Severe PTCH1 deficiency in cancer-prone Gorlin patient cells results in intrinsic radiosensitivity

Publication date: Available online 2 June 2018
Source:International Journal of Radiation Oncology*Biology*Physics
Author(s): Adeline Vulin, Melissa Sedkaoui, Sandra Moratille, Nicolas Sevenet, Pascal Soularue, Odile Rigaud, Laure Guibbal, Joshua Dulong, Penny Jeggo, J.F. Deleuze, Jérôme Lamartine, Michèle T. Martin
PurposeGorlin syndrome (or basal-cell nevus syndrome: BCNS) is a cancer-prone genetic disease in which hyper-susceptibility to secondary cancer and tissue reaction after radiotherapy is debated, as well as increased radiosensitivity at cellular level. It 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 Gorlin patients.Materials and MethodsThe radiosensitivity of fibroblasts from 6 Gorlin patients 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 (DDR) gene expression was characterized by real-time PCR 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 stable RNA interference. Gorlin cells were genotyped by two complementary sequencing methods.ResultsOnly cells from Gorlin patients presenting severe deficiency in PATCHED1 protein exhibited a significant increase in cellular radiosensitivity, affecting cell responses to both high and low radiation doses. For two of the radiosensitive cell strains, heterozygous mutations in the 5'end of PTCH1 gene explain PATCHED1 protein deficiency. In all sensitive cells, DDR 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.ConclusionsIn 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 Gorlin patients with PTCH1 mutations.

Teaser

Gorlin syndrome is a typical case of debated hyper-sensitivity to radiation, although it is well-recognized as a cancer-prone disorder. The present data reveal that only Gorlin cells presenting severe deficiency in PTCH1 gene expression exhibited significantly increased cellular radiosensitivity, and that the PATCHED1 protein had a direct role in regulating intrinsic radiosensitivity, after both high and low radiation doses. This may provide a basis for prognostic screens for radiosensitive Gorlin patients with PTCH1 mutations.


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