Sonia Cerdido Ochoa¹, Idoya Mª Martínez Vicente², Marta Abrisqueta González², Ana Lambertos Escudero², Cecilia María Herraiz Serrano², María Concepción Olivares Sánchez², CELIA JIMENEZ-CERVANTES FRIGOLS², JOSE CARLOS GARCIA-BORRON MARTINEZ²

(1) Universidad de Murcia, 30204, España (Región de Murcia)
(2) CONTROL MOLECULAR DE LA PROLIFERACIÓN Y DIFERENCIACIÓN, IMIB-Arrixaca, España

Mahogunin Ring Finger-1 (MGRN1) is an E3-ubiquitin ligase containing a RING Finger domain. The mahoganoid mutation, a loss-of-function mutation in the MGRN1 gene, was first described in mice and is associated, among other effects, with a pigmentation phenotype like that produced by gain-of-function mutations in MC1R, a melanoma susceptibility gene. Our group has shown that that lack of MGRN1 in mouse melanocytes and melanoma cells leads to aberrant progression through the cell cycle and alterations in genomic integrity with accumulation of DNA damage. The presence of genomic alterations in MGRN1-null cells suggests that activation of the TP53 tumour suppressor might account for impaired cell cycle progression. According with these precedents, the following aims were proposed: to analyse 1) the effects of MGRN1 ablation on the proliferation and genomic integrity of human melanoma cells and 2) the role of TP53 on the potential proliferation and cell cycle defects observed in human melanocytic cells lacking MGRN1

Characterization of the status of the TP53 gene in human melanoma cell lines expressing MGRN1; Ablation of MGRN1 expression in TP53-wildtype cells by CRISPR-Cas9; determination of proliferation rates by cell counting; flow cytometric analysis of cell cycle progression; analysis of genomic instability by comet assays and immunochemical techniques.

Using flow cytometric analysis, we found that absence of MGRN1 increases the fraction of cells in the S phase of the cell cycle of human melanoma cells wildtype for TP53, but not in TP53-mutated cells. However, the cellular models used do not have the same genetic background, thus excluding an interpretation of the results based exclusively on the status of TP53. TP53+/+ MGRN1-null cells show no variation in proliferation rate compared with control cells of the same genetic background, but do accumulate more steady-state basal DNA damage in the absence of external perturbations by genotoxic treatments. Surprisingly we did not detect alterations in susceptibility to damage induced by an exogenous genotoxic agent.

Repression of MGRN1 expression does not disrupt cell proliferation in human melanoma cells of TP53+/+ genetic background but increases their basal level of DNA damage in the absence of genotoxic agents.