RADIOBIOLOGICAL STUDIES OF HYBRID CELL LINES

这项研究的主要目的是利用人类杂交细胞， 研究辐射诱导肿瘤转化的机制。 为此，一种独特的体外肿瘤定量测定法， 已经使用HeLa X皮肤成纤维细胞杂交体开发了转化。 该测定利用了HeLa肿瘤相关基因的表达。 抗原，最近被确定为肠碱性磷酸酶（IAP）。 这种抗原在非致瘤性杂交体中被抑制，但在非致瘤性杂交体中被重新表达。 在自发性和辐射诱导的致瘤性 分离子，并且可以用作肿瘤转化的标记。 在下一个资助期的拟议研究中， 应用于定量研究处理化学品的影响， 修饰细胞间隙连接通讯（GJIC）的药物， 辐射诱导的转化频率，试图评估 这种现象作为肿瘤转化调节剂重要性 在这个系统中。 除了应用于定量研究外， HeLa x皮肤成纤维细胞系统提供了一个独特的机会， 肿瘤转化的分子和细胞变化， 特别是关于肿瘤抑制基因的作用 失活 11号染色体被确定为携带假定的 HeLa抑制基因负调控肿瘤发生和 IAP的表达。 因此，辐射诱导的致瘤细胞系 将使用信息性RFLP进行Southern分析， 11号染色体。 这种分析应首先确定损害是否 染色体11与致瘤性的诱导相关， 第二，通过建立一个共同的缺失区， 应该能够建立推定的抑制基因。 辐射- 诱导的致瘤细胞系已经显示出广泛水平的 IAP的表达。 因此，它们提供了一个独特的学习机会， IAP负调控因子损伤在肿瘤发生中的作用 在这个系统中的转换。 最后，研究表明， 致瘤表型的分离与gap的丢失有关 连接细胞间通讯，以及差距表达缺失 连接蛋白，连接蛋白43。 因此，我们建议研究 GJIC、连接蛋白43表达水平和IAP表达水平 在辐射诱导的致瘤细胞中， GJIC在这些细胞获得致瘤性中的损失。 此外，为了阐明GJIC和GJ基因的缺失是否 表达是获得致瘤性肿瘤的原因或结果， 表型，我们建议比较连接蛋白43转染的效果 另一方面是11号染色体的微细胞转移， 在GJIC、连接蛋白43、 表达、IAP表达和致瘤性。

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.