scholarly journals Chromatin Inactivation Precedes De Novo DNA Methylation during the Progressive Epigenetic Silencing of the RASSF1A Promoter

2005 ◽  
Vol 25 (10) ◽  
pp. 3923-3933 ◽  
Author(s):  
Maria Strunnikova ◽  
Undraga Schagdarsurengin ◽  
Astrid Kehlen ◽  
James C. Garbe ◽  
Martha R. Stampfer ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Epithelial Cells ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Mammary Epithelial Cells ◽  
Time Window ◽  
Cpg Island ◽  
Histone H3 ◽  
Mammary Epithelial

ABSTRACT Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.

scholarly journals PGN and LTA from Staphylococcus aureus Induced Inflammation and Decreased Lactation through Regulating DNA Methylation and Histone H3 Acetylation in Bovine Mammary Epithelial Cells

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 238 ◽  
Author(s):  
Yongjiang Wu ◽  
Jingbo Chen ◽  
Yawang Sun ◽  
Xianwen Dong ◽  
Zili Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Histone H3 ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
Hdac Activity ◽  
Histone H3 Acetylation ◽  
H3 Acetylation

Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common pathogens of mastitis, and S. aureus generally causes subclinical mastitis which is more persistent and resistant to treatment. Peptidoglycan (PGN) and lipoteichoic acid (LTA) are cell wall components of S. aureus. Although the roles of PGN and LTA in causing inflammation are well studied, the epigenetic mechanisms of the effects of PGN and LTA on the inflammation and lactation remain poorly understood. This study characterized the gene expression profiling by RNA sequencing and investigated DNA methylation and histone acetylation in relation to inflammation and lactation in the immortalized bovine mammary epithelial cell line (MAC-T). The cells were cultured for 24 h with neither PGN nor LTA (CON), PGN (30 μg/mL), LTA (30 μg/mL), and PGN (30 μg/mL) + LTA (30 μg/mL), respectively. The number of differentially expressed genes (DEGs) and the expression of proinflammatory factors including interleukin (IL)-1β, IL-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6 of the treatments increased in the following order: CON < PGN < LTA < PGN + LTA, and the DEGs mainly enriched on the cytokine-cytokine receptor interaction and chemokine signaling pathway. LTA and PGN + LTA induced hypomethylation of global DNA by suppressing DNA methyltransferase (DNMT) activity. PGN and LTA, alone or combined, decreased the mRNA expression of casein genes (CSN1S1, CSN2, and CSN3) and the expression of two caseins (CSN2 and CSN3), and reduced histone H3 acetylation by suppressing histone acetyltransferase (HAT) activity and promoting histone deacetylase (HDAC) activity. Collectively, this study revealed that PGN and LTA induced inflammation probably due to decreasing DNA methylation through regulating DNMT activity, and decreased lactation possibly through reducing histone H3 acetylation by regulating HAT and HDAC activity in bovine mammary epithelial cells.

scholarly journals Tumor Suppressor p16INK4ARegulates Polycomb-mediated DNA Hypermethylation in Human Mammary Epithelial Cells

2006 ◽  
Vol 281 (34) ◽  
pp. 24790-24802 ◽  
Author(s):  
Paul A. Reynolds ◽  
Mahvash Sigaroudinia ◽  
Giuseppe Zardo ◽  
Matthew B. Wilson ◽  
Geoffrey M. Benton ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Dna Hypermethylation ◽  
Human Mammary Epithelial Cells ◽  
Human Mammary Epithelial

scholarly journals Inactivation of p16 in Human Mammary Epithelial Cells by CpG Island Methylation

1998 ◽  
Vol 18 (4) ◽  
pp. 1793-1801 ◽  
Author(s):  
Scott A. Foster ◽  
David J. Wong ◽  
Michael T. Barrett ◽  
Denise A. Galloway
Keyword(s):  
Epithelial Cells ◽  
Kinase Inhibitor ◽  
Mammary Epithelial Cells ◽  
Cpg Island ◽  
Mammary Epithelial ◽  
Early Passage ◽  
Human Mammary Epithelial Cells ◽  
Human Mammary Epithelial ◽  
Cpg Island Methylation ◽  
P16 Expression

ABSTRACT Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.

scholarly journals Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland

1999 ◽  
Vol 112 (11) ◽  
pp. 1771-1783 ◽  
Author(s):  
A.D. Metcalfe ◽  
A. Gilmore ◽  
T. Klinowska ◽  
J. Oliver ◽  
A.J. Valentijn ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
De Novo ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Mammary Epithelial ◽  
Mammary Tissue ◽  
Pregnancy And Lactation

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

Up-Regulation of miR-195 Expression Leads to Decreased Expression of Basic Fibroblast Growth Factor in CLL Patients Treated with DNA Methylation Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3183-3183
Author(s):  
John D. Phillips ◽  
Ioana Pop ◽  
Ken Boucher ◽  
Margaret K. Yu
Keyword(s):  
Dna Methylation ◽  
Growth Factor ◽  
Tumor Suppressor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Dna Methyltransferase ◽  
Cpg Island ◽  
Fibroblast Growth ◽  
Substrate Inhibitor ◽  
Dna Methylation Inhibitors

Abstract A higher than age-expected DNA methylation index is predictive for early disease progression in patients with CLL (Yu et al. Leukemia Research 2006). We hypothesized that miRNA expression can also be silenced by promoter hypermethylation in CLL. Thus, methyl pool or DNA methyltransferase inhibitors can upregulate microRNAs with tumor suppressor characteristics by restoring the “normal” pattern of methylation. Results of patients treated with cladribine, a methyl substrate inhibitor, or 5-azacitidine, a DNA methyltransferase inhibitor were compared in 2 separate clinical protocols. The global DNA methylation decreased after treatment with cladribine or 5-azacitidine in 60% of the patients. At the 2006 ASH meeting, we reported on the consistent upregulation of miR-17-3p, miR-21, miR-29a, miR-29b, miR-29c, miR-30e, miR-104, miR-126, miR-128a, miR-130a, miR-141, miR-142-3p, miR-148a, miR-151, miR-199a, miR-199a*, and miR-301 by real-time PCR using the Early Access Human Panel from Applied Biosystems. Data from two patients on the cladribine protocol and 10 patients on the 5-azacitidine protocol were used for statistical analyses. Non-parametric Wilcoxon tests as well as t-tests were performed. Comparisons were made of the responders versus non-responders, and of cladribine versus 5-azacytidine. Since many of the miRNAs showed differences in the cladribine versus 5-azacytidine comparison, a second responder vs non-responder analysis was performed in which the two cladribine subjects were removed. MiR-195 was statistically more upregulated in the cladribine treated responder whereas miR-29c was statistically most upregulated in the 5-azacitidine treated patients (p=0.02). In the patients with global demethylation after treatment, upregulation of microRNA-195 was observed and directly correlated with regional demethylation of the CpG island, confirmed by bisulfite sequencing. Some of the predicted targets of miR-195 include bcl-2, CNOT6L, USP15, PADAH1B1, and ESRRG. MiR-195 may also have tumor suppressor characteristics as it also targets basic fibroblast growth factor (FGF-2), a gene important in CLL angiogenesis. There has been some evidence suggesting FGF-2 is an oncogene. For example, overexpression of FGF-2 isoforms facilitates growth of NIH 3T3 cells in low serum media and also mediates radioresistance of HeLa cells. FGF-2 is also protective against irradiation activation of p53 in the leukemia cells derived from patients with CLL. Although cladribine has been reported to downregulate FGF-2 by inhibiting adenosine deaminase, downregulation of FGF-2 at the transcript and protein levels was also observed in before and after treatment samples from patients treated with 5-azacytidine. We propose an alternative mechanism by which the FGF-2 transcript is degraded after binding to excess miR-195. In patients responsive to treatment with DNA methylation inhibitors, a regional decrease in the methylation status of the CpG island 5′ to miR-195 may lead to increased expression.

scholarly journals All Three Promoters of the Acetyl-Coenzyme A-Carboxylase α-encoding Gene Are Expressed in Mammary Epithelial Cells of Ruminants

2003 ◽  
Vol 51 (8) ◽  
pp. 1073-1081 ◽  
Author(s):  
Adrian Molenaar ◽  
Jianqiang Mao ◽  
Kim Oden ◽  
Hans-Martin Seyfert
Keyword(s):  
Epithelial Cells ◽  
Milk Fat ◽  
De Novo ◽  
Mammary Epithelial Cells ◽  
Molecular Probes ◽  
Mammary Epithelial ◽  
Encoding Gene ◽  
Rate Limiting ◽  
Acetyl Coenzyme A Carboxylase

The activity of the enzyme acetyl-CoA-carboxylase α (ACC-α) is rate limiting for the de novo synthesis of fatty acids. The encoding gene is expressed from three promoters in ruminants (PI-PIII). Their individual contribution to the formation of milk fat is unknown. Promoter-specific molecular probes were hybridized in situ to serial sections of mammary glands from cows and sheep to determine their developmental and spatial expression profile in the udder. We show that all three promoters are active in mammary epithelial cells (MECs) of udders from both species. This implies that, in principle, none of these promoters can be singled out as the key element controlling the ACC-α-related contribution to establishment of milk fat content, although the activity of PIII only is known to be disproportionally stimulated by lactation in MECs. We propose that all three promoters may be relevant for milk fat synthesis in cattle, whereas PII and PIII are crucial for milk fat formation in sheep. We show also that ACC-α synthesis is not strictly coupled to casein synthesis, particularly during pregnancy and involution.

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