[Expression establishment and functional analysis of breast cancer resistance protein with doxycycline induced tet regulating system in mouse fibroblast cell line PA317].
OBJECTIVE
Breast cancer resistance protein (BCRP), discovered in 1998, is a novel member of ATP-binding cassette (ABC) membrane transporters superfamily. This study was to establish the functional expression of BCRP with doxycycline (Dox) induced Tet regulating system in mouse fibroblast cell line PA317, provide an ideal experimental platform for understanding the mechanism of BCRP-mediated drug resistance, and develop effective methods to reverse the drug-resistance.


METHODS
Tet-on regulating plasmid was transferred into PA317 cells under the Dox induced Tet-on regulating system, and stable expression of Tet-on was established in PA317 cells through G418 selection. The response plasmid of recombinant pTRE-BCRP was transferred into positive PA317/Tet-on cells, and stable expression of BCRP was established through hygromycin selection. Six single cellular clones were taken and cultivated in amplification. Positive PA317/Tet-on/TRE-BCRP cells, which showed well dose-response in expression of BCRP with different concentration of Dox induction, were selected by both reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. The inhibitory effects of mitoxantrone on positive PA317/Tet-on/TRE-BCRP cells with various concentrations of Dox induction were detected by MTT method. Ko143, a specific inhibitor of BCRP, was used to detect the drug sensitivity of positive PA317/Tet-on/TRE-BCRP cells to mitoxantrone. Fluorescence intensity of remaining intracellular mitoxantrone in positive PA317/Tet-on/TRE-BCRP cells with different expression of BCRP was analyzed by flow cytometry (FCM).


RESULTS
The positive No.5 PA317/Tet-on /TRE-BCRP cell clone showed well dose-response in expression of BCRP with different concentration of Dox induction; its drug-resistance against mitoxantrone has positive correlation with BCRP expression (r=0.995, P=0.002); its drug sensitivity to mitoxantrone was significantly enhanced by Ko143 (P< 0.05). Furthermore, different expression of BCRP induced by various concentrations of Dox caused different intracellular mitoxantrone retention in this cell clone.


CONCLUSIONS
Functional expression of BCRP under Dox induced Tet regulating system was successfully established in PA317 cells, which provided an ideal experimental platform for further study of BCRP.
Yuan
Jian-Hui
JH
Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha,Hunan,410078, P.R.China.
He
Zhi-Min
ZM
Yu
Yan-Hui
YH
Chen
Zhu-Chu
ZC
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D004351:Drug Resistance; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008942:Mitoxantrone / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014162:Transfection
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D008942:Mitoxantrone; D013752:Tetracycline; D004318:Doxycycline