DNA methylome in pancreatic cancer identified novel promoter hyper-methylation in NPY and FAIM2 genes associated with poor prognosis in Indian patient cohort.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading cancers worldwide and has a poor survival, with a 5-year survival rate of only 8.5%. In this study we investigated altered DNA methylation associated with PDAC severity and prognosis. METHODS: Methylome data, generated using 450 K bead array, was compared between paired PDAC and normal samples in the TCGA cohort (n = 9) and our Indian cohort (n = 7). The total Indian Cohort (n = 75) was split into cohort 1 (n = 7), cohort 2 (n = 22), cohort 3 (n = 26) and cohort 4 (n = 20).Validation of differential methylation (6 selected CpG loci) and associated gene expression for differentially methylated genes (10 selected gDMs) were carried out in separate validation cohorts, using MSP, RT-PCR and IHC correlations between methylation and gene expression were observed in TCGA, GTEx cohorts and in validation cohorts. Kaplan-Meier survival analysis was done to study differential prognosis, during 2-5 years of follow-up. RESULTS: We identified 156 DMPs, mapped to 91 genes (gDMs), in PDAC; 68 (43.5%) DMPs were found to be differentially methylated both in TCGA cohort and our cohort, with significant concordance at hypo- and hyper-methylated loci. Enrichments of "regulation of ion transport", "Interferon alpha/beta signalling", "morphogenesis and development" and "transcriptional dysregulation" pathways were observed among 91 gDMs. Hyper-methylation of NPY and FAIM2 genes with down-regulated expression in PDAC, were significantly associated with poor prognosis in the Indian patient cohort. CONCLUSIONS: Ethnic variations among populations may determine the altered epigenetic landscape in the PDAC patients of the Indian cohort. Our study identified novel differentially methylated genes (mainly NPY and FAIM2) and also validated the previously identified differentially methylated CpG sites associated with PDAC cancer patient's survival. Comparative analysis of our data with TCGA and CPTAC cohorts showed that both NPY and FAIM2 hyper-methylation and down-regulations can be novel epigenetically regulated genes in the Indian patient population, statistically significantly associated with poor survival and advanced tumour stages.

E2F5 promotes prostate cancer cell migration and invasion through regulation of TFPI2, MMP-2 and MMP-9.

Previously, our laboratory demonstrated that a deregulated E2F5/p38/SMAD3 axis was associated with uncontrolled cellular proliferation in prostate cancer (PCa). Here, we investigate the role of E2F5 in PCa in further details. RNAi-mediated E2F5 knockdown and pathway-focused gene expression profiling in PC3 cells identified TFPI2 as a downstream target of E2F5. Manipulation of E2F5 expression was also found to alter MMP-2 and MMP-9 levels as detected by Proteome Profiler array, western blot and reverse transcription coupled quantitative polymerase chain reaction Site-directed mutagenesis, dual-luciferase assays and chromatin immunoprecipitation with anti-E2F5-IgG coupled with qPCR confirmed recruitment of E2F5 on TFPI2, MMP-2 and MMP-9 promoters. RNAi-mediated knockdown of E2F5 expression in PC3 caused a significant alteration of cell migration while that of TFFI2 resulted in a modest change. Abrogation of E2F5 and TFPI2 expression was associated with significant changes in the gelatinolytic activity of active forms of MMP-2 and MMP-9. Moreover, E2F5, MMP-2 and MMP-9 levels were elevated in biopsies of PCa patients relative to that of benign hyperplasia, while TFPI2 expression was reduced. MMP-9 was coimmunoprecipitated with anti-TFPI2-IgG in PCa tissue samples suggesting a direct interaction between the proteins. Finally, artemisinin treatment in PC3 cells repressed E2F5 along with MMP-2/MMP-9 while triggering TFPI2 expression which alleviated PC3 aggressiveness possibly through inhibition of MMP activities. Together, our study reinstates an oncogenic role of E2F5 which operates as a dual-function transcription factor for its targets TFPI2, MMP-2 and MMP-9 and promotes cellular invasiveness. This study also indicates a therapeutic potential of artemisinin, a natural compound which acts by correcting dysfunctional E2F5/TFPI2/MMP axis in PCa.

A novel hotspot and rare somatic mutation p.A138V, at TP53 is associated with poor survival of pancreatic ductal and periampullary adenocarcinoma patients.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is a cancer of the exocrine pancreas and 5-year survival rates remain constant at 7%. Along with PDAC, Periampullary Adenocarcinoma (PAC) accounts for 0.5-2% of all gastrointestinal malignancies. Genomic observations were well concluded for PDAC and PACs in western countries but no reports are available from India till now. METHODS: Targeted Next Generation Sequencing were performed in 8 (5 PDAC and 3 PAC) tumour normal pairs, using a panel of 412 cancer related genes. Primary findings were replicated in 85 tumour samples (31 PDAC and 54 PAC) using the Sanger sequencing. Mutations were also validated by ASPCR, RFLP, and Ion Torrent sequencing. IHC along with molecular dynamics and docking studies were performed for the p.A138V mutant of TP53. Key polymorphisms at TP53 and its associated genes were genotyped by PCR-RFLP method and association with somatic mutations were evaluated. All survival analysis was done using the Kaplan-Meier survival method which revealed that the survival rates varied significantly depending on the somatic mutations the patients harboured. RESULTS: Among the total 114 detected somatic mutations, TP53 was the most frequently mutated (41%) gene, followed by KRAS, SMAD4, CTNNB1, and ERBB3. We identified a novel hotspot TP53 mutation (p.A138V, in 17% of all patients). Low frequency of KRAS mutation (33%) was detected in these samples compared to patients from Western counties. Molecular Dynamics (MD) simulation and DNA-protein docking analysis predicted p.A138V to have oncogenic characteristics. Patients with p.A138V mutation showed poorer overall survival (p = 0.01). So, our finding highlights elevated prevalence of the p53p.A138V somatic mutation in PDAC and pancreatobiliary PAC patients. CONCLUSION: Detection of p.A138V somatic variant in TP53 might serve as a prognostic marker to classify patients. It might also have a role in determining treatment regimes. In addition, low frequency of KRAS hotspot mutation mostly in Indian PDAC patient cohort indicates presence of other early drivers in malignant transformation.

Differential methylation landscape of pancreatic ductal adenocarcinoma and its precancerous lesions.

BACKGROUND: Pancreatic cancer is one of the most lethal diseases with an incidence almost equal to the mortality. In addition to having genetic causes, cancer can also be considered an epigenetic disease. DNA methylation is the premier epigenetic modification and patterns of aberrant DNA methylation are recognized to be a common hallmark of human tumor. In the multistage carcinogenesis of pancreas starting from precancerous lesions to pancreatic ductal adenocarcinoma (PDAC), the epigenetic changes play a significant role. DATA SOURCES: Relevant studies for this review were derived via an extensive literature search in PubMed via using various keywords such as pancreatic ductal adenocarcinoma, precancerous lesions, methylation profile, epigenetic biomarkers that are relevant directly or closely associated with the concerned area of our interest. The literature search was intensively done considering a time frame of 20 years (1998-2018). RESULT: In this review we have highlighted the hypermethylation and hypomethylation of the precancerous PDAC lesions (pancreatic intra-epithelial neoplasia, intraductal papillary mucinous neoplasm, mucinous cystic neoplasm and chronic pancreatitis) and PDAC along with the potential biomarkers. We have also achieved the early epigenetic driver that leads to progression from precancerous lesions to PDAC. A bunch of epigenetic driver genes leads to progression of precancerous lesions to PDAC (ppENK, APC, p14/5/16/17, hMLH1 and MGMT) are also documented. We summarized the importance of these observations in therapeutics and diagnosis of PDAC hence identifying the potential use of epigenetic biomarkers in epigenetic targeted therapy. Epigenetic inactivation occurs by hypermethylation of CpG islands in the promoter regions of tumor suppressor genes. We listed all hyper- and hypomethylation of CpG islands of several genes in PDAC including its precancerous lesions. CONCLUSIONS: The concept of the review would help to understand their biological effects, and to determine whether they may be successfully combined with other epigenetic drugs. However, we need to continue our research to develop more specific DNA-demethylating agents, which are the targets for hypermethylated CpG methylation sites.

Genetic Alterations of Periampullary and Pancreatic Ductal Adenocarcinoma: An Overview.

Pancreatic Ductal AdenoCarcinoma (PDAC) is one of the most lethal malignancies of all solid cancers. Precancerous lesions for PDAC include PanIN, IPMNs and MCNs. PDAC has a poor prognosis with a 5-year survival of approximately 6%. Whereas Periampulary AdenoCarcinoma (PAC) having four anatomic subtypes, pancreatic, Common Bile Duct (CBD), ampullary and duodenum shows relative better prognosis. The highest incidence of PDAC has been reported with black with respect to white population. Similarly, incidence rate of PAC also differs with different ethnic populations. Several lifestyle, environmental and occupational exposures including long-term diabetes, obesity, and smoking, have been linked to PDAC, however, for PAC the causal risk factors were poorly described. It is now clear that PDAC and PAC are a multi-stage process resulting from the accumulation of genomic alterations in the somatic DNA of normal cells as well as inherited mutations. Approximately 10% of PDAC have a familial inheritance. Germline mutations in CDKN2A, BRCA2, STK11, PALB2, PRSS1, etc., as well as certain syndromes have been well associated with predisposition to PDAC. KRAS, CDKN2A, TP53 and SMAD4 are the 4 "mountains" (high-frequency driver genes) which have been known to earliest somatic alterations for PDAC while relatively less frequent in PAC. Our understanding of the molecular carcinogenesis has improved in the last few years due to extensive research on PDAC which was not well explored in case of PAC. The genetic alterations that have been identified in PDAC and different subgroups of PAC are important implications for the development of genetic screening test, early diagnosis, and prognostic genetic markers. The present review will provide a brief overview of the incidence and prevalence of PDAC and PAC, mainly, increased risk in India, the several kinds of risk factors associated with the diseases as well as required genetic alterations for disease initiation and progression.

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.

Predisposition to cancer caused by genetic and functional defects of mammalian Atad5.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

Deoxyribonucleic acid methylation driven aberrations in pancreatic cancer-related pathways.

Pancreatic cancer (PanCa) presents a catastrophic disease with poor overall survival at advanced stages, with immediate requirement of new and effective treatment options. Besides genetic mutations, epigenetic dysregulation of signaling pathway-associated enriched genes are considered as novel therapeutic target. Mechanisms beneath the deoxyribonucleic acid methylation and its utility in developing of epi-drugs in PanCa are under trails. Combinations of epigenetic medicines with conventional cytotoxic treatments or targeted therapy are promising options to improving the dismal response and survival rate of PanCa patients. Recent studies have identified potentially valid pathways that support the prediction that future PanCa clinical trials will include vigorous testing of epigenomic therapies. Epigenetics thus promises to generate a significant amount of new knowledge of biological and medical importance. Our review could identify various components of epigenetic mechanisms known to be involved in the initiation and development of pancreatic ductal adenocarcinoma and related precancerous lesions, and novel pharmacological strategies that target these components could potentially lead to breakthroughs. We aim to highlight the possibilities that exist and the potential therapeutic interventions.

Emerging role of non-invasive and liquid biopsy biomarkers in pancreatic cancer.

A global increase in the incidence of pancreatic cancer (PanCa) presents a major concern and health burden. The traditional tissue-based diagnostic techniques provided a major way forward for molecular diagnostics; however, they face limitations based on diagnosis-associated difficulties and concerns surrounding tissue availability in the clinical setting. Late disease development with asymptomatic behavior is a drawback in the case of existing diagnostic procedures. The capability of cell free markers in discriminating PanCa from autoimmune pancreatitis and chronic pancreatitis along with other precancerous lesions can be a boon to clinicians. Early-stage diagnosis of PanCa can be achieved only if these biomarkers specifically discriminate the non-carcinogenic disease stage from malignancy with respect to tumor stages. In this review, we comprehensively described the non-invasive disease detection approaches and why these approaches are gaining popularity for their early-stage diagnostic capability and associated clinical feasibility.

Possible Association Between Polymorphisms in ESR1, COL1A2, BGLAP, SPARC, VDR, and MMP2 Genes and Dental Fluorosis in a Population from an Endemic Region of West Bengal.

Dental fluorosis (DF) is the most prevalent form of fluorosis in India affecting millions of people all over the country. As estrogen receptor 1 (ESR1), collagen type 1 alpha 2 (COL1A2), bone γ-carboxyglutamic acid protein (BGLAP), secreted protein acidic and cysteine-rich (SPARC), vitamin D receptor (VDR), and matrix metallopeptidase 2 (MMP2) genes play critical roles in bone metabolism, bone formation, mineral metabolism, and mineralization, variants in these genes could influence susceptibility to DF. The present study was aimed at evaluating the association between 15 single-nucleotide polymorphisms (SNPs) in the six candidate genes (namely, ESR1, COL1A2, BGLAP, SPARC, VDR, and MMP2) and DF among 132 individuals (case = 71 and control = 61) living in a fluoride endemic region of West Bengal, India. No statistically significant association with disease risk was found when the genotypes and allele frequencies of each of the 15 SNPs was analyzed individually using odd's ratio with 95% confidence interval. "CC" and "AG" haplotypes of the COL1A2 gene showed a borderline association with DF. The present study is the first in India to use multifactor dimensionality reduction (MDR) analysis for identifying gene-gene and gene-environment interactions in fluorosis. The biomarker of serum fluoride showed a significant association with the disease state among the 17 attributes (15 SNPs and 2 biomarkers of urine fluoride and serum fluoride) (P value = 0.011). The best model of MDR analysis with maximized testing accuracy involved two SNPs from the ESR1 gene (rs9340799 and rs2077647) and one SNP from BGLAP gene (rs1543294) (P value < 0.0001).

Computational prediction of the molecular mechanism of statin group of drugs against SARS-CoV-2 pathogenesis.

Recently published clinical data from COVID-19 patients indicated that statin therapy is associated with a better clinical outcome and a significant reduction in the risk of mortality. In this study by computational analysis, we have aimed to predict the possible mechanism of the statin group of drugs by which they can inhibit SARS-CoV-2 pathogenesis. Blind docking of the critical structural and functional proteins of SARS-CoV-2 like RNA-dependent RNA polymerase, M-protease of 3-CL-Pro, Helicase, and the Spike proteins ( wild type and mutants from different VOCs) were performed using the Schrodinger docking tool. We observed that fluvastatin and pitavastatin showed fair, binding affinities to RNA polymerase and 3-CL-Pro, whereas fluvastatin showed the strongest binding affinity to the helicase. Fluvastatin also showed the highest affinity for the SpikeDelta and a fair docking score for other spike variants. Additionally, molecular dynamics simulation confirmed the formation of a stable drug-protein complex between Fluvastatin and target proteins. Thus our study shows that of all the statins, fluvastatin can bind to multiple target proteins of SARS-CoV-2, including the spike-mutant proteins. This property might contribute to the potent antiviral efficacy of this drug.

Human ELG1 regulates the level of ubiquitinated proliferating cell nuclear antigen (PCNA) through Its interactions with PCNA and USP1.

The level of monoubiquitinated proliferating cell nuclear antigen (PCNA) is closely linked with DNA damage bypass to protect cells from a high level of mutagenesis. However, it remains unclear how the level of monoubiquitinated PCNA is regulated. Here, we demonstrate that human ELG1 protein, which comprises an alternative replication factor C (RFC) complex and plays an important role in preserving genomic stability, as an interacting partner for the USP1 (ubiquitin-specific protease 1)-UAF1 (USP1-associated factor 1) complex, a deubiquitinating enzyme complex for PCNA and FANCD2. ELG1 protein interacts with PCNAs that are localized at stalled replication forks. ELG1 knockdown specifically resulted in an increase in the level of PCNA monoubiquitination without affecting the level of FANCD2 ubiquitination. It is a novel function of ELG1 distinct from its role as an alternative RFC complex because knockdowns of any other RFC subunits or other alternative RFCs did not affect PCNA monoubiquitination. Lastly, we identified a highly conserved N-terminal domain in ELG1 that was responsible for the USP1-UAF1 interaction as well as the activity to down-regulate PCNA monoubiquitination. Taken together, ELG1 specifically directs USP1-UAF1 complex for PCNA deubiquitination.

Spt2p defines a new transcription-dependent gross chromosomal rearrangement pathway.

Large numbers of gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. High mobility group 1 (HMG1) protein is a non-histone DNA-binding protein and is highly expressed in different types of tumors. The high expression of HMG1 could alter DNA structure resulting in GCRs. Spt2p is a non-histone DNA binding protein in Saccharomyces cerevisiae and shares homology with mammalian HMG1 protein. We found that Spt2p overexpression enhances GCRs dependent on proteins for transcription elongation and polyadenylation. Excess Spt2p increases the number of cells in S phase and the amount of single-stranded DNA (ssDNA) that might be susceptible to cause DNA damage and GCR. Consistently, RNase H expression, which reduces levels of ssDNA, decreased GCRs in cells expressing high level of Spt2p. Lastly, high transcription in the chromosome V, the location at which GCR is monitored, also enhanced GCR formation. We propose a new pathway for GCR where DNA intermediates formed during transcription can lead to genomic instability.

Evaluating the Association Between Dental Fluorosis and Polymorphisms in Bone Development and Mineralization Genes Among Population from a Fluoride Endemic Region of Eastern India.

Close to 12 million people in India are affected by more than the desirable level of fluoride in drinking water that could lead to dental, skeletal, and non-skeletal fluorosis. Dental fluorosis is a developmental defect that results in hypo-mineralization and pronounced porosity of enamel in the affected individuals. As estrogen receptor 1 (ESR1), collagen type 1 alpha 2 (COL1A2), bone γ-carboxyglutamic acid protein (BGLAP), and secreted protein acidic and cysteine rich (SPARC) genes are involved in bone development and mineralization, polymorphisms in these genes could be determining factors in influencing the risk to fluorosis among the exposed individuals in fluoride endemic areas. A case-control study was carried out among a total of 87 individuals (case = 36, control = 51) to examine the association between selected polymorphisms in the ESR1, COL1A2, BGLAP, and SPARC genes and risk of dental fluorosis from a fluoride endemic region of Eastern India. Altogether, 10 single nucleotide polymorphisms (SNPs) in ESR1 (rs2234693, rs2228480, rs3798577, rs2077647, and rs9340799), COL1A2 (rs42524, rs412777), BGLAP (rs1800247), and SPARC (rs6579885, rs4958278) genes were genotyped through PCR-RFLP in these subjects. The association of the SNPs for disease risk estimation was measured by odds ratio with 95% confidence interval. The risk genotypes of none of the 10 SNPs showed statistically significant association with risk of dental fluorosis. Frequencies of the haplotypes in the intragenic SNPs of the ESR1, COL1A2, and SPARC genes did not reveal any statistically significant difference between the case and control groups. The present study is the first of its kind from India that has attempted to investigate possible involvement of genetic factors in influencing the risk to fluorosis among the population from a fluoride endemic region.

SH3GL2 and CDKN2A/2B loci are independently altered in early dysplastic lesions of head and neck: correlation with HPV infection and tobacco habit.

To understand the association of candidate tumour suppressor genes SH3GL2, p16(INK4a), p14(ARF), and p15(INK4b) in the pathogenesis of head and neck squamous cell carcinoma (HNSCC), we studied the deletion, mutation, and methylation of these genes in 61 dysplastic lesions and 94 HNSCC samples. In mild dysplasia, SH3GL2, p16(INK4a), and p14(ARF) showed a higher frequency of overall alterations (60-70%) than in p15(INK4b) (40%). However, in subsequent stages of tumour progression, the alteration frequency of these genes did not change significantly. One novel mutation in common exon 2 of p16(INK4a)/p14(ARF) and three in exon 9 of SH3GL2 were seen. Concordance was seen in the expression of these genes with their molecular alterations. Deletions of INK4A-ARF and p15(INK4b) have a significant poor patient outcome. The alterations of p16(INK4a), p14(ARF), and p15(INK4b) were positively correlated with tobacco and inversely with HPV, while SH3GL2 alterations were independent of these factors. Based on aetiological factors, four tumour subtypes were recognized: HPV(-)tobacco(-) (I), HPV(+)tobacco(-) (II), HPV(-)tobacco(+) (III), and HPV(+)tobacco(+) (IV). Groups III and IV showed a high frequency of p16(INK4a)/p14(ARF)/p15(INK4b) alterations with significant poor patient outcome in comparison to group II. Our findings suggest that deregulation of SH3GL2-associated signalling and p16(INK4a)/p14(ARF)/p15(INK4b)-mediated G1-S/G2-M checkpoints of cell cycle are independent pathways for the development of early dysplastic lesions of the head and neck.

Hepatitis B Virus X Protein Upregulates hELG1/ ATAD5 Expression through E2F1 in Hepatocellular Carcinoma.

The precise mechanism by which HBx protein of hepatitis B virus (HBV) impacts on hepato-carcinogenesis remain largely elusive despite strong evidences for its' involvement in the process. Here, we have investigated the role of HBx on expression of a novel gene hELG1/ATAD5, which is required for genome maintenance and its' importance in hepatocarcinogenesis. This study has for the first time showed that the expression of this gene was significantly higher in human cancer such as HBV-associated hepatocellular carcinoma (HCC) and in different HCC cell lines compared to normal liver. In addition, a significant elevation in ATAD5 expression was also found in HBx transfected HCC cell lines implicating HBx mediated transcriptional regulation on ATAD5. Using different deletion mutant constructs of putative promoter, the active promoter region was first identified here and subsequently the regulatory region of HBx was mapped by promoter-luciferase assay. But ChIP assay with anti-HBx antibody revealed that HBx was not physically present in ATAD5 transcription machinery whereas anti-E2F1 antibody showed the presence of E2F1 in the complex. Luciferase assay with E2F1 binding site mutant had further confirmed it. Moreover, both loss-and gain-of-function studies of ATAD5 showed that ATAD5 could enhance HBV production in transfected cells whereas knock down of ATAD5 increased the sensitivity of HCC cell line to chemotherapeutics 5-fluorouracil. Overall, this data suggests that a positive feedback loop regulation between ATAD5 and HBV contributed to both viral replication and chemo-resistance of HCC cells.

Increased risk of oral leukoplakia and cancer among mixed tobacco users carrying XRCC1 variant haplotypes and cancer among smokers carrying two risk genotypes: one on each of two loci, GSTM3 and XRCC1 (Codon 280).

An individual's susceptibility to oral precancer and cancer depends not only on tobacco exposure but also on the genotypes/haplotypes at susceptible loci. In this hospital-based case-control study, 310 cancer patients, 197 leukoplakia patients, and 348 controls were studied to determine risk of the disease due to polymorphisms at three sites on XRCC1 and one site on XRCC3. Independently, variant genotypes on these loci did not modulate risk of leukoplakia and cancer except for the XRCC1 (codon 280) risk genotype in exclusive smokeless tobacco users with leukoplakia [odds ratios (OR), 2.4; 95% confidence intervals (CI), 1.0-5.7]. But variant haplotypes, containing one variant allele, on XRCC1 increased the risk of leukoplakia (OR, 1.3; 95% CI, 1.0-1.7). Among stratified samples, mixed tobacco users, carrying variant haplotypes, also had increased risk of both leukoplakia (OR, 2.2; 95% CI, 1.3-3.9) and cancer (OR, 1.9; 95% CI, 1.2-3.1). In a previous study on this population, it was shown that the GSTM3 (A/A) genotype increased the risk of oral leukoplakia and cancer among smokers, which has also been substantiated in this study with expanded sample sizes. The simultaneous presence of two risk genotypes in smokers, one on each of two loci, GSTM3 and XRCC1 (codon 280), increased the risk of cancer (OR, 2.4; 95% CI, 1.0-5.8). Again, smokers carrying two risk genotypes, one on each of two loci, GSTM3 and XRCC1 (codon 399), were also overrepresented in both leukoplakia and cancer populations (P(trend) = 0.02 and 0.04, respectively) but enhancement of risks were not observed; probably due to small sample sizes. Therefore, the presence of variant haplotypes on XRCC1 and two risk genotypes, one on each of two loci, GSTM3 and XRCC1, could be useful to determine the leukoplakias that might progress to cancer in a group of patients.

Polymorphism in CYP1A1 and CYP2E1 genes and susceptibility to leukoplakia in Indian tobacco users.

Inter-individual genetic differences may contribute to differences in susceptibility to human diseases triggered by environmental exposures. In this study, we investigated polymorphisms at two sites in the CYP1A1 and three sites in the CYP2E1 genes in 99 leukoplakia patients and 227 controls from one Indian population. The frequencies of genotypes at these polymorphic sites (MspI and Ileu/Val) in the CYP1A1 and (PstI, RsaI and DraI) in the CYP2E1 genes, were similar in patient and control groups. But the combined rare and heterozygous genotypes (CC+CD) at the DraI site in the CYP2E1 gene were over-represented among patients compared with controls (age-adjusted odds ratio (OR)=2.02, 95% confidence interval (CI)=1.21-3.35). Light tobacco smokers (i.e. <21 pack-year) and light tobacco chewers (i.e. <104 chewing-year) with a "rare" C allele at the DraI site had high risk of leukoplakia (OR=2.88, 95% CI=1.16-7.22; OR=2.94, 95% CI=1.15-7.65, respectively). The "mixed tobacco" users with "rare" C allele are more susceptible to the disease than "exclusive" tobacco smokers and chewers. The results indicate that the "rare" C allele at the DraI polymorphic site in CYP2E1 gene may enhance susceptibility to leukoplakia among tobacco users in this population. But the low sample size limited the power to precisely estimate the tobacco-genotype interactions.

DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity.

Genomic integrity depends on DNA replication, recombination and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of gamma-H2AX, 53BP1 and phosphorylated-ATM that mark chromosomal breaks. Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were upregulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.

Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination.

Gross chromosomal rearrangement (GCR) is a type of genomic instability associated with many cancers. In yeast, multiple pathways cooperate to suppress GCR. In a screen for genes that promote GCR, we identified MPH1, which encodes a 3'-5' DNA helicase. Overexpression of Mph1p in yeast results in decreased efficiency of homologous recombination (HR) as well as delayed Rad51p recruitment to double-strand breaks (DSBs), which suggests that Mph1p promotes GCR by partially suppressing HR. A function for Mph1p in suppression of HR is further supported by the observation that deletion of both mph1 and srs2 synergistically sensitize cells to methyl methanesulfonate-induced DNA damage. The GCR-promoting activity of Mph1p appears to depend on its interaction with replication protein A (RPA). Consistent with this observation, excess Mph1p stabilizes RPA at DSBs. Furthermore, spontaneous RPA foci at DSBs are destabilized by the mph1Delta mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.