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

Kalyani Khanra,Nandan Bhattacharyya,Anindita Chakraborty

doi:10.7314/apjcp.2015.16.18.8177

Kalyani Khanra, Nandan Bhattacharyya + Show 1 more

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https://doi.org/10.7314/apjcp.2015.16.18.8177

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Abstract

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 (polβ Δ208-304) specific for ovarian cancer. Pol β Δ208-304 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. Polβ Δ208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H2O2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol β Δ208-304 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.

Highlights

Human body is being exposed to different kinds of carcinogens, radiation every day
When PolβΔ208−304 cells were treated with H2O2 of different concentrations (0.01-1mM), almost 50% cell died in control cells after treatment with 0.5mM H2O2 for 6 hrs whereas cell death rate is much higher (66%) in PolβΔ208−304. cell line
The results show that in NMU (100μM) and Methyl methanesulfonate (MMS) (250μM) treatment the DNA fragmentation is prominent in 6 hrs in PolβΔ208−304 cells whereas the fragmentation was detected in 9 hrs in treated HeLa cells

Summary

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. 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

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