title: Functional study of the novel multidrug resistance gene HA117 and its comparison to multidrug resistance gene 1.
abstract: BACKGROUND
  The novel gene HA117 is a multidrug resistance (MDR) gene expressed by all-trans retinoic acid-resistant HL-60 cells. In the present study, we compared the multidrug resistance of the HA117 with that of the classical multidrug resistance gene 1 (MDR1) in breast cancer cell line 4T1.
  
  
  METHODS
  Transduction of the breast cancer cell line 4T1 with adenoviral vectors encoding the HA117 gene and the green fluorescence protein gene (GFP) (Ad-GFP-HA117), the MDR1 and GFP (Ad-GFP-MDR1) or GFP (Ad-GFP) was respectively carried out. The transduction efficiency and the multiplicity of infection (MOI) were detected by fluorescence microscope and flow cytometry. The transcription of HA117 gene and MDR1 gene were detected by reverse transcription polymerase chain reaction (RT-PCR). Western blotting analysis was used to detect the expression of P-glycoprotein (P-gp) but the expression of HA117 could not be analyzed as it is a novel gene and its antibody has not yet been synthesized. The drug-excretion activity of HA117 and MDR1 were determined by daunorubicin (DNR) efflux assay. The drug sensitivities of 4T1/HA117 and 4T1/MDR1 to chemotherapeutic agents were detected by Methyl-Thiazolyl-Tetrazolium (MTT) assay.
  
  
  RESULTS
  The transducted efficiency of Ad-GFP-HA117 and Ad-GFP-MDR1 were 75%-80% when MOI was equal to 50. The transduction of Ad-GFP-HA117 and Ad-GFP-MDR1 could increase the expression of HA117 and MDR1. The drug resistance index to Adriamycin (ADM), vincristine (VCR), paclitaxel (Taxol) and bleomycin (BLM) increased to19.8050, 9.0663, 9.7245, 3.5650 respectively for 4T1/HA117 and 24.2236, 11.0480, 11.3741, 0.9630 respectively for 4T1/MDR1 as compared to the control cells. There were no significant differences in drug sensitivity between 4T1/HA117 and 4T1/MDR1 for the P-gp substrates (ADM, VCR and Taxol) (P < 0.05), while the difference between them for P-gp non-substrate (BLM) was statistically significant (P < 0.05). DNR efflux assay confirmed that the multidrug resistance mechanism of HA117 might not be similar to that of MDR1.
  
  
  CONCLUSIONS
  These results confirm that HA117 is a strong MDR gene in both HL-60 and 4T1 cells. Furthermore, our results indicate that the MDR mechanism of the HA117 gene may not be similar to that of MDR1.
authors.affiliation: Laboratory of Oncology, Affiliated Children's Hospital, Chongqing Medical University, No,136, Zhongshan 2nd Road, Yuzhong District, Chongqing 86 400014, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhao
authors.forename: Xianqing
authors.initials: X
authors.lastname: Jin
authors.forename: Youhua
authors.initials: Y
authors.lastname: Xu
authors.forename: Yuxia
authors.initials: Y
authors.lastname: Guo
authors.forename: Rui
authors.initials: R
authors.lastname: Liang
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Guo
authors.forename: Tingfu
authors.initials: T
authors.lastname: Chen
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Sun
authors.forename: Xionghui
authors.initials: X
authors.lastname: Ding
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D001761:Bleomycin / Q000008:administration & dosage; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D004317:Doxorubicin / Q000008:administration & dosage; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D017239:Paclitaxel / Q000008:administration & dosage; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000008:administration & dosage
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D001761:Bleomycin; D014750:Vincristine; D004317:Doxorubicin; D017239:Paclitaxel