Screening Drug Resistance Genes Of Glioma And The Establishment And Identification Of Glioma Cell Line TLC1

Objective Malignant gliomas are the most common primary brain tumors inadults.In clinical,chemotherapy is one of the major methods for treatingmalignant glioma.However,the clinical effectiveness of chemotherapy is oftenlimited by the emergence of drug-resistant tumor cells.One of the most criticalissues to be solved in regard to cancer chemotherapy is the need to establish amethod for predicting efficacy or toxicity of anticancer drugs for individual patients.To this end,two experimental approaches were used in this study:1.To determinethose genes whose expression were changed in cells with acquired resistance toVM-26,2.To analyze the genes which were differentially expressing between theclinical drug sensitive and drug resistance individuals.This double strategy was todiscover genes that were differentially expressing once the resistance had beenestablished and had already suffered changes in their expression naturally.Method1.Six fresh glioma specimens obtained immediately after surgical resectionwere primary cultured.After divided into two groups according their inhibitionrates by Cytotoxicity Assays,cDNA microarray was used to identify differentexpressing genes among those tissues.2.Glioma cell line SHG44 was successive exposure to increasing amounts ofVM-26 for up to 4 months and then cells were cultured in DMEM,in which theconcentration of VM-26 was 4.5ng/4000 cells for 1 month.Cell proliferation andcytotoxicity were serially monitored and cDNA microarray was used to identifydifferent expressing genes between the parent cell and VM-26 resistant cellSHG44/VM-26.3.Established a new glioma cell line by primary culture and identified it. Result1.There were 3 glioma cases in the drug resistance group and 3 glioma casesin the drug sensitive group.After the cDNA microarray analysis,data wasclustered by SAM software,21 genes were screened out as different expressinggenes,6 of them were up regulated and 15 of them were down regulated.Althoughthe high expression of gene HDAC1 was authenticated by semi-quantitative PCR,there was no statistic difference between the two groups.2.The IC₅₀ of drug resistance cell SHG44/VM-26 is 52.06μg/ml,which is52.58 times higher than that of parent cell SHG44,whose IC₅₀ is 0.99μg/ml.Thedoubling time of SHG44/VM-26 is 48.9 hours,longer than that of SHG44,whosedoubling time is 25.6 hours.The number of different expressing genes screened bycDNA microarray was 121,37 of them were up regulated and 84 of them were downregulated.The high expressions of gene MDR1,NGFR,HSP22 and the lowexpressions of gene CX IX,CDKN3,NADE were authenticated bysemi-quantitative PCR.The expressions of MDR1,NGFR,HSP22 inSHG44/VM-26 were 3.92,1.81,2.06 times higher than that in SHG44 respectively,and the expressions of CX IX,CDKN3,NADE in SHG44/VM-26 were 0.13,0.18,0.81 times of that in SHG44.3.A new glioma cell line was established and had been passaged more than80 generations at present.Doubling time of it is 38.5 hours and the rate of colonyforming is 5.13%.The result of FCM demonstrated this glioma cell line washypodiploid.It had the infiltration activity.Conclusion The high expression of MDR1 certificate that both the establishmentof drug resistant cell line SHG44/VM-26 and the cDNA microarray analysis systemare stable.High expressions of NGFR,HSP22 and low expressions of CX IX, CDKN3,NADE are related to the drug resistance.Whether the high expression ofHDAC1 is correlate to drug resistance can not be confirmed in this study.