Characterization and Regulation of High Risk and Maligna
高风险和恶性疾病的特征和监管
基本信息
- 批准号:6756285
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:CD95 molecule all trans retinol apoptosis breast neoplasms cancer prevention cell bank /registry cell growth regulation cell line clinical trials comparative genomic hybridization drug interactions gene expression growth inhibitors human genetic material tag human subject interferon gamma mammary epithelium microarray technology neoplasm /cancer pharmacology neoplastic cell neoplastic growth patient oriented research tamoxifen tissue resource /registry transforming growth factors women's health
项目摘要
The antiestrogen tamoxifen and vitamin A-related compounds, the retinoids, each have strong antiproliferative effects on breast cancer cells. In combination these agents act synergistically to further inhibit growth, representing an important means for enhancing their antiproliferative actions. This has resulted in increased efficacy from both a therapeutic and chemopreventive standpoint. The present study was undertaken to define this synergism and determine the mechanism of action of this enhanced activity in combination. The effect of 4-hydroxytamoxifen (TAM) and all-trans retinoic acid (AT) in combination on proliferation of MCF-7 breast cancer cells was studied in vitro. It was found that TAM and AT, in combination, acted synergistically to cause a time?dependent and dose?dependent inhibition of MCF-7 cell growth. Both TAM and AT each blocked cell cycle progression throughout 7 days of treatment, but without any synergistic or additive effect on this process. TAM and AT acted synergistically, however, to stimulate apoptosis, with increased DNA fragmentation and downregulation of bcl-2 protein expression in a manner temporally equivalent to the synergistic inhibition of growth. The negative growth factor transforming growth factor beta (TGF) is secreted by these cells and was studied as a potential mediator of the synergistic effects of TAM + AT on apoptosis. While TAM but not AT stimulated TGF1 secretion, in combination TAM and AT acted synergistically to induce a 5 fold increase in TGF1 secretion over 72 hours. TGF1 alone had no apoptotic effects on these cells, but TGF1 in combination with AT acted synergistically to inhibit growth, to downregulate bcl-2 protein expression, and to stimulate apoptosis of these cells in a manner analogous to that noted for TAM + AT. Co-incubation of TAM + AT with anti-TGF antibody partially blocked the enhanced apoptotic effects but did not abolish the synergistic downregulation of bcl-2 by the agents in combination. Together, these findings indicate that the mechanism of the synergistic antiproliferative action of TAM +AT on MCF-7 cells has two components: a.) the downregulation of bcl-2 by AT/TAM; and b.) a novel autocrine component, the enhanced secretion of TGF and the subsequent synergistic interaction of TGF with AT to stimulate apoptosis. Because AT is also a naturally occurring hormone, these findings may have broad implications for regulation of breast cancer cell growth.
AT regulation of another important mediator of apoptosis, Fas antigen, is being studied. Fas antigen, a cell surface protein and member of the TNF receptor family, is a mediator of apoptosis in multiple cell types, but is not expressed in MCF-7 breast cancer cells. AT and interferon-gamma (IFN), in combination, act synergistically to inhibit growth of these cells, which encouraged us to examine their potential regulation of Fas-antigen expression. We found that AT and IFN in combination act synergistically to induce Fas protein expression in MCF-7 cells in a time-dependent and dose-dependent manner over 48 hours. Induction of Fas mRNA was assessed by quantitative RT-PCR. AT + IFN synergistically induced Fas mRNA within 6 hours, indicating transcriptional regulation of Fas antigen expression. Following induction of Fas antigen, treatment with Fas antibody alone resulted in minimal cellular growth inhibition. Coincubation of Fas antibody with cycloheximide resulted in a significant decrease in cell viability and stimulation of apoptosis between 6 and 12 hours of treatment. This was associated with characteristic morphologic changes of apoptosis and DNA fragmentation, and indicated the presence of endogenous inhibitors to Fas signaling in these cells. Inhibition to FAS signaling was not associated with the expression of FLIPS or FLIPL. The lack of expression of caspase 3 protein and the strong expression of bcl-2 protein confirmed the presence of a type II FAS signaling pathway, and strongly suggested that bcl-2 expression was responsible for inhibition of FAS mediated apoptosis in these cells. Confirmatory studies to test the ability of Bcl-2 antisense oligonucleotides to enhance Fas-mediated apoptosis are in progress. The induction of Fas-mediated apoptosis by AT + IFN suggests important new treatment strategies for breast cancer, and further clarifies the anntiproliferative actions of these two naturally occurring agents.
The cellular, metabolic and molecular changes which occur in a normal breast epithelial cell in the development of a breast cancer are poorly understood and need to be defined. An understanding of these changes are important for defining the carcinogenic pathway of breast cancer, for identification of prognostic biomarkers for risk assessment, identification of biomarkers for selection and monitoring of chemoprevention agents, and for the development and evaluation of new chemoprevention drugs and chemoprevention strategies. To further define these characteristics we have developed high risk breast epithelial cell lines from normal high risk tissue from women with invasive breast cancer. These cell lines have normal breast epithelial cell morphology and express cytokeratins 14 and 18, and thus contain both basal and luminal cell types. We examined growth and metabolic regulation by retinoids, vitamin-A related compounds which are naturally occuring and which have chemopreventive and therapeutic effects for breast cancer. Studies to date have shown that these cells secrete the negative growth factor transforming growth factor beta (TGF), predominantly in the isomeric form TGF2. All-trans retinoic acid (AT) stimulates secretion of TGF2, primarily in the latent form, and inhibits growth through blockade of cell cycle progression without stimulating apoptosis. Active TGF2 has strong antiproliferative effects on these breast epithelial cells, indicating secreted TGF may have an important paracrine or endocrine regulatory role in women at risk for breast cancer. Gene expression profiles and their regulation by AT and TGF are being studied by cDNA microarray. This will allow identification of distinctive patterns of global gene expression between these two prominent naturally occurring antiproliferative agents. Confirmation of expression differences will be confirmed with duplicate array and quantitative RT-PCR. To identify potential changes in the carcinogenic pathway of the high risk cells, gene expression profiles of high risk cells will also be compared with those of normal nonrisk breast epithelial cells using the reference cell line MCF10A as an internal standard. In this manner important differences in the progression to the high risk state can be identified, as well as characterization of their regulation by important chemopreventive modulators.
Two clinical trials which compliment these studies have been initiated. The first trial is Protocol 02-C-0077, Characterization of High Risk Breast Duct Epithelium by Cytology, Breast Duct Endoscopy, and cDNA Gene Expression Profile. This is a collaborative study in which we will characterize the molecular, cytologic, and architectural changes in breast ductal epithelium associated with high risk for sporadic breast cancer. Breast ductal epithelial cells will be collected by breast duct lavage from the contralateral breast in postmenopausal women with ipsilateral breast cancer. These cells will be compared to ductal epithelial cells from the breast of female postmenopausal normal volunteers who are not at increased risk for breast cancer. Ductal epithelial cell specimens will be analyzed cytologically for the presence of hyperplasia, atypia, or in situ changes, and correlated with ductal architecture determined by duct endoscopy. The gene expression profile of normal and high risk ductal epithelial cells will be studied by cDNA microarray to determine changes in gene expression associated with increased risk for breast cancer. Additional molecular profiling experiments which will be performed include proteomic tissue lysate arrays, and fluorescence in situ hybridization with DNA probes for commonly altered oncogenes and tumor suppressor genes on cytological preparations. Postmenopausal women (normal volunteers) who subsequently receive estrogen replacement therapy will be reexamined three months after initiation of therapy by cytology and cDNA microarray to determine the effects of estrogens on cytology and gene expression of normal breast duct epithelium. This study of high risk epithelium may allow identification of early changes in the transformation of breast epithelium, as well as to facilitate identification of molecular markers for prognosis and risk assessment, and for the selection and evaluation of chemopreventive agents.
The second trial is Protocol 02-C-00-144. Establishment of Normal Breast Epithelial Cell Cultures, and a High Risk Cell Line and Tissue Repository from Breast Tissue from Women at High Risk for Breast Cancer. This is a collaborative study which is designed to promote identification and understanding of phenotypic, metabolic, and molecular changes which occur in high risk or premalignant breast epithelial cells. In this study a repository will be created of high risk breast epithelial tissues and cell lines from all major sites of breast tissue at increased risk for breast cancer. These will include: normal tissue adjacent to ta breast cancer, normal tissue in the same breast at a site distant from the breast cancer, from the contralateral normal breast, from women with a strong family history of breast cancer (including BRCA1 and BRCA2 mutation carriers), from women without a history of breast cancer but with a Gail model risk estimate of breast cancer of > 1.67%, or from women with prior definitive mediastinal irradiation for lymphoma. Normal tissue will also be acquired from normal women not at increased risk for breast cancer. Short-term epithelial and fibroblast cell lines will be developed from this tissue. These cell lines and respective tissues will be used to establish a repository of high risk breast cell lines and breast tissues which can be used to further define the carcinogenic pathway for breast cancer, including the cellular, metabolic, and molecular characteristics, for cDNA and tissue arrays, and to develop a drug discovery program for chemoprevention agents using a cell line screen of high risk breast epithelial cells. It is planned that this will become a major resources for the study of premalignant changes in breast cancer. This diversification of cell line and tissue material will provide excellent complimentation to ongoing laboratories studies examining high risk breast epithelial cells, and to the clincial protocol described above characterizing high risk breast duct epithelium from the contralateral breast.
抗雌激素他莫昔芬和维生素A相关的化合物,类视视视网麻素,每个化合物对乳腺癌细胞具有强大的抗增殖作用。结合起来,这些代理协同作用以进一步抑制生长,代表增强其抗增殖作用的重要手段。从治疗性和化学预防的角度来看,这导致了提高的功效。本研究的目的是定义这种协同作用,并确定这种增强活性组合的作用机理。在体外研究了4-羟基莫昔芬(TAM)和全反式视黄酸(AT)对MCF-7乳腺癌细胞增殖的影响。发现TAM和AT结合起来协同作用,导致时间?依赖和剂量?依赖于MCF-7细胞生长的抑制作用。在整个7天的治疗过程中,TAM和每个阻塞细胞周期的进展均未对此过程进行任何协同或添加剂效应。然而,TAM和在ACT的协同作用以刺激凋亡,而DNA碎片增加和Bcl-2蛋白表达的下调,其方式与生长的协同抑制在时间上等同于。这些细胞分泌了负生长因子转化生长因子β(TGF),并作为TAM + AT对凋亡的协同作用的潜在介体进行了研究。虽然TAM但在刺激的TGF1分泌中却没有,但在TAM联合和以协同作用时,在72小时内诱导TGF1分泌的5倍增加。仅TGF1对这些细胞没有凋亡作用,但是TGF1与AT结合起作用,以协同作用以抑制生长,下调Bcl-2蛋白表达,并以类似于TAM +的方式刺激这些细胞的凋亡。 TAM +与抗TGF抗体的共孵育部分阻止了增强的凋亡作用,但并未消除药剂组合中Bcl-2的协同下调。总之,这些发现表明TAM +AT ON MCF-7细胞的协同抗增殖作用的机理具有两个组成部分:a。)AT/TAM的Bcl-2下调; b。)一种新型的自分泌成分,TGF的分泌增强以及TGF与AT的随后的协同相互作用,以刺激凋亡。因为AT也是一种天然发生的激素,所以这些发现可能对调节乳腺癌细胞生长具有广泛的影响。
在调节另一个重要的凋亡介质时,正在研究FAS抗原。 FAS抗原是一种细胞表面蛋白,也是TNF受体家族的成员,是多种细胞类型中凋亡的介体,但在MCF-7乳腺癌细胞中未表达。 AT和干扰素 - 伽马(IFN)结合起作用,协同作用以抑制这些细胞的生长,这鼓励我们检查它们对Fas抗原表达的潜在调节。我们发现,在组合中的IFN和IFN在48小时内以时间依赖性和剂量依赖性方式协同诱导MCF-7细胞中的FAS蛋白表达。通过定量RT-PCR评估FAS mRNA的诱导。 AT + IFN在6小时内协同诱导FAS mRNA,表明FAS抗原表达的转录调节。诱导FAS抗原后,单独使用FAS抗体的治疗导致细胞生长抑制最少。 FAS抗体与环己酰亚胺的共同结合导致细胞活力显着降低,并在6至12小时的治疗之间刺激凋亡。这与细胞凋亡和DNA碎片化的特征形态变化有关,并表明内源性抑制剂在这些细胞中存在FAS信号传导。对FAS信号的抑制与翻转或FLIPL的表达无关。 caspase 3蛋白的表达不足和Bcl-2蛋白的强表达证实了II型FAS信号通路的存在,并强烈建议Bcl-2表达是抑制这些细胞中FAS介导的凋亡的原因。确认性研究以测试Bcl-2反义寡核苷酸增强FAS介导的细胞凋亡的能力。 AT + IFN诱导FAS介导的凋亡提出了重要的乳腺癌新治疗策略,并进一步阐明了这两种天然发生的药物的阳离子作用。
乳腺癌发育中正常乳腺上皮细胞中发生的细胞,代谢和分子变化知之甚少,需要定义。对这些变化的理解对于定义乳腺癌的致癌途径至关重要,对于鉴定风险评估的预后生物标志物,用于选择和监测化学预防剂的生物标志物以及新化学预防药物的开发和评估。为了进一步定义这些特征,我们从正常高风险组织的高风险乳房上皮细胞系中产生了来自浸润性乳腺癌的女性。这些细胞系具有正常的乳腺上皮细胞形态,并表达细胞角蛋白14和18,因此含有基底和腔细胞类型。我们检查了类维生素类似的生长和代谢调节,自然发生的维生素-A相关化合物,这些化合物对乳腺癌具有化学预防和治疗作用。迄今为止的研究表明,这些细胞分泌负生长因子转化生长因子β(TGF),主要是在异构体形式TGF2中。全反式视黄酸(AT)刺激TGF2的分泌,主要是潜在形式,并通过阻断细胞周期进展而抑制生长而不会刺激细胞凋亡。活性TGF2对这些乳腺上皮细胞具有强大的抗增生作用,表明分泌的TGF可能在患有乳腺癌风险的女性中具有重要的旁分泌或内分泌调节作用。 cDNA微阵列研究了基因表达谱及其对AT和TGF的调节。这将允许鉴定这两种自然发生的抗增生剂之间的全球基因表达的独特模式。表达差异的确认将通过重复的阵列和定量RT-PCR确认。为了确定高风险细胞的致癌途径的潜在变化,也将使用参考细胞系MCF10A作为内标将高风险细胞的基因表达谱与正常非风险乳腺上皮细胞的基因表达谱进行比较。以这种方式,可以确定在向高风险状态的进展中的重要差异,并通过重要的化学预防调节剂来表征其调节。
已经开始了两项补充这些研究的临床试验。第一个试验是方案02-C-0077,通过细胞学,乳腺导管内窥镜检查和cDNA基因表达谱的高风险乳腺导管上皮表征。这是一项协作研究,我们将表征与零星乳腺癌高风险相关的乳腺导管上皮细胞的分子,细胞学和结构变化。乳腺导管上皮细胞将通过同侧乳腺癌的绝经后妇女的对侧乳房的乳房导管灌洗来收集。这些细胞将与绝经后正常志愿者的乳房的导管上皮细胞进行比较,这些志愿者没有增加乳腺癌的风险。导管上皮细胞样品将在细胞学上分析,以了解增生,非洲典型或原位变化,并与通过导管内窥镜确定的导管结构相关。 cDNA微阵列将研究正常和高风险导管上皮细胞的基因表达谱,以确定与乳腺癌风险增加有关的基因表达变化。将进行的其他分子分析实验包括蛋白质组织裂解物阵列,以及与DNA探针的原位杂交,用于在细胞学制备中常见的致癌基因和肿瘤抑制基因。后来的妇女(正常志愿者)随后接受雌激素替代疗法后三个月在通过细胞学和cDNA微阵列开始治疗后三个月,以确定雌激素对雌激素对正常乳腺导管上皮的细胞学和基因表达的影响。这项对高风险上皮的研究可以允许鉴定乳腺上皮转化的早期变化,并促进鉴定用于预后和风险评估的分子标记,以及选择和评估化学预防药物。
第二个试验是协议02-C-00-144。建立正常的乳房上皮细胞培养物,以及来自乳腺癌高风险的乳房组织的高风险细胞系和组织仓库。这是一项协作研究,旨在促进对表型,代谢和分子变化的识别和理解,这些变化发生在高风险或前乳腺上皮细胞中。在这项研究中,将为乳腺癌所有主要部位的高风险乳房上皮组织和细胞系创建一个存储库,乳腺癌风险增加。这些将包括:与TA乳腺癌相邻的正常组织,远离乳腺癌的部位的同一乳房中的正常组织,远离对侧正常乳房,来自具有较强乳腺癌家族史的女性(包括BRCA1和BRCA2突变携带者) ),来自没有乳腺癌史的女性,但具有盖尔模型估计> 1.67%的估计,或者来自先前具有淋巴瘤纵隔纵隔辐照的女性。正常妇女也将获得正常组织,而不是乳腺癌风险增加。短期上皮和成纤维细胞系将从该组织开发。这些细胞系和各自的组织将用于建立一个高风险乳腺细胞系和乳腺组织的存储库,可用于进一步定义乳腺癌的致癌途径,包括细胞,代谢和分子特征,用于cDNA和组织阵列,并使用高风险乳房上皮细胞的细胞线筛选为化学预防剂开发药物发现程序。据计划,这将成为研究乳腺癌前改变的主要资源。细胞系和组织材料的这种多样化将为正在进行的实验室研究提供出色的补充,以检查高风险乳房上皮细胞,以及上面描述的clincial方案,这些方案表征了对侧乳房的高风险乳腺导管上皮。
项目成果
期刊论文数量(0)
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会议论文数量(0)
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{{ truncateString('DAVID DANFORTH', 18)}}的其他基金
Characterization and Regulation of High Risk and Maligna
高风险和恶性疾病的特征和监管
- 批准号:
7331396 - 财政年份:
- 资助金额:
-- - 项目类别:
Characterization and Regulation of High Risk and Maligna
高风险和恶性疾病的特征和监管
- 批准号:
6947445 - 财政年份:
- 资助金额:
-- - 项目类别:
Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer
乳腺癌风险中乳腺导管上皮的分子特征
- 批准号:
8763680 - 财政年份:
- 资助金额:
-- - 项目类别:
Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer
乳腺癌风险中乳腺导管上皮的分子特征
- 批准号:
8938390 - 财政年份:
- 资助金额:
-- - 项目类别:
Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer
乳腺癌风险中乳腺导管上皮的分子特征
- 批准号:
9344100 - 财政年份:
- 资助金额:
-- - 项目类别:
Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer
乳腺癌风险中乳腺导管上皮的分子特征
- 批准号:
10926568 - 财政年份:
- 资助金额:
-- - 项目类别:
TAMOXIFEN AND RETINOID REGULATION OF BREAST EPITHELIAL CELL GROWTH AND METABOLISM
他莫昔芬和维A酸对乳腺上皮细胞生长和代谢的调节
- 批准号:
6290764 - 财政年份:
- 资助金额:
-- - 项目类别:
High Risk /Malignant Breast Epithelium--Character. /Reg.
高风险/恶性乳腺上皮——特征。
- 批准号:
6558360 - 财政年份:
- 资助金额:
-- - 项目类别:
Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer
乳腺癌风险中乳腺导管上皮的分子特征
- 批准号:
10702991 - 财政年份:
- 资助金额:
-- - 项目类别:
Molecular Characterization of Breast Duct Epithelium at Risk for Breast Cancer
乳腺癌风险中乳腺导管上皮的分子特征
- 批准号:
7969760 - 财政年份:
- 资助金额:
-- - 项目类别:
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ALDH1a2 (RALDH2) in a Murine Oral Cavity Squamous Cell Carcinoma Model
小鼠口腔鳞状细胞癌模型中的 ALDH1a2 (RALDH2)
- 批准号:
8649168 - 财政年份:2014
- 资助金额:
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Aldo-keto reductase family 1 member B10 AKR1B10 in pancreatic carcinogenesis
醛酮还原酶家族 1 成员 B10 AKR1B10 在胰腺癌发生中的作用
- 批准号:
8220421 - 财政年份:2012
- 资助金额:
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