title: Establishment of hepatocellular carcinoma multidrug resistant monoclone cell line HepG2/mdr1.
abstract: BACKGROUND
  The multidrug resistance (MDR) associated with the expression of the mdr1 gene and its product P-glycoprotein is a major factor in the prognosis of hepatocellular carcinoma cell (HCC) patients treated with chemotherapy. Our study was to establish a stable HCC MDR cell line where a de novo acquisition of multidrug resistance specifically related to overexpression of a transgenic mdr1.
  
  
  METHODS
  The 4.5-kb mdr1 cDNA obtained from the plasmid pHaMDR1-1 was cloned into the PCI-neo mammalian expression vector, later was transferred by liposome to human hepatocarcinoma cell line HepG2. Then the transfected HepG2 cells resisting G418 were clustered and cultured and the specific fragment of mdr1 cDNA, mRNA and the P-glycoprotein (Pgp) in these HepG2 cells were detected by PCR, RT-PCR and flow cytometry, respectively. The accumulation of the daunorubicin was determinated by flow cytometry simultaneously. The nude mice model of grafting tumour was established by injecting subcutaneously HepG2/mdr1 cells in the right axilla. When the tumour diameter reached 5 mm, adriamycin was injected into peritoneal cavity. The size and growth inhibition of tumour were evaluated.
  
  
  RESULTS
  The mdr1 expression vector was constructed successfully and the MDR HCC line HepG2/mdr1 developed. The PCR analysis showed that the specific fragment of mdr1 cDNA in HepG2/mdr1 cells, but not in the control group HepG2 cells. Furthermore, the content of the specific fragment of mdr1 mRNA and Pgp expression in HepG2/mdr1 cells were (59.7 +/- 7.9)% and (12.28 +/- 2.09)%, respectively, compared with (16.9 +/- 3.2)% and (3.07 +/- 1.06)% in HepG2 cells. In the nude mice HCC model, the tumour genes of both groups were identified. After ADM therapy, the mean size of HepG2 cell tumours was significantly smaller than HepG2/mdr1 cell tumours.
  
  
  CONCLUSIONS
  The approach using the transfer of mdr1 cDNA may be applicable to the development of MDR hepatocarcinoma cell line, whose MDR mechanism is known. This would provide the experimental basis of MDR research.
authors.affiliation: Department of General Surgery, Second Hospital, Chongqing University of Medical Sciences, Chongqing, China.
authors.forename: Yong-Bing
authors.initials: YB
authors.lastname: Chen
authors.forename: Mao-Lin
authors.initials: ML
authors.lastname: Yan
authors.forename: Jian-Ping
authors.initials: JP
authors.lastname: Gong
authors.forename: Ren-Pin
authors.initials: RP
authors.lastname: Xia
authors.forename: Li-Xin
authors.initials: LX
authors.lastname: Liu
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.forename: Shi-Chun
authors.initials: SC
authors.lastname: Lu
authors.forename: Jing-Guang
authors.initials: JG
authors.lastname: Zhang
authors.forename: Dao-Bing
authors.initials: DB
authors.lastname: Zeng
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Xie
authors.forename: Jia-Yin
authors.initials: JY
authors.lastname: Yang
authors.forename: Lü-Nan
authors.initials: LN
authors.lastname: Yan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D016685:Mitomycin / Q000494:pharmacology / Q000627:therapeutic use; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016685:Mitomycin; D004317:Doxorubicin