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Competitive nested polymerase chain reaction for quantification of human MDR1 gene expression
F Grünebach1, E U Griese, K Schumacher
1Department of Hematology, Oncology and Immunology, Robert-Bosch-Hospital, Stuttgart, Germany.
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 of MDR1 mRNA. This assay uses a transcript generated in vitro as an internal standard which is later coamplified together with the MDR1 cDNA. Both cDNAs exhibit the same MDR1 primer sites but differ in the length of the amplicon. For a second round of amplification we applied nested MDR1 primers and were successful in improving the sensitivity of this competitive PCR system. This test for characterizing the MDR1 expression offers high sensitivity and specificity and is therefore of great clinical relevance. It should be useful in improving monitoring and design of chemotherapy.
Insights
A new competitive PCR assay quantifies multidrug resistance (MDR) gene expression. This method accurately measures MDR1 mRNA levels, aiding chemotherapy monitoring and design.
Area of Science:
- Molecular Biology
- Oncology
- Biochemistry
Background:
- Multidrug resistance (MDR) is a major challenge in chemotherapy, characterized by cellular resistance to various drugs.
- MDR involves biochemical changes like increased glutathione, altered nuclear enzymes (e.g., topoisomerase II), enhanced DNA repair, and P-glycoprotein (gp-170) overexpression.
- P-glycoprotein, encoded by the MDR1 gene, actively pumps drugs out of cells, reducing intracellular drug accumulation and causing resistance.
Purpose of the Study:
- To develop a highly sensitive and specific assay for absolute quantification of MDR1 mRNA.
- To provide a tool for characterizing multidrug resistance gene expression in clinical settings.
Main Methods:
- Development of a competitive polymerase chain reaction (PCR) assay.
- Utilized an in vitro-generated transcript as an internal standard for coamplification with MDR1 cDNA.
- Employed nested MDR1 primers in a second amplification round to enhance assay sensitivity.
Main Results:
- The competitive PCR assay allows for absolute quantification of MDR1 mRNA.
- Nested primers significantly improved the sensitivity of the competitive PCR system.
- The assay demonstrated high sensitivity and specificity for MDR1 expression analysis.
Conclusions:
- The developed competitive PCR assay is a valuable tool for precise MDR1 mRNA quantification.
- This assay has significant clinical relevance for monitoring chemotherapy effectiveness.
- It can aid in the improved design and optimization of chemotherapy regimens.
