Severe PATCHED1 deficiency in cancer-prone Gorlin patient cells results in intrinsic radiosensitivity

PURPOSE: Gorlin syndrome (or basal-cell nevus syndrome) is a cancer-prone genetic disease in which hypersusceptibility to secondary cancer and tissue reaction after radiation therapy is debated, as is increased radiosensitivity at cellular level. Gorlin syndrome results from heterozygous mutations in the PTCH1 gene for 60% of patients, and we therefore aimed to highlight correlations between intrinsic radiosensitivity and PTCH1 gene expression in fibroblasts from adult patients with Gorlin syndrome. METHODS AND MATERIALS: The radiosensitivity of fibroblasts from 6 patients with Gorlin syndrome was determined by cell-survival assay after high (0.5-3.5 Gy) and low (50-250 mGy) γ-ray doses. PTCH1 and DNA damage response gene expression was characterized by real-time polymerase chain reaction and Western blotting. DNA damage and repair were investigated by γH2AX and 53BP1 foci assay. PTCH1 knockdown was performed in cells from healthy donors by using stable RNA interference. Gorlin cells were genotyped by 2 complementary sequencing methods. RESULTS: Only cells from patients with Gorlin syndrome who presented severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For 2 of the radiosensitive cell strains, heterozygous mutations in the 5' end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DNA damage response pathways (ATM, CHK2, and P53 levels and activation by phosphorylation) were deregulated after irradiation, whereas DSB repair recognition was unimpaired. Furthermore, normal cells with RNA interference-mediated PTCH1 deficiency showed reduced survival after irradiation, directly linking this gene to high- and low-dose radiosensitivity. CONCLUSIONS: In the present study, we show an inverse correlation between PTCH1 expression level and cellular radiosensitivity, suggesting an explanation for the conflicting results previously reported for Gorlin syndrome and possibly providing a basis for prognostic screens for radiosensitive patients with Gorlin syndrome and PTCH1 mutations

Concomitant heterochromatinisation and down-regulation of gene expression unveils epigenetic silencing of RELB in an aggressive subset of chronic lymphocytic leukemia in males

<p>Abstract</p> <p>Background</p> <p>The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both <it>in vitro </it>and <it>in vivo</it>, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes.</p> <p>Methods</p> <p>Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent <it>in situ </it>labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test.</p> <p>Results</p> <p>Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, <it>RELB </it>was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells.</p> <p>Conclusion</p> <p>The molecular defects in the silencing of <it>RELB </it>involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic <it>RELB </it>silencing as a new marker of the progressive disease in males.</p

Occurrence of Chronic Stage Chikungunya in the General Population of Martinique during the First 2014 Epidemic: A Prospective Epidemiological Study

International audienceChronic stage chikungunya (CHIK), defined by persisting symptoms more than 3 months after initial diagnosis of acute infection, is frequent. However, its burden and impact have rarely been described prospectively in a general population during an ongoing epidemic in the Caribbean. From January 2014 to January 2015, a severe CHIK outbreak occurred in Martinique. Our objective was to describe epidemiological characteristics and outcomes of chronic stage CHIK in its local population. Participants, clinically diagnosed with probable CHIK infection, were included prospectively by general practitioners during the epidemic's peak from April to October 2014. All identified cases benefited from a follow-up phone call 3 months or more after initial diagnosis during which they were interrogated about persisting clinical signs, past and ongoing treatment, and quality of life. Five hundred and nine subjects participated in the study. Mean age at initial diagnosis was 43.2 ± 23.6 years with a female-male ratio of 1.98. Two hundred participants (39.3%) had probable chronic stage CHIK: 98.5% still experienced pain at least 3 months after acute infection, with 84.3% of reported joint pains; 21.2% were woken up by the pain; 47.2% felt depressed/anxious; and 31.3% experienced memory/concentration disorders. Resumption of daily activity and work was complicated for 55.8% and 36.2% of cases. Persistent impact on morbidity, health outcomes, psychological, and economic aspects further underline the crucial role of community-based medicine and the necessity of an evidence-based multidisciplinary approach toward chronic stage CHIK identification, management, and follow-up in this particular world regio

Occurrence of Chronic Stage Chikungunya in the General Population of Martinique during the First 2014 Epidemic: A Prospective Epidemiological Study

International audienceChronic stage chikungunya (CHIK), defined by persisting symptoms more than 3 months after initial diagnosis of acute infection, is frequent. However, its burden and impact have rarely been described prospectively in a general population during an ongoing epidemic in the Caribbean. From January 2014 to January 2015, a severe CHIK outbreak occurred in Martinique. Our objective was to describe epidemiological characteristics and outcomes of chronic stage CHIK in its local population. Participants, clinically diagnosed with probable CHIK infection, were included prospectively by general practitioners during the epidemic's peak from April to October 2014. All identified cases benefited from a follow-up phone call 3 months or more after initial diagnosis during which they were interrogated about persisting clinical signs, past and ongoing treatment, and quality of life. Five hundred and nine subjects participated in the study. Mean age at initial diagnosis was 43.2 ± 23.6 years with a female-male ratio of 1.98. Two hundred participants (39.3%) had probable chronic stage CHIK: 98.5% still experienced pain at least 3 months after acute infection, with 84.3% of reported joint pains; 21.2% were woken up by the pain; 47.2% felt depressed/anxious; and 31.3% experienced memory/concentration disorders. Resumption of daily activity and work was complicated for 55.8% and 36.2% of cases. Persistent impact on morbidity, health outcomes, psychological, and economic aspects further underline the crucial role of community-based medicine and the necessity of an evidence-based multidisciplinary approach toward chronic stage CHIK identification, management, and follow-up in this particular world regio