Editorial Cite this article: Hegde M L. A Perspective on Chromosomal Double Strand Break Markers ... more Editorial Cite this article: Hegde M L. A Perspective on Chromosomal Double Strand Break Markers in Mammalian Cells. J J Rad Oncol. 2014, 1(1): 003. The DNA damage response (DDR) in mammalian cells is a complex and highly orchestrated signaling process that ϐ the DNA damage sites [1,2]. The most lethal type of DNA damage is the double-strand break (DSB), which is gener-ated by ionizing radiation (IR), radiomimetic drugs and also drugs of DNA topoisomerase 2 poison family [3]. DSBs are also endogenously caused during replication of single-strand breaks generated by reactive oxygen species or at stalled replication forks [4,5]. While IR is extensive-ly used in treating various cancers, IR-induced DSBs and other genomic lesions can cause mutations and gross genome rearrangements in normal cells as well, possibly leading to secondary leukemia and other cancers. Fortu-nately, multiple DSB repair (DSBR) systems, in coordina-tion with cell cy...
Reactive oxygen species (ROS), continuously generated as by-products of respiration, inflict more... more Reactive oxygen species (ROS), continuously generated as by-products of respiration, inflict more damage on the mitochondrial (mt) than on the nuclear genome because of the nonchromatinized nature and proximity to the ROS source of the mitochondrial genome. Such damage, particularly single-strand breaks (SSBs) with 5′-blocking deoxyribose products generated directly or as repair intermediates for oxidized bases, is repaired via the base excision/SSB repair pathway in both nuclear and mt genomes. Here, we show that EXOG, a 5′-exo/endonuclease and unique to the mitochondria unlike FEN1 or DNA2, which, like EXOG, has been implicated in the removal of the 5′-blocking residue, is required for repairing endogenous SSBs in the mt genome. EXOG depletion induces persistent SSBs in the mtDNA, enhances ROS levels, and causes apoptosis in normal cells but not in mt genome-deficient rho0 cells. Thus, these data show for the first time that persistent SSBs in the mt genome alone could provide the...
The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6... more The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6-methylguanine (m6Gua), was analyzed during in vitro replication of m6Gua-containing synthetic polydeoxynucleotides by T4 and T5 phage DNA polymerases and Escherichia coli DNA polymerase I. When poly(dT,m6dG) and poly(dC,m6dG) with covalently attached primers were replicated, O6-methylguanine paired with either thymine or cytosine but with a much higher preference for thymine. dCTP and dTTP acted as competitive inhibitors of each other during DNA synthesis. O6-Methylguanine also directed incorporation of dAMP by T5 DNA polymerase. This dAMP incorporation was not inhibited by dTTP. Contrary to theoretical predictions that the m6dG X dT pair should be comparable to the dA X dT pair, the presence of m6dG in the template inhibited DNA synthesis. Based on Kappm values, E. coli DNA polymerase I showed a much higher preference for dTMP incorporation over dCMP opposite m6dG in the template than ...
Among the many basic alkylated sites produced in DNA by simple alkylating mutagens, Oâ¶-alkylgua... more Among the many basic alkylated sites produced in DNA by simple alkylating mutagens, Oâ¶-alkylguanine is believed to be the major promutagenic lesion. Using Oâ¶-methyl dGTP as a substrate during in vitro synthesis of biologically active T7 DNA we have shown that the incorporation of the alkylated nucleotide leads to increased reversion of an amber mutation in the phage DNA. We have shown that Oâ¶-methylguanine has a strong preference for pairing with thymine over cytosine during in vitro replication of synthetic DNA templates with T4 and T5 phage DNA polymerases and E. coli DNA polymerase I. The degree of preference depends not only on the DNA polymerase used but possibly on other factors as well, including the divalent metal ion cofactors. The alkylated base in the template DNA also inhibits chain elongation of the product strand. While Oâ¶-methyl dGTP can act as a substrate for DNA polymerases, as an analog of dATP, the alkylated nucleotide is incorporated only infrequently and...
To determine the role of norepinephrine (NE) on DNA damage and reactive oxygen species (ROS) gene... more To determine the role of norepinephrine (NE) on DNA damage and reactive oxygen species (ROS) generation in ovarian surface epithelial cells. Non-tumorigenic, immortalized ovarian surface epithelial cells were treated with NE, bleomycin, and bleomycin followed by NE. The comet assay was performed on each treatment group to determine the amount of single and double-strand breaks induced by treatments. ROS levels for each treatment group were measured using the H2DCF-DA fluorescence assay. Finally, RNA transcripts were measured for each treatment group with regards to the expression of DNA repair and oxidative stress genes. The mean tail moment of untreated cells was significantly greater than that of cells treated with NE (p=0.02). The mean tail moment of cells treated with bleomycin was significantly greater than that of cells treated with bleomycin followed by NE (p<0.01). Treatment with NE resulted in significantly less ROS generation than in untreated cells (p<0.01). NE treatment after hydrogen peroxide treatment resulted in a noticeable decrease in ROS generation. Genes associated with oxidative stress were upregulated in cells treated with bleomycin, however this upregulation was blunted when bleomycin-treated cells were treated subsequently with NE. NE is associated with decreased DNA damage and ROS production in ovarian surface epithelial cells. This effect is protective in the presence of the oxidative-damaging agent bleomycin. These results suggest an additional physiologic role for the stress hormone NE, in protecting ovarian surface epithelial cells from oxidative stress.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
The ability to repair promutagenic damage resulting from exposure to carcinogens is a critical fa... more The ability to repair promutagenic damage resulting from exposure to carcinogens is a critical factor in determining quantitative relationships in carcinogenesis, including the target cell for neoplasia. One major pathway for the repair of alkylating agent-induced DNA damage involves removal of alkylated bases by N-methylpurine-DNA-glycosylase (MPG), the first enzyme in base excision repair. We have measured the expression level of MPG mRNA in liver, lung, and kidney of Sprague-Dawley rats as a function of age. A quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) method was used to measure cellular MPG mRNA. MPG mRNA was readily detectable in each tissue analyzed and the age-dependent and tissue specific expressions were not statistically different. The lowest amount of mRNA was measured in preweanling liver and the highest amounts were found in preweanling lung and kidney. Since MPG is reported to be responsible for excision of 1,N(6)-ethenoadenine and N(2),3-et...
Radiation-induced genome damage clusters contain DNA double-strand breaks (DSB), and more frequen... more Radiation-induced genome damage clusters contain DNA double-strand breaks (DSB), and more frequent oxidized bases and single-strand breaks. Secondary DSBs could result during repair of the clustered base lesions. All DSBs contain nonligatable termini that require processing. While nonhomologous end joining (NHEJ) is the predominant DSB repair (DSBR) pathway, our initial studies suggest that the secondary DSBs are repaired via alternative end-joining (Alt-EJ) processes. While current DSBR studies mostly examine single DSBs with undamaged termini, we have generated a novel linearized reporter (GFP) plasmid containing damaged DSB termini mimicking IR-induced damage to recapitulate in vivo DSBR by in-cell and in vitro repair assays. After in-cell repair using mammalian cells containing the plasmid substrate, and in vitro assays with nuclear extracts, we recovered the circularized plasmids for amplification in E. coli. Sequencing of the repaired plasmid molecules indicates that repair oc...
Excessive accumulation of pro-oxidant metals, observed in affected brain regions, has consistentl... more Excessive accumulation of pro-oxidant metals, observed in affected brain regions, has consistently been implicated as a contributor to the brain pathology including neurodegenerative diseases and acute injuries such as stroke. Furthermore, the potential interactions between metal toxicity and other commonly associated etiological factors, such as misfolding/aggregation of amyloidogenic proteins or genomic damage, are poorly understood. Decades of research provide compelling evidence implicating metal overload in neurological diseases and stroke. However, the utility of metal toxicity as a therapeutic target is controversial, possibly due to a lack of comprehensive understanding of metal dyshomeostasis-mediated neuronal pathology. In this article, we discuss the current understanding of metal toxicity and the challenges associated with metal-targeted therapies.
The objective of this study was to compare the efficacy and side-effects of two regimens of vagin... more The objective of this study was to compare the efficacy and side-effects of two regimens of vaginal misoprostol for second trimester voluntary medical termination of pregnancy (MTP) according to the MTP Act of India. A randomized trial was conducted in 185 women from January 2007 to September 2008. Women in group 1 were given vaginal misoprostol 400 microg every 6 h for a maximum of four doses. Women in group 2 were given vaginal misoprostol 400 microg every 12 h for a maximum of four doses. Our primary outcome measure was induction abortion interval. Secondary outcome measures were success rate, side-effects and completeness of procedure. Results were calculated applying Fisher's exact test, chi-square test, Z test and calculating the P value using an alpha level of 0.05 for Type I error. The mean induction abortion interval in group 1 (12.59 h) was significantly shorter (P < 0.001) than that in the group 2 (16.41 h). The percentage of women who achieved successful abortion ...
Journal of health, population, and nutrition, 2005
This case-control study was conducted in the Cardiology Department of Medical College, Kolkata, I... more This case-control study was conducted in the Cardiology Department of Medical College, Kolkata, India, during 2000-2001, to explore the link between stressful life events and subsequent myocardial infarction (MI). One hundred consecutive confirmed MI patients were selected as a case group. One hundred age-, sex- and income-matched controls were selected from visitors other than relatives who attended these patients. The subjects were interviewed and asked to rate 61 life events with a number between 0 and 20. They also noted which of these they had experienced in the last one year. The main exposure variables included life events as per E.S. Paykel, smoking, alcohol consumption, chewing of tobacco, marital status, literacy, employment, and monthly per-capita income. The results showed that an MI patient was likely to experience 4.16 stressful life events, which were twice as much as the control group (2.24). The total stress score was the highest for serious personal illness followe...
Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontane... more Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural an...
Progress in nucleic acid research and molecular biology, 1983
ABSTRACT Oâ¶-Methylguanine, produced in DNA by simple alkylating carcinogens, is considered to b... more ABSTRACT Oâ¶-Methylguanine, produced in DNA by simple alkylating carcinogens, is considered to be the primary mutagenic Lesion and is implicated in carcinogenesis induced by these agents. Its ultimate biological effect is determined both by its persistence in DNA and by its miscoding potential during replication and transcription. Polydeoxynucleotides containing Oâ¶-methylguanine were synthesized for use as substrates for repair enzymes and as templates for DNA polymerases. Replication was carried out with T5 phage DNA polymerase under standard condition. The removal of mⶠdG from DNA in mammalian cells is similar to the repair pathway previously established for E. coli in that (1) demethylation of mâ¶dG occurs in situ without alteration or removal of the resulting guanine residue, (2) the enzyme repair appears to react stoichiometrically with the substrate, and (3) it appears that the expression of the methyl transferase activity is under regulatory control. Levels of the activity were found to vary widely in mammalian cells. Incorporation of mâ¶dG during DNA synthesis may be a significant factor in the mutagenesis caused by this agent. The base-pairing properties of mâ¶dG during in vitro DNA replication support the proposed role of mâ¶dG in the mutagenesis caused by alkylating carcinogens in vivo. Oâ¶-Methylguanine base pairs preferentially with thymine during DNA synthesis leading to GC ..--&gt;.. AT transition mutation. However, dT and dC act as competitive inhibitors during incorporation opposite mâ¶dG, therefore, the mutagenic potential of mâ¶dG is dependent on the relative intracellular concentrations of these precursors. Different DNA polymerases recognize mâ¶dG differently depending on whether it is a substrate (mâ¶dGTP) or in the template (mâ¶dG) for DNA replication. The relative mutagenic potentials of mâ¶dGTP and mâ¶dG in the cell are therefore dependent on the cell type as well as on the concentration of the substrates. (ERB)
1. Ultraviolet irradiation of double stranded DNA in vitro caused strand separation, as judged by... more 1. Ultraviolet irradiation of double stranded DNA in vitro caused strand separation, as judged by the susceptibility of the DNA to degradation by an endonuclease from Neurospora crassa that is specific for single strands, by its behavior on elution from hydroxylapatite and by its sedimentation properties. The extent of degradation by the endonuclease was almost linearly proportional to the ultraviolet dose up to 2.8 • 10 s J/m 5, when 45% of the DNA was degraded. Increased DNA concentration and high concentrations of Na ÷ and Mg 2÷ were inhibitory to degradation. The pH of the DNA solution had no significant effect.
Human breast carcinoma (MCF7-MLNr) cells resistant to the bifunctional drugs t_-phenylalanine mus... more Human breast carcinoma (MCF7-MLNr) cells resistant to the bifunctional drugs t_-phenylalanine mustard (L-PAM, 5-fold resistance), mechlorethamine (9-fold), cisplatin (3-fold), and BCNU (3-fold) were used to investigate the role of DNA repair in the development of resistance to alkylating agents. We have previously shown that neither L-PAM transport and metabolism nor glutathione-associated enzymes were altered in MCF7-MLNr cells, compared to the sensitive cells MCF7-WT. This study shows that treatment of pRSV-CAT plasmid with L-PAM at concentrations up to 1 PM proportionally inhibit the expression of chloramphenicol acetyl transferase (CAT) activity, while higher concentrations abolished CAT activity. pRSV-CAT reactivation was significantly increased when plasmid was transfected into MCF7-MLNr cells, compared to MCF7-WT cells. This indicates that resistant cells have more efficient capacity to recognize and repair L-PAM induced DNA damage. The mRNA expression of DNA nucleotide excision repair genes ERCCl, XPD (ERCCZ), XPB (ERCC3), and polymerase p was found to be similar in both the MCF7-WT and MCF7-MLNr cells. Western blot analysis also reveals no difference in the expression of ERCCl, AP endonuclease, poly (ADP-ribose) polymerase, and alkyl-iV-purine-DNA glycosylase Abbreviations: ANPG,alkyl-N-purine-DNA glycosylase; AP endonuclease, apurinic site endonuclease; BCNU, 1,3-bis(2-chloroethyl) nitrosourea (carmustine); cisplatin, cisdiamminedichloroplatinum (II); L-PAM, L-phenylalanine mustard (melphalan); WT, wild type cells; ERCCl, excision-repair cross-complementing CHO mutant cell line of complementation group 1; XPD (ERCC2), excision-repair cross-complementing CHO mutant cell line of complementation group 2 and XP group D; XPB (ERCC3), excision-repair cross-complementing CHO mutant cell line of complementation group 3 and XP group B; MDR, multidrug resistance; MGMT, 06-methylguanine-DNA methyltransferase; MLNr, cells selected for resistance to melphalan; CAT, chloramphenicol acetyl transferase; PARP, poly (ADP-ribose) polymerase; XP, xeroderma pigmentosum MUSICA.McGILL.CA. 0921-8777/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 092l-8777(95)00022-4 180 L. Yen et al. /Mutaiion proteins. The lack of correlation between enhanced host cell reactivation capacity in resistant cells, and the expression of these specific DNA repair genes suggests that proteins encoded by these genes are not rate limiting steps for resistance to bi-functional alkylating drugs in human breast cancer cells.
A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was d... more A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was developed for measuring mRNA concentration, in rodent cells, of theN-methylpurine–DNA glycosylase (MPG), a ubiquitous DNA repair protein responsible for the removal ofN-alkylpurines and ethenoadducts of adenine, guanine, and cytosine from DNA. The method, applicable for quantitation of any mRNA, is based on the standard approach of comparing the relative
In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylat... more In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylating drug resistance of tumor cells, we utilized the A2780(100) line, that was selected by repeated exposure of A2780 cell line (human ovarian carcinoma line) to chlorambucil (CBL). A2780(100) was 5–10-fold more resistant to nitrogen mustards (IC50 of 50–60 μM) and other DNA crosslinking agents, e.g.,
Editorial Cite this article: Hegde M L. A Perspective on Chromosomal Double Strand Break Markers ... more Editorial Cite this article: Hegde M L. A Perspective on Chromosomal Double Strand Break Markers in Mammalian Cells. J J Rad Oncol. 2014, 1(1): 003. The DNA damage response (DDR) in mammalian cells is a complex and highly orchestrated signaling process that ϐ the DNA damage sites [1,2]. The most lethal type of DNA damage is the double-strand break (DSB), which is gener-ated by ionizing radiation (IR), radiomimetic drugs and also drugs of DNA topoisomerase 2 poison family [3]. DSBs are also endogenously caused during replication of single-strand breaks generated by reactive oxygen species or at stalled replication forks [4,5]. While IR is extensive-ly used in treating various cancers, IR-induced DSBs and other genomic lesions can cause mutations and gross genome rearrangements in normal cells as well, possibly leading to secondary leukemia and other cancers. Fortu-nately, multiple DSB repair (DSBR) systems, in coordina-tion with cell cy...
Reactive oxygen species (ROS), continuously generated as by-products of respiration, inflict more... more Reactive oxygen species (ROS), continuously generated as by-products of respiration, inflict more damage on the mitochondrial (mt) than on the nuclear genome because of the nonchromatinized nature and proximity to the ROS source of the mitochondrial genome. Such damage, particularly single-strand breaks (SSBs) with 5′-blocking deoxyribose products generated directly or as repair intermediates for oxidized bases, is repaired via the base excision/SSB repair pathway in both nuclear and mt genomes. Here, we show that EXOG, a 5′-exo/endonuclease and unique to the mitochondria unlike FEN1 or DNA2, which, like EXOG, has been implicated in the removal of the 5′-blocking residue, is required for repairing endogenous SSBs in the mt genome. EXOG depletion induces persistent SSBs in the mtDNA, enhances ROS levels, and causes apoptosis in normal cells but not in mt genome-deficient rho0 cells. Thus, these data show for the first time that persistent SSBs in the mt genome alone could provide the...
The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6... more The kinetics of incorporation of deoxynucleotide precursors directed by the promutagenic base, O6-methylguanine (m6Gua), was analyzed during in vitro replication of m6Gua-containing synthetic polydeoxynucleotides by T4 and T5 phage DNA polymerases and Escherichia coli DNA polymerase I. When poly(dT,m6dG) and poly(dC,m6dG) with covalently attached primers were replicated, O6-methylguanine paired with either thymine or cytosine but with a much higher preference for thymine. dCTP and dTTP acted as competitive inhibitors of each other during DNA synthesis. O6-Methylguanine also directed incorporation of dAMP by T5 DNA polymerase. This dAMP incorporation was not inhibited by dTTP. Contrary to theoretical predictions that the m6dG X dT pair should be comparable to the dA X dT pair, the presence of m6dG in the template inhibited DNA synthesis. Based on Kappm values, E. coli DNA polymerase I showed a much higher preference for dTMP incorporation over dCMP opposite m6dG in the template than ...
Among the many basic alkylated sites produced in DNA by simple alkylating mutagens, Oâ¶-alkylgua... more Among the many basic alkylated sites produced in DNA by simple alkylating mutagens, Oâ¶-alkylguanine is believed to be the major promutagenic lesion. Using Oâ¶-methyl dGTP as a substrate during in vitro synthesis of biologically active T7 DNA we have shown that the incorporation of the alkylated nucleotide leads to increased reversion of an amber mutation in the phage DNA. We have shown that Oâ¶-methylguanine has a strong preference for pairing with thymine over cytosine during in vitro replication of synthetic DNA templates with T4 and T5 phage DNA polymerases and E. coli DNA polymerase I. The degree of preference depends not only on the DNA polymerase used but possibly on other factors as well, including the divalent metal ion cofactors. The alkylated base in the template DNA also inhibits chain elongation of the product strand. While Oâ¶-methyl dGTP can act as a substrate for DNA polymerases, as an analog of dATP, the alkylated nucleotide is incorporated only infrequently and...
To determine the role of norepinephrine (NE) on DNA damage and reactive oxygen species (ROS) gene... more To determine the role of norepinephrine (NE) on DNA damage and reactive oxygen species (ROS) generation in ovarian surface epithelial cells. Non-tumorigenic, immortalized ovarian surface epithelial cells were treated with NE, bleomycin, and bleomycin followed by NE. The comet assay was performed on each treatment group to determine the amount of single and double-strand breaks induced by treatments. ROS levels for each treatment group were measured using the H2DCF-DA fluorescence assay. Finally, RNA transcripts were measured for each treatment group with regards to the expression of DNA repair and oxidative stress genes. The mean tail moment of untreated cells was significantly greater than that of cells treated with NE (p=0.02). The mean tail moment of cells treated with bleomycin was significantly greater than that of cells treated with bleomycin followed by NE (p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.01). Treatment with NE resulted in significantly less ROS generation than in untreated cells (p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.01). NE treatment after hydrogen peroxide treatment resulted in a noticeable decrease in ROS generation. Genes associated with oxidative stress were upregulated in cells treated with bleomycin, however this upregulation was blunted when bleomycin-treated cells were treated subsequently with NE. NE is associated with decreased DNA damage and ROS production in ovarian surface epithelial cells. This effect is protective in the presence of the oxidative-damaging agent bleomycin. These results suggest an additional physiologic role for the stress hormone NE, in protecting ovarian surface epithelial cells from oxidative stress.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
The ability to repair promutagenic damage resulting from exposure to carcinogens is a critical fa... more The ability to repair promutagenic damage resulting from exposure to carcinogens is a critical factor in determining quantitative relationships in carcinogenesis, including the target cell for neoplasia. One major pathway for the repair of alkylating agent-induced DNA damage involves removal of alkylated bases by N-methylpurine-DNA-glycosylase (MPG), the first enzyme in base excision repair. We have measured the expression level of MPG mRNA in liver, lung, and kidney of Sprague-Dawley rats as a function of age. A quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) method was used to measure cellular MPG mRNA. MPG mRNA was readily detectable in each tissue analyzed and the age-dependent and tissue specific expressions were not statistically different. The lowest amount of mRNA was measured in preweanling liver and the highest amounts were found in preweanling lung and kidney. Since MPG is reported to be responsible for excision of 1,N(6)-ethenoadenine and N(2),3-et...
Radiation-induced genome damage clusters contain DNA double-strand breaks (DSB), and more frequen... more Radiation-induced genome damage clusters contain DNA double-strand breaks (DSB), and more frequent oxidized bases and single-strand breaks. Secondary DSBs could result during repair of the clustered base lesions. All DSBs contain nonligatable termini that require processing. While nonhomologous end joining (NHEJ) is the predominant DSB repair (DSBR) pathway, our initial studies suggest that the secondary DSBs are repaired via alternative end-joining (Alt-EJ) processes. While current DSBR studies mostly examine single DSBs with undamaged termini, we have generated a novel linearized reporter (GFP) plasmid containing damaged DSB termini mimicking IR-induced damage to recapitulate in vivo DSBR by in-cell and in vitro repair assays. After in-cell repair using mammalian cells containing the plasmid substrate, and in vitro assays with nuclear extracts, we recovered the circularized plasmids for amplification in E. coli. Sequencing of the repaired plasmid molecules indicates that repair oc...
Excessive accumulation of pro-oxidant metals, observed in affected brain regions, has consistentl... more Excessive accumulation of pro-oxidant metals, observed in affected brain regions, has consistently been implicated as a contributor to the brain pathology including neurodegenerative diseases and acute injuries such as stroke. Furthermore, the potential interactions between metal toxicity and other commonly associated etiological factors, such as misfolding/aggregation of amyloidogenic proteins or genomic damage, are poorly understood. Decades of research provide compelling evidence implicating metal overload in neurological diseases and stroke. However, the utility of metal toxicity as a therapeutic target is controversial, possibly due to a lack of comprehensive understanding of metal dyshomeostasis-mediated neuronal pathology. In this article, we discuss the current understanding of metal toxicity and the challenges associated with metal-targeted therapies.
The objective of this study was to compare the efficacy and side-effects of two regimens of vagin... more The objective of this study was to compare the efficacy and side-effects of two regimens of vaginal misoprostol for second trimester voluntary medical termination of pregnancy (MTP) according to the MTP Act of India. A randomized trial was conducted in 185 women from January 2007 to September 2008. Women in group 1 were given vaginal misoprostol 400 microg every 6 h for a maximum of four doses. Women in group 2 were given vaginal misoprostol 400 microg every 12 h for a maximum of four doses. Our primary outcome measure was induction abortion interval. Secondary outcome measures were success rate, side-effects and completeness of procedure. Results were calculated applying Fisher's exact test, chi-square test, Z test and calculating the P value using an alpha level of 0.05 for Type I error. The mean induction abortion interval in group 1 (12.59 h) was significantly shorter (P < 0.001) than that in the group 2 (16.41 h). The percentage of women who achieved successful abortion ...
Journal of health, population, and nutrition, 2005
This case-control study was conducted in the Cardiology Department of Medical College, Kolkata, I... more This case-control study was conducted in the Cardiology Department of Medical College, Kolkata, India, during 2000-2001, to explore the link between stressful life events and subsequent myocardial infarction (MI). One hundred consecutive confirmed MI patients were selected as a case group. One hundred age-, sex- and income-matched controls were selected from visitors other than relatives who attended these patients. The subjects were interviewed and asked to rate 61 life events with a number between 0 and 20. They also noted which of these they had experienced in the last one year. The main exposure variables included life events as per E.S. Paykel, smoking, alcohol consumption, chewing of tobacco, marital status, literacy, employment, and monthly per-capita income. The results showed that an MI patient was likely to experience 4.16 stressful life events, which were twice as much as the control group (2.24). The total stress score was the highest for serious personal illness followe...
Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontane... more Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural an...
Progress in nucleic acid research and molecular biology, 1983
ABSTRACT Oâ¶-Methylguanine, produced in DNA by simple alkylating carcinogens, is considered to b... more ABSTRACT Oâ¶-Methylguanine, produced in DNA by simple alkylating carcinogens, is considered to be the primary mutagenic Lesion and is implicated in carcinogenesis induced by these agents. Its ultimate biological effect is determined both by its persistence in DNA and by its miscoding potential during replication and transcription. Polydeoxynucleotides containing Oâ¶-methylguanine were synthesized for use as substrates for repair enzymes and as templates for DNA polymerases. Replication was carried out with T5 phage DNA polymerase under standard condition. The removal of mⶠdG from DNA in mammalian cells is similar to the repair pathway previously established for E. coli in that (1) demethylation of mâ¶dG occurs in situ without alteration or removal of the resulting guanine residue, (2) the enzyme repair appears to react stoichiometrically with the substrate, and (3) it appears that the expression of the methyl transferase activity is under regulatory control. Levels of the activity were found to vary widely in mammalian cells. Incorporation of mâ¶dG during DNA synthesis may be a significant factor in the mutagenesis caused by this agent. The base-pairing properties of mâ¶dG during in vitro DNA replication support the proposed role of mâ¶dG in the mutagenesis caused by alkylating carcinogens in vivo. Oâ¶-Methylguanine base pairs preferentially with thymine during DNA synthesis leading to GC ..--&gt;.. AT transition mutation. However, dT and dC act as competitive inhibitors during incorporation opposite mâ¶dG, therefore, the mutagenic potential of mâ¶dG is dependent on the relative intracellular concentrations of these precursors. Different DNA polymerases recognize mâ¶dG differently depending on whether it is a substrate (mâ¶dGTP) or in the template (mâ¶dG) for DNA replication. The relative mutagenic potentials of mâ¶dGTP and mâ¶dG in the cell are therefore dependent on the cell type as well as on the concentration of the substrates. (ERB)
1. Ultraviolet irradiation of double stranded DNA in vitro caused strand separation, as judged by... more 1. Ultraviolet irradiation of double stranded DNA in vitro caused strand separation, as judged by the susceptibility of the DNA to degradation by an endonuclease from Neurospora crassa that is specific for single strands, by its behavior on elution from hydroxylapatite and by its sedimentation properties. The extent of degradation by the endonuclease was almost linearly proportional to the ultraviolet dose up to 2.8 • 10 s J/m 5, when 45% of the DNA was degraded. Increased DNA concentration and high concentrations of Na ÷ and Mg 2÷ were inhibitory to degradation. The pH of the DNA solution had no significant effect.
Human breast carcinoma (MCF7-MLNr) cells resistant to the bifunctional drugs t_-phenylalanine mus... more Human breast carcinoma (MCF7-MLNr) cells resistant to the bifunctional drugs t_-phenylalanine mustard (L-PAM, 5-fold resistance), mechlorethamine (9-fold), cisplatin (3-fold), and BCNU (3-fold) were used to investigate the role of DNA repair in the development of resistance to alkylating agents. We have previously shown that neither L-PAM transport and metabolism nor glutathione-associated enzymes were altered in MCF7-MLNr cells, compared to the sensitive cells MCF7-WT. This study shows that treatment of pRSV-CAT plasmid with L-PAM at concentrations up to 1 PM proportionally inhibit the expression of chloramphenicol acetyl transferase (CAT) activity, while higher concentrations abolished CAT activity. pRSV-CAT reactivation was significantly increased when plasmid was transfected into MCF7-MLNr cells, compared to MCF7-WT cells. This indicates that resistant cells have more efficient capacity to recognize and repair L-PAM induced DNA damage. The mRNA expression of DNA nucleotide excision repair genes ERCCl, XPD (ERCCZ), XPB (ERCC3), and polymerase p was found to be similar in both the MCF7-WT and MCF7-MLNr cells. Western blot analysis also reveals no difference in the expression of ERCCl, AP endonuclease, poly (ADP-ribose) polymerase, and alkyl-iV-purine-DNA glycosylase Abbreviations: ANPG,alkyl-N-purine-DNA glycosylase; AP endonuclease, apurinic site endonuclease; BCNU, 1,3-bis(2-chloroethyl) nitrosourea (carmustine); cisplatin, cisdiamminedichloroplatinum (II); L-PAM, L-phenylalanine mustard (melphalan); WT, wild type cells; ERCCl, excision-repair cross-complementing CHO mutant cell line of complementation group 1; XPD (ERCC2), excision-repair cross-complementing CHO mutant cell line of complementation group 2 and XP group D; XPB (ERCC3), excision-repair cross-complementing CHO mutant cell line of complementation group 3 and XP group B; MDR, multidrug resistance; MGMT, 06-methylguanine-DNA methyltransferase; MLNr, cells selected for resistance to melphalan; CAT, chloramphenicol acetyl transferase; PARP, poly (ADP-ribose) polymerase; XP, xeroderma pigmentosum MUSICA.McGILL.CA. 0921-8777/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 092l-8777(95)00022-4 180 L. Yen et al. /Mutaiion proteins. The lack of correlation between enhanced host cell reactivation capacity in resistant cells, and the expression of these specific DNA repair genes suggests that proteins encoded by these genes are not rate limiting steps for resistance to bi-functional alkylating drugs in human breast cancer cells.
A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was d... more A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was developed for measuring mRNA concentration, in rodent cells, of theN-methylpurine–DNA glycosylase (MPG), a ubiquitous DNA repair protein responsible for the removal ofN-alkylpurines and ethenoadducts of adenine, guanine, and cytosine from DNA. The method, applicable for quantitation of any mRNA, is based on the standard approach of comparing the relative
In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylat... more In a systematic study to elucidate the involvement of pro- and anti-apoptotic proteins in alkylating drug resistance of tumor cells, we utilized the A2780(100) line, that was selected by repeated exposure of A2780 cell line (human ovarian carcinoma line) to chlorambucil (CBL). A2780(100) was 5–10-fold more resistant to nitrogen mustards (IC50 of 50–60 μM) and other DNA crosslinking agents, e.g.,
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