A new PCR MIMIC strategy to quantify low mdr1 mRNA levels in drug resistant cell lines and AML blast samples

• Published in: Leukemia research Vol. 23; no. 7; pp. 653 - 663
• Main Authors: Illmer, Thomas, Schaich, Markus, Oelschlägel, Uta, Nowak, Ralf, Renner, Ulf, Ziegs, Barbara, Subat, Sabine, Neubauer, Andreas, Ehninger, Gerhard
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.07.1999; Elsevier Science
• Subjects: Acute Disease; Adolescent; Adult; Aged; AML; Antigens, CD34 - analysis; Antineoplastic agents; Antineoplastic Agents, Phytogenic - administration & dosage; Antineoplastic Agents, Phytogenic - pharmacology; Antineoplastic Combined Chemotherapy Protocols - therapeutic use; ATP Binding Cassette Transporter, Subfamily B, Member 1 - biosynthesis; Binding, Competitive; Biological and medical sciences; CD 34; Chemotherapy; Chromosome Banding; Cytarabine - administration & dosage; Cytogenetics; Daunorubicin - administration & dosage; DNA, Complementary - genetics; Drug Resistance, Multiple - genetics; Drug Resistance, Neoplasm - genetics; Etoposide - administration & dosage; Etoposide - pharmacology; Gene Expression Regulation, Leukemic; Genes, MDR; Humans; K 562; K562 Cells - drug effects; K562 Cells - metabolism; Leukemia, Myeloid - drug therapy; Leukemia, Myeloid - genetics; Leukemia, Myeloid - metabolism; Leukemia, Myeloid - pathology; MDR1; Medical sciences; Middle Aged; MIMIC; Mitoxantrone - administration & dosage; Neoplasm Proteins - biosynthesis; Neoplastic Stem Cells - drug effects; Neoplastic Stem Cells - metabolism; Oligonucleotide Probes - metabolism; Pharmacology. Drug treatments; Quantitative PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Messenger - analysis; RNA, Messenger - genetics; RNA, Neoplasm - analysis; RNA, Neoplasm - genetics; Sensitivity and Specificity; Tumor Cells, Cultured - drug effects; Tumor Cells, Cultured - metabolism; AML; MBC, mononuclear blood cells; CsA, cylosporin A; P-gp, P-glycoprotein; AML, acute myeloid leukemia; K 562; Quantitative PCR; CD 34; LRP, lung resistance protein; MDR1; MIMIC; rT, reverse transcriptase; CR, complete remission; MRP, multi drug resistance associated protein; Cytogenetics; PCR, polymerase chain reaction; RNA-directed DNA polymerase; Enzyme; Transferases; Acute; P Glycoprotein; Exploration; Genotype; Malignant hemopathy; Gene expression; Polymerase chain reaction; Competitive fixation; Nucleotidyltransferases; Chemotherapy; Phenotype; Treatment; Multiple resistance; Genetics; Technique; Molecular biology; Negative therapeutic reaction; Quantitative analysis; Acute myelocytic leukemia
• ISSN: 0145-2126; 1873-5835
• DOI: 10.1016/S0145-2126(99)00076-4

Determination of the MDR-phenotype in patients suffering from AML is an important hallmark of treatment outcome but is often complicated by technical problems in P-gp assessment. A PCR-MIMIC strategy was employed to construct PCR-fragments for a competitive and quantitative mdr1 reverse transcription-PCR-assay. Using K562 cells, which had been selected for drug resistance to the epipodophyllotoxin VP16, a stepwise increase of mdr1 levels depending on the concentration of VP 16 was shown with the MIMIC technique. Comparison of mdr1 levels in drug selected K562 cells with the corresponding levels for P-gp and functional data indicated a mRNA threshold that has to be exceeded for the full expression of the MDR-phenotype. Subsequently mdr1 levels of 34 samples of de novo acute myeloid leukemia were determined with the PCR-MIMIC strategy. Ten patient samples could be identified with elevated mdr1 levels which were substantially lower than the levels observed in the MDR-cell line K 562 0.7 μM VP16. Outcome analysis revealed that eight of the ten patients had an unfavourable prognosis and did not achieve CR after induction chemotherapy. Coexpression of mdr1 and CD 34 was not associated with CR in all examined cases. Moreover all these patients had unfavourable cytogenetic aberrations. These data indicate a sensitive technique with applicability in patient material.