Biology subjects

Parsons, J. L.

Publications and source records attributed to Parsons, J. L..

2 recordsLinked to original sources

bioRxiv2018

Characterisation of deubiquitylating enzymes in the cellular response to high-LET ionising radiation and complex DNA damage

PurposeIonising radiation, particular high linear energy transfer (LET) radiation, can induce complex DNA damage (CDD) where two or more DNA lesions are induced in close proximity which contributes significantly to the cell killing effects. However knowledge of the enzymes and mechanisms involved in co-ordinating the recognition and processing of CDD in cellular DNA are currently lacking.\n\nMethods and MaterialsAn siRNA screen of deubiquitylation enzymes was conducted in HeLa cells irradiated with high-LET -particles or protons, versus low-LET protons and x-rays, and cell survival monitored by clonogenic assays. Candidates whose depletion led to decreased cell survival specifically in response to high-LET radiation were validated in both HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells, and the association with CDD repair was confirmed by using an enzyme modified neutral comet assay.\n\nResultsDepletion of USP6 decreased cell survival specifically following high-LET -particles and protons, but not by low-LET protons or x-rays. USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly(ADP-ribose) polymerase-1 (PARP-1). This phenotype was mimicked using the PARP inhibitor olaparib.\n\nConclusionUSP6 controls cell survival in response to high-LET radiation by stabilising PARP-1 protein levels which is essential for CDD repair. We also describe synergy between CDD induced by high-LET protons and PARP inhibition in effective cancer cell killing.

cancer biology↗

bioRxiv2018

MicroRNA-184 Is Induced By Store-Operated Calcium Entry And Regulates Early Keratinocyte Differentiation

Extracellular calcium (Ca2+) and store-operated Ca2+ entry (SOCE) govern homeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1, 25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+-dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a 4-fold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a 3-fold rise in levels of cyclin E and doubled the levels of {gamma}H2AX, a marker of DNA double strand breaks. The p21 cyclin-dependent kinase (CDK) inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to a SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

molecular biology↗