| Multidrug resistance (MDR) describes a variety of strategies that rumor cells develop to evade the cytotoxic effects of anti-cancer drugs. Tumor cells that have become MDR show decreased cellular sensitivity to the drugs employed in chemotherapy as well as a broad spectrum of drugs without obvious common targets or structural homology. MDR is a major obstacle to the successful treatment of tumors.The mechanisms that have been found to confer drug resistance include high expression of drug efflux pump, altered cellular drug pharmacokinetics, increased drug detoxification, increased DNA damage repair, suppression of drug-induced apoptosis, upregulation of lipids and other biochemical changes. However, to date, no mechanism can be generalized, preclinical and clinical evidence from interventions aimed at any single resistance mechanism has been disappointing. Therefore, it's not surprising that two or more mechanisms often co-exist to confer MDR phenotypes. In addition, the increasing evidence of genetic diversity in drug resistant cell lines and clinical resistance allows us to assume there's a intricate molecularnetwork that controls the sensitivity of tumor cells to chemotherapeutic agents.These concepts suggest searching for more molecules associated with MDR will be necessary in order to understand the mechanisms of MDR and further design new therapeutic regimens to circumvent drug resistance. So, we used a modified subtractive hybridization method to identify up-regulated genes from vincristine (VCR) or adriamycin (ADR) resistant human gastric adenocarcinoma cell lines that derived from the cell line SGC7901.We obtained 200 clones containing cDNA insertion fragments from the drug resistant cancer cells. After restrictive enzyme digestion, 86 clones containing cDNA fragments of 0.5kb to 1.5kb were identified. Among the 86 clones, 63 clones (23 clones from SGC7901/VCR cells; 40 clones from SGC7901/ADR cells.) displayed negative signals by the reverse dot blot analysis.After sequencing and a BLAST search, 39% (9 clones) from SGC7901/VCR cells were identical as known genes in GenBank, 35% (8 clones) matched with the sequences in dbEST or identical to putative genes, and 26% (6 clones) were novel ESTs or putative genes. For SGC7901/ADR cell line, 44% (18 clones) were identical to known genes, 28% (11 clones) matched with the sequences in dbEST or identical to putative genes, and 28% (11 clones) were novel ESTs or putative genes.Among the 27 known genes, including metabolic enzymes, oncogenes, signal transduction genes, cell cycle genes, transcription factors and transporters, two have been well described related to drug resistance of human cancers. One is thioredoxin, the other two are thymidylate synthetase (TYMS) and high-mobility group (nonhistone chromosomal) protein isoforms I and Y.Among the genes with undefined functions, PTD001 (AF077051) mRNA from SGC7901/ADR cells containing a complete ORF (open reading frame) was analyzed. Semi-quantitative RT-PCR assay confirmed it was upregulated in drug-resistant cells, and the polyclonal antibody against the induced protein did recognize a protein of about 25kDa in mammalian cells, suggesting it was a real gene. Notably, immunofluorescent staining demonstrated PTD001 protein was nearly twice upregulated in drug-resistant SGC7901/ADR cells compared with drug-sensitive SGC7901 cells.The results suggest that this strategy is efficient for large-scale cloning of differentially expressed genes in drug resistant cells. Further characterization of these genes will shade light on the understanding of molecular mechanisms of multidrug resistance of human cancer cells. |
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