Competitive nested polymerase chain reaction for quantification of humanMDR1 gene expression

Abstract

Tumor cell resistance to cytotoxic drugs is considered one of the major obstacles to successful chemotherapy. Multidrug resistance (MDR) describes the simultaneous expression of cellular resistance to a wide range of structurally and functionally unrelated drugs. The development of the multidrug resistance phenotype is accompanied by multiple morphological and biochemical changes: (a) increased glutathione levels in the cytoplasm, (b) modified levels of enzymes in the nucleus, particularly topoisomerase II, (c) increased DNA repair capacity and (d) overexpression of the (human)MDR1 gene encoding a transmembrane efflux pump (P-glycoprotein, gp-170), which leads to decreased intracellular accumulation and therefore to resistance to a variety of cytotoxic drugs. In this report we describe a competitive polymerase chain reaction (PCR) assay for the absolute quantification ofMDR1 mRNA. This assay uses a transcript generatedin vitro as an internal standard which is later coamplified together with theMDR1 cDNA. Both cDNAs exhibit the sameMDR1 primer sites but differ in the length of the amplicon. For a second round of amplification we applied nestedMDR1 primers and were successful in improving the sensitivity of this competitive PCR system. This test for characterizing theMDR1 expression offers high sensitivity and specificity and is therefore of great clinical relevance. It should be useful in improving monitoring and design of chemotherapy.