Correlation of chloroethylnitrosourea resistance with ERCC-2 expression in human tumor cell lines as determined by quantitative competitive polymerase chain reaction.
We have developed a method to quantitate ERCC-2 gene expression in tumor cell lines. A mutant ERCC-2 DNA fragment (1-bp mutation) is used as a competitive DNA template in a coamplification PCR reaction with cDNA obtained by reverse transcribing DNase-free total RNA from six human tumor cell lines. The PCR products are separated on agarose gel by virtue of their differential banding pattern upon restriction enzyme digestion. Densitometric readings of the PCR products from a negative film of the gel are used to establish a linear regression curve, which in turn is used to quantitate ERCC-2 levels. Beta-actin expression is similarly quantitated. Normalized ERCC-2 gene expression (either to beta-actin or to total RNA) correlates with cytotoxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea or (2-chloroethyl)-3-sarcosinamide-1-nitrosourea, suggesting that ERCC-2 may play an important role in drug resistance in these cell lines. This method is reliable and can be used to quantitate gene expression in clinical tumor specimens.
Chen
Z P
ZP
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Montreal Quebec, Canada.
Malapetsa
A
A
Marcantonio
D
D
Mohr
G
G
Brien
S
S
Panasci
L C
LC
D000970:Antineoplastic Agents / Q000633:toxicity; D001483:Base Sequence; D002330:Carmustine / Q000031:analogs & derivatives* / Q000633:toxicity*; D004265:DNA Helicases; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins; D004351:Drug Resistance; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D051759:Xeroderma Pigmentosum Group D Protein
D000970:Antineoplastic Agents; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011506:Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; C044565:2-((((2-chloroethyl)nitrosoamino)carbonyl)amino)propanamide; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human; D002330:Carmustine