Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.

In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2'-3'-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.

Curcumin suppresses gastric tumor cell growth via ROS-mediated DNA polymerase γ depletion disrupting cellular bioenergetics.

BACKGROUND: Curcumin, as a pro-apoptotic agent, is extensively studied to inhibit tumor cell growth of various tumor types. Previous work has demonstrated that curcumin inhibits cancer cell growth by targeting multiple signaling transduction and cellular processes. However, the role of curcumin in regulating cellular bioenergetic processes remains largely unknown. METHODS: Western blotting and qRT-PCR were performed to analyze the protein and mRNA level of indicated molecules, respectively. RTCA, CCK-8 assay, nude mice xenograft assay, and in vivo bioluminescence imaging were used to visualize the effects of curcmin on gastric cancer cell growth in vitro and in vivo. Seahorse bioenergetics analyzer was used to investigate the alteration of oxygen consumption and aerobic glycolysis rate. RESULTS: Curcumin significantly inhibited gastric tumor cell growth, proliferation and colony formation. We further investigated the role of curcumin in regulating cellular redox homeostasis and demonstrated that curcumin initiated severe cellular apoptosis via disrupting mitochondrial homeostasis, thereby enhancing cellular oxidative stress in gastric cancer cells. Furthermore, curcumin dramatically decreased mtDNA content and DNA polymerase γ (POLG) which contributed to reduced mitochondrial oxygen consumption and aerobic glycolysis. We found that curcumin induced POLG depletion via ROS generation, and POLG knockdown also reduced oxidative phosphorylation (OXPHOS) activity and cellular glycolytic rate which was partially rescued by ROS scavenger NAC, indiating POLG plays an important role in the treatment of gastric cancer. Data in the nude mice model verified that curcumin treatment significantly attenuated tumor growth in vivo. Finally, POLG was up-regulated in human gastric cancer tissues and primary gastric cancer cell growth was notably suppressed due to POLG deficiency. CONCLUSIONS: Together, our data suggest a novel mechanism by which curcumin inhibited gastric tumor growth through excessive ROS generation, resulting in depletion of POLG and mtDNA, and the subsequent disruption of cellular bioenergetics.