RADIOBIOLOGICAL STUDIES OF HYBRID CELL LINES

杂交细胞系的放射生物学研究

• 批准号: 2089805
• 负责人: JOHN L REDPATH
• 金额: $ 21.66万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-03-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2089805
• 关键词: DNA damage; HeLa cells; alkaline phosphatase; athymic mouse; cell cell interaction; cell population study; fibroblasts; fibronectins; gap junctions; gene induction /repression; human genetic material tag; hybrid cells; karyotype; membrane channels; neoplasm /cancer genetics; neoplasm /cancer pharmacology; nutrition related tag; phorbols; radiation carcinogenesis; radiation genetics; radiation related neoplasm /cancer; retinoids; transfection; tumor antigens; tumor suppressor genes

The major objective of this research is to utilize human hybrid cells to investigate mechanisms of radiation-induced neoplastic transformation. To this end, a unique quantitative assay for in vitro neoplastic transformation has been developed using HeLa x skin fibroblast hybrids. This assay takes advantage of the expression of the HeLa tumor-associated antigen, recently identified as intestinal alkaline phosphatase (IAP). This antigen is suppressed in the non-tumorigenic hybrids but is re- expressed in both spontaneous and radiation-induced tumorigenic segregant, and can be used as a marker for neoplastic transformation. In the proposed studies for the next funding period, this system will be applied to quantitative studies dealing with the influence of chemical agents which modify gap junction intercellular communication (GJIC) on radiation-induced transformation frequency in an attempt to assess the importance of this phenomenon as a modulator of neoplastic transformation in this system. Apart from the application to quantitative studies, the HeLa x skin fibroblast system affords a unique opportunity to investigate the molecular and cellular changes underlying neoplastic transformation, particularly with respect to the role of tumor-suppressor gene inactivation. Chromosome 11 has been identified as carrying the putative HeLa suppressor gene which negatively regulates both tumorigenicity and the expression of IAP. Thus, radiation-induced tumorigenic cell lines will be subjected to Southern analysis using informative RFLP's for chromosome 11. This analysis should, firstly, establish whether damage to chromosome 11 correlates with induction of tumorigenicity, and secondly, by establishment of a common deleted region, the locus of the putative suppressor gene should be able to be established. Radiation- induced tumorigenic cell lines have been shown a wide range of levels of expression of IAP. They therefore afford a unique opportunity to study the role of damage to the negative regulator of IAP in neoplastic transformation in this system. Finally, it has been shown that segregation of the tumorigenic phenotype is associated with loss of gap junction intercellular communication, and loss of expression of the gap junction protein, connexin 43. Thus, we propose to examine the extent of GJIC, level of connexin 43 expression and level of expression of IAP in radiation-induced tumorigenic cells in order to establish the role of loss of GJIC in the acquisition of tumorigenicity by these cells. Furthermore, in an attempt to clarify whether loss of GJIC and GJ gene expression is a cause or an effect of acquisition of the tumorigenic phenotype, we propose to compare the effects of connexin 43 transfection on the one hand, and microcell transfer of chromosome 11 on the other, into tumorigenic hybrids on the endpoints of GJIC, connexin 43 expression, IAP expression, and tumorigenicity.

这项研究的主要目标是利用人类杂交细胞 探讨辐射诱发肿瘤转化的机制。 为此，一种独特的体外肿瘤定量检测方法 已经使用HeLa x皮肤成纤维细胞杂交体进行了转化。 本实验利用HeLa肿瘤相关基因的表达 抗原，新近鉴定为肠道碱性磷酸酶(IAP)。 这种抗原在非致瘤杂交瘤中被抑制，但被重新激活。 在自发和辐射诱发的致癌中均有表达 分离剂，可作为肿瘤转化的标志物。 在下一个供资期间的拟议研究中，这一制度将是 应用于处理化学物质影响的定量研究 修饰缝隙连接细胞间通讯(GJIC)的药物 辐射诱导的转化频率，试图评估 这一现象作为肿瘤转化调节器的重要性 在这个系统中。除了在定量研究方面的应用外， HeLa x皮肤成纤维细胞系统为研究提供了一个独特的机会 肿瘤转化背后的分子和细胞变化， 尤其是关于肿瘤抑制基因的作用 失活。11号染色体已被确认携带有推定的 HeLa抑制基因负性调节致瘤性和致瘤性 IAP的表达。因此，辐射诱导的致瘤细胞系 将使用信息丰富的RFLP进行Southern分析 11号染色体。这项分析首先应该确定 11号染色体的突变与肿瘤的诱发有关，并且 其次，通过建立一个共同的缺失区， 推测的抑癌基因应该能够被建立。辐射- 诱导的致瘤细胞系已被显示出广泛的水平 IAP的表达。因此，他们提供了一个独特的学习机会 IAP负性调节因子损伤在肿瘤中的作用 在这个系统中的转变。最后，事实证明， 致瘤表型的分离与GAP的丢失有关 连接细胞间通讯和缝隙表达的丧失 连接蛋白，连接蛋白43。因此，我们建议研究一下 GJIC、Cx43表达水平和IAP表达水平 在辐射诱导的致瘤细胞中的作用 在这些细胞获得致瘤性的过程中GJIC的丢失。 此外，为了阐明GJIC和GJ基因的缺失是否 表达是致癌物获得的原因或结果 表型，我们建议比较间隙连接蛋白43的转染效果。 一方面，11号染色体的微细胞转移， 在GJIC的末端转化为致瘤杂交体，连接蛋白43 表达、IAP表达和致瘤性。