TRANSFORMING FUNCTIONS OF MUTANT COLON CANCER ONCOGENES

这项提议的目标是在人类结肠上皮细胞中定义 5个基因在结直肠癌中的转化活性 是体细胞突变的常见部位。这些基因是P53、K-ras、MCC、 APC和DCC。具体地说，我们将测试这些更改后的形式 可能的结肠癌基因及其致癌能力 一种独特的未转化结肠腺瘤细胞系VACO-1的研究进展 330.VACO-330是从人类良性结肠腺瘤建立的，它保留了 良性结肠上皮的许多特征。VACO-330不会致癌 在裸鼠体内，不能锚定独立生长在软组织中 琼脂，具有良好分化的上皮形态。这就是增长 依赖因子，需要转化生长因子-α刺激生长。我们 研究表明，VACO-330携带一个突变型和一个野生型P53等位基因， 只携带生殖系ras等位基因，并在很低水平上表达mcc， APC和DCC成绩单。结肠中存在常见的基因改变 癌症包括K-ras-p53、MCC和APC基因突变，以及 P53、DCC、APC和MCC基因缺失。目前仍存在的主要问题 1)恶性表型的功能特征是什么？ 这些改变的基因；2)这些改变的基因是显性的还是 隐性的，编码活跃的癌基因还是无效的抑癌基因？我们 将通过研究从腺瘤到 转染人VACO-330细胞的肿瘤细胞 五个基因。一般的方法将是按顺序确定 分析VACO-330中哪些等位基因已经突变，哪些仍然存在 野生型。然后我们将：1)关闭VACO-330中的野生型表达 类型等位基因(检测抑制活性)，2)添加外源突变 VACO-330野生型等位基因(显性致癌分析 活性)，以及3)向VACO-330突变体添加外源野生型等位基因 等位基因(检测抑制活性)。VACO-330的研究进展 由转基因基因诱导的将通过检测 转染体：获得致瘤性和锚定 独立生长，形态和生长速度的变化，以及 从转化生长因子刺激的要求中获得独立性 阿尔法。 建议进行的具体研究如下：1)通过转染法确定 共表达突变型K-ras和突变型P53等位基因的促癌活性 VACO-330进展；2)为了确定VACO-330 APC的基因， DCC和MCC等位基因；3)为了确定DCC的抑制基因活性， 反义构建体法检测APC和MCC的表达 这将关闭这些转录本在VACO-330中的表达；4) 检测转基因野生型MCC、APC和APC的抑制活性 通过检测它们逆转结肠癌的能力；5)确定 特异性突变体MCC和APC的显性转化活性 转染法从结肠癌中克隆的等位基因 将这些突变的MCC和APC基因克隆到VACO-330中。

The goal of this proposal is to define in human colon epithelium the transforming activity of five genes which in human colorectal cancers are common sites of somatic mutation. These genes are p53, K-ras, MCC, APC, and DCC. Specifically, we will test altered forms of these putative colon cancer genes for their ability to cause malignant progression of a unique nontransformed colon adenoma cell line, VACO- 330. VACO-330, established from a benign human colon adenoma, retains many features of a benign colon epithelium. VACO-330 is nontumorigenic in the nude mouse, incapable of anchorage independent growth in soft agar, and has well differentiated epithelial morphology. It is growth factor dependent, requiring for growth stimulation by TGF-alpha. We have shown that VACO-330 bears one mutant and one wild type p53 allele, bears only germ line ras alleles, and expresses at very low levels MCC, APC, and DCC transcripts. Common genetic alterations present in colon cancers include mutations of the K-ras-p53, MCC and APC genes, and deletions of p53, DCC, APC and MCC genes. Major questions now remaining are: 1) what functional features of the malignant phenotype are imparted by these altered genes; 2) are these altered genes dominant or recessive, encoding active oncogenes or inactive suppressor genes? We will answer these questions by study of progression from adenoma to carcinoma in VACO-330 transfected with additional altered forms of these five genes. The general approach will be to determine by sequence analysis which alleles are in VACO-330 already mutant and which remain wild type. We will then: 1) turn off in VACO-330 expression of the wild type alleles (assaying suppressor activities), 2) add exogenous mutant alleles to the VACO-330 wild type alleles (assaying dominant oncogenic activity), and 3) add exogenous wild type alleles to the VACO-330 mutant alleles (assaying suppressor activities). Progression of VACO-330 induced by transfected genes will be determined by assay of transfectants for: acquisition of tumorigenicity and anchorage independent growth, for changes in morphology and growth rate, and for acquisition of independence from the requirement for stimulation by TGF- alpha. Specific studies proposed are: 1) To determine by transfection the activity of coexpressed mutant K-ras and mutant p53 alleles in promoting VACO-330 progression; 2) To determine the genotype of the VACO-330 APC, DCC and MCC alleles; 3) To determine suppressor gene activity of DCC, APC, and MCC by assay of progression induced by antisense constructs which will turn off expression of these transcripts in VACO-330; 4) To determine the suppressor activity of transfected wild type MCC, APC and DCC by assay of their ability to revert colon neoplasia; 5) To determine the dominant transforming activity of specific mutant MCC and APC alleles which have been cloned from colon carcinomas by transfecting these mutant MCC and APC cDNAs into VACO-330.