Nilabja Sikdar

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.

Glutathione S-transferase M3 (A/A) genotype as a risk factor for oral cancer and leukoplakia among Indian tobacco smokers.

Polymorphism in glutathione S‐transferase (GST) genes, causing variations in enzyme activities, may influence susceptibility to oral cancer and leukoplakia in smokers and/or smokeless tobacco users. In this case‐control study consisting of 109 leukoplakia and 256 oral cancer patients and 259 controls, genotype frequencies at GSTM1, GSTT1, GSTM3 and GSTP1 loci were determined by polymerase chain reaction‐restriction fragment length polymorphism methods and analyzed by multiple logistic regression to determine the risks of the diseases. There were no significant differences in the distributions of GSTM1, GSTM3 and GSTT1 genotypes in patients and controls when all individuals were compared. In contrast, frequencies of ile/ile genotype at codon 105 and variant val‐ala haplotype of GSTP1 was significantly higher (OR = 1.5; 95% CI = 1.0–2.0) and lower (OR = 1.4; 95% CI = 1.0–1.9) in oral cancer patients compare to controls, respectively. The impacts of all genotypes on risks of oral cancer and leukoplakia were also analyzed in patients with different tobacco habits and doses. Increased risks of cancer and leukoplakia were observed in tobacco smokers with GSTM3 (A/A) genotype (OR = 2.0, 95% CI = 1.0–4.0; OR = 2.0, 95% CI = 1.0–4.4, respectively). So, GSTM3 (A/A) genotype could become one of the markers to know which of the leukoplakia would be transformed into cancer. Heavy tobacco chewing (> 124 chewing‐year) increased the risk of cancer in individuals with GSTT1 homozygous null genotype (OR = 3.0; 95% CI = 1.0–9.8). Furthermore, increased lifetime exposure to tobacco smoking (> 11.5 pack‐year) increased the risk of leukoplakia in individuals with GSTM1 homozygous null genotype (OR = 2.4; 95% CI = 1.0–5.7). It may be suggested that polymorphisms in GSTP1, GSTM1, GSTM3 and GSTT1 genes regulate risk of cancer and leukoplakia differentially among different tobacco habituals.

Polymorphisms at XPD and XRCC1 DNA repair loci and increased risk of oral leukoplakia and cancer among NAT2 slow acetylators.

Polymorphisms at N‐acetyl transferase 2 locus (NAT2) lead to slow, intermediate and rapid acetylation properties of the enzyme. Improper acetylation of heterocyclic and aromatic amines, present in tobacco, might cause DNA adduct formation. Generally, DNA repair enzymes remove these adduct to escape malignancy. But, tobacco users carrying susceptible NAT2 and DNA repair loci might be at risk of oral leukoplakia and cancer. In this study, 389 controls, 224 leukoplakia and 310 cancer patients were genotyped at 5 polymorphic sites on NAT2 and 3 polymorphic sites on each of XRCC1 and XPD loci by PCR‐RFLP method to determine the risk of the diseases. None of the SNPs on these loci independently could modify the risk of the diseases in overall population but variant genotype (Gln/Gln) at codon 399 on XRCC1 and major genotype (Lys/Lys) at codon 751 on XPD were associated with increased risk of leukoplakia and cancer among slow acetylators, respectively (OR = 4.2, 95% CI = 1.2–15.0; OR = 1.6, 95% CI = 1.1–2.3, respectively). Variant genotype (Asn/Asn) at codon 312 on XPD was also associated with increased risk of cancer among rapid and intermediate acetylators (OR = 1.9, 95% CI = 1.2–2.9). Variant C‐G‐A haplotype at XRCC1 was associated with increased risk of leukoplakia (OR = 1.7, 95% CI = 1.2–2.4) but leukoplakia and cancer in mixed tobacco users (OR = 3.1, 95% CI = 1.4–7.1, OR = 2.4, 95% CI = 1.1–5.4, respectively) among slow acetylators. Although none of the 3 loci could modulate the risk of the diseases independently but 2 loci in combination, working in 2 different biochemical pathways, could do so in these patient populations.

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 individuals 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 (Ptrend = 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.

Increased risk of oral cancer in relation to common Indian mitochondrial polymorphisms and Autosomal GSTP1 locus

Polymorphisms at mitochondrial (mt) loci could modulate the risk of diseases including cancers. Here the mtDNA polymorphisms at 12,308 nucleotide pairs (np), 11,467 np, 10,400 np, and 10,398 np were studied to examine the association with the risk of oral cancer and leukoplakia, alone and in combination with polymorphisms at the GST loci.

Predisposition of antituberculosis drug induced hepatotoxicity by cytochrome P450 2E1 genotype and haplotype in pediatric patients

784 Journal of Gastroenterology and Hepatology 21 (2006) 781–786

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, p16INK4a, p14ARF, and p15INK4b 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, p16INK4a, and p14ARF showed a higher frequency of overall alterations (60–70%) than in p15INK4b (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 p16INK4a/p14ARF 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 p15INK4b have a significant poor patient outcome. The alterations of p16INK4a, p14ARF, and p15INK4b 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 p16INK4a/p14ARF/p15INK4b alterations with significant poor patient outcome in comparison to group II. Our findings suggest that deregulation of SH3GL2‐associated signalling and p16INK4a/p14ARF/p15INK4b‐mediated G1–S/G2–M checkpoints of cell cycle are independent pathways for the development of early dysplastic lesions of the head and neck. Copyright

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 mph1Δ mutation. Therefore, Mph1p promotes GCR formation by partially suppressing HR, likely through its interaction with RPA.

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.

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 γ-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 up-regulated 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.