G2/M Arrest Sensitises Erythroid Leukemia Cells to TRAIL-induced Apoptosis

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CtIP Mutations Cause Seckel and Jawad Syndromes

Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5) but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2). Here, we report two mutations in the CtIP (RBBP8) gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation

The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles

Cost-effectiveness of microendoscopic discectomy versus conventional open discectomy in the treatment of lumbar disc herniation: a prospective randomised controlled trial [ISRCTN51857546]

BACKGROUND: Open discectomy is the standard surgical procedure in the treatment of patients with long-lasting sciatica caused by lumbar disc herniation. Minimally invasive approaches such as microendoscopic discectomy have gained attention in recent years. Reduced tissue trauma allows early ambulation, short hospital stay and quick resumption of daily activities. A comparative cost-effectiveness study has not been performed yet. We present the design of a randomised controlled trial on cost-effectiveness of microendoscopic discectomy versus conventional open discectomy in patients with lumbar disc herniation. METHODS/DESIGN: Patients (age 18–70 years) presenting with sciatica due to lumbar disc herniation lasting more than 6–8 weeks are included. Patients with disc herniation larger than 1/3 of the spinal canal diameter, or disc herniation less than 1/3 of the spinal canal diameter with concomitant lateral recess stenosis or sequestration, are eliglible for participation. Randomisation into microendoscopic discectomy or conventional unilateral transflaval discectomy will take place in the operating room after induction of anesthesia. The length of skin incision is equal in both groups. The primary outcome measure is the functional assessment of the patient, measured by the Roland Disability Questionnaire for Sciatica, at 8 weeks and 1 year after surgery. We will also evaluate several other outcome parameters, including perceived recovery, leg and back pain, incidence of re-operations, complications, serum creatine kinase, quality of life, medical consumption, absenteeism and costs. The study is a randomised prospective multi-institutional trial, in which two surgical techniques are compared in a parallel group design. Patients and research nurses are kept blinded of the allocated treatment during the follow-up period of 2 years. DISCUSSION: Currently, open discectomy is the golden standard in the surgical treatment of lumbar disc herniation. Whether microendoscopic discectomy is more cost-effective than unilateral transflaval discectomy has to be determined by this trial

DNA-PK-Dependent RPA2 Hyperphosphorylation Facilitates DNA Repair and Suppresses Sister Chromatid Exchange

Hyperphosphorylation of RPA2 at serine 4 and serine 8 (S4, S8) has been used as a marker for activation of the DNA damage response. What types of DNA lesions cause RPA2 hyperphosphorylation, which kinase(s) are responsible for them, and what is the biological outcome of these phosphorylations, however, have not been fully investigated. In this study we demonstrate that RPA2 hyperphosphorylation occurs primarily in response to genotoxic stresses that cause high levels of DNA double-strand breaks (DSBs) and that the DNA-dependent protein kinase complex (DNA-PK) is responsible for the modifications in vivo. Alteration of S4, S8 of RPA2 to alanines, which prevent phosphorylations at these sites, caused increased mitotic entry with concomitant increases in RAD51 foci and homologous recombination. Taken together, our results demonstrate that RPA2 hyperphosphorylation by DNA-PK in response to DSBs blocks unscheduled homologous recombination and delays mitotic entry. This pathway thus permits cells to repair DNA damage properly and increase cell viability

Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s) underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI) methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition.</p> <p>Methods</p> <p>To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis.</p> <p>Results</p> <p>Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99) between the total number of hypermethylated CGIs and GI₅₀values (<it>i.e</it>., increased drug resistance) following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells.</p> <p>Conclusion</p> <p>Selective epigenetic disruption of distinct biological pathways was observed during development of platinum resistance in ovarian cancer. Integrated analysis of DNA methylation and gene expression may allow for the identification of new therapeutic targets and/or biomarkers prognostic of disease response. Finally, our results suggest that epigenetic therapies may facilitate the prevention or reversal of transcriptional repression responsible for chemoresistance and the restoration of sensitivity to platinum-based chemotherapeutics.</p

Dna polymerase η modulates replication fork progression and dna damage responses in platinum-treated human cells

Human cells lacking DNA polymerase eta (pol eta) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of pol eta in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking pol eta than in cells expressing pol eta. This provides the first direct evidence that pol eta modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated pol eta-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of pol eta. Pol eta-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy

Dna polymerase η modulates replication fork progression and dna damage responses in platinum-treated human cells

Human cells lacking DNA polymerase eta (pol eta) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of pol eta in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking pol eta than in cells expressing pol eta. This provides the first direct evidence that pol eta modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated pol eta-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of pol eta. Pol eta-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy