Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells

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 non-dividing 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.     

小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提     

Elevated expression of the estrogen receptor prevents the down-regulation of p21Waf1/Cip1 in hormone dependent breast cancer cells

Expression of an estrogen receptor alpha (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol-induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP-ER) hindered entry of estrogen treated MCF-7 cells into S phase of the cell cycle. In analyses of key cell-cycle regulating proteins, we observed that GFP-ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP-ER expressing cells but not in non-GFP-ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down-regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase.     

Cell transformation by the E5/E8 protein of bovine papillomavirus type 4. p27(Kip1), Elevated through increased protein synthesis is sequestered by cyclin D1-CDK4 complexes.

The E5/E8 hydrophobic protein of BPV-4 is, at only 42 residues, the smallest transforming protein identified to date. Transformation of NIH-3T3 cells by E5/E8 correlates with up-regulation of both cyclin A-associated kinase activity and, unusually, p27(Kip1) (p27) but does not rely on changes in cyclin E or cyclin E-CDK2 activity. Here we have examined how p27 is prevented from functioning efficiently as a CDK2 inhibitor, and we investigated the mechanisms used to achieve elevated p27 expression in E5/E8 cells. Our results show that normal subcellular targeting of p27 is not subverted in E5/E8 cells, and p27 retains its ability to inhibit both cyclin E-CDK2 and cyclin A-CDK activities upon release from heat-labile complexes. E5/E8 cells also have elevated levels of cyclins D1 and D3, and high levels of nuclear p27 are tolerated because the inhibitor is sequestered within an elevated pool of cyclin D1-CDK4 complexes, a significant portion of which retain kinase activity. In agreement with this, pRB is constitutively hyperphosphorylated in E5/E8 cells in vivo. The increased steady-state level of p27 is achieved largely through an increased rate of protein synthesis and does not rely on changes in p27 mRNA levels or protein half-life. This is the first report of enhanced p27 synthesis as the main mechanism for increasing protein levels in continuously cycling cells. Our results are consistent with a model in which E5/E8 promotes a coordinated elevation of cyclin D1-CDK4 and p27, as well as cyclin A-associated kinase activity, which act in concert to allow continued proliferation in the absence of mitogens.     

Estrogen receptor activation at serine 305 is sufficient to upregulate cyclin D1 in breast cancer cells

Recent studies have shown that p21-activated kinase 1 (Pak1) phosphorylates estrogen receptor-alpha (ER alpha) at Ser 305 and also promotes its transactivation function. Here, we sought to investigate whether substitution of serine 305 in ER with glutamic acid (ER alpha-S305E), which mimics the phosphorylation state, would influence the status of ER-target genes. To explore this possibility, we generated clones overexpressing ER alpha-S305E in ER-negative MDA-MB-231 cells and analyzed the status of ER-regulated genes using a gene array. Results indicated that the expression of ER alpha-S305E is sufficient to upregulate the expression of a few but not all ER-regulated genes, i.e., cyclin D1 and zinc finger protein 147 (estrogen-responsive finger protein), while there was no significant change in the expression of remaining genes on the array. In addition, we found an increased expression as well as nuclear accumulation of cyclin D1 protein in MDA-MB-231 cells expressing ER alpha-S305E as compared to the level of cyclin D1 in MDA-MB-231 cells expressing WT-ER alpha or pcDNA. Furthermore, ER alpha-S305E, but not mutation of ER alpha-S305 to alanine, enhanced the cyclin D1 promoter activity. These findings suggest that ER alpha activation at S305 is sufficient to upregulate the expression of cyclin D1, an ER-regulated gene that is implicated in the progression of breast cancer. Phosphorylation of ER alpha by Pak1 or its upstream regulators could upregulate the expression of a subset of ER-target genes in a ligand-independent manner and hence, might contribute toward the development of hormone independence in breast cancer cells.     

Primary and compensatory roles for RB family members at cell cycle gene promoters that are deacetylated and downregulated in doxorubicin-induced senescence of breast cancer cells

When treated with DNA-damaging chemotherapy agents, many cancer cells, in vivo and in vitro, undergo a terminal growth arrest and acquire a senescence-like phenotype. We investigated the molecular basis for this in breast cancer cells following a 2-hour treatment with 1 muM doxorubicin. Treated cells arrested in G1 and G2 phases of the cell cycle, with concomitant reductions in S-phase and G2-M regulatory genes. p53 and p21 protein levels increased within hours after treatment and were maintained for 5 to 6 days but were reduced 8 days posttreatment, though the cells remained growth arrested. Levels of p130 rose after drug treatment, and it was the primary RB family member recruited to the S-phase promoters cyclin A and PCNA and G2-M promoters cyclin B and cdc2, remaining present for the entire 8-day time period. In contrast, p107 protein and promoter occupancy levels declined sharply after drug treatment. RB was recruited to only the PCNA promoter. In MCF-7 cells with p130 knockdown, p107 compensated for p130 loss at all cell cycle gene promoters examined, allowing cells to retain the growth arrest phenotype. Cells with p130 and p107 knockdown similarly arrested, while cells with knockdown of all three family members failed to downregulate cyclin A and cyclin B. These results demonstrate a mechanistic role for p130 and compensatory roles for p107 and RB in the long-term senescence-like growth arrest response of breast cancer cells to DNA damage.     

IRX5 prompts genomic instability in colorectal cancer cells

The Iroquois homeobox gene 5 (IRX5), one of the members of the Iroquois homeobox family, has been identified to correlate with worse prognosis in many cancers, including colorectal cancer (CRC). In this study, upregulation of IRX5 revealed a great reduction in the proliferation of CRC colorectal cancer cell line SW480 and DLD-1, which was accompanied by G1/S arrest, increased expression in cyclin E1, P21, and P53 and a decrease in cyclin A2, B1, and D1. Furthermore, IRX5-mediated an increase expression of RH2A protein, the biomarker of DNA damage. Consequently, the SA-β-gal level is higher in IRX5-overexpression cells compared to control ones, which showed elevated DNA damage triggered cellular senescence. Recapitulating the above findings, IRX5 exhibited higher levels of genomic instability. IRX5 may be a perspective target for cancer therapy and it deserves further investigation.     

Cyclin E expression and proliferation in breast cancer.

Cyclin E is a part of the cell cycle machinery and aberrantly expressed in several malignancies including breast cancer. Since cyclin E is cell cycle specifically expressed, we wanted to examine the relation between proliferation and expression of cyclin E with special attention to tumours with overexpression of the protein. Seventy-four breast tumours were analysed for the expression of cyclin E by immunohistochemistry and Western blotting and related to the growth fraction determined by Ki-67. Significant correlations were obtained between the growth fraction, the percentage of cyclin E positive cells, the intensity of cyclin E and total amount of cyclin E determined by Western blotting. The majority of the tumours had less cyclin E than Ki-67 positive cells indicating a conserved cell cycle specific expression of the protein which further was supported by flow cytometric analysis of breast cancer cell lines. The cell cycle specificity of cyclin E was found even in tumours with inactivated retinoblastoma protein (pRB) demonstrating the existence of a pRB independent regulation of cyclin E. A fraction of the tumours had considerably elevated cyclin E levels that were not in relation to the proliferative activity as observed for the other tumours. These tumours were in general highly proliferative and considered to overexpress cyclin E. Patients with tumours of high proliferative activity, high total cyclin E levels or disproportionally elevated cyclin E expressions in relation to proliferation had significantly increased risk of death in breast cancer, whereas the intensity of the immunohistochemical cyclin E staining did not affect the survival.     

Cyclin E as molecular marker in the management of breast cancer: a review

Over the past decade numerous molecular markers have been identified that may play a role in breast carcinogenesis and prognosis. The most commonly used markers in clinical practice are the estrogen receptor, progesterone receptor and HER-2/neu. Recent studies found cyclin E to be a promising prognostic indicator in breast cancer and examined its potential as a target for therapy. Further studies demonstrated that cyclin E levels were periodic during the cell cycle, with levels of protein peaking in the G1 phase. This peak in cyclin E levels also correlated with maximum enzymatic function of the cyclin E-cdk2 complex, suggesting a critical role of cyclin E in regulating G1 to S-phase transition. Studies examining the relevance of cyclin E alterations in breast cancer have shown gene amplifications in some breast cancer cell lines, data that provide strong support for the role of cyclin E in breast carcinogenesis. It is believed that the most significant cyclin E alteration is post-translational cleavage of full-length cyclin E into low molecular weight forms that are hyperactive compared to the 50-kDa, full-length protein and correlate with increasing stage and grade of breast cancer. The role of cyclin E in the prognosis and therapy of breast cancer is reviewed according to recent publications.     

The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3

Mixed-lineage kinase 3 (MLK3) activates mitogen-activated protein kinase (MAPK) signaling pathways and has important functions in migration, invasion, proliferation, tumorigenesis, and apoptosis. We investigated the role of the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) in the regulation of MLK3 protein levels. We show that CHIP interacts with MLK3 and, together with the E2 ubiquitin-conjugating enzyme UbcH5 (UbcH5a, -b, -c, or -d), ubiquitinates MLK3 in vitro. CHIP or Hsp70 overexpression promoted endogenous MLK3 ubiquitination and induced a decline in MLK3 protein levels in cells with Hsp90 inhibition. Furthermore, CHIP overexpression caused a proteasome-dependent reduction in exogenous MLK3 protein. Geldanamycin (GA), heat shock, and osmotic shock treatments also reduced the level of MLK3 protein via a CHIP-dependent mechanism. In addition, CHIP depletion in ovarian cancer SKOV3 cells increased cell invasion, and the enhancement of invasiveness was abrogated by small interfering RNA (siRNA)-mediated knockdown of MLK3. Thus, CHIP modulates MLK3 protein levels in response to GA and stress stimuli, and CHIP-dependent regulation of MLK3 is required for suppression of SKOV3 ovarian cancer cell invasion.