Differential modulation of cancer-related genes by mitochondrial DNA haplogroups and the STING DNA sensing system.

Activation of the Simulator of Interferon Genes (STING) system by mitochondrial (mt) DNA can upregulate type 1 interferon genes and enhance immune responses to combat bacterial and viral infections. In cancers, the tumor-derived DNA activates STING leading to upregulation of IFN-beta and induction of antitumor T cells. The entire mtDNA from the cell lines was sequenced using next-generation sequencing (NGS) technology with independent sequencing of both strands in both directions, allowing identification of low-frequency heteroplasmy SNPs. There were 15 heteroplasmy SNPs showing a range from 3.4% to 40.5% occurrence in the K cybrid cell lines. Three H haplogroup cybrids possessed SNP heteroplasmy that ranged from 4.39% to 30.7%. The present study used qRT-PCR to determine if cybrids of H and K haplogroups differentially regulate expression levels of five cancer genes (BRAC1, ALK, PD1, EGFR, and HER2) and seven STING subunits genes (CGAS, TBK1, IRF3, IκBa, NFκB, TRAF2, and TNFRSF19). Some cybrids underwent siRNA knockdown of STING followed by qRT-PCR in order to determine the impact of STING on gene expression. Rho0 (lacking mtDNA) ARPE-19 cells were used to determine if mtDNA is required for the expression of the cancer genes studied. Our results showed that (a) K cybrids have lower expression levels for BRAC1, ALK, PD1, EGFR, IRF3, and TNFRSF19 genes but increased transcription for IκBa and NFκB compared to H cybrids; (b) STING KD decreases expression of EGFR in both H and K cybrids, and (c) PD1 expression is negligible in Rho0 cells. Our findings suggest that the STING DNA sensing pathway may be a previously unrecognized pathway to target modulation of cancer-related genes and the PD1 expression requires the presence of mtDNA.

European mtDNA Variants Are Associated With Differential Responses to Cisplatin, an Anticancer Drug: Implications for Drug Resistance and Side Effects.

Background: Cisplatin, a powerful antitumor agent, causes formation of DNA adducts, and activation of apoptotic pathways. Presently, cisplatin resistance develops in up to 70% of patients but the underlying molecular mechanism(s) are unclear and there are no markers to determine which patients will become resistant. Mitochondria play a significant role not only in energy metabolism but also retrograde signaling (mitochondria to nucleus) that modulates inflammation, complement, and apoptosis pathways. Maternally inherited mitochondrial (mt) DNA can be classified into haplogroups representing different ethnic populations that have diverse susceptibilities to diseases and medications. Methods: Transmitochondrial cybrids, where all cell lines possess identical nuclear genomes but either the H (Southern European) or J (Northern European) mtDNA haplogroups, were treated with cisplatin and analyzed for differential responses related to viability, oxidative stress, and expression levels of genes associated with cancer, cisplatin-induced nephrotoxicity and resistance, apoptosis and signaling pathways. Results: The cisplatin-treated-J cybrids showed greater loss of cell viability along with lower levels of reactive oxygen species and mitochondrial membrane potential compared to cisplatin-treated-H cybrids. After cisplatin treatment, J cybrids showed increased gene expression of BAX, CASP3, and CYP51A, but lower levels of SFRP1 compared to untreated-J cybrids. The cisplatin-treated-H cybrids had elevated expression of CDKN1A/P21, which has a role in cisplatin toxicity, compared to untreated-H cybrids. The cisplatin-treated H had higher transcription levels of ABCC1, DHRS2/HEP27, and EFEMP1 compared to cisplatin-treated-J cybrids. Conclusions: Cybrid cell lines that contain identical nuclei but either H mtDNA mitochondria or J mtDNA mitochondria respond differently to cisplatin treatments suggesting involvement of the retrograde signaling (from mitochondria to nucleus) in the drug-induced cell death. Varying toxicities and transcription levels of the H vs. J cybrids after cisplatin treatment support the hypothesis that mtDNA variants play a role in the expression of genes affecting resistance and side effects of cisplatin.

Increased expression of ApoE and protection from amyloid-beta toxicity in transmitochondrial cybrids with haplogroup K mtDNA.

Mitochondrial (mt) DNA haplogroups, defined by specific single nucleotide polymorphism (SNP) patterns, represent populations of diverse geographic origins and have been associated with increased risk or protection of many diseases. The H haplogroup is the most common European haplogroup while the K haplogroup is highly associated with the Ashkenazi Jewish population. Transmitochondrial cybrids (cell lines with identical nuclei, but mtDNA from either H (n=8) or K (n=8) subjects) were analyzed by the Seahorse flux analyzer, quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry (IHC). Cybrids were treated with amyloid-ß peptides and cell viabilities were measured. Other cybrids were demethylated with 5-aza-2'-deoxycytidine (5-aza-dC) and expression levels for APOE and NFkB2 were measured. Results show K cybrids have (a) significantly lower mtDNA copy numbers, (b) higher expression levels for MT-DNA encoded genes critical for oxidative phosphorylation, (c) lower Spare Respiratory Capacity, (d) increased expression of inhibitors of the complement pathway and important inflammasome-related genes; and (e) significantly higher levels of APOE transcription that were independent of methylation status. After exposure to amyloid-ß1-42 peptides (active form), H haplogroup cybrids demonstrated decreased cell viability compared to those treated with amyloid-ß42-1 (inactive form) (p<0.0001), while this was not observed in the K cybrids (p=0.2). K cybrids had significantly higher total global methylation levels and differences in expression levels for two acetylation genes and four methylation genes. Demethylation with 5-aza-dC altered expression levels for NFkB2, while APOE transcription patterns were unchanged. Our findings support the hypothesis that mtDNA-nuclear retrograde signaling may mediate expression levels of APOE, a key factor in many age-related diseases. Future studies will focus on identification of the mitochondrial-nuclear retrograde signaling mechanism(s) contributing to these mtDNA-mediated differences.

Mitochondrial DNA variants can mediate methylation status of inflammation, angiogenesis and signaling genes.

Mitochondrial (mt) DNA can be classified into haplogroups representing different geographic and/or racial origins of populations. The H haplogroup is protective against age-related macular degeneration (AMD), while the J haplogroup is high risk for AMD. In the present study, we performed comparison analyses of human retinal cell cybrids, which possess identical nuclei, but mtDNA from subjects with either the H or J haplogroups, and demonstrate differences in total global methylation, and expression patterns for two genes related to acetylation and five genes related to methylation. Analyses revealed that untreated-H and -J cybrids have different expression levels for nuclear genes (CFH, EFEMP1, VEGFA and NFkB2). However, expression levels for these genes become equivalent after treatment with a methylation inhibitor, 5-aza-2'-deoxycytidine. Moreover, sequencing of the entire mtDNA suggests that differences in epigenetic status found in cybrids are likely due to single nucleotide polymorphisms (SNPs) within the haplogroup profiles rather than rare variants or private SNPs. In conclusion, our findings indicate that mtDNA variants can mediate methylation profiles and transcription for inflammation, angiogenesis and various signaling pathways, which are important in several common diseases.

Human retinal transmitochondrial cybrids with J or H mtDNA haplogroups respond differently to ultraviolet radiation: implications for retinal diseases.

BACKGROUND: It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation. METHODOLOGY/PRINCIPAL FINDINGS: Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids. CONCLUSION/SIGNIFICANCE: In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Mitochondrial DNA variants mediate energy production and expression levels for CFH, C3 and EFEMP1 genes: implications for age-related macular degeneration.

BACKGROUND: Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). METHODOLOGY/PRINCIPAL FINDINGS: Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

Loss of mitochondrial membrane potential triggers the retrograde response extending yeast replicative lifespan.

In the budding yeast Saccharomyces cerevisiae, loss of mitochondrial DNA (rho(0)) can induce the retrograde response under appropriate conditions, resulting in increased replicative lifespan (RLS). Although the retrograde pathway has been extensively elaborated, the nature of the mitochondrial signal triggering this response has not been clear. Mitochondrial membrane potential (MMP) was severely reduced in rho(0) compared to rho(+) cells, and RLS was concomitantly extended. To examine the role of MMP in the retrograde response, MMP was increased in the rho(0) strain by introducing a mutation in the ATP1 gene, and it was decreased in rho(+) cells by deletion of COX4. The ATP1-111 mutation in rho(0) cells partially restored the MMP and reduced mean RLS to that of rho(+) cells. COX4 deletion decreased MMP in rho(+) cells to a value intermediate between rho(+) and rho(0) cells and similarly increased RLS. The increase in expression of CIT2, the diagnostic gene for the retrograde response, seen in rho(0) cells, was substantially suppressed in the presence of the ATP1-111 mutation. In contrast, CIT2 expression increased in rho(+) cells on deletion of COX4. Activation of the retrograde response results in the translocation of the transcription factor Rtg3 from the cytoplasm to the nucleus. Rtg3-GFP translocation to the nucleus was directly observed in rho(0) and rho(+)cox4Δ cells, but it was blunted in rho(0) cells with the ATP1-111 mutation. We conclude that a decrease in MMP is the signal that initiates the retrograde response and leads to increased RLS.

HRAS1 and LASS1 with APOE are associated with human longevity and healthy aging.

The search for longevity-determining genes in human has largely neglected the operation of genetic interactions. We have identified a novel combination of common variants of three genes that has a marked association with human lifespan and healthy aging. Subjects were recruited and stratified according to their genetically inferred ethnic affiliation to account for population structure. Haplotype analysis was performed in three candidate genes, and the haplotype combinations were tested for association with exceptional longevity. An HRAS1 haplotype enhanced the effect of an APOE haplotype on exceptional survival, and a LASS1 haplotype further augmented its magnitude. These results were replicated in a second population. A profile of healthy aging was developed using a deficit accumulation index, which showed that this combination of gene variants is associated with healthy aging. The variation in LASS1 is functional, causing enhanced expression of the gene, and it contributes to healthy aging and greater survival in the tenth decade of life. Thus, rare gene variants need not be invoked to explain complex traits such as aging; instead rare congruence of common gene variants readily fulfills this role. The interaction between the three genes described here suggests new models for cellular and molecular mechanisms underlying exceptional survival and healthy aging that involve lipotoxicity.

A novel role of peroxin PEX6: suppression of aging defects in mitochondria.

Yeast cells become older with each division, but their daughters are born young. Mutational analysis shows that maintenance of this age asymmetry requires segregation of a complement of active mitochondria to daughters and that this process breaks down in older mother cells. This decline has implications for stem cell aging in higher organisms. PEX6, a peroxisome biogenesis gene, has been isolated as a multicopy suppressor of an atp2 age asymmetry mutant. Suppression depended on the presence of particular amino acid residues in Atp2p, and required adenosine triphosphate (ATP) binding and/or ATP hydrolysis activity of Pex6p. Extra copies of PEX6 corrected the deficit in Atp2p in mitochondria in the mutant by improving its import kinetics, resulting in near normal mitochondrial inheritance by daughter cells. The novel function of Pex6p described here may provide insights into peroxisomal and mitochondrial disorders and into metabolic diseases in general.

Nuclear gene expression changes due to mitochondrial dysfunction in ARPE-19 cells: implications for age-related macular degeneration.

PURPOSE: To measure changes in nuclear gene expression resulting from mitochondrial dysfunction in retinal pigment epithelial cells. METHODS: ARPE-19 retinal pigment epithelial cells were depleted of their mitochondrial (mt)DNA by passaging in a low concentration of ethidium bromide. Loss of mitochondrial DNA was determined by uridine auxotrophy and quantitative real-time polymerase chain reaction of isolated DNA. Loss of mitochondrial membrane potential was estimated by uptake of JC-1. Changes in nuclear gene expression were determined by quantitative real-time reverse transcription-polymerase chain reaction of isolated total RNA from ethidium-bromide-treated and untreated cells. Morphologic and phenotypic changes were determined by phase-contrast microscopy, sensitivity to the oxidant tert-butyl hydroperoxide (tBH), and invasion assay. RESULTS: ARPE-19 cells became auxotrophic for growth on uridine after eight passages in 50 ng/mL ethidium bromide. Quantitative PCR revealed almost complete loss of mitochondrial DNA (rho(0) cells). Uptake of JC-1 was reduced in the rho(0) cells, indicating reduction of mitochondrial membrane potential. Quantitative RT-PCR measured increased expression of genes coding for drusen components, lipid transport, extracellular matrix components, and responses to inflammation in the rho(0) cells. The rho(0) cells also exhibited an increased sensitivity to killing by tBH and increased migration and invasion through solubulized basement membrane-coated tissue culture inserts. CONCLUSIONS: ARPE-19 cells respond to loss of mitochondrial function by changes in nuclear gene expression that resemble changes observed in age-related macular degeneration. The results lead to the hypothesis that loss of mitochondrial function with age and resultant changes in nuclear gene expression may explain some of the changes in the macula that are associated with the known clinical manifestations of age-related macular degeneration.

Common and cell type-specific responses of human cells to mitochondrial dysfunction.

In yeast, mitochondrial dysfunction activates a specific pathway, termed retrograde regulation, which alters the expression of specific nuclear genes and results in increased replicative life span. In mammalian cells, the specific nuclear genes induced in response to loss of mitochondrial function are less well defined. This study characterizes responses in nuclear gene expression to loss of mitochondrial DNA sequences in three different human cell types: T143B, an osteosarcoma-derived cell line; ARPE19, a retinal pigment epithelium cell line; and GMO6225, a fibroblast cell population from an individual with Kearns-Sayre syndrome (KSS). Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure gene expression of a selection of glycolysis, TCA cycle, mitochondrial, peroxisomal, extracellular matrix, stress response, and regulatory genes. Gene expression changes that were common to all three cell types included up-regulation of GCK (glucokinase), CS (citrate synthase), HOX1 (heme oxygenase 1), CKMT2 (mitochondrial creatine kinase 2), MYC (v-myc myelocytomatosis viral oncogene homolog), and WRN (Werner syndrome helicase), and down-regulation of FBP1 (fructose-1, 6-bisphosphatase 1) and COL4A1 (collagen, type IV, alpha 1). RNA interference experiments show that induction of MYC is important in rho0 cells for the up-regulation of glycolysis. In addition, a variety of cell type-specific gene changes was detected and most likely depended upon the differentiated functions of the individual cell types. These expression changes may help explain the response of different tissues to the loss of mitochondrial function due to aging or disease.

Prohibitins and Ras2 protein cooperate in the maintenance of mitochondrial function during yeast aging.

The yeast Saccharomyces cerevisiae has a finite replicative life span. Yeasts possess two prohibitins, Phb1p and Phb2p, in similarity to mammalian cells. These proteins are located in the inner mitochondrial membrane, where they are involved in the processing of newly-synthesized membrane proteins. We demonstrate that the elimination of one or both of the prohibitin genes in yeast markedly diminished the replicative life span of cells that lack fully-functional mitochondria, while having no effect on cells with functioning mitochondria. This deleterious effect was suppressed by the deletion of the RAS2 gene. The expression of PHB1 and PHB2 declined gradually up to 5-fold during the life span. Cells in which PHB1 was deleted in conjunction with the absence of a mitochondrial genome displayed remarkable changes in mitochondrial morphology, distribution, and inheritance. This loss of mitochondrial integrity was not seen in cells devoid of PHB1 but possessing an intact mitochondrial genome. In a subset of the cells, the changes in mitochondrial integrity were associated with increased production of reactive oxygen species, which co-localized with the altered mitochondria. The mitochondrial deficits described above were all suppressed by deletion of RAS2. Our data, together with published information, are interpreted to provide a unified view of the role of the prohibitins in yeast aging. This model posits that the key initiating event is a decline in mitochondrial function, which leads to progressive oxidative damage that is exacerbated in the absence of the prohibitins. This aggravation of the initial damage is ameliorated by the suppression of the production of mitochondrial proteins in the absence of Ras2p signaling of mitochondrial biogenesis.

Expression of metallothionein isoforms in human chorioretinal complex.

PURPOSE: To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the human chorioretinal complex. METHODS: Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression. RESULTS: Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT II > MT I > MT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina. CONCLUSIONS: The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.