TRANSFORMING FUNCTIONS OF MUTANT COLON CANCER ONCOGENES

该提案的目标是在人类结肠上皮中定义 人大肠癌中5个基因的转化活性 是体细胞突变的常见位点 这些基因是p53，K-ras，MCC， APC和DCC。 具体地说，我们将测试这些改变的形式， 假定的结肠癌基因，因为它们能够引起恶性肿瘤， 一个独特的非转化结肠腺瘤细胞系，VACO- 330. VACO-330，从一个良性的人类结肠腺瘤，保留 良性结肠上皮的许多特征。 VACO-330无致瘤性 在裸鼠中，不能在软组织中锚定独立生长 琼脂，并具有分化良好的上皮形态。 这是增长 因子依赖性，需要TGF-α的生长刺激。 我们 已经显示VACO-330携带一个突变型和一个野生型p53等位基因， 仅具有生殖系ras等位基因，并且以非常低的水平表达MCC， APC和DCC转录物。 结肠中常见的遗传改变 癌症包括K-ras-p53、MCC和APC基因的突变， 缺失p53、DCC、APC和MCC基因。 现在剩下的主要问题 是：1）恶性表型的哪些功能特征被赋予 （2）这些变异的基因是显性的，还是 隐性的，编码活性癌基因或非活性抑制基因？ 我们 我将通过研究从腺瘤到腺瘤的进展来回答这些问题。 在VACO-330中转染了另外改变形式的这些 五个基因 一般的方法是按顺序确定 分析VACO-330中哪些等位基因已经突变， 野生型 然后，我们将：1）在VACO-330中关闭野生型表达 型等位基因（测定抑制活性），2）添加外源突变体 VACO-330野生型等位基因（测定显性致癌基因 活性），和3）将外源野生型等位基因添加到VACO-330突变体中 等位基因（测定抑制活性）。 VACO-330的进展 将通过以下测定来确定转染基因诱导的 转染子用于：获得致瘤性和锚定 独立生长，形态和生长速率的变化，以及 从TGF-β刺激的需要中获得独立性， α的 建议的具体研究是：1）通过转染来确定 共表达的突变K-ras和突变p53等位基因在促进 VACO-330进展; 2）为了确定VACO-330 APC的基因型， DCC和MCC等位基因：3）检测DCC抑制基因活性， APC和MCC通过反义构建体诱导的进展的测定 这将关闭VACO-330中这些转录物的表达; 4） 测定转染的野生型MCC、APC和 DCC通过测定其逆转结肠肿瘤形成的能力; 5）确定 特异性突变MCC和APC的显性转化活性 已经从结肠癌中克隆的等位基因， 将这些突变MCC和APC cDNA导入VACO-330。

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.