Repair analysis of 4-hydroperoxycyclophosphamide-induced DNA interstrand crosslinking in the c-myc gene in 4-hydroperoxycyclophosphamide-sensitive and-resistant medulloblastoma cell lines |
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Authors: | Qing Dong Nancy Bullock Francis Ali-Osman O. Michael Colvin Darell D. Bigner Henry S. Friedman |
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Affiliation: | (1) Department of Pathology, Duke University Medical Center, 27710 Durham, NC, USA;(2) Department of Pediatrics, Duke University Medical Center, 27710 Durham, NC, USA;(3) Department of Experimental Pediatrics, M.D. Anderson Cancer Center, University of Texas, 77030 Houston, TX, USA;(4) The Johns Hopkins Oncology Center, Johns Hopkins University, 21205 Baltimore, MD, USA;(5) Preuss Laboratory for Brain Tumor Research, Duke University Medical Center, 27710 Durham, NC, USA |
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Abstract: | Cyclophosphamide is one of the most active agents in the treatment of medulloblastoma. However, development of resistance to this alkylator frequently occurs and is the harbinger of tumor progression and death. In order to understand the biochemical basis of this resistance, we generated a panel of medulloblastoma cell lines in our laboratory that were resistant to 4-hydroperoxycyclophosphamide (4-HC). Previously, we have shown that elevated levels of aldehyde dehydrogenase and glutathione mediate cellular resistance to 4-HC. The present study was conducted to identify the third unknown mechanism mediating the resistance of cell line D283 Med (4-HCR) to 4-HC, testing the hypothesis that this resistance is mediated by an increased repair of DNA interstrand crosslinks (ICLs). The doses of 4-HC that produced a one- and two-log cell kill of D283 Med cells were 25 and 50 M, respectively, compared with values of 125 and 165 M in D283 Med (4-HCR), the resistant cell line. The formation and disappearance of 4-HC-induced DNA ICLs at the c-myc gene were subsequently studied by DNA denaturing/renaturing gel electrophoresis and Southern blot analysis. 4-HC-induced DNA ICLs in the c-myc gene exhibited a dose-dependent relationship. The percentage of the c-myc gene that was crosslinked was approximately 1–3% at a dose of 100 M. More than 50% of the DNA crosslinking in D283 Med (4-HCR) cells was removed by 6 h after drug treatment, whereas, in D283 Med cells, more than 90% of the DNA crosslinking was still present at 6 h. These findings suggest that the increased repair of DNA ICLs in D283 Med (4-HCR) may contribute significantly to its resistance to 4-HC.This work was supported by NIH Grants CA 11898, CA 56115, NS 30245, NS 20093; and ACS Grant DHP-67E |
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Keywords: | 4-Hydroperoxycyclophosphamide c-myc DNA interstrand crosslinks |
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