.73, P < 0.0001). The analysis in the two main histotypes revealed a significant increase of MMP9 mRNA in adenocarcinomas in comparison to normal tissues (P = 0.006) but not in squamous cell carcinomas, while TIMP-1 mRNA showed a significative increase both in adenocarcinomas and in squamous cell carcinoma samples (P = 0.02 and 0.01, respectively). Both MMP9 and TIMP-1 mRNAs were significantly correlated to lymphnode invasion and cancer stage. Survival analysis revealed that high levels of expression of MMP9 mRNA, but not of TIMP-1, were significantly associated to an unfavourable outcome in NSCLC patients in toto (P = 0.017). In addition our results showed that high levels of MMP9 expression are of independent prognostic impact in operable NSCLC. Our data seem to demonstrate a simultaneous and coordinated up-regulation of MMP9 and TIMP-1 expression at the mRNA level in NSCLC, even if this phenomenon seems variable according to the histotype. In addition, the increase of MMP9/TIMP-1 ratio may reflect an unbalance of their production in affected tissues. The increased expression of the two mRNAs, even not necessarily equate their enzymatic activities, seems to parallel a major cancer aggressiveness.

[T4 tracheo-bronchial carcinoma: clinical evaluation of 48 sleeve pneumonectomies]. / Carcinoma tracheobronchiale T4. Bilancio clinico di 48 sleeve pneumonectomies.

The aim of the study was to verify the indications for surgery in T4 tracheo-bronchial carcinomas. Forty-eight tracheal-sleeve pneumonectomies for T4 bronchogenic carcinoma were performed in our unit from 1986 to 2003. The patients were 42 males and 6 females. A postero-lateral thoracotomy was preferred (46 right, 2 left). Bronchial reimplantation was performed additionally (tracheal-sleeve lobectomy) in 2 patients on the right side. The morbidity was 25% and the mortality 6.2% (1 acute respiratory distress syndrome, 1 myocardial infarction, 1 anastomotic fistula). Twenty-three cases were sT4N2M0, 14 sT4N1M0, and 11 sT4N0M0. The sT4N2M0 and sT4N1M0 cases were not associated with more than 3 year survival, despite adjuvant therapies; sT4N0M0 squamous cell carcinomas, on the other hand, had > 40% 10-year survival with no adjuvant therapy. Associated prosthetic replacement of the superior vena cava neither affected the risk nor improved the prognosis. Surgery for T4 tracheo-bronchial carcinoma appears feasible for well differentiated sT4N0 squamous cell carcinomas; at more advanced stages this procedure is no more than a dangerous form of palliation.