Development and clinical application of the molecular diagnostic tests to select anti-cancer drugs

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Drug resistance is a major obstacle in chemotherapy of leukemia patients. Since the treatment responsiveness is different among patients, the therapy should be tailored to specific subtypes of diseases such as defined by drug resistance. To screen candidate genes putatively involved in the drug resistance, we analyzed gene expression profiling in drug-resistant leukemia cells using a commercially available cDNA microarray, Human Cancer CHIP version 3.0 (TaKaRa Biotechnology). This carries probe sets for 630 complementary DNAs designed to monitor genes of which expression is assumed to be involved in the biological process of cancer. A pair of RNA samples was extracted from trimetrexate-resistant leukemia MOLT-3 cells (MOLT-3/TMQ200) and sensitive cells, and independently fluorescence-labeled with Cy3 or Cy5 dye during reverse-transcription reaction. A sample mixture was competitively hybridized with the targets spotted onto the microarray, and a raw digital image was scanned and computed with Scan Array 4000 (General Scanning). In addition to overexpression of known genes as molecular mechanisms such as MDR1, there appeared overexpression of novel genes that are involved in DNA repair or gene transcription such as ATM. There were many down-regulated genes, including those involved in cell adhesion and intracellular signal transduction. Another experiment for gene expression analysis of MOLT-3 resistant to idarubicin showed decreased expression of topoisomerase inhibitor II alfa and increased expression of GS3955 protein (putative serine/threonine kinase). These results were confirmed by Northern blot analysis. This specific gene expression profiling comprised of those up-regulated and down-regulated may be indicative of cells with resistance to drugs, and its detection may predict the response. Gene expression profiling analysis will clarify molecular mechanisms of drug resistance by screening of novel genes and annotation that are involved in it.