Comparison of in vitro vs. in vivo formation of cis-platin DNA adducts
Comparison of in vitro vs. in vivo formation of cis-platin DNA adducts
INTRODUCTION: More than 50% of the compounds used in chemotherapy damage DNA. The most effective chemotherapeutic compounds induce DNA lesions that are slowly repaired, blocking DNA replication and cell cycle over a relatively long period of time after treatment. However cell cycle arrest induces events that promote DNA repair, making cells resistant to chemotherapeutic drugs. Among the most difficult DNA lesions to repair are DNA interstrand-crosslinks. Cis-platin induces multiple forms of DNA lesions: 1) intra-strand crosslinks (96%), 2) inter-strand crosslinks (1%), 3) mono adducts (~2%) and 4) DNA-protein crosslinks (<1%). The type of lesion formed by cis-platin depends on the DNA sequence. However, the torsion of DNA within the cell nucleus could also influence the DNA damage induced by cis-platin. Since in eukaryotic cells the DNA is heavily folded in a structure called chromatin, we propose that chromatin influences the type of DNA lesions induced by cis-platin.
HYPOTHESIS: In the cell, DNA undergoes periodical torsions within a structure called chromatin. We hypothesize that cis-platin induced DNA lesions in naked DNA in vitro are different than those induced in chromatin in vivo.
GOAL: To compare the formation of cis-platin induced DNA lesions in vitro vs. in vivo.
METHOD: The student will isolate genomic DNA from yeast cells. The DNA will be damaged in vitro using increasing amounts of cis-platin and then sheared to obtain a population of fragmented DNA having, on average, a length of ~1 kbp. Thereafter, the damaged DNA will be analyzed by HPLC to determine the type of DNA lesions. In parallel, yeast cells will be treated with cis-platin. The DNA will be isolated, sheared and analyzed by HPLC. The results will be compared and the percentage for each type of DNA lesion determined for both, in vitro and in vivo treatments.
In the laboratory we have the know-how to treat yeast cells with DNA damaging agents (Toussaint M and Conconi A (2006). High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents. NATURE Prot 1, 1922-1928). Moreover, Dr Richard Wagner - Department of radiobiology, Faculty of medicine, University of Sherbrooke - is a DNA chemist that studies DNA lesions by HPLC (Cadet J, Douki T, Ravanat JL, Wagner JR (2012). Measurement of oxidatively generated base damage to nucleic acids in cells: Facts and artifacts. Bioanal. Rev. 4, 55-74).
