NOVEL MYOEPITHELIAL CELL LINE TO SUPPORT 1 BREAST C

支持 1 个乳腺癌的新型肌上皮细胞系

• 批准号: 2414465
• 负责人: Sanford Howard Barsky
• 金额: $ 7.45万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-22 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2414465
• 关键词: DNA; SCID mouse; apoptosis; athymic mouse; breast neoplasms; cell growth regulation; cell sorting; clinical research; clone cells; extracellular matrix; female; histogenesis; human subject; mitogens; myoepithelial cell; neoplastic cell; northern blottings; nucleosomes; paracrine; preneoplastic state; xenotransplantation

The growth of primary breast carcinoma and pre-cancerous cells in culture has proved difficult, and, as a result, there is a paucity of both short term and immortal primary breast carcinoma cell lines. Primary breast carcinoma cultures, though able to be initiated in selective media, undergo terminal differentiation, senescence, or apoptosis after only a few passages. The reasons for this are unclear. Because primary breast carcinomas and -pre-cancerous diseases states (DCIS) arise in via in the setting of surrounding myoepithelial cells, cells which exert important paracrine effects through both direct cell-cell interactions and through synthesis of a basement membrane extracellular matrix, we hypothesized that the growth of primary breast carcinomas and precancerous cells in vitro might still be dependent on this type of myoepithelial interaction. Using a novel human myoepithelial cell line, HMS-1, and a urea/guanidine hydrochloride gel extract (Humatrix) of HMS-X, a transplantable human xenograft of HMS-1 which secretes a copious extracellular matrix, we have demonstrated in preliminary experiments dramatic effects on primary breast carcinoma cell morphogenesis. Using HMS-1 as a feeder layer, specific induction of spheroid formation occurred in 5 different cases of primary breast cancer. Using the extracted gel, Humatrix, specific induction of primary breast carcinoma cell glandular morphogenesis also occurred. This small grant proposal will exploit the effects of HMS-1 and Humatrix on primary DCIS and primary breast carcinomas to establish both short term and long term cultures which hopefully will lead to immortalization with the help of athymic/Scid mice. The initial observations of glandular and spheroid morphogenesis will be expanded with objective studies of the effects of HMS-l and Humatrix on primary breast carcinoma mitogenesis, morphogenesis, genetic stability, and apoptosis. Mitogenesis studies will exploit Ki-67 antigen expression, bromodeoxyuridine uptake, and flow cytometric studies. Morphogenesis studies will exploit known breast differentiation markers including gross cystic fluid protein-IS, milk fat globule-2, laminin, type IV collagen, BRST-2, CU18, casein, other milk proteins and cadherins. Measurements of genetic stability will include karyotype analyses. Apoptosis studies will include measurements of nucleosomal DNA laddering, nuclear condensation, and expression of the apoptosis-associated genes SGP-2 and ICE (interleukin-1 converting enzyme) by Northern blot. If the studies of this pilot proposal prove successful in demonstrating a myoepithelial paracrine effect on primary breast carcinoma growth, a subsequent R01 application will address the molecular mechanism of this effect by exploiting a subtractive expression library approach.

原发性乳腺癌及癌前病变细胞的体外生长 事实证明，这是困难的，因此，这两个短期的缺乏， 长期和永生原代乳腺癌细胞系。原发性乳腺 癌培养物，虽然能够在选择性培养基中起始， 经历终末分化、衰老或凋亡， 几个通道。其原因尚不清楚。因为原发性乳腺癌 癌和癌前疾病状态（DCIS）出现在通过在 周围肌上皮细胞的设置，发挥重要作用的细胞 旁分泌效应通过直接的细胞-细胞相互作用和通过 基底膜细胞外基质的合成，我们假设 原发性乳腺癌和癌前细胞的生长 体外可能仍然依赖于这种类型的肌上皮相互作用。 使用新的人肌上皮细胞系HMS-1和尿素/胍， HMS-X的盐酸凝胶提取物（Humatrix），一种可移植的人 异种移植的HMS-1分泌丰富的细胞外基质，我们有 在初步实验中证明了对原发性乳腺癌的显著影响 癌细胞形态发生使用HMS-1作为饲养层， 在5种不同的原发性肝癌病例中， 乳腺癌使用提取的凝胶Humatrix，特异性诱导 原发性乳腺癌细胞也发生腺形态发生。这 一项小型赠款提案将利用HMS-1和Humatrix的作用， 原发性DCIS和原发性乳腺癌， 以及长期的文化，这些文化有望导致永生， 无胸腺/Scid小鼠的帮助。腺泡和腺泡的初步观察结果表明， 球状体形态发生将扩大与客观的研究， HMS-1和Humatrix对原发性乳腺癌有丝分裂的影响， 形态发生、遗传稳定性和凋亡。有丝分裂研究将 利用Ki-67抗原表达、溴脱氧尿苷摄取和流动 细胞计数研究。形态发生研究将利用已知的乳房 分化标志物包括大体囊液蛋白-IS、乳脂 球蛋白-2、层粘连蛋白、IV型胶原蛋白、BRST-2、CU 18、酪蛋白、其他牛奶 蛋白质和钙粘蛋白。遗传稳定性的测量将包括 核型分析细胞凋亡研究将包括测量 核小体DNA梯状化、核浓缩和 糖尿病相关基因SGP-2和ICE（白细胞介素-1转换酶） 用北方印迹法。如果这项试验计划的研究成功， 在证明肌上皮旁分泌对原发性乳腺癌的影响方面， 癌生长，随后的R 01应用将解决分子 通过利用消减表达文库来研究这种效应的机制 approach.