REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

Clinical primary flexor tendon repair and rehabilitation: The Bern Experience

In this chapter we present our experience with treatment of zone 2 flexor tendon repair using a six-strand repair technique combined with postoperative place-and-hold exercise. The six-strand Lim/Tsai repair technique combined with place-and-hold exercises demonstrated better digital function compared to a two-strand repair without place-and-hold exercises. Range of motion in the Lim/Tsai repair group appeared to be increased without a higher rate of ruptures but with a shorter rehabilitation period. The fact that the two groups differed in both suture techniques and rehabilitation programs made it impossible to know whether the better results in the group of Lim/Tsai were due to the six-strand repair or the place-and-hold exercises or both. Despite the obvious benefit of early active mobilization, an active motion protocol may not always be possible to apply in a substantial number of patients due to concomitant injuries, the quality of the surgical repair or patient factors (swelling, pain, limited compliance). Since August 2006 a staged rehabilitation program (“stop and go”) was introduced within our unit using early active controlled flexion (green), place-and-hold (yellow), or passive flexion exercises (red) introduced by Kleinert-Duran. Our experience using the six-strand suture repair technique and “stop and go” is outlined.

MET inhibition in tumor cells by PHA665752 impairs homologous recombination repair of DNA double strand breaks

Abnormal activation of cellular DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems has broad implications for both cancer biology and treatment. Recent studies suggest a potential link between DNA repair and aberrant activation of the hepatocyte growth factor receptor Mesenchymal-Epithelial Transition (MET), an oncogene that is overexpressed in numerous types of human tumors and considered a prime target in clinical oncology. Using the homologous recombination (HR) direct-repeat direct-repeat green fluorescent protein ((DR)-GFP) system, we show that MET inhibition in tumor cells with deregulated MET activity by the small molecule PHA665752 significantly impairs in a dose-dependent manner HR. Using cells that express MET-mutated variants that respond differentially to PHA665752, we confirm that the observed HR inhibition is indeed MET-dependent. Furthermore, our data also suggest that decline in HR-dependent DNA repair activity is not a secondary effect due to cell cycle alterations caused by PHA665752. Mechanistically, we show that MET inhibition affects the formation of the RAD51-BRCA2 complex, which is crucial for error-free HR repair of double strand DNA lesions, presumably via downregulation and impaired translocation of RAD51 into the nucleus. Taken together, these findings assist to further support the role of MET in the cellular DNA damage response and highlight the potential future benefit of MET inhibitors for the sensitization of tumor cells to DNA damaging agents.

Coupling of mutated Met variants to DNA repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

3CAPS – a structural AP-site analogue as a tool to investigate DNA base excision repair

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP-sites. With its 3′-phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′-deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

Laparoscopic incisional hernia repair is feasible and safe after liver transplantation

Incisional hernia is a common complication after liver transplantation. The current study evaluated incidence and risk factors for incisional hernia and compared laparoscopic and open hernia repair in terms of feasibility and outcome.

Assessment of in vivo genotoxicity of the rodent carcinogen furan: evaluation of DNA damage and induction of micronuclei in mouse splenocytes

In recent years, several surveys have highlighted the presence of the rodent carcinogen furan in a variety of food items. Even though the evidence of carcinogenicity of furan is unequivocal, the underlying mechanism has not been fully elucidated. In particular, the role of genotoxicity in furan carcinogenicity is still not clear, even though this information is considered pivotal for the assessment of the risk posed by the presence of low doses of furan in food. In this work, the genotoxic potential of furan in vivo has been investigated in mice, under exposure conditions similar to those associated with cancer onset in the National Toxicology Program long-term bioassay. To this aim, male B6C3F1 mice were treated by gavage for 4 weeks with 2, 4, 8 and 15 mg furan/kg b.w./day. Spleen was selected as the target organ for genotoxicity assessment, in view of the capability of quiescent splenocytes to accumulate DNA damage induced by repeat dose exposure. The induction of primary DNA damage in splenocytes was evaluated by alkaline single-cell gel electrophoresis (comet assay) and by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX). The presence of cross-links was probed in a modified comet assay, in which cells were irradiated in vitro with gamma-rays before electrophoresis. Chromosome damage was quantitated through the detection of micronuclei in mitogen-stimulated splenocytes using the cytokinesis-block method. Micronucleus induction was also assessed with a modified protocol, using the repair inhibitor 1-beta-arabinofuranosyl-cytosine to convert single-strand breaks in micronuclei. The results obtained show a significant (P < 0.01) increase of gamma-H2AX foci in mitogen-stimulated splenocytes of mice treated with 8 and 15 mg furan/kg b.w. and a statistically significant (P < 0.001) increases of micronuclei in binucleated splenocytes cultured in vitro. Conversely, no effect of in vivo exposure to furan was observed when freshly isolated quiescent splenocytes were analysed by immunofluorescence and in comet assays, both with standard and radiation-modified protocols. These results indicate that the in vivo exposure to furan gives rise to pre-mutagenic DNA damage in resting splenocytes, which remains undetectable until it is converted in frank lesions during the S-phase upon mitogen stimulation. The resulting DNA strand breaks are visualized by the increase in gamma-H2AX foci and may originate micronuclei at the subsequent mitosis.

A new histology scoring system for the assessment of the quality of human cartilage repair: ICRS II

A reliable and reproducible method is needed to assess cartilage repair.

Repair of orbital floor fractures using bioresorbable poly-L/DL-lactide plates

To assess the long-term clinical and radiologic findings after insertion of a bioresorbable polylactide plates P(L/DL)LA 70/30 implant (PolyMax) in the repair of orbital floor and wall defects, with special focus on stability and clinical signs of foreign-body reaction.

Craniofacial morphology in complete unilateral cleft lip and palate patients consecutively treated with 1-stage repair of the cleft

OBJECTIVE: To retrospectively evaluate the craniofacial morphology of children with a complete unilateral cleft lip and palate treated with a 1-stage simultaneous cleft repair performed in the first year of life. METHODS: Cephalograms and extraoral profile photographs of 61 consecutively treated patients (42 boys, 19 girls) who had been operated on at 9.2 (SD, 2.0) months by a single experienced surgeon were analyzed at 11.4 (SD, 1.5) years. The noncleft control group comprised 81 children (43 boys and 38 girls) of the same ethnicity at the age of 10.4 (SD, 0.5) years. RESULTS: In children with cleft, the maxilla and mandible were retrusive; the palatal and mandibular planes were more open, and sagittal maxillomandibular relationship was less favorable in comparison to noncleft control subjects. Soft tissues in patients with cleft reflected retrusive morphology of hard tissues--subnasal and supramental regions were less convex, profile was flatter, and nasolabial angle was more acute relative to those of the control subjects. CONCLUSIONS: Craniofacial morphology after 1-stage repair was deviated in comparison with noncleft control subjects. However, the degree of deviation was comparable with that found after treatment with alternative surgical protocols.

Evaluation of cartilage repair tissue after matrix-associated autologous chondrocyte transplantation using a hyaluronic-based or a collagen-based scaffold with morphological MOCART scoring and biochemical T2 mapping: preliminary results

In cartilage repair, bioregenerative approaches using tissue engineering techniques have tried to achieve a close resemblance to hyaline cartilage, which might be visualized using advanced magnetic resonance imaging.

Quantitative T2 mapping of knee cartilage: differentiation of healthy control cartilage and cartilage repair tissue in the knee with unloading - initial results

Purpose: To prospectively determine on T2 cartilage maps the effect of unloading during a clinical magnetic resonance (MR) examination in the postoperative follow-up of patients after matrix-associated autologous chondrocyte transplantation (MACT) of the knee joint. Materials and Methods: Ethical approval for this study was provided by the local ethics commission, and written informed consent was obtained. Thirty patients (mean age, 35.4 years +/- 10.5) with a mean postoperative follow-up period of 29.1 months +/- 24.4 were enrolled. A multiecho spin-echo T2-weighted sequence was performed at the beginning (early unloading) and end (late unloading) of the MR examination, with an interval of 45 minutes. Mean and zonal region of interest T2 measurements were obtained in control cartilage and cartilage repair tissue. Statistical analysis of variance was performed. Results: The change in T2 values of control cartilage (early unloading, 50.2 msec +/- 8.4; late unloading, 51.3 msec +/- 8.5) was less pronounced than the change in T2 values of cartilage repair tissue (early unloading, 51.8 msec +/- 11.7; late unloading, 56.1 msec +/- 14.4) (P = .024). The difference between control cartilage and cartilage repair tissue was not significant for early unloading (P = .314) but was significant for late unloading (P = .036). Zonal T2 measurements revealed a higher dependency on unloading for the superficial cartilage layer. Conclusion: Our results suggest that T2 relaxation can be used to assess early and late unloading values of articular cartilage in a clinical setting and that the time point of the quantitative T2 measurement affects the differentiation between native and abnormal articular cartilage. (c) RSNA, 2010.

Long-term follow-up of open and laparoscopic repair of large incisional hernias

Long-term results after laparoscopic repair of large incisional hernias remain to be determined. The aim of this prospective study was to compare early and late complications between laparoscopic repair and open repair in patients with large incisional hernias.