Comparative evaluation by semiquantitative reverse transcriptase polymerase chain reaction of MDR1, MRP and GSTp gene expression in breast carcinomas

Abstract

Summary Identification and quantitative evaluation of drug resistance markers are essential to assess the impact of multidrug resistance (MDR) in clinical oncology. The MDR1 gene confers pleiotropic drug resistance in tumour cells, but other molecular mechanisms are also involved in drug resistance. In particular, the clinical pattern of expression of the other MDR-related genes is unclear and their interrelationships are still unknown. Here, we report standardization of the procedures used to determine a reliable method of semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) using a standard series of drug-sensitive and increasingly resistant cell lines to evaluate the expression of three MDR-related genes, i.e. MDR1 (multidrug resistance gene 1), MRP (multidrug resistance related protein) and GSTp (glutathione-S-transferase p), reported to be endogenous standard genes for normalization of mRNAs. A total of 74 breast cancer surgical biopsies, obtained before any treatment, were evaluated by this method. When compared with classical clinical and laboratory findings, GSTp mRNA level was higher in diploid tumours. However, the main finding of our study suggests a clear relationship between two of these MDR-related gene expressions, namely GSTp and MRP. This finding provides new insight into human breast tumours, which may possibly be linked to the glutathione conjugate carrier function of MRP. Well defined semiquantitative RT-PCR procedures can therefore constitute a powerful tool to investigate MDR phenotype at mRNA levels of different related genes in small and precious tumour biopsy specimens.

Prognostic impact of multidrug resistance gene expression on the management of breast cancer in the context of adjuvant therapy based on a series of 171 patients

“…In the previous study, published in 1998, based on a series of 74 patients, we observed a significant positive correlation between MRP1 and GSTpi expression (Lacave et al, 1998). This correlation was not confirmed in the present study, possibly because of a more scattered distribution of expression levels.…”

“…The conditions and methodological aspects of the end point rt -PCR procedures used in this study have been previously described in detail (Lacave et al, 1998). Although real-time PCR has been established as the gold standard rt -PCR procedure for gene expression studies since the end of the 1990s, most patients described here were included in the mid-1990s, at a time when real-time PCR was not routinely performed in the majority of laboratories.…”

“…In a previous study, we used a semiquantitative rt -PCR method to evaluate multidrug resistance gene expression in surgical breast cancer biopsies (Lacave et al, 1998). The primary objective of the present study was to complete this preliminary study by evaluating the clinical impact of the expression of these genes on the management of breast cancer in a series of 171 patients receiving adjuvant chemotherapy.…”

Molecular and functional MDR1‐Pgp and MRPs expression in human glioblastoma multiforme cell lines

“…Positive controls of specific gene expression were obtained with total RNA extracts from the HL60/DNR cell line for MDR1, 22 MCF7R cell line for MRP1, 23 human liver for MRP2 and MRP3, human lung for MRP4 and human skeletal muscle for MRP5. 7 Amplification of cDNA of ␤-actin, phospho-gluco kinase (PGK), and ␤ 2 -microglobulin (␤ 2 M) was used as an internal control for MDR1, MRP1 and MRP2-5, respectively.…”

“…Concerning MDR1 and MRP1 gene expression analysis, the RT-PCR method used has been already reported by our laboratory. 23 In order to check the accuracy of the RT-PCR method for MRP2-5 genes, PCR products of positive controls were sequenced on both strands using an ABI 310 genetic analyzer (PE Applied Biosystems, Inc., Courtaboeuf, France) and the BigDye Terminator sequencing kit (PE Applied Biosystems) according to the manufacturer's instructions. Sequence analysis using Sequence Analysis 2.1.1. software (PE Applied Biosystems) yielded the expected and previously published sequences (GenBank accession numbers U49248, AF083552, U83660 and U83661 for MRP2, MRP3, MRP4 and MRP5 genes, respectively).…”