Genomic Maintenance & Repair

Monday, October 17, 2005 3:00 PM-5:00 PM Exhibit Hall

(PP308) Detection of oxidative clustered DNA lesions in human cancerous cell lines.

Holt, Stewart*,1, Francisco, Dave1, Hada, Megumi1, Georgakilas, Alexandros1, 1 Biology Department, Greenville, NC, USA

ABSTRACT- One of the main etiological hypotheses in the promotion of mutagenesis, carcinogenesis and genomic instability is the accumulation of DNA damage and mutations due to infidelity or deficiencies of the repair mechanisms against endogenous or exogenous sources of DNA damage. Higher levels of endogenous oxidative stress, DNA damage and/or defective DNA repair have been reported in different malignancies and tumors. Oxidative clustered DNA lesions (OCDL) i.e., two or more closely spaced DNA lesions (within a short DNA fragment of 1-10 base pairs) including AP sites and oxidized purines/pyrimidines or single strand breaks (SSBs), are expected to challenge the cellular repair mechanisms and have a high mutagenic potential. To date there is limited data on the possible role of OCDL and in general complex DNA damage in the promotion of cancer. Two human cancerous cells lines (and their controls), NALM-6 lymphoblasts (acute lymphoblastic leukemia, ALL) and MCF-7 epithelial cells (breast cancer) have been used as our models. For the detection of OCDL (bi-stranded and adjacent) we have applied sensitive new adaptations of electrophoresis techniques like neutral or alkaline Pulsed Field Gel Electrophoresis (PFGE), neutral or alkaline Single Cell Gel Electrophoresis (SCGE) and by using different human or E. coli repair enzymes we have been able to detect the levels of endogenous and radiation-induced (low doses: 0-4 Gy) OCDL in NALM-6 or MCF-7 and compare them with their controls. In order to assess the role of OCDL in the promotion of genomic instability, we have also started detecting the levels of chromosomal aberrations in all cell lines using the techniques of multiplex FISH and BAND (mFISH and mBAND) with or without exposure to ionizing radiation. Our preliminary results suggest a strong association between clustered DNA damage and genomic instability expressed as increased levels of OCDL and chromosomal aberrations in both cancerous cell lines.

Key words: Clustered DNA damage, Repair, Chromosomal Aberrations, Cancer

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2005 RRS