title: [Screening of the drug resistance-associated gene in HepG2 cell line transfected with aldose reductase like gene-1 (ARL-1)].
abstract: OBJECTIVE
  Aldose reductase like gene-1 overexpresses in hepatocellular carcinomas; it might be the drug resistance- associated gene to anti-tumor drugs containing carbonyl groups on hepatocellular carcinomas. This study was designed to establish human hepatocellular carcinoma cell line (HepG2) transfected with aldose reductase like gene-1 (ARL-1), then to observe the changes of drug resistance to anti-tumor drugs with carbonyl group and screen the drug resistance-associated genes through cDNA chip.
  
  
  METHODS
  PBK/ARL-1 plasmid was transfected to HepG2 cells by liposome to establish HepG2 monoclonal cells with high expression of ARL-1. The HepG2 cells with high expression of ARL-1 were determined by reverse transcription-polymerase chain reaction (RT-PCR),flow cytometric analysis (FCA), and immunohistochemical staining. MTT assay and cell apoptosis analysis were performed to determine the drug resistance ability of the cells to Adriamycin (ADM) and mytomycin (MMC), which contain carbonyl group. 5-fluorouracil (5-FU) that has no carbonyl group was used as control. Then two kinds of cDNA probes were made from HepG2 cells and ARL-HepG2 cells and labeled with Cy3 and Cy5 dyes. The probes were hybridized to the human liver cDNA chip and differentially expressed genes from the two cells were screened. The genes could be involved in drug resistance were confirmed by RT-PCR and Western blot.
  
  
  RESULTS
  Compared with HepG2 cells, HepG2 cells transfected with ARL-1 gene have an increase in expression of ARL-1. Drug resistance ability of HepG2 cells transfected with ARL-1 gene to ADM and MMC increased 2.6 and 3.47 folds, respectively (t=6.39, P< 0.05 in ADM group; t=30.06, P< 0.01 in MMC group). Drug resistance to 5-FU had no statistical difference between them (t=0.684,P >0.05 in 5-FU group). 15 genes down-regulated and 9 genes up-regulated after transfected with ARL-1 gene were found by cDNA chip scanned. Some differentially displayed genes such as ubiquitin and MDR confirmed by RT-PCR and Western blot were consistent to the results of cDNA chip.
  
  
  CONCLUSIONS
  The HepG2 cells with high expression of ARL-1 have more drug resistance to anti-tumor drugs with carbonyl group. Up-regulated MDR expression and down-regulated ubiquitin expression may contribute to the drug resistance of HepG2 cells.
authors.affiliation: Centre of Molecular Biology, Nanhua University, Hengyang, Hunan, PR China. XDY7@263.net
authors.forename: Xiang-Dong
authors.initials: XD
authors.lastname: Yang
authors.forename: Da-Nian
authors.initials: DN
authors.lastname: Tang
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.forename: De-Liang
authors.initials: DL
authors.lastname: Cao
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism; D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000074408:Aldo-Keto Reductases; D000970:Antineoplastic Agents / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004354:Drug Screening Assays, Antitumor; D005472:Fluorouracil / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D016685:Mitomycin / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D012334:RNA, Neoplasm / Q000032:analysis; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D012334:RNA, Neoplasm; D016685:Mitomycin; D004317:Doxorubicin; C120471:AKR1B10 protein, human; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D005472:Fluorouracil