SEMIAUTOMATED PCR METHOD FOR QUANTITATING MDR1 EXPRESSION

The MDR1 gene is involved in drug resistance in many hematopoietic and solid tumors. The Quantitative PCR system 5000(TM)(QPCR-5000; Perkin- Elmer) is a new instillment system that uses electrochemiluminescence to automatically quantitate polymerase chain reaction (PCR) products. A comparative study between radioactively labeled PCR (P-32-PCR) and Q PCR was performed to analyze the MDR1 gene expression in the drug-resi stant (Doxorubicin) cell lines Dox40, Dox6, the parental cell line 822 6/S, CEM Dox1 and three acute myeloid leukemia (AML) patient samples. Using tile Dox40 and Dox6 resistant cell lines, we coilzpared the sens itivities of QPCR and P-32-PCR. A strong signal was obtained from QPCR at 20 to 25 cycles (which is in the linear range for quantitation), w hile a weak signal was obtained using P-32-PCR at the same cycle numbe r: Dilution experiments gave better precision with the QPCR than with the radioactive method. AML samples were studied with the MDRI-specifi c MAbs MRK16 and 4E3, and the efflux function was analyzed using Rh-12 3 retention in the absence or presence of verapamil. The three samples showed high (D = 0.79), medium (D = 0.52) and negative (D = 0.08) p-g lycoprotein (P-gp) levels and correlated with efflux function. The MDR 1/beta 2-M mRNA ratios for P-32-PCR were 0.41, 0.40 and 0.12, respecti vely, and were 0.127, 0.097 and 0.028, respectively, for QPCR. There w ere significant differences between the samples with high and medium P -gp levels comparing tile two methods. Very low levels of MDR1 in CEM Dox1 cells could be detected only by QPCR. In conclusion, QPCR was Sou nd to be more reproducible, accurate and sensitive than P-32-PCR.