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.
Malapetsa
A
A
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Québec, Canada.
Noë
A J
AJ
Poirier
G G
GG
Desnoyers
S
S
Berger
N A
NA
Panasci
L C
LC
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