Negative regulation of MDR1 promoter activity in MCF-7, but not in multidrug resistant MCF-7/Adr, cells by cross-coupled NF-κb/p65 and c-Fos transcription factors and their interaction with the CAAT region

Besim Ogretmen, Ahmad Safa

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81 Citations (Scopus)

Abstract

In this study, the possible involvement of repressor protein(s) in suppressing MDR1 promoter activity in the sensitive MCF-7 human breast cancer cell line and its drug resistant variant MCF-7/Adr was investigated. RT-PCR revealed that MDR1 mRNA is under detectable levels in MCF-7, while it is highly expressed in MCF-7/Adr cells. After treatment of MCF-7 cells with cycloheximide (CHX), MDR1 mRNA reached detectable levels, suggesting that MDR1 mRNA expression might be controlled by a labile negative regulatory protein(s) in MCF-7 cells. In electrophoretic mobility shift assays (EMSA) using a 5'-end-labeled 241 bp MDR1 promoter DNA fragment (residues -198 to +43) as a probe, one protein complex that specifically binds to the CAAT region of the MDR1 promoter was detected in MCF-7, but not MCF-7/Adr. In addition, following transient transfections of MCF-7 and MCF-7/Adr cells with a pGL3-Basic plasmid construct containing a CAAT-deleted MDR1 promoter DNA fragment, a significant increase in luciferase activity was observed compared to the 241 bp MDR1 promoter in MCF-7 but not MCF-7/Adr cells. Moreover, a ds CAAT oligomer, cloned upstream of the SV-40 promoter in the pGL3-Promoter vector, resulted in a 70-80% decrease in luciferase activity in MCF-7 cells. To identify the CAAT binding protein complex, EMSA and SDS-PAGE were performed. Two proteins with molecular masses of about 65 and 60 kDa were detected by silver staining. Western blot analysis revealed that this complex consists of NF-κB/p65 and c-Fos transcription factors. Moreover, incubating MCF-7 nuclear extracts with antibodies specific for NF-κB/p65 or c-Fos in EMSAs almost completely inhibited formation of the complex, supporting the association of NF-κB/p65 and c-Fos. Therefore, this study provides evidence that molecular interplay between the NF-κB/p65 and c-Fos transcription factors exhibits a negative regulatory function on MDR1 promoter by interacting with the CAAT region in MCF-7.

Original languageEnglish (US)
Pages (from-to)2189-2199
Number of pages11
JournalBiochemistry
Volume38
Issue number7
DOIs
StatePublished - Feb 16 1999
Externally publishedYes

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MCF-7 Cells
Electrophoretic mobility
Transcription Factors
Luciferases
Messenger RNA
Assays
Repressor Proteins
Proteins
DNA
Molecular mass
Electrophoretic Mobility Shift Assay
Cycloheximide
Silver
Oligomers
Carrier Proteins
Plasmids
Cells
Association reactions
Silver Staining
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{36f71d6623c4481fa25da625b44e6954,
title = "Negative regulation of MDR1 promoter activity in MCF-7, but not in multidrug resistant MCF-7/Adr, cells by cross-coupled NF-κb/p65 and c-Fos transcription factors and their interaction with the CAAT region",
abstract = "In this study, the possible involvement of repressor protein(s) in suppressing MDR1 promoter activity in the sensitive MCF-7 human breast cancer cell line and its drug resistant variant MCF-7/Adr was investigated. RT-PCR revealed that MDR1 mRNA is under detectable levels in MCF-7, while it is highly expressed in MCF-7/Adr cells. After treatment of MCF-7 cells with cycloheximide (CHX), MDR1 mRNA reached detectable levels, suggesting that MDR1 mRNA expression might be controlled by a labile negative regulatory protein(s) in MCF-7 cells. In electrophoretic mobility shift assays (EMSA) using a 5'-end-labeled 241 bp MDR1 promoter DNA fragment (residues -198 to +43) as a probe, one protein complex that specifically binds to the CAAT region of the MDR1 promoter was detected in MCF-7, but not MCF-7/Adr. In addition, following transient transfections of MCF-7 and MCF-7/Adr cells with a pGL3-Basic plasmid construct containing a CAAT-deleted MDR1 promoter DNA fragment, a significant increase in luciferase activity was observed compared to the 241 bp MDR1 promoter in MCF-7 but not MCF-7/Adr cells. Moreover, a ds CAAT oligomer, cloned upstream of the SV-40 promoter in the pGL3-Promoter vector, resulted in a 70-80{\%} decrease in luciferase activity in MCF-7 cells. To identify the CAAT binding protein complex, EMSA and SDS-PAGE were performed. Two proteins with molecular masses of about 65 and 60 kDa were detected by silver staining. Western blot analysis revealed that this complex consists of NF-κB/p65 and c-Fos transcription factors. Moreover, incubating MCF-7 nuclear extracts with antibodies specific for NF-κB/p65 or c-Fos in EMSAs almost completely inhibited formation of the complex, supporting the association of NF-κB/p65 and c-Fos. Therefore, this study provides evidence that molecular interplay between the NF-κB/p65 and c-Fos transcription factors exhibits a negative regulatory function on MDR1 promoter by interacting with the CAAT region in MCF-7.",
author = "Besim Ogretmen and Ahmad Safa",
year = "1999",
month = "2",
day = "16",
doi = "10.1021/bi982236+",
language = "English (US)",
volume = "38",
pages = "2189--2199",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "7",

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TY - JOUR

T1 - Negative regulation of MDR1 promoter activity in MCF-7, but not in multidrug resistant MCF-7/Adr, cells by cross-coupled NF-κb/p65 and c-Fos transcription factors and their interaction with the CAAT region

AU - Ogretmen, Besim

AU - Safa, Ahmad

PY - 1999/2/16

Y1 - 1999/2/16

N2 - In this study, the possible involvement of repressor protein(s) in suppressing MDR1 promoter activity in the sensitive MCF-7 human breast cancer cell line and its drug resistant variant MCF-7/Adr was investigated. RT-PCR revealed that MDR1 mRNA is under detectable levels in MCF-7, while it is highly expressed in MCF-7/Adr cells. After treatment of MCF-7 cells with cycloheximide (CHX), MDR1 mRNA reached detectable levels, suggesting that MDR1 mRNA expression might be controlled by a labile negative regulatory protein(s) in MCF-7 cells. In electrophoretic mobility shift assays (EMSA) using a 5'-end-labeled 241 bp MDR1 promoter DNA fragment (residues -198 to +43) as a probe, one protein complex that specifically binds to the CAAT region of the MDR1 promoter was detected in MCF-7, but not MCF-7/Adr. In addition, following transient transfections of MCF-7 and MCF-7/Adr cells with a pGL3-Basic plasmid construct containing a CAAT-deleted MDR1 promoter DNA fragment, a significant increase in luciferase activity was observed compared to the 241 bp MDR1 promoter in MCF-7 but not MCF-7/Adr cells. Moreover, a ds CAAT oligomer, cloned upstream of the SV-40 promoter in the pGL3-Promoter vector, resulted in a 70-80% decrease in luciferase activity in MCF-7 cells. To identify the CAAT binding protein complex, EMSA and SDS-PAGE were performed. Two proteins with molecular masses of about 65 and 60 kDa were detected by silver staining. Western blot analysis revealed that this complex consists of NF-κB/p65 and c-Fos transcription factors. Moreover, incubating MCF-7 nuclear extracts with antibodies specific for NF-κB/p65 or c-Fos in EMSAs almost completely inhibited formation of the complex, supporting the association of NF-κB/p65 and c-Fos. Therefore, this study provides evidence that molecular interplay between the NF-κB/p65 and c-Fos transcription factors exhibits a negative regulatory function on MDR1 promoter by interacting with the CAAT region in MCF-7.

AB - In this study, the possible involvement of repressor protein(s) in suppressing MDR1 promoter activity in the sensitive MCF-7 human breast cancer cell line and its drug resistant variant MCF-7/Adr was investigated. RT-PCR revealed that MDR1 mRNA is under detectable levels in MCF-7, while it is highly expressed in MCF-7/Adr cells. After treatment of MCF-7 cells with cycloheximide (CHX), MDR1 mRNA reached detectable levels, suggesting that MDR1 mRNA expression might be controlled by a labile negative regulatory protein(s) in MCF-7 cells. In electrophoretic mobility shift assays (EMSA) using a 5'-end-labeled 241 bp MDR1 promoter DNA fragment (residues -198 to +43) as a probe, one protein complex that specifically binds to the CAAT region of the MDR1 promoter was detected in MCF-7, but not MCF-7/Adr. In addition, following transient transfections of MCF-7 and MCF-7/Adr cells with a pGL3-Basic plasmid construct containing a CAAT-deleted MDR1 promoter DNA fragment, a significant increase in luciferase activity was observed compared to the 241 bp MDR1 promoter in MCF-7 but not MCF-7/Adr cells. Moreover, a ds CAAT oligomer, cloned upstream of the SV-40 promoter in the pGL3-Promoter vector, resulted in a 70-80% decrease in luciferase activity in MCF-7 cells. To identify the CAAT binding protein complex, EMSA and SDS-PAGE were performed. Two proteins with molecular masses of about 65 and 60 kDa were detected by silver staining. Western blot analysis revealed that this complex consists of NF-κB/p65 and c-Fos transcription factors. Moreover, incubating MCF-7 nuclear extracts with antibodies specific for NF-κB/p65 or c-Fos in EMSAs almost completely inhibited formation of the complex, supporting the association of NF-κB/p65 and c-Fos. Therefore, this study provides evidence that molecular interplay between the NF-κB/p65 and c-Fos transcription factors exhibits a negative regulatory function on MDR1 promoter by interacting with the CAAT region in MCF-7.

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