EGR1促进前列腺增生上皮细胞系 BPH-1的增殖

早期生长反应基因1（early growth response gene 1, EGR1）属于锌指结构的转录因子,表达受多种因素调节,其蛋白至少参与对30种以上靶基因的调控. EGR1在前列腺癌中作为癌基因其表达量与肿瘤的恶性程度成正比,而在良性前列腺增生（benign prostatic hyperplasia, BPH）中其机制和功能尚不明确. EGR1在大鼠和人BPH组织中表达升高, 提示EGR1在BPH进程中发挥重要作用. 通过构建EGR1表达载体,以及EGR1稳定转染的良性前列腺增生上皮细胞系BPH-1,可见过表达EGR1的BPH-1细胞增殖能力升高. 通过转染siRNA将EGR1表达抑制,BPH 1细胞的增殖水平下降. 雌激素在BPH疾病进程中发挥重要作用, 在BPH 1细胞中,雌二醇 (estradiol, E2) 能促进EGR1的核迁移,从而激活其转录活性. 在EGR1稳定转染的BPH 1细胞系中胰岛素样生长因子2 (insulin like growth factor 2, IGF2) 的表达上调,表明EGR1可以调控IGF2的表达. 同时发现,E2可上调BPH-1细胞中 IGF2的表达,而将EGR1敲除后上调效果消失, 说明E2通过EGR1来调节IGF2的表达. E2对EGR1及其靶基因的调节可能是E2参与影响BPH的重要环节. 本文为进一步研究E2和EGR1在BPH中作用奠定了基础.     

Polycomb CBX7 has a unifying role in cellular lifespan

In contrast to cancer cells and embryonic stem cells, the lifespan of primary human cells is finite. After a defined number of population doublings, cells enter in an irreversible growth-arrested state termed replicative senescence. Mutations of genes involved in immortalization can contribute to cancer. In a genetic screen for cDNAs bypassing replicative senescence of normal human prostate epithelial cells (HPrEC), we identified CBX7, a gene that encodes a Polycomb protein, as shown by sequence homology, its interaction with Ring1 and its localization to nuclear Polycomb bodies. CBX7 extends the lifespan of a wide range of normal human cells and immortalizes mouse fibroblasts by downregulating expression of the Ink4a/Arf locus. CBX7 does not inter-function or colocalize with Bmi1, and both can exert their actions independently of each other as shown by reverse genetics. CBX7 expression is downregulated during replicative senescence and its ablation by short-hairpin RNA (shRNA) treatment inhibited growth of normal cells though induction of the Ink4a/Arf locus. Taken together, these data show that CBX7 controls cellular lifespan through regulation of both the p16(Ink4a)/Rb and the Arf/p53 pathways.     

Human heme oxygenase: cell cycle-dependent expression and DNA microarray identification of multiple gene responses after transduction of endothelial cells

The purpose of the present study was to examine the role of human heme oxygenase (human HO-1) in cell cycle progression following exposure to heme or human HO-1 gene transfer and to identify target genes associated with human HO-1-meditated increases in cell cycle progression using cDNA microarray technology. Heme-induced robust human HO-1 expression in quiescent human microvessel endothelial cells cultured in 1% FBS and the levels of human HO-1 expression progressively declined without a change in the cell cyclin. To identify genes regulated by human HO-1 in the cell cycle, human endothelial cells were transduced with a retroviral vector encoded with human HO-1 gene or an empty vector. Transgene expression and functionality of the recombinant protein were assessed by Western blotting, enzyme activity, carbon monoxide, cGMP production, and cell cycle analysis. Human cDNA gene array and quantitative real-time RT-PCR were used to identify both known and novel differentially expressed genes in cells overexpressing human HO-1. Major findings were upregulation of several genes associated with cell cycle progression, including cyclin E and D; downregulation of cyclin-dependent kinase inhibitors p21 and p27, cyclin-dependent kinases 2, 5, and 6, and monocyte chemoattractant protein-1; and upregulation of growth factors, including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor I (VEGFRI), endothelial growth factor (EGF) and hepatic-derived growth factor (HDGF). These findings identify an array of gene responses to overexpression of human HO-1 and elucidate new aspects of human HO-1 signaling involved in cell growth.     

A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.     

Androgens down-regulate myosin light chain kinase in human prostate cancer cells

Androgens play a major role in the growth and survival of primary prostate tumors. The molecular mechanisms involved in prostate cancer progression are not fully understood but genes that are regulated by androgens clearly influence this process. We searched for new androgen-regulated genes using the Affymetrix GeneChip Human Genome U95 Set in the androgen-sensitive LNCaP prostate cancer cell line. Analysis of gene expression profiles revealed that myosin light chain kinase (MLCK) mRNA levels were markedly down-regulated by the synthetic androgen R1881. The microarray data were confirmed by ribonuclease protection assays. RNA and protein analyses revealed that LNCaP cells express both long (non-muscle) and short (smooth muscle) isoforms, and that both isoforms are down-regulated by androgens. Taken together, these data identify MLCK as a novel downstream target of the androgen signalling pathway in prostate cells.