RESUMO
Retinitis pigmentosa (RP) is the most common cause of hereditary blindness, and may occur in isolation as a non-syndromic condition or alongside other features in a syndromic presentation. Biallelic or monoallelic mutations in one of eight genes encoding pre-mRNA splicing factors are associated with non-syndromic RP. The molecular mechanism of disease remains incompletely understood, limiting opportunities for targeted treatment. Here we use CRISPR and base edited PRPF6 and PRPF31 mutant cell lines, and publicly-available data from human PRPF31 +/- patient derived retinal organoids and PRPF31 siRNA-treated organotypic retinal cultures to confirm an enrichment of differential splicing of microtubule, centrosomal, cilium and DNA damage response pathway genes in these cells. We show that genes with microtubule/centrosome/centriole/cilium gene ontology terms are enriched for weak 3' and 5' splice sites, and that subtle defects in spliceosome activity predominantly affect efficiency of splicing of these exons. We suggest that the primary defect in PRPF6 or PRPF31 mutant cells is microtubule and centrosomal defects, leading to defects in cilium and mitotic spindle stability, with the latter leading to DNA damage, triggering differential splicing of DNA damage response genes to activate this pathway. Finally, we expand understanding of "splicing factor RP" by investigating the function of TTLL3, one of the most statistically differentially expressed genes in PRPF6 and PRPF31 mutant cells. We identify that TTLL3 is the only tubulin glycylase expressed in the human retina, essential for monoglycylation of microtubules of the cilium, including the retinal photoreceptor cilium, to prevent cilium degeneration and retinal degeneration. Our preliminary data suggest that rescue of tubulin glycylation through overexpression of TTLL3 is sufficient to rescue cilium number in PRPF6 and PRPF31 mutant cells, suggesting that this defect underlies the cellular defect and may represent a potential target for therapeutic intervention in this group of disorders.
RESUMO
RESUMEN Introducción: en el mundo, el cáncer de próstata es la principal causa de muerte en hombres. Algunas evidencias sugieren que los ácidos grasos omega-3 reducen la viabilidad de las células tumorales mientras que los ácidos omega-6 promueven su proliferación; al respecto, otros estudios han mostrado resultados controvertidos. Objetivo: evaluar los efectos citotóxicos, genotóxicos y anticlonogénicos de ácidos omega-3: α-linolénico (ALA), eicosapentaenoico (EPA) y docosahexaenoico (DHA), omega-6: linoleico (LA), araquidónico (AA) y omega-9: oleico (OA) en células de cáncer de próstata (PC-3). Metodología: se evaluó el efecto sobre la viabilidad celular relativa mediante las pruebas de MTT y Azul de Tripano, el efecto genotóxico mediante intercambios de cromátidas hermanas (ICHs) y ensayo cometa, y el efecto anticlonogénico in vitro, en diferentes concentraciones (25, 50, 100 y 150 µM) de seis ácidos grasos omega en células de cáncer de próstata (PC-3). Resultados: la viabilidad relativa por MTT mostró valores ≤ IC50 con las concentraciones mayores (100 y 150 µM) para los ácidos grasos omega-3 EPA y DHA y omega-6 AA (150 µM), mientras que la viabilidad relativa, evaluada con Azul de Tripano, con estos mismos ácidos, redujeron la viabilidad a 0 %. DHA y EPA mostraron efecto genotóxico y la disminución de la clonogenicidad celular (p < 0,01). Por otro lado, LA y AA disminuyeron la viabilidad relativa observada con Azul de Tripano, sugiriendo diferentes mecanismos de acción de los ácidos grasos en la membrana celular. Conclusión: los resultados mostraron que los ácidos grasos omega-3, EPA, DHA, y omega-6, AA, disminuyen la formación de colonias, reducen la viabilidad celular y aumentan el efecto genotóxico respecto al control no tratado, en el modelo in vitro de células tumorales de próstata PC-3.
SUMMARY Introduction: Prostate cancer is the main cause of cancer related deaths in men worldwide. Previous studies have suggested that omega-3 fatty acids reduce cell viability in tumour cells, whereas omega-6 fatty acids increase clonogenicity. Nevertheless, other reports have shown controversial results. Objective: Evaluate cytotoxicity, genotoxicity and clonogenicity in a prostate cancer derived human cell line (PC-3), treated with fatty acids omega-3: α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); omega-6: linoleic acid (LA) and arachidonic acid (AA); omega-9: oleic acid (OA). Methods: The tests included (a) cytotoxicity assays by MTT and Trypan Blue; (b) genotoxicity evaluation by the sister-chromatid exchanges technique (SCE) and the DNA-comet assay; and (c) in vitro clonogenic assay of six fatty acids in prostate cancer cell (PC-3) at different concentrations (25 µM, 50 µM, 100 µM and 150 µM). Results: The cell viability by MTT data showed ≤ IC50 values for the omega-3 EPA and DHA and omega-6 AA fatty acids at the two major concentrations (100 µM and 150 µM). Moreover, the same fatty acids viability values dropped to 0 % with Trypan Blue test. EPA and DHA showed genotoxic effect and a clonogenic cell decrease (p<0,01). The latter test also revealed a viability diminishment for LA and AA, suggesting different mechanisms of action of fatty acids on cell membrane. Conclusion: The in vitro evaluation revealed that EPA, DHA and AA reduce the clonogenicity and cell viability of prostate tumour cells and cause genotoxicity in prostate tumour derived PC-3 cells.
