ISSN 1007-7626 CN 11-3870 / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2012 2 28 2 152 ~ 157 p53 * 414000 breast cancer resistance protein BCRP ATP p53 wild type p53wt-p53 MCF-7 wt-p53 -κb nuclear factor-κbnf-κb BCRP p53 Saos-2 wtp53 BCRP p53 wt-p53 BCRP MatInspector BCRP p53 IκB Saos-2 NF-κB Saos-2 NF-κB p53 BCRP p53 Saos-2 BCRP NF-κB p53 -κb R730 Q753 Transcriptional Regulation of Breast Cancer Resistance Protein BCRP by p53 in p53-null Saos-2 cells WU Xin-Gang * PENG Shu-BinL Si-PingZHANG Nian-FengZOU Jin Department of Basic Medical SciencesYueyang Higher Vocational and Technical CollegeYueyang 414000Hunan China Abstract Breast cancer resistance protein BCRP is a member of the ATP-binding cassette ABC transporter superfamily BCRP confers drug resistance in cancer by transporting chemotherapeutic agents such as mitoxantronetopotecanand methotrexate Although a recent study demonstrated that wild type p53 wt-p53 may suppress BCRP expression through the nuclear factor-κb NF-κB pathway in breast cancer cell line MCF-7which expresses wt-p53 at low levelthe detailed molecular mechanisms of transcriptional regulation on BCRP remain unclear Herewe set out to reveal the exogenous p53's role on the expression of BCRP In the human osteosarcoma cell line Saos-2a p53-null cell linetransient transfection assays showed that the BCRP expression was activated by wt-p53 but not p53 mutants p53 R175H and p53 R248W We further co-transfected the p53 expression plasmid with BCRP luciferase promoter reporter construct into the Saos-2 celland the results revealed that wt-p53 may facilitate the BCRP promoter activity Howeverwe did not find any p53 binding site by applying MatInspector StrikinglyNF-κB activity inhibition downplayed the activation effect of BCRP promoter activity by wtp53 These results suggested that transcriptional activation of BCRP by wt-p53 is NF-κB- dependent Key words breast cancer resistance protein BCRP p53 nuclear factor-κb NF-κB 2011-11-22 2011-12-27 No YZ1104G * Tel 13873069242 E-mail wu_alwin@ yahoo com cn Received November 22 2011 Accepted December 27 2011 Supported by Yueyang Higher Vocational and Technical College No YZ1104G * Corresponding author Tel 13873069242 E-mail wu_alwin@ yahoo com cn
2 p53 153 multidrug resistancemdr p53 BCRP MDR ATP P P- 1 glycoproteinp-gp multidrug resistance proteinmrp breast 1 1 cancer resistance proteinbcrp Saos-2 ATCC BCRP 1998 Doyle 10% RPMI-1640 Gibco BRL MCF-7 / AdrVp 37 5% CO 2 pc53-sn3 pc53-175 pc53-248 1 2 p53 p53 p53 R175H p53 R248W pbcrp-luc 50% p53 BCRP pbabe / 60% IκBα NF-κB IκBα 70% p53 MCF-7 1 2 p53 NF-κB BCRP Primer 3 3 p53 Saos-2 Invitrogen Table 1 Table 1 Primers used to perform reverse transcription PCR Primer Sequence 5' 3' Length of PCR product / bp p53 Forward CCAGCCAAAGAAGAAACCAC 194 Reverse TATGGCGGGAGG TAGACTGA BCRP Forward CACCTTATTGGCCTCAGGAA 206 Reverse CCTGCTTGGAAGGCTCTATG β-actin Forward ACCGTGGAGAAGAGCTACGA 309 Reverse GTACTTGCGCTCAGAAGGAG 1 3 Lipofectamine 2000 Invitrogen 1 d 24 2 μg 10 5 / RNA 90% ~ 95% - 70 100 μl 1 5 RT-PCR 5 μl 5 min 20 RevertAid Fermantas min 4 ~ 6 h RNA 1 5 μg Oligo dt 15 1 μl 48 h 1 4 RNA 70% 4 7 500 r / min 5 min DEPC 0 5 μg / μl RNase-free 12 μl 70 5 min 5 min 5 TRIzol Gibco BRL 4 μl 10 mmol / L dntp 2 μl RNA PBS TRIzol 1 μl 20 U 37 5 min MMV 1 5 ml EP 5 min 1 μl 15U 42 60 min 70 10 10 min 4 12 000 r / min min RT-PCR 1 μl 10 PCR 15 min Mg 2 + 2 μl 2 mmol / L dntp 2-20 20 min 4 12 000 r / min 20 minμl 1 μl 1μL Taq
154 28 0 5 μl 20 μl PCR 94 5 min 94 1 min 55 1 min72 1 min 30 72 10 min 1 5% 2 1 6 PBS 1 000 2 1 p53 Saos-2 BCRP r / min 10 min PBS p53 Saos-2 150 mmol / L NaCl 50 mmol / L Tris-HCl ph8 0 0 1% NP-40 1mmol / L PMSF 50 ~ 100 μl 30 min 30 s 4 12 000 r / min 30 min Bio-Rad - 70 1 7 Western 50 μg 5 Fig 1 Effects of exogenous p53 on BCRP 0 35 mol / L Tris-HClpH6 8 36% expression p53-null Saos-2 cells were transfected 0 012% 10 28% SDS 5% with 2μg of p53 expression plasmids or empty vectors β- 5 min 10% SDS pcmv-bam-neo After 48 hoursthe cells were 100 V 2 h 4 100 V harvested A Total RNA was extracted First-strand 80 min PBST 5% 2 cdna was synthesized with 1 5 μg of total RNA and a BCRP cdna fragment 206 bp was amplified by PCR h p53 Santa Cruz 1 1 000 β-actin was used as an internal control B Cell BCRP ALEXIS 1 100 β- lysates were prepared Proteins were separated by 10% Actin Sigma 1 10 000 5% SDS-PAGEtransferred onto nitrocelluloseand 1 h PBST HRP immunoblotted with an anti-bcrp monoclonal antibody ZYMED 1 1 000 BXP-21and followed by ECL detection β-actin was ZYMED 1 1 000 5% used as a loading control 1 h PBST SuperSignal Pierce 1 8 1 d 10 5 / Saos-2 Fig 2 24 90% ~ 95% p53 BCRP p53 48 h 0 3 μg p53 BCRP 1 PBS 200 μl 0 3 μg p53 BCRP Promega Saos-2 EP 4 12 000 r / min 10 min p53 BCRP 20 μl 100 μl 2 3 NF-κB p53 BCRP Promega Lumi-Scint 3 1 9 SPSS 14 0 t BCRP mrna p53 BCRP Fig 1 2 2 p53 BCRP p53 BCRP IκB-α Bioscan 10 s NF-κB NF-κB 30 s 50 μl 50 μl 2 β- Fig 3 NF-κB 1 33 mg ONPG 200 mmol / L p53 BCRP Na 2 HPO 4 2 mmol / L MgCl 2 100 mmol / L β- PBS ph 7 3 405 nm 630 nm 3 ELx800 Bio-Tek BCRP ABC
2 p53 155 Fig 2 Effects of exogenous p53 on BCRP promoter activity A Saos-2 cells were transfected with different dose of wild type p53 wt-p53 expression plasmids pc53-sn3 and BCRP luciferase promoter reporter construct The total concentration of DNA was adjusted to 1 μg with empty vector pcmv-bam-neo Luciferase activity was measured after 48 hours of transfection and normalized by β-galactosidase activity Values are expressed as the percentage of the relative luciferase activity 100% in cell extract from Saos-2 cells that were transfected with empty vector Values are mean ± SE n = 3 Statistical significance compared with control * P < 0 05 ** P < 0 01 B Saos-2 cells were transfected with 0 3μg of wt-p53 or mutant p53 expression plasmids and BCRP luciferase promoter reporter construct Luciferase activity was measured after 48 hours of transfection Values are mean ± SE n = 3 Statistical significance compared with control * P < 0 05 Fig 3 NF-κB plays an important role in p53- mediated BCRP repression Saos-2 cells were transfected with wt-p53 expression plasmids or empty vector BCRP luciferase promoter reporter construct and different dose of a dominant-negative IκB-α mutant expression plasmid pbabe / IκB Luciferase activity was measured after 48 hours Values are expressed as the percentage of the relative luciferase activity 100% in cell extract from Saos-2 cells that were transfected with empty vector and without pbabe-iκb Values are mean ± SE n = 3 Statistical significance compared with control * P < 0 05 ** P < 0 01 populationsp SP 1 2 BCRP 4 ~ 6 BCRP estrogen response elementere progesterone response elementpre BCRP BCRP -1 hypoxiainducible factor 1HIF-1 7 / HIF-2 8 BCRP hypoxia response elementhre PTEN / PI3K / AKT 9 MEK-ERK-RSK 10 11 IL-1βIL-6 TNF-α c-myc 12 k-ras 13 cjun 14 MSX2 15 -β TGF-β 16 17 18 BCRP 3 p53 MCF-7 p53 BCRP MDR p53 Saos-2 9- SN38 p53 BCRP p53 Imatinib - p53 R175H - Gefitinib Erlotinib BCRP DNA p53 R248W BCRP BCRP side p53
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