COMPETITIVE NESTED POLYMERASE CHAIN-REACTION FOR QUANTIFICATION OF HUMAN MDR1 GENE-EXPRESSION

Tumor cell resistance to cytotoxic drugs is considered one of the majo r obstacles to successful chemotherapy. Multidrug resistance (MDR) des cribes the simultaneous expression of cellular resistance to a wide ra nge of structurally and functionally unrelated drugs. The development of the multidrug resistance phenotype is accompanied by multiple morph ological and biochemical changes: (a) increased glutathione levels in the cytoplasm, (b) modified levels of enzymes in the nucleus, particul arly topoisomerase II, (c) increased DNA repair capacity and (d) overe xpression of the (human) MDR1 gene encoding a transmembrane efflux pum p (P-glycoprotein, gp-170), which leads to decreased intracellular acc umulation and therefore to resistance to a variety of cytotoxic drugs. In this report we describe a competitive polymerase chain reaction (P CR) assay for the absolute quantification of MDR1 mRNA. This assay use s a transcript generated in vitro as an internal standard which is lat er coamplified together with the MDR1 cDNA. Both cDNAs exhibit the sam e MDR1 primer sites but differ in the length of the amplicon. For a se cond round of amplification we applied nested MDR1 primers and were su ccessful in improving the sensitivity of this competitive PCR system. This test for characterizing the MDR1 expression offers high sensitivi ty and specificity and is therefore of great clinical relevance. It sh ould be useful in improving monitoring and design of chemotherapy.