Whole-genome duplication in the Multicellularity Long Term Evolution Experiment.

Whole-genome duplication (WGD) is widespread across eukaryotes and can promote adaptive evolution 1-4 . However, given the instability of newly-formed polyploid genomes 5-7 , understanding how WGDs arise in a population, persist, and underpin adaptations remains a challenge. Using our ongoing Multicellularity Long Term Evolution Experiment (MuLTEE) 8 , we show that diploid snowflake yeast ( Saccharomyces cerevisiae ) under selection for larger multicellular size rapidly undergo spontaneous WGD. From its origin within the first 50 days of the experiment, tetraploids persist for the next 950 days (nearly 5,000 generations, the current leading edge of our experiment) in ten replicate populations, despite being genomically unstable. Using synthetic reconstruction, biophysical modeling, and counter-selection experiments, we found that tetraploidy evolved because it confers immediate fitness benefits in this environment, by producing larger, longer cells that yield larger clusters. The same selective benefit also maintained tetraploidy over long evolutionary timescales, inhibiting the reversion to diploidy that is typically seen in laboratory evolution experiments. Once established, tetraploidy facilitated novel genetic routes for adaptation, playing a key role in the evolution of macroscopic multicellular size via the origin of evolutionarily conserved aneuploidy. These results provide unique empirical insights into the evolutionary dynamics and impacts of WGD, showing how it can initially arise due to its immediate adaptive benefits, be maintained by selection, and fuel long-term innovations by creating additional dimensions of heritable genetic variation.

Constitutive expression of the Type VI Secretion System carries no measurable fitness cost in Vibrio cholerae.

The Type VI Secretion System (T6SS) is a widespread and highly effective mechanism of microbial warfare; it confers the ability to efficiently kill susceptible cells within close proximity. Due to its large physical size, complexity, and ballistic basis for intoxication, it has widely been assumed to incur significant growth costs in the absence of improved competitive outcomes. In this study, we precisely examine the fitness costs of constitutive T6SS firing in the bacterium Vibrio cholerae. We find that, contrary to expectations, constitutive expression of the T6SS has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5% (95% CI) relative to a T6SS- control. Mathematical modeling of microbial populations demonstrates that, due to clonal interference, constitutive expression of the T6SS will often be neutral, with little impact on evolutionary outcomes. Our findings underscore the importance of precisely measuring the fitness costs of microbial social behaviors and help explain the prevalence of the T6SS across Gram-negative bacteria.

Mitophagy initiates retrograde mitochondrial-nuclear signaling to guide retinal pigment cell heterogeneity.

Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial (RPE) cell heterogeneity is a well-recognized but understudied pathogenic factor. Due to the daily phagocytosis of photoreceptor outer segments, unique photo-oxidative stress, and high metabolism for maintaining vision, the RPE has robust macroautophagy/autophagy, and mitochondrial and antioxidant networks. However, the autophagy subtype, mitophagy, in the RPE and AMD is understudied. Here, we found decreased PINK1 (PTEN induced kinase 1) in perifoveal RPE of early AMD eyes. PINK1-deficient RPE have impaired mitophagy and mitochondrial function that triggers death-resistant epithelial-mesenchymal transition (EMT). This reprogramming is mediated by novel retrograde mitochondrial-nuclear signaling (RMNS) through superoxide, NFE2L2 (NFE2 like bZIP transcription factor 2), TXNRD1 (thioredoxin reductase 1), and phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase) that induced canonical transcription factors ZEB1 (zinc finger E-box binding homeobox 1) and SNAI1 (Snail family transcriptional repressor 1) and an EMT transcriptome. NFE2L2 deficiency disrupted RMNS that paradoxically normalized morphology but decreased function and viability. Thus, RPE heterogeneity is defined by the interaction of two cytoprotective pathways that is triggered by mitophagy function. By neutralizing the consequences of impaired mitophagy, an antioxidant dendrimer tropic for the RPE and mitochondria, EMT (a recognized AMD alteration) was abrogated to offer potential therapy for early AMD, a stage without treatment.Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AMD: age-related macular degeneration; CCCP: cyanide m-chlorophenyl hydrazone; CDH1: cadherin 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; DHE: dihydroethidium; D-NAC: N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSEA: Gene Set Enrichment Analysis; HSPD1: heat shock protein family D (Hsp60) member 1; IVT: intravitreal; KD: knockdown; LMNA, lamin A/C; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-l-cysteine; NQO1: NAD(P)H quinone dehydrogenase 1; NFE2L2: NFE2 like bZIP transcription factor 2; O2-: superoxide anion; OCR: oxygen consumption rate; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; RMNS: retrograde mitochondrial-nuclear signaling; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SNAI1: snail family transcriptional repressor 1; TJP1: tight junction protein 1; TPP-D-NAC: triphenyl phosphinium and N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; Trig: trigonelline; TXNRD1: thioredoxin reductase 1; VIM: vimentin; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1.

Illuminating a new path to multicellularity.

A new species of multicellular bacteria broadens our understanding of prokaryotic multicellularity and provides insight into how multicellular organisms arise.

On authorship in science: power, misconduct, responsibility and accountability.

The discovery of a case of data manipulation resulting in retraction of a high-impact paper revived conversations around scientific misconduct in India. Such malpractice is neither new nor rare. When it is discovered, there is a tendency to push the blame onto a junior author. But what makes one eligible to be an author in a scientific manuscript? In a case of misconduct, which authors must take the blame, and how do we hold them accountable? In this essay, I use the case of the recent retraction mentioned above to highlight the contentious nature of authorship in science.

Nrf2 deficiency decreases NADPH from impaired IDH shuttle and pentose phosphate pathway in retinal pigmented epithelial cells to magnify oxidative stress-induced mitochondrial dysfunction.

The nuclear factor-erythroid 2-related factor-2 (Nrf2), a major antioxidant transcription factor, is decreased in several age-related diseases including age-related macular degeneration (AMD), the most common cause of blindness among the elderly in western society. Since Nrf2's mito-protective response is understudied, we investigated its antioxidant response on mitochondria. Control and Nrf2-deficient retinal pigmented epithelial (RPE) cells were compared after treating with cigarette smoke extract (CSE). Mitochondrial antioxidant abundance and reactive oxygen species (ROS) were quantified. Mitochondrial function was assessed by TMRM assay, NADPH, electron transport chain activity, and Seahorse. Results were corroborated in Nrf2-/- mice and relevance to AMD was provided by immunohistochemistry of human globes. CSE induced mitochondrial ROS to impair mitochondrial function. H2 O2 increase in particular, was magnified by Nrf2 deficiency, and corresponded with exaggerated mitochondrial dysfunction. While Nrf2 did not affect mitochondrial antioxidant abundance, oxidized PRX3 was magnified by Nrf2 deficiency due to decreased NADPH from decreased expression of IDH2 and pentose phosphate pathway (PPP) genes. With severe CSE stress, intrinsic apoptosis was activated to increase cell death. PPP component TALDO1 immunolabeling was decreased in dysmorphic RPE of human AMD globes. Despite limited regulation of mitochondrial antioxidant expression, Nrf2 influences PPP and IDH shuttle activity that indirectly supplies NADPH for the TRX2 system. These results provide insight into how Nrf2 deficiency impacts the mitochondrial antioxidant response, and its role in AMD pathobiology.

Mitochondrial calcium at the synapse.

Mitochondria are dynamic organelles, which serve various purposes, including but not limited to the production of ATP and various metabolites, buffering ions, acting as a signaling hub, etc. In recent years, mitochondria are being seen as the central regulators of cellular growth, development, and death. Since neurons are highly specialized cells with a heavy metabolic demand, it is not surprising that neurons are one of the most mitochondria-rich cells in an animal. At synapses, mitochondrial function and dynamics is tightly regulated by synaptic calcium. Calcium influx during synaptic activity causes increased mitochondrial calcium influx leading to an increased ATP production as well as buffering of synaptic calcium. While increased ATP production is required during synaptic transmission, calcium buffering by mitochondria is crucial to prevent faulty neurotransmission and excitotoxicity. Interestingly, mitochondrial calcium also regulates the mobility of mitochondria within synapses causing mitochondria to halt at the synapse during synaptic transmission. In this review, we summarize the various roles of mitochondrial calcium at the synapse.

The impact of lipids, lipid oxidation, and inflammation on AMD, and the potential role of miRNAs on lipid metabolism in the RPE.

The accumulation of lipids within drusen, the epidemiologic link of a high fat diet, and the identification of polymorphisms in genes involved in lipid metabolism that are associated with disease risk, have prompted interest in the role of lipid abnormalities in AMD. Despite intensive investigation, our understanding of how lipid abnormalities contribute to AMD development remains unclear. Lipid metabolism is tightly regulated, and its dysregulation can trigger excess lipid accumulation within the RPE and Bruch's membrane. The high oxidative stress environment of the macula can promote lipid oxidation, impairing their original function as well as producing oxidation-specific epitopes (OSE), which unless neutralized, can induce unwanted inflammation that additionally contributes to AMD progression. Considering the multiple layers of lipid metabolism and inflammation, and the ability to simultaneously target multiple pathways, microRNA (miRNAs) have emerged as important regulators of many age-related diseases including atherosclerosis and Alzheimer's disease. These diseases have similar etiologic characteristics such as lipid-rich deposits, oxidative stress, and inflammation with AMD, which suggests that miRNAs might influence lipid metabolism in AMD. In this review, we discuss the contribution of lipids to AMD pathobiology and introduce how miRNAs might affect lipid metabolism during lesion development. Establishing how miRNAs contribute to lipid accumulation in AMD will help to define the role of lipids in AMD, and open new treatment avenues for this enigmatic disease.

Oxidative Stress Induces an Interactive Decline in Wnt and Nrf2 Signaling in Degenerating Retinal Pigment Epithelium.

AIMS: Cells have evolved a highly sophisticated web of cytoprotective systems to neutralize unwanted oxidative stress, but are challenged by unique modern day stresses such as cigarette smoking and ingestion of a high-fat diet (HFD). Age-related disease, such as age-related macular degeneration (AMD), the most common cause of blindness among the elderly in Western societies, develops in part, when oxidative stress overwhelms cytoprotective systems to injure tissue. Since most studies focus on the protection by a single protective system, the aim of this study was to investigate the impact of more than one cytoprotective system against oxidative stress. RESULTS: Wingless (Wnt) and nuclear factor-erythroid 2-related factor 2 (Nrf2), two fundamental signaling systems that are vital to cell survival, decline after mice are exposed to chronic cigarette smoke and HFD, two established AMD risk factors, in a bidirectional feedback loop through phosphorylated glycogen synthase kinase 3 beta. Decreased Wnt and Nrf2 signaling leads to retinal pigment epithelial dysfunction and apoptosis, and a phenotype that is strikingly similar to geographic atrophy (GA), an advanced form of AMD with no effective treatment. INNOVATION: This study is the first to show that chronic oxidative stress from common modern day environmental exposures reduces two fundamental and vital cytoprotective networks in a bidirectional feedback loop, and their decline leads to advanced disease phenotype. CONCLUSION: Our data offer new insights into how combined modern oxidative stresses of cigarette smoking and HFD contribute to GA through an interactive decline in Wnt and Nrf2 signaling. Antioxid. Redox Signal. 29, 389-407.

The impact of oxidative stress and inflammation on RPE degeneration in non-neovascular AMD.

The retinal pigment epithelium (RPE) is a highly specialized, unique epithelial cell that interacts with photoreceptors on its apical side and with Bruch's membrane and the choriocapillaris on its basal side. Due to vital functions that keep photoreceptors healthy, the RPE is essential for maintaining vision. With aging and the accumulated effects of environmental stresses, the RPE can become dysfunctional and die. This degeneration plays a central role in age-related macular degeneration (AMD) pathobiology, the leading cause of blindness among the elderly in western societies. Oxidative stress and inflammation have both physiological and potentially pathological roles in RPE degeneration. Given the central role of the RPE, this review will focus on the impact of oxidative stress and inflammation on the RPE with AMD pathobiology. Physiological sources of oxidative stress as well as unique sources from photo-oxidative stress, the phagocytosis of photoreceptor outer segments, and modifiable factors such as cigarette smoking and high fat diet ingestion that can convert oxidative stress into a pathological role, and the negative impact of impairing the cytoprotective roles of mitochondrial dynamics and the Nrf2 signaling system on RPE health in AMD will be discussed. Likewise, the response by the innate immune system to an inciting trigger, and the potential role of local RPE production of inflammation, as well as a potential role for damage by inflammation with chronicity if the inciting trigger is not neutralized, will be debated.

Expression of GULP1 in bronchial epithelium is associated with the progression of emphysema in chronic obstructive pulmonary disease.

BACKGROUND: GULP1 functions as a cytoplasmic adaptor protein involved in the engulfment of apoptotic cells. The aim of this study was to investigate the expression and/or promoter methylation of GULP1 in the bronchial tissue and the lung parenchyma of chronic obstructive pulmonary disease (COPD) patients and control subjects without COPD (non-smokers and smokers). METHODS: Using a case-control design, we selected a group of 15 subjects with non-small cell lung carcinoma (NSCLC), 15 subjects with COPD, 9 subjects of without COPD (4 non-smokers and 5 smokers) as controls. We studied the expression of GULP1 in the bronchial tissue and the lung parenchyma by means of immunohistochemistry (IHC). To understand the mechanistic aspect of GULP1 expression in COPD and NSCLC, we performed quantitative methylation specific PCR (QMSP) in cases and controls of COPD and NSCLC. RESULTS: Our IHC analysis revealed that GULP1 was not expressed in pneumocytes or alveolar macrophages of COPD patients, however, GULP1 expression was detected at different levels in bronchial epithelium. GULP1 expression statistically correlated with severity of COPD-emphysema (p = 0.001, χ2 test). GULP1 promoter methylation was not observed by QMSP assay in any of the samples thereby excluding the role of promoter methylation in differential expression of GULP1 in COPD and NSCLC. CONCLUSIONS: This study provides preliminary observations on the differences in GULP1 expression in different cellular components of lung tissues from COPD and control subjects. Our findings suggest a potential role for GULP1 in the pathogenesis and progression of COPD-emphysema that warrants further investigation.

Lipids, oxidized lipids, oxidation-specific epitopes, and Age-related Macular Degeneration.

Age-related Macular Degeneration (AMD) is the leading cause of blindness among the elderly in western societies. While antioxidant micronutrient treatment is available for intermediate non-neovascular disease, and effective anti-vascular endothelial growth factor treatment is available for neovascular disease, treatment for early AMD is lacking due to an incomplete understanding of the early molecular events. The role of lipids, which accumulate in the macula, and their oxidation, has emerged as an important factor in disease development. These oxidized lipids can either directly contribute to tissue injury or react with amine on proteins to form oxidation-specific epitopes, which can induce an innate immune response. If inadequately neutralized, the inflammatory response from these epitopes can incite tissue injury during disease development. This review explores how the accumulation of lipids, their oxidation, and the ensuing inflammatory response might contribute to the pathogenesis of AMD. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder .

Pentraxin 3 recruits complement factor H to protect against oxidative stress-induced complement and inflammasome overactivation.

The discovery that genetic abnormalities in complement factor H (FH) are associated with an increased risk for age-related macular degeneration (AMD), the most common cause of blindness among the elderly, raised hope of new treatments for this vision-threatening disease. Nonetheless, over a decade after the identification of this important association, how innate immunity contributes to AMD remains unresolved. Pentraxin 3 (PTX3), an essential component of the innate immunity system that plays a non-redundant role in controlling inflammation, regulates complement by interacting with complement components. Here, we show that PTX3 is induced by oxidative stress, a known cause of AMD, in the retinal pigmented epithelium (RPE). PTX3 deficiency in vitro and in vivo magnified complement activation induced by oxidative stress, leading to increased C3a, FB, and C3d, but not C5b-9 complex formation. Increased C3a levels, resulting from PTX3 deficiency, raised the levels of Il1b mRNA and secretion of activated interleukin (IL)-1ß by interacting with C3aR. Importantly, PTX3 deficiency augmented NLRP3 inflammasome activation, resulting in enhanced IL-1ß, but not IL-18, production by the RPE. Thus, in the presence of PTX3 deficiency, the complement and inflammasome pathways worked in concert to produce IL-1ß in sufficient abundance to, importantly, result in macrophages accumulating in the choroid. These results demonstrate that PTX3 acts as an essential brake for complement and inflammasome activation by regulating the abundance of FH in the RPE, and provide critical insights into the complex interplay between oxidative stress and innate immunity in the early stages of AMD development. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Association of DNA sequence variation in mitochondrial DNA polymerase with mitochondrial DNA synthesis and risk of oral cancer.

Enzymes responsible for mitochondrial (mt) DNA synthesis and transcription are encoded by nuclear genome and inherited mutations in these genes may play important roles in enhancing risk of precancer and cancer. Here, genetic variations in 23 functionally relevant tagSNPs in 6 genes responsible for mtDNA synthesis and transcription were studied in 522 cancer and 241 precancer (i.e. leukoplakia) patients and 525 healthy controls using Illumina Golden Gate assay to explore association with risk of oral precancer and cancer. Two SNPs, rs41553913 at POLRMT and rs9905016 at POLG2, significantly increased risk of oral leukoplakia and cancer, respectively, at both genotypic and allelic levels. Gene-environment interaction models also revealed that tobacco habits and SNPs at POLG2 and TFAM may modulate risk of both leukoplakia and cancer. In silico analysis of published data-set also revealed that variant heterozygote (TC) significantly increased transcription of POLG2 compared to wild genotype (p=0.03). Cancer tissues having variant allele genotypes (TC+CC) at POLG2 contained 1.6 times (p<0.01) more mtDNA compared to cancer tissues having wild genotype (TT). In conclusion, polymorphisms at POLG2 and POLRMT increased risk of oral cancer and leukoplakia, respectively, probably modulating synthesis and activity of the enzymes. Enhanced synthesis of mtDNA in cancer tissues may have implication in carcinogenesis, but the mechanism is yet to be explored.

Sequence and expression variations in 23 genes involved in mitochondrial and non-mitochondrial apoptotic pathways and risk of oral leukoplakia and cancer.

Oral cancer is usually preceded by pre-cancerous lesion and related to tobacco abuse. Tobacco carcinogens damage DNA and cells harboring such damaged DNA normally undergo apoptotic death, but cancer cells are exceptionally resistant to apoptosis. Here we studied association between sequence and expression variations in apoptotic pathway genes and risk of oral cancer and precancer. Ninety nine tag SNPs in 23 genes, involved in mitochondrial and non-mitochondrial apoptotic pathways, were genotyped in 525 cancer and 253 leukoplakia patients and 538 healthy controls using Illumina Golden Gate assay. Six SNPs (rs1473418 at BCL2; rs1950252 at BCL2L2; rs8190315 at BID; rs511044 at CASP1; rs2227310 at CASP7 and rs13010627 at CASP10) significantly modified risk of oral cancer but SNPs only at BCL2, CASP1and CASP10 modulated risk of leukoplakia. Combination of SNPs showed a steep increase in risk of cancer with increase in "effective" number of risk alleles. In silico analysis of published data set and our unpublished RNAseq data suggest that change in expression of BID and CASP7 may have affected risk of cancer. In conclusion, three SNPs, rs1473418 in BCL2, rs1950252 in BCL2L2 and rs511044 in CASP1, are being implicated for the first time in oral cancer. Since SNPs at BCL2, CASP1 and CASP10 modulated risk of both leukoplakia and cancer, so, they should be studied in more details for possible biomarkers in transition of leukoplakia to cancer. This study also implies importance of mitochondrial apoptotic pathway gene (such as BCL2) in progression of leukoplakia to oral cancer.

SNP rs1049430 in the 3'-UTR of SH3GL2 regulates its expression: Clinical and prognostic implications in head and neck squamous cell carcinoma.

Single nucleotide polymorphisms (SNPs) in the 3'-UTR region are emerging cis-regulatory factors associated with the occurrences of several human diseases. SH3GL2, which is located at chromosome 9p21-22, is associated with hyperplastic/mildly dysplastic lesions of the head and neck and has a long 3'-UTR with multiple SNPs. The aim of the present study was to determine the susceptible allele(s) in the 3'-UTR SNPs of SH3GL2 in head and neck squamous cell carcinoma (HNSCC). First, we screened the genotypes of all SNPs located in the 3'-UTR of SH3GL2 in 110 controls and 147 cases in Indian populations by sequencing. A SNP (rs1049430:>G/T) that showed only heterozygosity was further confirmed by genotyping with an Illumina GoldenGate platform in 530 controls and 764 cases. Genotype-specific survival analysis of the HNSCC patients was performed. In addition, genotype-specific mRNA stability, isoform expression and protein expression were analyzed. SNP rs1049430 was not associated with disease occurrence, but it was associated with poor patient outcome. The G allele was associated with decreased SH3GL2 mRNA stability, differential splicing and low protein expression. Thus, our data demonstrate that the presence of the susceptible G allele in SNP rs1049430 is associated with the inactivation of SH3GL2 and could be used as a prognostic marker of HNSCC.

Involvement of epigenetics and EMT-related miRNA in arsenic-induced neoplastic transformation and their potential clinical use.

Exposure to toxicants leads to cumulative molecular changes that overtime increase a subject's risk of developing urothelial carcinoma. To assess the impact of arsenic exposure at a time progressive manner, we developed and characterized a cell culture model and tested a panel of miRNAs in urine samples from arsenic-exposed subjects, urothelial carcinoma patients, and controls. To prepare an in vitro model, we chronically exposed an immortalized normal human bladder cell line (HUC1) to arsenic. Growth of the HUC1 cells was increased in a time-dependent manner after arsenic treatment and cellular morphology was changed. In a soft agar assay, colonies were observed only in arsenic-treated cells, and the number of colonies gradually increased with longer periods of treatment. Similarly, invaded cells in an invasion assay were observed only in arsenic-treated cells. Withdrawal of arsenic treatment for 2.5 months did not reverse the tumorigenic properties of arsenic-treated cells. Western blot analysis demonstrated decreased PTEN and increased AKT and mTOR in arsenic-treated HUC1 cells. Levels of miR-200a, miR-200b, and miR-200c were downregulated in arsenic-exposed HUC1 cells by quantitative RT-PCR. Furthermore, in human urine, miR-200c and miR-205 were inversely associated with arsenic exposure (P = 0.005 and 0.009, respectively). Expression of miR-205 discriminated cancer cases from controls with high sensitivity and specificity (AUC = 0.845). Our study suggests that exposure to arsenic rapidly induces a multifaceted dedifferentiation program and miR-205 has potential to be used as a marker of arsenic exposure as well as a maker of early urothelial carcinoma detection.

D-loop somatic mutations and ∼5 kb "common" deletion in mitochondrial DNA: important molecular markers to distinguish oral precancer and cancer.

Apart from genomic DNA, mutations at mitochondrial DNA (mtDNA) have been hypothesized to play vital roles in cancer development. In this study, ∼5 kb deletion and D-loop mutations in mtDNA and alteration in mtDNA content were investigated in buccal smears from 104 healthy controls and 74 leukoplakia and 117 cancer tissue samples using Taqman-based quantitative assay and re-sequencing. The ∼5 kb deletion in mtDNA was significantly less (9.8 and 10.5 folds, P < 0.0001) in cancer tissues compared to control and leukoplakia tissues, respectively. On the other hand, somatic mutations in D-loop, investigated in 54 controls, 50 leukoplakias and 56 cancer patients, were found to be significantly more in cancer tissues, but not in leukoplakia tissues, compared to control (Z-score = 5.4). MtDNA contents were observed to be significantly more in leukoplakia (2.1 folds, P = 0.004) and cancer (1.6 folds, P = 0.03) tissues compared to control tissues. So, D-loop somatic mutations and ∼5 kb deletion patterns could be used as distinguishing markers between precancer and cancer tissues. This observation further suggests that somatic mutations in D-loop may facilitate carcinogenesis and cancer cells with less ∼5 kb deletion, i.e., intact mtDNA, may become resistant to apoptosis.

Comprehensive SNP scan of DNA repair and DNA damage response genes reveal multiple susceptibility loci conferring risk to tobacco associated leukoplakia and oral cancer.

Polymorphic variants of DNA repair and damage response genes play major role in carcinogenesis. These variants are suspected as predisposition factors to Oral Squamous Cell Carcinoma (OSCC). For identification of susceptible variants affecting OSCC development in Indian population, the "maximally informative" method of SNP selection from HapMap data to non-HapMap populations was applied. Three hundred twenty-five SNPs from 11 key genes involved in double strand break repair, mismatch repair and DNA damage response pathways were genotyped on a total of 373 OSCC, 253 leukoplakia and 535 unrelated control individuals. The significantly associated SNPs were validated in an additional cohort of 144 OSCC patients and 160 controls. The rs12515548 of MSH3 showed significant association with OSCC both in the discovery and validation phases (discovery P-value: 1.43E-05, replication P-value: 4.84E-03). Two SNPs (rs12360870 of MRE11A, P-value: 2.37E-07 and rs7003908 of PRKDC, P-value: 7.99E-05) were found to be significantly associated only with leukoplakia. Stratification of subjects based on amount of tobacco consumption identified SNPs that were associated with either high or low tobacco exposed group. The study reveals a synergism between associated SNPs and lifestyle factors in predisposition to OSCC and leukoplakia.

Variant haplotypes at XRCC1 and risk of oral leukoplakia in HPV non-infected samples.

BACKGROUND: One of the mechanisms in human papillomavirus (HPV)-related carcinogenesis is inhibition of DNA repair by HPV oncoprotein. In this study, we investigated whether polymorphisms at XRCC1, one of the DNA repair loci, could modulate the risk of tobacco-related leukoplakia and cancer in HPV-infected individuals. METHODS: Tissue DNA from 83 oral cancer, 91 leukoplakia and 100 healthy controls were screened for HPV 16/18 infection and polymorphisms at XRCC1 by PCR-RFLP to estimate the risk of diseases independently and jointly. RESULTS: Human papillomavirus infection was significantly associated with increased risk of leukoplakia and cancer (OR = 2.8, 95% CI = 1.2-6.5 and OR = 5.5, 95% CI = 1.6-19, respectively). Independently, genotypes at three polymorphic sites on XRCC1 did not modulate the risk of diseases but pooled variant haplotypes increased the risk of leukoplakia in overall and HPV non-infected (OR = 1.8, 95% CI = 1.2-2.8; OR = 2.2, 95% CI = 1.2-4.0, respectively) samples but not that of cancer. CONCLUSION: The association between variant haplotypes at XRCC1 and risk of leukoplakia is pronounced in non-infected individuals since HPV oncoprotein could inhibit directly the DNA repair activity of XRCC1. But more samples of leukoplakia and cancer are essential to validate these results.