Cloning, And Functional Analysis Of The Gene Differentially Expressed In Multidrug Resistant Gastric Adenocarcinoma Cells

Multidrug resistance (MDR) is the main cause of the failure of chemotherapy on gastric cancer. Several molecules, such as P-gp, MRP, LRP, BCRP, GST, PKC and TopoII, have been confirmed associated with MDR of tumor cells. However, the expression levels of these MDR molecules aren't high in all of gastric cancer cell lines or tissues, which suggests other unknown molecules and mechanisms underlying gastric cancer MDR. To better understand the molecular mechanisms of multidrug resistance (MDR) of human cancers, our lab isolated differentially expressed genes from drug-resistant human gastric adenocarcinoma cell lines using a polymerase chain reaction-based subtractive hybridization technique and differentially display polymerase chain reaction technique, and identify up-regulated genes from vincristine (VCR) or adriamycin (ADR) resistant human gastric adenocarcinoma cell lines that derived from the sensitive cell line SGC7901. The two drug-resistant cell lines were selected with a single anti-cancer drug, which also displayed crossresistance to other anti-cancer drugs such as cisplatin, etoposide, mitomycin C and 5-fluorouracil (5-FU). Six genes are selected including TBP1P, NPD017, TTC-1, ME3, MRS2L, MAD2 P and MAD2 from these differentially expressed genes to find the key molecule,which may play an important role in multidrug resistance of gastric cancer.Aims: To clone the six selected genes differentially expressed in multidrug resistant gastric adenocarcinoma cells, and to analyze the functions and possible mechanisms of these genes involving MDR; we also want to probe whether the different chemodrug induced multidrug resistance gastric cell lines have the same mechanisms to form their multidrug resistance.Methods: (1) RT-PCR, DNA sequencing and gene cloning techniques were performed to obtain the encoding cDNA of the six selected genes and construct the sense and antisense eukaryotic expressed vectors of the six genes. (2) Gene transfection, RNA dot blot, Northern blot and Western blot, were used to obtain and identify the six individual genes transfected gastric cancer cell lines. (3) In vitro drug sensitive assay was used to detected chemodrugs sensitivity of the six individual genes transfected gastric cancer cell lines; and flow cytometry were used to detected the effects of the six genes on drug efflux. (4) Electron microscopy and laser cofocal microscopy were performed to examine morphologic change in multidrug resistance gastric cell lines.Results:(1) The encoding cDNA of the six genes and the eukaryotic expressed vectors were obtained. Upregulated expression of ME3, NPD017 and MAD2 3 in parental sensitive gastric cancer cell line SGC7901 increased the resistance to VCR; upregulated expression of ME3, TTC-1, TBPIP and MAD2β increased the resistance to ADR , furthermore, upregulated expression of TBPIP, NPD017, TTC-1, MAD2β and MRS2L decreased the adriamycin accumulation in transfected cells, however, upregulated ME3 expression increased drug accumulation. (2) Laser cofocal microscopy show a reversal cytosolic. nuclear drug distribution in gastric caner drug resistance cell lines comparing with sensitive cell line SGC7901. (3) Electronmicroscopy shows there exist obviously morphologic difference in subcellularly ultramicroscopic structure between drug resistance and sensitive cell lines. The morphology of nuclear menbrane in drug resistance cell is unregulated and unsmoothed, and the junction between cells is tighter than sensitive cell, and intercellular canaliculus formed between drug resistance cells. (4) RT-PCR was performed to obtain the encoding cDNA of MAD2 gene. A smaller PCR product was amplified. DNA sequencing confirmed that the smaller PCR product was a new alternative splicing form of MAD2 gene , and named it MAD2β , then cloned it into eukaryotic expression vector pcDNA3.1 in forward direction. SGC7901 was transfected with MAD2 3 . Positive clones were selected by G418. Intracellular ADR intensity and the sensitivity of gastric cancer cells to chemodrugs were...