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The TFAP2C transcription factor has been shown to downregulate transcription of the universal cell cycle inhibitor p21clp (CDKN1A). In examining the mechanism of TFAP2C-mediated repression, we have identified a ternary complex at the proximal promoter containing TFAP2C, the oncoprotein Myc, and the trimethylated lysine 4 of histone H3 (H3K4me3) demethylase, KDM5B. We demonstrated that while TFAP2C and Myc can downregulate the CDKN1A promoter independently, KDM5B acts as a corepressor dependent on the other two proteins. All three factors collaborate for optimal CDKN1A repression, which requires the AP-2 binding site at '111/'103 and KDM5B demethylase activity. Silencing of TFAP2C-KDM5B-Myc led to increased H3K4me3 at the endogenous promoter and full induction of CDKN1A expression. Coimmunoprecipitation assays showed that TFAP2C and Myc associate with distinct domains of KDM5B and the TFAP2C C-terminal 270 amino acids (aa) are required for Myc and KDM5B interaction. Overexpression of all three proteins resulted in forced S-phase entry and attenuation of checkpoint activation, even in the presence of chemotherapy drugs. Since each protein has been linked to poor prognosis in breast cancer, our findings suggest that the TFAP2CMyc- KDM5B complex promotes cell cycle progression via direct CDKN1A repression, thereby contributing to tumorigenesis and therapy failure. [ABSTRACT FROM AUTHOR]

Objectives: Adenoviral vectors are used in transferring exogenous genes to a variety of cells and tissue types both in vitro and in vivo. Gene expression changes induced by an E1/E3-defective adenovirus vector have been studied in human mammary epithelial cells by comparing the gene expression profile in infected and uninfected cells. Methods: The human mammary epithelial cell line HB2 was infected with an E1/E3-defective adenovirus type 5 vector. Total RNA was extracted from infected and uninfected cells 24 and 72 h after infection and subjected to microarray analysis using the Affymetrix U133A genomic chip system. Semiquantitative RT-PCR confirmed the regulation of genes observed by microarray analysis. Results: The microarray analysis showed 24 and 95 transcripts to be regulated 24 and 72 h after infection, respectively. A relatively high number of genes involved in innate and inflammatory host immune responses, including interleukin-8, interleukin-6, NF-κB2, RELB and fos, were induced. As expected from an E1-defective virus, changes in the expression of genes involved in the G1-S transition and in the activation of cell proliferation were not detected. Conclusion: Our study provides insight into the host transcriptional response following transduction of an adenoviral vector into mammary epithelial cells. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The MUC1 protein is a highly O-glycosylated transmembrane molecule that is expressed at the luminal surface of most glandular epithelial cells and is upregulated in carcinomas. Here, we report the effect of the activation of the c-ErbB2 → Ras pathway on the expression of the MUC1 gene in the nontumorigenic mammary cell lines MTSV1-7 and HB2 and in the malignant cell lines T47D and ZR75. Endogenous levels of MUC1 mRNA and protein in HB2 clones permanently overexpressing c-ErbB2 or V12-H-Ras were markedly reduced compared with levels in the parental cell lines. Furthermore, in transient transfection assays, the transcription of a CAT reporter construct driven by the MUC1 promoter was inhibited when cotransfected with a c-ErbB2 or a V12-H-ras expressing vector. Transient transfections using mutant forms of the ras oncogene, and the inhibitor chemical wortmannin, indicated that the pathway activated by c-ErbB2 proceeds via activation of Ras and that the Raf and phosphoinositide 3-kinase pathways are involved. Finally, cotransfection assays using a reporter gene driven by the MUC1 promoter carrying abolishing mutations in some of the cis-acting elements showed that a GC box at -99/-91 is crucial for responsiveness to c-ErbB2 inhibition of transcription. [ABSTRACT FROM AUTHOR]

The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the Jarid1B protein we examined the effects of deletion of the gene in mice. Systemic knock out of Jarid1b resulted in early embryonic lethality, whereas mice not expressing the related Jarid1A gene are viable and fertile. A second mouse strain expressing a Jarid1b gene with the ARID domain deleted was viable and fertile but displayed a mammary phenotype, where terminal end bud development and side branching was delayed at puberty and in early pregnancy. Since development of terminal end buds are completely dependent on signalling from the estrogen receptor (ERα), we investigated the expression of a target gene (progesterone receptor) in the ∆ARID mouse and found levels to be reduced as compared to wild-type. JARID1B is widely expressed in ER+ breast cancers and breast cancer cell lines, and interaction with ERα was demonstrated by co-immunoprecipitations in cells transfected with tagged ERα and JARID1B genes. Down-regulation of expression of JARID1B using shRNAi in MCF-7 cells resulted in a dramatic decrease in E2 stimulated tumour growth in nude mice. The data demonstrate a specific role for Jarid1B in early embryonic development, in the development and differentiation of the normal mammary gland, and in estrogen induced growth of ER+ breast cancer.