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[Proc Amer Assoc Cancer Res, Volume 45, 2004]


Cellular, Molecular, and Tumor Biology 51: DNA Damage and Repair III

Abstract #2664

The effect of cisplatin adducts on double strand break repair by non-homologous end joining

Christine P. Diggle, Johanne Bentley, Margaret A. Knowles and Anne E. Kiltie

Cancer Research UK Clinical Centre, Leeds, United Kingdom

Chemoradiation is the combined treatment of patients with chemotherapy agents, such as cisplatin, and radiotherapy. It is now the standard treatment in several cancer types, and the subject of clinical trials in others. Radiotherapy treatment alone causes many types of DNA damage, the most lethal is the double strand break (DSB). If not repaired a single DSB can produce cellular death. The presence of a cisplatin adduct close to a DSB could physically hinder the cisplatin removal and repair by the nucleotide excision repair pathway, or the cisplatin adduct could hinder the DSB repair pathways. Repair of DSBs can occur via several pathways, one of which is the non-homologous end joining (NHEJ) pathway. In the basic NHEJ pathway a Ku heterodimer binds to the free ends of the broken DNA. DNA-PKcs is then recruited to the Ku heterodimer to form the DNA-PK holoenzyme. The Ku heterodimer translocates along the DNA strand as the DNA passes through its ring-like shape. The DNA-PK holoenzyme has kinase activity, which may be important in phosphorylating proteins in this pathway. Finally, XRCC4 stimulates DNA ligase IV to join the broken ends. Previous work has shown that cisplatinated DNA may decrease the rate of multiple exogenous Ku heterodimers translocating along the DNA strand and lowers the DNA-PK kinase activity. The objective of the present work was to examine the effect cisplatin adduct formation has on NHEJ proficiency in a more physiological environment, using cellular extracts and much larger DNA fragments of 3 kb in size. Double strand breaks were created by restriction enzyme digestion of plasmid DNA. This linearised DNA was treated with cisplatin and incubated with cellular extracts that have NHEJ activity. The presence of cisplatin dramatically reduced the level of NHEJ in a cisplatin dose dependent manner. As the cisplatin molecule interacts with the G bases of the DNA, we designed a new DNA substrate, where surrounding the restriction enzyme site there were minimal G bases for 25 base pairs. A second substrate was produced which in addition had a single GTG motif 10 bases from the DSB. A cisplatin adduct binding to this sequence produces an intra strand cisplatin adduct which can distort the local DNA structure. The DNA from the two new substrates was linearised with restriction enzyme, treated with cisplatin and subject to NHEJ. Once again a dose dependent effect was seen in the ability of cisplatin treated DNA to perform NHEJ. However, the presence of a GTG adduct had relatively little effect on NHEJ. To conclude, we have shown that the ability to perform NHEJ in a physiological environment was reduced if the DNA was cisplatinated. However the presence of a single additional adduct had minimal impact on NHEJ levels.







HOME HELP FEEDBACK HOW TO CITE ABSTRACTS ARCHIVE CME INFORMATION SEARCH
Cancer ResearchClinical Cancer Research
Cancer Epidemiology Biomarkers & PreventionMolecular Cancer Therapeutics
Molecular Cancer ResearchCancer Prevention Research
Cancer Prevention Journals PortalCancer Reviews Online
Annual Meeting Education BookMeeting Abstracts Online
Copyright © 2004 by the American Association for Cancer Research.