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Correlation of chloroethylnitrosourea resistance with ERCC-2 expression in human tumor cell lines as determined by quantitative competitive polymerase chain reaction.
- Source :
-
Cancer research [Cancer Res] 1996 Jun 01; Vol. 56 (11), pp. 2475-8. - Publication Year :
- 1996
-
Abstract
- 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.
- Subjects :
- Antineoplastic Agents toxicity
Base Sequence
DNA Primers chemistry
Humans
Molecular Sequence Data
Polymerase Chain Reaction methods
RNA, Messenger genetics
Tumor Cells, Cultured
Xeroderma Pigmentosum Group D Protein
Carmustine analogs & derivatives
Carmustine toxicity
DNA Helicases
DNA-Binding Proteins
Drug Resistance
Gene Expression Regulation, Neoplastic
Proteins genetics
Transcription Factors
Subjects
Details
- Language :
- English
- ISSN :
- 0008-5472
- Volume :
- 56
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Cancer research
- Publication Type :
- Academic Journal
- Accession number :
- 8653679