Identification of a 116 kDa protein able to bind 1,3-bis(2-chloroethyl)-1-nitrosourea-damaged DNA as poly(ADP-ribose) polymerase.
SKI-1 is a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant glioma cell line and SK-MG-1 is a BCNU-sensitive glioma cell line. Both cell lines do not express O6-methylguanine-DNA methyl transferase (MGMT) and exhibit comparable levels of 3-methyladenine DNA glycosylase. In order to detect DNA binding proteins involved in alternative DNA repair mechanisms of BCNU damage, we performed Southwestern analysis using a DNA probe damaged with BCNU and nuclear protein extracts from SKI-1 and SK-MG-1 cell lines. Both cell lines express a protein of M(r) 116,000 that is able to bind to BCNU-damaged DNA with higher specificity than to undamaged DNA. This protein was identified as poly(ADP-ribose) polymerase (PARP). Using glioma extracts depleted of PARP or using antibody to block the DNA binding domain of PARP no other protein binding to BCNU-treated probe was observed. Addition of methoxyamine, an inhibitor of DNA strand breaks, led to a significant reduction of PARP binding to BCNU-treated DNA. BCNU treatment of both glioma cell lines led to reduced nicotinamide adenine dinucleotide levels, indicating activation of PARP. Thus, the recognition and binding of PARP to BCNU-induced DNA nicks with concomitant PARP activation may be important processes that are involved in the initial stage of DNA repair of BCNU lesions in glial cells.
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Québec, Canada.
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Malapetsa
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Noë
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Poirier
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Desnoyers
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Berger
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Panasci
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D015139:Blotting, Southern; D015153:Blotting, Western; D002330:Carmustine / Q000494:pharmacology*; D004249:DNA Damage; D015342:DNA Probes; D004260:DNA Repair / Q000502:physiology*; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D019008:Drug Resistance, Neoplasm; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005910:Glioma / Q000201:enzymology; D006801:Humans; D009243:NAD / Q000032:analysis; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
D018906:Antineoplastic Agents, Alkylating; D015342:DNA Probes; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D002330:Carmustine