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Tej Patel, Cristina M Kenney, Catherine Y Liu, Marilyn Chwa, Shari R Atilano, Jon Lucas Norman; Anticancer Drug Cisplatin Differentially Affects Retina Pigment Epithelial Cells Depending on Mitochondrial Backgrounds. Invest. Ophthalmol. Vis. Sci. 2016;57(12):244.
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© ARVO (1962-2015); The Authors (2016-present)
Mitochondrial (mt) DNA can be classified into haplogroups based upon single nucleotide polymorphism patterns that define racial populations. The anticancer drug, cisplatin, is known to cause toxicity to the retina, including retinal pigmentary changes. Our hypothesis is that retinal pigment epithelial (RPE) cells with the same nuclear DNA but containing different mtDNA backgrounds are differentially affected by cisplatin.
Cytoplasmic hybrids (cybrids), cell lines with identical nuclear genomes but different mitochondrial genomes, are created by fusion of platelets (with mtDNA but no nuclear DNA) collected from various individuals to RPE cells devoid of mtDNA (H haplotype, 3 cell lines; J haplotype, 3 cell lines). H and J cybrids were treated for 48 hours with 25 or 50 µM cisplatin and cell viabilities were measured using trypan blue exclusion. H and J cybrids were also treated with 40µM Cisplatin for 48 hours and the mitochondrial membrane potential (ΔΨm) was measured to test for early apoptotic changes. QPCR was used to analyze cellular gene expression changes. Untreated cultures served as the controls. Statistical analysis was done using GraphPad Prism 5.
With 25 uM and 50 uM cisplatin treatment, J cybrids show decreased viability to 65% (p=0.043) and 42% (p=0.002) of untreated, respectively. H cybrids had cell viability that was less effected by cisplatin: 87% (p=0.58) and 62% (p=0.051) of untreated H-cybrids, respectively. Cisplatin-treated and untreated H-cybrids had similar ΔΨm: 5.3% ± 7.8% (p=0.5). In contrast, treated J-cybrids s showed a 24.0% ± 4.1% (p<0.0001) decrease in ΔΨm compared to untreated. Comparison of gene expression in J-treated versus H-treated cybrids showed increase in apoptotic gene BAX (p=0.03, fold 1.4), but a decrease in genes such as SFRP1 (p=0.0001, fold 0.21), DHRS2 (p=0.041, fold 0.58) and EFEMP1 (p=0.009, fold 0.45).
Retinal Pigment Epithelial cells with identical nuclear DNA but H versus J mtDNA haplogroups show significant differences in cell viability, mitochondrial membrane potential and cellular gene expression levels when treated with the cancer drug cisplatin. We show that mitochondrial DNA changes alone can affect cellular response. These findings may help identify individuals who may be more susceptible to cisplatin retinal toxicity depending on their mitochondrial background.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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