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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.

SUBMITTER: Zhao L

PROVIDER: S-EPMC2912836 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Functional study of the novel multidrug resistance gene HA117 and its comparison to multidrug resistance gene 1.

Zhao Lihua L Jin Xianqing X Xu Youhua Y Guo Yuxia Y Liang Rui R Guo Zhenhua Z Chen Tingfu T Sun Yanhui Y Ding Xionghui X

Journal of experimental & clinical cancer research : CR 20100719

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 20642825

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Project description:EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His6) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-β-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (Kd ) and maximum specific one-site binding (Bmax ) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.

Dataset Information

Functional study of the novel multidrug resistance gene HA117 and its comparison to multidrug resistance gene 1.

Publications

Functional study of the novel multidrug resistance gene HA117 and its comparison to multidrug resistance gene 1.

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