HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress

The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta (polβΔ208−304) specific for ovarian cancer. Pol βΔ208-304 has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. PolβΔ 208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H 2 O 2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol βΔ 208-304 transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.


Introduction
Human body is being exposed to different kinds of carcinogens, radiation every day.These constant bombardments are causing the DNA damage both in the nucleus and in the mitochondria.In order to manage these high frequencies of DNA damage, nature has developed different DNA repair mechanisms that can be run in parallel to repair the DNA.Base Excision Repair (BER) pathway is one of these pathways, that highly involves the repair of alkylating agent mediated DNA damage by single nucleotide repair pathway (short-patch repair pathway) and the other 2-8 nt repair pathway or long patch repair pathway (Sobol et al., 1996;Fortini et al., 2003;Slupphaug et al., 2003).
The loss of BER activity due to the inefficient function of any of these BER proteins may lead to or further contributes to the development of cancer, neurodegenerative disorders; etc (Singhal e al., 1995;Wilson et al., 2000;Idris et al., 2002).In order to repair efficiently, the activities of BER proteins are tightly regulated.DNA polymerase β is the smallest enzyme in BER pathway.It belongs to the X family of polymerase (Wilson et al., 2000).It is a single copy 34kb gene consisting of 14 exons (Sobol et al., 1996).It is located

RESEARCH ARTICLE
HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress Kalyani Khanra 1 Anindita Chakraborty 2 , Nandan Bhattacharyya 1 * in chromosome no 8p12 (Beard and Wilson, 2006).DNA Pol β encodes a 39 kDa protein consisting of 335 amino acids with two subunits, small subunit of 8 kDa (1 amino acids residues to 87 amino acids) and large subunit of 31 kDa (88 amino acids residues to 335 amino acids) connected by a protease sensitive hinge region (Beard & Wilson, 2000).The catalytic property of DNA polymerase β resides in the Carboxyl terminal region of the 31 kDa domain, whereas the amino terminal 8 kDa domain shows a single strand DNA binding activity.This small subunit have single strand DNA binding activity and dRP lyase activity encoded by exons I-IV, its activity is necessary to direct Pol β to short gaps possessing a 5'-phosphate termini (Wilson et al., 2000;Sobol and Wilson, 2001).The large subunit has double strand DNA binding activity and catalytic activity.
DNA polymerase beta is linked to cancer which was detected in different cell lines and tumor tissue sample of cancer (Bhattacharyya et al., 1999a;Bhattacharyya et al., 1999b;Chen et al., 2000;Bhattacharyya et al., 2001;Chen et al., 2002;Khanra et al., 2012aKhanra et al., , 2012bKhanra et al., , 2012d)).It was claimed that 30% cases of cancer polymerase beta have mutation (Starcevic et al., 2004;Lang et al., 2007).In gastric cancer patients out of 20 samples 6 (30%) have pol beta mutation (Iwanaga et al., 1999).In colorectal cancer 6 in 8 patients (75%) have polymerase beta mutation (Lang et al., 2004).In breast cancer, 26%, esophageal cancer, 40% have mutation (Dong et al., 2002).Almost 36% tumors express polymerase beta variant in which deletion of exon 11 is highly common.This variant of polymerase beta is expressed in colorectal, breast, lungs adenocarcinomas (Sweasy et al., 2005).In colorectal cancer K289M (Sweasy et al., 2005) was detected which has less BER activity.Expression of cancer-associated polβ variants in mouse cells could lead to a series of cancer associated phenotypes, including an increased mutation frequency and the induction of cellular transformation (Sweasy et al., 2005).The gastric cancer associated variant E295K has a phenotype that could be related with etiology of cancer (Lang et al., 2007).This variant is unable to catalyze DNA synthesis but have DNA binding and dRP lyase activity same as wild type (WT) polβ (Lang et al., 2007).Lang et al (2007) experimentally showed that this mutant when expressed in MEFs, conferred a dominantnegative phenotype and sensitivity to MMS, induced more sister chromatid exchanges (SECs) per nucleus than Wild type (WT) and it causes cellular transformation that could lead to tumorigenesis or tumor progression (Lang et al., 2004).Another Gastric cancer associated variant Leu22Pro (L22P) exhibits little dRP lyase activity but retain its polymerase activity.In 22 residues of DNA polβ has no direct contact with the DNA, L22P variant has reduced DNA binding affinity.The L22P variant protein is deficient in base excision repair (Dalal et al., 2008).In another Gastric cancer variant (cys239arg), cys 239 is located in inflexible loop region that when altered reduced the accuracy of DNA synthesis Dobashi et al., 1994.In prostate cancer, I260M variant is located in hinge region of polymerase beta (Dalal et al., 2005).Ile260 is a key residue of the hydrophobic hinge.This hinge influences the geometry of the DNA within the polymerase active site that is important for accurate DNA synthesis and important for the closing of the polymerase and important for conformation change after substrate binding and fidelity of protein, results inaccurate DNA synthesis when replaced by Methionine.Characterization of the I260M variant shows that it has a functional phenotype that could be linked to the etiology or malignant progression of human cancer (Dalal et al., 2005).Another DNA polβ variant D246V has been reported which is less efficient than WT for incorporation of AZT in a recessed primer template (Kosa et al., 1999).This Asp residues at position 246 helps to maintain the proper positioning of the DNA within the active site of DNA polβ.Valine at the tip of the loop results in mis-incorporation resulting from an altered or misaligned DNA structure within the active site (Kosa et al., 1999;Dalal et al., 2004).Bhattacharyya et al (1999) reported three variant of polβ in squamous, non-small or large cell carcinomas.The common variant was large deletion of 87 bp of exon 11 which lies in catalytic part of polymerase beta.Another variant of 140bp deletion along with addition of exon alpha was detected.Functional study of 87 bp deleted DNA polβ shows that it acts as dominant negative manner.This variant is unable to bind properly to double strand DNA and has lost repair activity (Bhattacharyya et al., 1997;Bhattacharyya et al., 2000) thus acts as a dominant negative manner.In addition, when this variant is expressed in Mouse Embryonic fibroblast (MEFs) cell line, which induced tumor occurrence in nude mice (Sweasy et al., 2005).So above evidence indicate that Polβ variants are linked to etiology of cancer.
Genomic stability is disrupted by environmental carcinogen as well as metabolically produced oxidative stress, metabolic by-product, errors during DNA replication and DNA recombination contribute to the mutations.It is estimated that BER repairs 10 4 lesions per cell per day and this proofreading is vital to avoid genomic instability (Lindahl, 1993;Yamtich and Sweasy, 2010).By and large, under normal circumstances, this immense error burden is successfully dealt with by the highly coordinated cellular DNA repair mechanisms thus maintaining genomic integrity.
Alkylating agents are mono-functional or bi-functional (two reactive groups) carcinogen that are able to form intra-chain and inter-chain cross-links on DNA directly.Nucleotides can be alkylated at several different positions in nitrogen and oxygen and it's biological consequences are diverse (Friedbarg EC 2003).MMS, NMU are mono alkylating agent directly incorporated in DNA.MMS is an SN2 alkylating agent that interacts with nucleophilic centers forms 7-methylguanine and 3'-methyladenine adducts in DNA (Hoffman & Hecht 1985;Gu et al., 2010).NMU add methyl group to DNA.Endogenous alkylating agent O6-meG interferes base pairing and cause G:C to A:T transition mutations.Other endogenous alkylating agents 7-methylguanines (7meG) are unstable and spontaneously degrade to AP sites create single strand break (Ochs et al., 1999;Oshima et al., 2003).It was detected that Polβ knockout cell lines are highly sensitive to methylating agents.
A UV (100-400 nm) ray is a strong mutagenic agent which creates various lesions.UV A (320-400 nm) is mutagenic consists mostly of single strand breaks in DNA (Mouret et al., 2006;Kong et al., 2009).UVB (295-320 nm) is involved in direct formation of thymine dimer or other pyrimidine dimer double strand DNA breaks.UV-C (100-295 nm), can generate hydroxyl (•OH) and oxygen radicals(•O2), •OH mainly attacks DNA integrity by removing hydrogen from the deoxyribose backbone or addition of double bonds to DNA bases or resulting base modification including multitude of altered bases, base loss, and strand breaks.The most common lesions created by UV are cyclobutane pyrimidine dimer (CPDs) and Pyrimidine-pyrimidine (6-4) photoproducts (6,4-PP) at the adjacent pyrimidine.The formation of a pyrimidine dimer causes a bend in the DNA helix and DNA polymerase cannot read the DNA template as a result DNA replication is prevented by these pyrimidine dimer, which leads to mutation.The formation of a pyrimidione (6-4) photoproduct mimics an abasic site (Douki et al., 2003).
In our lab we have reported some point mutations in the genomic DNA of ovarian tumor samples (Khanra et al., 2012a,bc).These mutations were detected at exon intron junction region of exon 8, exon 9 and exon 11 by SSCP analysis (Khanra et al., 2012c).We also have detected cDNA mutation in these samples by RT-PCR DOI:http://dx.doi.org/10.7314/APJCP.2015.16.18.8177HeLa Cells Containing a Truncated Form of DNA Polymerase beta are Sensitized to Alkylating Agents and Western blot analysis (Khanra et al., 2012d).One of the cDNA mutations have deletion of exon 11-13 which lies in catalytic part of DNA polymerase beta.Exon 11 consists of 29 amino acids in the region of 208-236, Exon 12 consists of 21 amino acids in the region of 237-258, exon 13 is 45 amino acids which resides within amino acid residues 259-304.
In this report, the functional consequence of the loss of three exons of polβ in response to alkylating agents such as Methyl methanesulfonate (MMS) and N-methyl-Nnitrosoureaa (NMU) and oxidative stress inducing agents such as UV & H 2 O 2 have been studied.The present variant of polβ detected in ovarian cancer (Khanra et al., 2012d) has lost exon 11-13 that catalytic part is responsible for dNTP selection, nucleotidyl transferase activity and it would be interesting to determine the function of this mutant in response to alkylating agent, UV and oxidative agents.

Cloning of truncated form of DNA polymerase beta in PCDNAIneo vector
PolβΔ208-304 cDNA was amplified from tumor RNA sample using primers of full length polβ cDNA (Khanra et al., 2012d) and cloned in a vector pcDNAIneo (Invitrogen).The orientation and sequence of the mutated product was confirmed by sequencing before transecting vector pcDNAI/neo in HeLa cell line.

Transfection of PolβΔ 208−304 in HeLa cell line and stable cell line preparation
HeLa cells were cultured in DMEM medium with 10% fetal calf serum (Gibco; USA) and 100 U/mL penicillin and 100U/mL streptomycin at 37°C in a humidified atmosphere with 5% CO 2 in an incubator (SANYO, Japan).Cells were transfected by calcium phosphate coprecipitation method and Transfected cells were selected in G418 containing medium 100μg/μl.

RT-PCR analysis for expression of truncated from of Polβ
RNAs were isolated from PolβΔ 208−304 transfected cell line, HeLa cell line and only vector transfected cell line without PolβΔ 208−304 and RT-PCR was done to detect the expression of PolβΔ 208−304 in transcriptional level.As per manufacturers' instruction RNAs were isolated using RNA isolation kit (Trisure, Bioline).To eliminate the possibility of genomic DNA contamination, reaction was also set up without reversed transcriptase enzyme

Western blot analysis for detecting expression of truncated DNA polymerase beta
After preparing stable cell line, cell lysates were made and western blot (Bhattacharyya et al., 2001) was done to confirm transfection of PolβΔ 208−304 in cell line.Total cell lysates were isolated from transfected cell lines and control cell lines by using lysis buffer containing 10mM Tris-HCl (PH 8), 1mM EDTA, 400mM NaCl, 2mM beta mercaptoethanol, freshly prepared 1mM PMSF, and protease inhibitors (Roche applied Science).Proteins were denatured in 900 C with SDS loading dye separated on 12.5 % SDS-PAGE gel.Detection of protein was done by using anti-Pol β monoclonal primary antibody (Novus) by diluting 1:5000 in Phosphate-Buffered Saline (PBS) containing 0.05% Tween-20 and 5% nonfat, dry milk.Incubation with secondary antibodies (SC-2020; 1:10,000) and detection reaction were performed according to manufacturer's instruction (PIERCE).

Study of Cytotoxicity
2.5.1.Alkylating agent (MMS, NMU) and UV sensitivity assay by MTT: HeLa cell and PolβΔ 208−304 transfected cells are plated in 96 well plates.After 12-24 hrs cell medium was discarded.Cells were treated with alkylating agent (NMU, MMS) of different concentrations.Then after 24hrs 5mg/ml concentration of MTT (dissolved in PBS) were added in each well.After 4 hours MTT was removed from each well and 100μl DMSO was added in each well.Absorbency was measured in 540 nm (Hermann et al., 1994;Hirsh et al., 2009).The sensitivity of PolβΔ 208−304 in chemical treatment was detected by calculating rate of survival using following formula: inhibition rate (%) = (1 -OD A1/A0) x100.Here A1 is absorbency of treated cells, and A0 is absorbency of control cell without treatment.Cell plated in above mentioned protocol and treated after 24 hrs with MMS different doses in Serum free medium.All the assays were done in triplicate.

H 2 O 2 , UV sensitivity detection by MTT assay
C e l l s u r v i v a b i l i t y w a s a n a l y z e d u s i n g a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay (Sigma, MO, USA) after treatment with H202, UV (Krahn et al., 2004).HeLa cells and PolβΔ 208−304 transfected cells were plated onto 96 well plates (1500/well) and after 24hrs medium was discarded and serum free medium was added before 1 hr of treatment.H 2 O 2 of different concentrations (12μM -1000μM) was added, medium was discarded after 1 hour and new medium added after 12 and half hours cells treated with 10 μl MTT dissolved in PBS.After 3 and half hours, MTT was removed from each well and 100μl DMSO was added to each well.
For UV radiation experiment, cells were exposed to different doses of UV light (Stratagene UV-C bulbs, 254 nm) at a dose in between 5-50 J/m 2 .Then 100μl of serum free medium was added to each well.After 22 hours of treatment, 10μl MTT (dissolved in PBS) was added with a final concentration of 0.5mg /ml.After 4 hours, medium was removed from each well and 100μl DMSO was added to each well.Absorbency measured at 540 nM.All the assays were done in triplicate.

Colony forming assay
Exponentially grown cells were seeded in 35 mm dishes (300/ml) and next day treated with UV at 5-15 J/ m 2 , MMS of 0.125-2μM, NMU of 1-100 μM, H 2 O 2 of 0.05 to 2 μM concentrations.After treatment, the cells were washed with PBS and fresh medium was added and cultured in complete medium for 2 weeks.After 14 days of incubation, colonies were fixed with ethanol, stained with Giemsa (25% in ethanol), counted and calculated as percent of colony following this formula: Untreated/ controls x100.All the assays were done in triplicate.

Study of apoptosis
Sensitivity study by DNA fragmentation assay against MMS, NMU, H 2 O 2 , and UV: DNA PolβΔ 208−304 cells (5x105 cells) are seeded in 100mm dish.To assess the fragmentation of cellular DNA into the characteristic apoptotic ladder, cells were treated with the mutagen (UV, NMU, MMS, and H 2 O 2 ) of different time (6, 9, 12 h) post treatment incubation.Then cells were harvested by trypsinization and lysed in ice-cold cell lysis buffer (10 mM Tris, pH 7.5; 1 mM EDTA; 0.5% Triton X-100).Then lysed cells were incubated with 0.1 % SDS and 300 mg/ml of Proteinase K at 56°C overnight.Aqueous phases were extracted with phenol/chloroform and the DNA samples were precipitated with cold ethanol in the presence of ½ volume of 10M ammonium acetate, dried and dissolved by heating at 55oC in TE buffer before loading in a gel.DNA subjected to electrophoresis using 1.8 % agarose in Tris-borate buffer.DNA was visualized by staining with ethidium bromide and photographed in gel.

Caspase activation detection
For caspase 3, 9 activation assays, whole cell extracts were made from cells treated with UV and H 2 O 2 with either for different time period or concentrations.Cell lysates were prepared by using lysis buffer containing 20mM Tris-HCl pH 8, 150mM NaCl, 1mM of EDTA, 1mM EGTA and Protease inhibitor (Protease inhibitor mini kit; Roche applied Bioscience).Proteins were electrophoretically separated on 12.5% gel SDS-PAGE and transferred onto nitrocellulose membrane.The membrane was preincubated in TBST with 5% blocker (Fat free milk) for 30 mins.Then washed with TBST for 5 mins and incubated in primary antibody of caspase 3, caspase 9 (Cell signaling cat#9668, #9505; dilution 1:1000) for overnight as per manufacturer's instruction.Membrane was rinsed with TBST for 1 hr by changing TBST 4-5 times and incubated afterwards with HRP conjugated secondary antibody with 2.5 % blocker (Bio-Rad; cat#170-6516, Caspase3-Goat anti-mouse IgG; 1:1000; Cell signaling, cat #7074; HRP conjugate anti rabbit antibody 1:1000 conc.) for 1hr.After washing with TBST, bands were visualized by using Super Signal west pico (Pierce, IL).

Statistical analysis
Data were presented as mean ± standard deviation (SD).Analysis of variance (ANOVA) followed by Bonferroni's test was used to determine the significant differences between groups.Values of P less than 0.05 were considered significant.

Western blot analysis to detect expression of truncated product
PCR products were cloned in pcDNAIneo vector (Figure 1

Study of apoptosis
Sensitivity study by DNA fragmentation assay against MMS, NMU, H 2 O 2 , and UV: After treatment with toxic agent, DNA fragmentation was detected as small bands in a laddering pattern in 1.8% agarose gel.The results show that in NMU (100μM) and MMS (250μM) treatment the DNA fragmentation is prominent in 6 hrs in PolβΔ 208−304 cells whereas the fragmentation was detected in 9 hrs in treated HeLa cells.In higher doses of chemical treatment transfected cells are showing more degradation of DNA.

Caspase activation detection
To gain further insight into whether caspases are involved in alkylating agent mediated apoptotic signals, we determined the activation of caspase-9 and -3 in HeLa and PolβΔ 208−304 cells.Binding of cytochrome C to Apaf-1 results in the cleavage of procaspase-9, which in turn activates caspase-3.In order to determine whether caspases are activated we measured the changes in caspases-9 and -3 activity in HeLa and PolβΔ 208−304 cells after treatment.
In this study we noticed that after 6 hrs of treatment with NMU and MMS treatment PolβΔ 208−304 transfected cell activation started where no activation is observed in control cell line (Figure 6A, B, C, D).In the present study we also noticed UV and H 2 O 2 induced activation of caspase-3 in both HeLa and polβΔ cells, but the activation rate is same in PolβΔ 208−304 cells compared to HeLa cells (Figure 7).As shown in Figure 7, the activation of caspase 3 occurred at the dose of 15 J/m 2 in HeLa cell, whereas the same activation was detected in PolβΔ 208−304 cells at same dose.
Caspase 3 are activated at 6 hrs of UV treatment in case of HeLa control and PolβΔ 208−304 cell line indicate this cell line is sensitive to UV treatment but not due to transfection of polβΔ, the activation occurs as early as 6 hrs.Therefore this result suggests that PolβΔ 208−304 are not sensitive to UV treatment.In case of H 2 O 2 after 6hrs of (500μM H 2 O 2 ) treatment caspase activation occurs in control and transfected cell line.

Discussion
PolβΔ 208−304 variant has large deletion of 97 amino acids in the catalytic part of amino acids residues 208 to 304.The variant of 716 bp has deletion in Exon 11-13.The exon 11 has nucleotidyl selection activity; exons 12 and 13 have dNTPs selection activity (Idris et al., 2006).Hence deletion of this part may affect polymerizing activity but single strand binding and double strand binding activity presumed to be remained same that have nucleotidyl transferase activity and dNTPs selection activity.In this variant of DNA polβ one of the three aspartates at position 256 (Asp190, Asp192, and Asp256) necessary for catalysis reaction is missing which is located at exon 12 [24][25][26] and Amino acids Tyr271, Phe272, Asn 279 and Arg283 located in catalytic domain that have role in minor groove interaction thus maintaining the fidelity of the polymerization are also missing (Dong et al., 2002;Sweasy et al., 2005;Lang et al., 2007).The fidelity of polymerization will also be affected due to deletion of Exon 13.Bhattacharyya and Banerjee (1997) have shown that 87 bp deletion of polβ in breast, colon, and lung cancer has a dominant negative (DN) effect (Bhattacharyya and Banerjee 1997).As the alteration is heterozygous, this variant will compete with WT polβ for DNA binding, thus may act as dominant negative manner.The hindrance of the function of wild-type polβ may sensitize the cancer cells to the alkylating agent and thus may the target of an effective therapy.
The Polβ-DNA complex binds to the incoming nucleoside triphosphate (dNTP) and place it within the active site.Subsequently, conformational changes occur, the incorrect dNTP binding results in a poor fit, and disrupt DNA repair mechanism.As this newly identified mutant lost dNTPs selection region it may incorporate wrong bases that may results poor fitting or no repair.
It has been shown that Mouse embryonic fibroblast cells deficient in polβ are highly sensitive to alkylating agents.The lesions produced by these agents are repaired by BER, thus confirming the role of polb in this repair pathway (Sobol et al., 1996;Ochs et al., 1999).If this variant is a dominant negative one, then it is thought to bind to glycosilase and AP endonuclease treated DNA sites hence may block access of wild type polβ.If this hypothesis is true, then we may expect an increased sensitivity of polβΔ 208−304 variant expressing HeLa cells to alkylating drugs.Significant increase in alkylating drug sensitivity was observed in the polβΔ cells suggesting inhibition of BER (Figure 2,3,4).
The MTT assay results have shown that the polβΔ 208−304 cells are more sensitive to MMS as well as NMU (Figure 2.a &b).In this study, the MMS, NMU treatment of polβΔ 208−304 cells have showed enhanced DNA fragmentation than that of HeLa cells.DNA laddering of polβΔ 208−304 cells was apparent at lower doses of MMS, NMU (Figure 5).
If we assume that polβΔ 208−304 is a dominant negative mutant, then these cells are not expected to be more sensitive to UV irradiation.The nucleotide excision repair (NER) pathway repairs DNA damage resulting from UVC irradiation which produces primarily pyrimidine dimmers.Polβ is not involved in this process.No significant difference in UVC sensitivity could be observed between HeLa cells and HeLa polβΔ 208−304 cells (Figure 2d, 4b).This data also indicates that the presumed dominant negative mutant of polβ does not interfere with NER.
Like ionizing radiation, causes oxidation at abasic sites, purines and pyrimidines, and SSBs occurs by H 2 O 2 treatment (Li et al., 1997).H 2 O 2 -treated polβ null and normal cells show no difference between repair of SSBs, indicating polβ independent repair of DNA damage (Peus et al., 1999).The oxidized AP sites may not be effectively excised by polβ after H 2 O 2 treatment (Pelle et al., 1990).In our study, polβΔ208-304 cells are showing same sensitivity as that of the HeLa control cell line.Therefore, the data also indicates that radiation and H 2 O 2 induced damage is being repaired by polβ-independent long patch repair pathway.
As this variant has its 8-kDa ss DNA binding domain is intact, it will bind to the SSBs and the 5'-deoxyribose phosphate will be removed by the dRP lyase activity of polβ.But as the catalytic domain is not intact, the sealing of the abasic site may not be possible.Therefore SSBs will be accumulated that may trigger of cell death particularly apoptosis.Therefore, we measured the mitochondrial damage related caspases such as caspase 9 and caspase 3.In addition, as the activation of caspase 9 and caspase 3 activity leads to PARP degradation, we have compared the PARP degradation pattern in these two cell lines.The first step for the reactive oxygen species (ROS) mediated apoptosis is that cytochrome c binds to Apaf thus forming a complex called apoptosome which recruits and activates procaspase 9 Then caspase 9 cleaves inactive procaspase 3 to form active caspase 3. We thus examined the effects of MMS, NMU, H 2 O 2 and UV on the processing of caspase 9 and caspase 3, which are directly activated by caspase 9 (Hussain et al., 1995;Lawley et al., 2000).The results of caspase assay also indicate more activation of caspases in polβΔ 208−304 transfected cell line.
In conclusion, we have identified a variant form of polβ, which is specific for ovarian cancer, has a potential to be a dominant negative mutant.This presumed to be polβ DN mutant, has intact N-terminal DNA binding domain may be disrupting the function of the WT polβ by binding to the specific damaged DNA (AP sites).As this DN mutant lost it's catalytic domain partially thereby lost the catalytic ability.In addition, when this DN mutant binds to the gap it may also hinder the binding of other DNA polymerases.Hence it may inhibit both SP BER and LP BER.Similar observation was made (Clairmont and Sweasy, 1996;Clairmont et al., 1998) where a polβ mutant containing yeast cells are sensitive to alkylating agent but not to UV indicating an involvement in BER but not in NER.Another study by Bhattacharyya and Banerjee showed an heterozygous variant form without catalytic domain exhibited substantially decreased BER activity and increased sensitivity to MNNG (Bhattacharyya and Banerjee, 1997).The alkylated DNA is not exclusively repaired by the BER pathway but mismatch repair is also involved in the removal of damage resulting from alkylating agents (deWind et al., 1999).But in this case the polβΔ 208−304 construct also blocks this escape route.
The activation of caspase 9 became apparent and progressed at least up to 6 hours (Figure 7) after alkylating agent treatment in polβΔ 208−304 than control cell line.polβΔ 208−304 transfected cells susceptible to chemotherapeutic agent like alkylating agent.This would represent a clinical significance if translated into clinical setting because resistance to radiation or chemo during the relapse of the disease could be potentially overcome by this approach.Hence hopefully in the near future, strategies based on RNAi will be ready for preclinical or clinical trials.
.a).Presence of a variant was confirmed by digestion with restriction enzyme (Fig1.b).The variant clones were transfected into HeLa cell and the stable cell lines were generated.The expression of the variant product was confirmed by RT-PCR (Figure 1.c) and western blot analysis (Figure 1.d).For further analyses, Alkylating agent sensitivity detection by MTT assay Cytotoxicity of PolβΔ 208−304 cells were determined and compared with the control HeLa cells and HeLa transfected with vector alone (data not shown) by the MTT assay.Cell cytotoxicity assay indicate that transfected cell are more sensitive to NMU and MMS than control cell line.Treatment of PolβΔ 208−304 cells and control cells with MMS (1μM -750μM) results in the decrease of the percentage of cell survival.Treatment of this cell lines with 10 μM MMS resulted in the 89% cell survival compared to 94% cell survival noticed in HeLa control cells.In 250μM doses of MMS, 41% control cells are dead when same concentration of MMS, resulted in 70 % of cell death in transfected cell line.These results showed that the PolβΔ 208−304 cells were more sensitive to MMS treatment than parental HeLa cell line (Figure 2.a).Similar trend were observed when PolβΔ 208−304 cells were treated with NMU (1-500μM) (Figure 2.a).The results show that almost 50% HeLa cells died after treatment with 250μM NMU (Figure 2.b) in control cell whereas 89% PolβΔ 208−304 transfected cells died at the same concentration of NMU.Treatment resulted in the death of 21%, 33%, 51%, and 75% of cell death at concentrations of 50μM, 100μM, and 250μM, 500uM respectively in HeLa cells where PolβΔ 208−304 exhibited 34%, 65%, 91%, 98% of cell death at the same concentrations of NMU.These data indicated that PolβΔ 208−304 could increase the sensitivity towards NMU and MMS at a significant level.

Figure 1 .
Figure 1. a Schematic Construction of recombinant plasmid inserted with PolβΔ 208−304 .b Cloning of PolβΔ 208−304 in vector.M; Marker, L1; vector without insert, L2; vector with insert of 716bp.c RT-PCR product of PolβΔ 208−304 transfected cell line and normal cell line and vector alone.M; Marker, L1 and L2: RT product of PolβΔ 208−304 transfected cell line 1 and cell line ; L3: RT-PCR product of control cell line and L4: Hela cell transfected with vector alone.d Detection of Expression of DNA polβ by western blot analysis.L1and L2: PolβΔ 208−304 transfected cell line 1 and 2 respectively.L3: Hela cell line without transfection

Figure 2 .Figure 4 .
Figure 2. a & b Effects of Carcinogen on Cell Viability were Examined by Using the MTT (thiazolyl blue) assay.This assay monitors conversion of MTT to insoluble purple formazan in a reaction catalyzed by mitochondrial dehydrogenases in viable cells.-s-denotes HeLa cells, whereas -n-indicates HeLapolβΔ cells.a; Graphically represent Cell viability along with treatment of MMS of different concentration.b; Cell viability with different concentration of NMU.c; Cell viability with different concentration of H2O2, d; Cell viability with different concentration of UV.The data represent mean±SD (n=3)

Figure 5 .C
Figure 5. a&b: Study of Apoptosis by DNA Fragmentation after Treating with UV(10 J/m 2 ).a; DNA laddering after treatment with UV in HeLa cell line b; DNA laddering after treatment with UV in pol βΔ208-304 cell line respectively.In this figure, C indicates control, L1indicate UV treatment after 6 hrs, L2 indicate UV treatment after 9 hrs, L3 indicate UV treatment after 12hrs, L4indicate UV treatment after 24hrs.c &d: Study of apoptosis by DNA laddering after treatment with NMU (100μM).c; the DNA laddering after treatment with doses of NMU in HeLa cell line d; DNA laddering after treatment with NMU in pol βΔ208-304 cell line respectively.C indicate Control, L1 indicate NMU treatment after 6 hrs, L2 indicate NMU treatment after 9hrs, L3 indicate NMU treatment after 12hrs, L4 indicate NMU treatment after 24hrs.e &f: DNA laddering showing after treatment with MMS (250μM).e; the DNA laddering after treatment with doses of MMS in HeLa cell line h; DNA laddering after treatment with MMS in pol βΔ208-304 cell line respectively.1 indicates control; 2 indicate MMS treatment after 3 hrs; 3 indicate MMS treatment after 6hrs; 4 indicate MMS treatment after 9hrs; 5 indicate MMS treatment after 12hrs.g &h Study of apoptosis by DNA fragmentation assay g; the DNA laddering after treatment with different doses of H 2 O 2 in HeLa cell line h; DNA laddering after treatment with H 2 O 2 in pol βΔ208-304 cell line respectively.C indicate control, L1 indicate treatment of 25μM H 2 O 2 ,L2 indicate 50μM H 2 O 2 , L3 indicate 100 μM H 2 O 2

Figure 6 .
Figure 6.Study of Apoptosis by Caspase assay Detected by Western blot Analysis in HeLa Cell Line and PolβΔ208−304 Cell Line of Different Time Period.Here C indicate cell line of without treatment, 6hrs, 9hrs, 12hrs indicate cell line with treatment after 6hrs, 9hrs, 12hrs respectively.A: caspase 3 activation by treatment with MMS (100µM).B. Caspase 9 activation by MMS (100 µM) treatment.C: Caspase 3 activation by NMU (250µM) treatment.D: Caspase 9 activation by NMU (250µM) treatment Wild type (WT) polβ consists of 335 amino acid residues.The variant form has a deletion of 97 amino acids in catalytic part.The sensitivity of this new cell line, PolβΔ 208−304 , was tested by MTT assay after treating cells with alkylating agents and oxidative stress generating agent UV, H 2 O 2 .PolβΔ 208−304 cells exhibit greater sensitivity to alkylating agent and less sensitivity towards H 2 O 2 .PolβΔ 208−304 has deletion of exon 11-13.