Down-regulation of UCRP and UBE2L6 in BRCA2 knocked-down human breast cells

Tripathi, Manish Kumar; Chaudhuri, Gautam

doi:10.1016/j.bbrc.2004.12.142

Cited by 26 publications

(29 citation statements)

References 38 publications

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“…HMEC cells were grown in medium purchased from Clonetics under their recommended conditions. Other cells were maintained and grown in ATCC recommended media and conditions, as described before (12,13). Resting or nondividing cells are defined as the cells that are in a 100% confluent state of growth in a culture flask or the cells that are arrested mostly at the G 0 phase by serum starvation.…”

Section: Methodsmentioning

confidence: 99%

“…End Point RT-PCR Analysis-Total RNA was isolated from cells and analyzed for specific transcripts by RT-PCR analysis (13). ␤-Actin was used as a loading control.…”

Section: Methodsmentioning

confidence: 99%

“…Real-time RT-PCR Analysis-Quantitation of BRCA2 and SLUG transcripts were performed using a TaqMan assay (Applied Biosystems) as described before (13). 18 S rRNA was used as an endogenous control (product 4319413E).…”

Section: Methodsmentioning

confidence: 99%

“…Anti-SLUG antiserum was custom-developed (Antibody Solutions, Mountain View, CA) and purified. Proteins from cell lysate were analyzed by polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane, as described before (13). Membranes were blotted with anti-SLUG antibody or anti-actin antibody (Sigma) and then incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

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Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

Tripathi

Misra

Khedkar

et al. 2005

Journal of Biological Chemistry

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137

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The expression of the breast cancer susceptibility protein BRCA2 is highly regulated in human breast, ovary, and pancreatic cells. BRCA2 is not expressed in the nondividing cells, and expression is cell cycle stage-dependent and is elevated in the sporadic cancer cells. Mutational analysis of the upstream sequence of the human BRCA2 gene revealed an E2-box-containing silencer at the ؊701 to ؊921 position. The E2-box is essential for the cell-cycle stage-dependent activity of the silencer. We affinity-purified a 29-kDa silencer-binding protein (SBP) from the nuclear extracts of human breast cells BT-549 and MDA-MB-231. We explored whether the E2-box-binding repressor protein SLUG, which is of similar molecular size, is involved in the silencing process. Supershift assay with the purified SBP and anti-SLUG antibody revealed the identity of the SBP as SLUG. We found that silencer is inactive in the human breast cancer cells such as MDA-MB-468 and MCF-7 that do not express SLUG, further suggesting the involvement of SLUG in the BRCA2 gene silencing. Inducible expression of human SLUG in the dividing MDA-MB-468 cells reduced BRCA2 RNA levels with the activation of the silencer. Furthermore, small interfering RNA-mediated knockdown of SLUG mRNA in the BT-549 cells caused inhibition of the silencer function. Chromatin immunoprecipitation assays suggested that SLUG mediates its action by recruiting C-terminal-binding protein-1 (CtBP-1) and histone deacetylase-1 (HDAC-1) at the silencer E2-box. The general HDAC inhibitor, trichostatin A, inhibited the SLUG-mediated regulation of the silencer function. It thus appears that SLUG is a negative regulator for BRCA2 gene expression.

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Section: Methodsmentioning

confidence: 99%

“…End Point RT-PCR Analysis-Total RNA was isolated from cells and analyzed for specific transcripts by RT-PCR analysis (13). ␤-Actin was used as a loading control.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

Tripathi

Misra

Khedkar

et al. 2005

Journal of Biological Chemistry

Self Cite

137

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“…Human breast carcinoma MDA-MD-468 and BT-549 cells were maintained following standard ATCC recommended media. All cells were maintained in a humidified CO 2 (5%) incubator at 37 °C [18,19]. BT-549 cells were isolated from a 72-year-old Caucasian breast cancer patient (ATCC).…”

Section: Cellsmentioning

confidence: 99%

Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells

Tripathi

Misra

Chaudhuri

2005

Biochemical and Biophysical Research Communications

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Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process. KeywordsInvasiveness; Breast tumor; SLUG; Cytokeratin 8; Cytokeratin 19; Metallothionein 1G; Metallothionein 1L; siRNA; Microarray A serious gap in our understanding of cancer concerns malignancy and metastasis. We have yet to clearly identify gene defects that specifically permit cells to invade surrounding tissues, spread through the body, and form metastases. Local invasiveness requires a break down of the mechanisms that normally hold epithelial cells together. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. One of the known genotypic differences between these two cells is that BT-549 expresses the zinc finger repressor protein SLUG but the MDA-MB-468 cells do not [1]. Hence, we tested whether any gene overexpressed in the MDA-MB-468 cells is regulated by SLUG.We paid particular attention to SLUG because it is implicated as one of the determinants of tumor cell invasiveness [2][3][4][5][6]. This protein is a member of a superfamily of transcriptional regulators that are implicated in the formation of mesoderm and neural crest, as well as in We then overexpressed SLUG in MDA-MB-468 cells from a doxycycline-inducible promoter and compared the gene expression profile in the presence or absence of the inducer. We identified that cytokeratins 8 and 19 genes are repressed when SLUG expression is induced. We have verified the role of SLUG in regulating these cytokeratins by siRNA-mediated knock down of SLUG in BT-549 cells. We also report here our preliminary characterization of the promoters of these cytokeratin genes as it is regulated by SLUG exp...

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ISG15‐dependent Regulation

Haas

2007

Protein Degradation Series

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Down-regulation of UCRP and UBE2L6 in BRCA2 knocked-down human breast cells

Cited by 26 publications

References 38 publications

Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

Regulation of BRCA2 Gene Expression by the SLUG Repressor Protein in Human Breast Cells

Negative regulation of the expressions of cytokeratins 8 and 19 by SLUG repressor protein in human breast cells

ISG15‐dependent Regulation

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