Studies on Molecular Diagnostic Tests for Drug Resistance in Leukemia Cells: Molecular Mechanisms of Drug Resistance Analyzed through Genomic Instability and Its application to Molecular Diagnostic Tests

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Various gene alterations are involved in drug resistance in leukemia cells. In order to understand the mechanism that underlies emergence of cells with such gene alterations in human leukemia, we performed clonal analysis of the gene expression of mutant dihydrofolate reductase (DHFR) and mdr1 in trimetrexate-resistant human leukemia MOLT-3 cells. 70- and 60-fold trimetrexate-resistant sublines were developed in the presence or the absence of exogenous supply of thymidine (MOLT-3/TMQィイD270ィエD2/Th+, MOLT-3/TMQィイD260ィエD2/Th-, respectively). All of the clonal lines from the two sublines expressed the mutated DHFR mRNA, with a base change (T→C) at the second position of codon 31, as well as the wild type mRNA, in accordance with cross-resistance to methotrexate. On the other hand, mdr1 mRNA expression was demonstrated by reverse-transcription polymerase chain reaction only in clonal lines from MOLT-3/TMQィイD270ィエD2/Th+ cells. Polymerase chain reaction-based assay for separate microsatellites, mfd27 and mfd41, demonstrated genomic instability among clonal and subclonal lines of MOLT-3. The clonal analysis of polymorphic microsatellites also suggested that emergence of MDR in trimetrexate-resistant MOLT-3 cells in thymidine was not only heterogeneous but also fluently expanding among clones. Genomic instability may have a role in establishment and clonal evolution of drug resistance in leukemia cells. The cells would be useful as an in vitro model system for studies on mechanism of clinical drug resistance. To assess the status of the mismatch repair system, we analyzed the integrity of the DNA mismatch repair genes, including hMLH1 and hMSH2, using immunohistochemistry and polymerase chain reaction. However, we have so far not identified any alteration of expression or structure of the genes. Molecular bases behind the genomic instability and drug resistance would provide informative markers for molecular diagnostic tests for evaluation of drug resistance.