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MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERalpha) in regulation of the MUC1 gene expression.

Zaretsky JZ, Barnea I, Aylon Y, Gorivodsky M, Wreschner DH, Keydar I - Mol. Cancer (2006)

Bottom Line: The effect of different MUC1 promoter regions on MUC1 gene expression was monitored.Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed.It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. josephz@post.tau.ac.il

ABSTRACT

Background: The MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter.

Results: Using TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERalpha, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed.

Conclusion: Analysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERalpha in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy.

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Expression of the MUC1 isoform specific mRNA in mouse DA3 cells transfected with plasmids pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 and ppolyIII. DA3 cells were transfected with plasmid DNA. Cells transfected with ppolyIII were used as negative controls. DA3 cells stably transfected with MUC1/SEC, MUC1/TM and MUC1/Y cDNA were used as positive control. Total RNA was extracted 48 hrs after transfection and cDNA was synthesized. PCR amplification of MUC1 isoform specific fragments were performed using isoform specific primers. PCR products were separated by electrophoresis on 1.2% agarose gel and stained with ethidium bromide. A – Schematic structure of the pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 plasmids and the table of the MUC1/SEC, MUC1/TM and MUC1/Y mRNA expression in transfected DA3 cells. B – RT-PCR of the MUC1 isoform specific RNA extracted from DA3 cells transiently transfected with indicated plasmids. Lane M-DNA marker; lanes 1, 4, 7, 10 and 13 (negative control) – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11 and 14 (negative control) – PCR performed with MUC1/TM specific primers; lanes 3, 6, 9, 12 and 15 (negative control) – PCR performed with MUC1/Y specific primers. Positive control: DA3 cells stably transfected with MUC1 isoform specific cDNA. Lane SEC – cells expressed MUC1/SEC; lane TM – cells expressed MUC1/TM and lane Y – cells expressed MUC1/Y RNAs.
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Figure 1: Expression of the MUC1 isoform specific mRNA in mouse DA3 cells transfected with plasmids pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 and ppolyIII. DA3 cells were transfected with plasmid DNA. Cells transfected with ppolyIII were used as negative controls. DA3 cells stably transfected with MUC1/SEC, MUC1/TM and MUC1/Y cDNA were used as positive control. Total RNA was extracted 48 hrs after transfection and cDNA was synthesized. PCR amplification of MUC1 isoform specific fragments were performed using isoform specific primers. PCR products were separated by electrophoresis on 1.2% agarose gel and stained with ethidium bromide. A – Schematic structure of the pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 plasmids and the table of the MUC1/SEC, MUC1/TM and MUC1/Y mRNA expression in transfected DA3 cells. B – RT-PCR of the MUC1 isoform specific RNA extracted from DA3 cells transiently transfected with indicated plasmids. Lane M-DNA marker; lanes 1, 4, 7, 10 and 13 (negative control) – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11 and 14 (negative control) – PCR performed with MUC1/TM specific primers; lanes 3, 6, 9, 12 and 15 (negative control) – PCR performed with MUC1/Y specific primers. Positive control: DA3 cells stably transfected with MUC1 isoform specific cDNA. Lane SEC – cells expressed MUC1/SEC; lane TM – cells expressed MUC1/TM and lane Y – cells expressed MUC1/Y RNAs.

Mentions: In our previous investigations [11] we employed CAT assays to evaluate the activity of a truncated human MUC1 promoter. In this study, we examined the role of different domains within the full-length MUC1 promoter in expression of MUC1 gene. The plasmid, Dpr, based on the ppolyII backbone was constructed to contain the full length (-2872/+1) human MUC1 promoter and genomic sequence of the human MUC1 gene including exons and introns. Using this plasmid we generated three sequential deletions from the 5'-end of the MUC1 promoter: pDprΔ2154 which lacks 2154 base pairs of promoter; pDprΔ2446 and pDprΔ2839 carrying only 426 and 33 residual nucleotides of the promoter sequence, respectively. These plasmids were used in transient transfection assays (Fig. 1A).


MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERalpha) in regulation of the MUC1 gene expression.

Zaretsky JZ, Barnea I, Aylon Y, Gorivodsky M, Wreschner DH, Keydar I - Mol. Cancer (2006)

Expression of the MUC1 isoform specific mRNA in mouse DA3 cells transfected with plasmids pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 and ppolyIII. DA3 cells were transfected with plasmid DNA. Cells transfected with ppolyIII were used as negative controls. DA3 cells stably transfected with MUC1/SEC, MUC1/TM and MUC1/Y cDNA were used as positive control. Total RNA was extracted 48 hrs after transfection and cDNA was synthesized. PCR amplification of MUC1 isoform specific fragments were performed using isoform specific primers. PCR products were separated by electrophoresis on 1.2% agarose gel and stained with ethidium bromide. A – Schematic structure of the pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 plasmids and the table of the MUC1/SEC, MUC1/TM and MUC1/Y mRNA expression in transfected DA3 cells. B – RT-PCR of the MUC1 isoform specific RNA extracted from DA3 cells transiently transfected with indicated plasmids. Lane M-DNA marker; lanes 1, 4, 7, 10 and 13 (negative control) – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11 and 14 (negative control) – PCR performed with MUC1/TM specific primers; lanes 3, 6, 9, 12 and 15 (negative control) – PCR performed with MUC1/Y specific primers. Positive control: DA3 cells stably transfected with MUC1 isoform specific cDNA. Lane SEC – cells expressed MUC1/SEC; lane TM – cells expressed MUC1/TM and lane Y – cells expressed MUC1/Y RNAs.
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Related In: Results  -  Collection

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Figure 1: Expression of the MUC1 isoform specific mRNA in mouse DA3 cells transfected with plasmids pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 and ppolyIII. DA3 cells were transfected with plasmid DNA. Cells transfected with ppolyIII were used as negative controls. DA3 cells stably transfected with MUC1/SEC, MUC1/TM and MUC1/Y cDNA were used as positive control. Total RNA was extracted 48 hrs after transfection and cDNA was synthesized. PCR amplification of MUC1 isoform specific fragments were performed using isoform specific primers. PCR products were separated by electrophoresis on 1.2% agarose gel and stained with ethidium bromide. A – Schematic structure of the pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 plasmids and the table of the MUC1/SEC, MUC1/TM and MUC1/Y mRNA expression in transfected DA3 cells. B – RT-PCR of the MUC1 isoform specific RNA extracted from DA3 cells transiently transfected with indicated plasmids. Lane M-DNA marker; lanes 1, 4, 7, 10 and 13 (negative control) – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11 and 14 (negative control) – PCR performed with MUC1/TM specific primers; lanes 3, 6, 9, 12 and 15 (negative control) – PCR performed with MUC1/Y specific primers. Positive control: DA3 cells stably transfected with MUC1 isoform specific cDNA. Lane SEC – cells expressed MUC1/SEC; lane TM – cells expressed MUC1/TM and lane Y – cells expressed MUC1/Y RNAs.
Mentions: In our previous investigations [11] we employed CAT assays to evaluate the activity of a truncated human MUC1 promoter. In this study, we examined the role of different domains within the full-length MUC1 promoter in expression of MUC1 gene. The plasmid, Dpr, based on the ppolyII backbone was constructed to contain the full length (-2872/+1) human MUC1 promoter and genomic sequence of the human MUC1 gene including exons and introns. Using this plasmid we generated three sequential deletions from the 5'-end of the MUC1 promoter: pDprΔ2154 which lacks 2154 base pairs of promoter; pDprΔ2446 and pDprΔ2839 carrying only 426 and 33 residual nucleotides of the promoter sequence, respectively. These plasmids were used in transient transfection assays (Fig. 1A).

Bottom Line: The effect of different MUC1 promoter regions on MUC1 gene expression was monitored.Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed.It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. josephz@post.tau.ac.il

ABSTRACT

Background: The MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter.

Results: Using TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERalpha, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed.

Conclusion: Analysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERalpha in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy.

Show MeSH
Related in: MedlinePlus