INTERACTION OF ANTITUMOR AGENTS WITH ACTIVATED ONCOGENES

该应用程序的总体目标是确定DNA损伤是否 由临床上使用的烷基化抗肿瘤剂产生， 改变激活的癌基因的表达， 致瘤表型的维持。 使用Maxam-Gilbert DNA测序方法学初步研究将确定 氮芥（HN-2）、L-苯丙氨酸氮芥（L-PAM）和两种 环磷酰胺衍生物（4-HC和C2）具有DNA序列 在克隆的编码区攻击的偏好，从几个 激活的癌基因 这种DNA损伤对 受损癌基因序列的转录将在 体外RNA转录系统。 序列选择性 然后通过以下方法在治疗的肿瘤细胞中研究所述试剂： 确定癌基因和其他基因中的烷基化模式 关键的细胞基因因抗肿瘤药物的不同而不同， 剂. 这些实验也将使修复的研究 在激活的癌基因中的DNA损伤。 然后药物将 研究了DNA损伤特性，细胞毒性，如通过 集落形成试验和对癌基因表达的影响（如 通过斑点和北方印迹分析测量 这些癌基因被激活的细胞系。 具有单个 拷贝过表达的癌基因将与具有 同一癌基因的扩增拷贝，以确定是否抗- 肿瘤因子对癌基因失活或多或少有效 和细胞杀伤取决于癌基因拷贝数。 从这些 研究表明，抗肿瘤药物可能被选择 用于对抗特定的致癌基因，因为 特别是抗肿瘤剂，以有效地降低肿瘤的发生， 肿瘤细胞中的致癌基因。

The overall goal of this application is to determine if DNA damage produced by clinically used alkylating anti-tumor agents can alter the expression of activated oncogenes which are critical for the maintenance of the tumorigenic phenotype. Using Maxam-Gilbert DNA sequencing methodology initial studies will determine if nitrogen mustard (HN-2), L-phenylalanine mustard (L-PAM), and two cyclophosphamide derivatives (4-HC and C2) have DNA sequence preferences for attack in cloned coding regions from several activated oncogenes. The effects of this DNA damage on transcription of the damaged oncogene sequence will be studied in an in vitro RNA transcription system. The sequence selectivity of the agents will then be studied in treated tumor cells by determining if alkylation patterns in oncogenes and other critical cellular genes differ with the different anti-tumor agents. These experiments will also allow the study of the repair of DNA damage in activated oncogenes. The drugs will then be studied for DNA damaging properties, cytotoxicity as measured by colony formation assays, and effects on oncogene expression (as measured by Dot and Northern blot analysis) in human tumor cell lines having these oncogenes activated. Cell lines having single copy over-expressed oncogenes will be compared to cell lines having amplified copies of the same oncogene to determine if the anti- tumor agents are more, or less, effective at oncogene inactivation and cell killing depending on the oncogene copy number. From these studies it is hoped that anti-tumor agents might be selected for use against specific oncogenes due to the ability of the particular anti-tumor agent to efficiently inactivate critical oncogenes in the tumor cell.