COLON CANCER CELL GROWTH INHIBITION USING ANTISENSE OLIGOS AGAINST CRYPTO

使用针对 CRYPTO 的反义寡核苷酸抑制结肠癌细胞生长

• 批准号: 3804858
• 负责人: M P SELVAM
• 金额: --
• 依托单位: CENTER BIOLOGICS EVALUATION RESEARCH TRANSFUSION
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804858
• 关键词: antineoplastics; antisense nucleic acid; cytotoxicity; neoplastic cell; neoplastic transformation; oligonucleotides; thiophosphate; tissue /cell culture; transfection

Cripto is a gene recently identified and cloned from a human embryonal carcinoma cell line. The human cripto cDNA is 2020 base pairs long with an open reading frame of 564 base pairs. The open reading frame encodes for a protein of 188 amino acids that contains a control portion of 37 amino acids which shares a structural cysteine rich motif in common with other members of the EGF super gene family, such as human EGF, human TGF a and human amphiregulin. It has been demonstrated previously that the over expression of EGF or TGF a cDNAs in fibroblasts and epithelial cells can lead to transformation in vitro and in certain cases to tumorigenicity in vivo. Preliminary experiments have shown that transfection of human cripto cDNA expression vector in which transcription is under the control of RSV- LTR into mouse NIH-3T3 fibroblasts induces the formation of transformed foci. Since cripto and TGFa are expressed in undifferentiated human embryonal carcinoma cell line in which they may function as autocrine growth factors, and since a portion of the 'cripto' protein shares the homology to EGF & TGFa, we have decided to make antisense oligos against the CRIPTO cDNA sequence, in order to show the ability to shut off the function of this factor. As a preliminary step in this project, we are screening 3 different phosphorothioate oligos in antisense, sense and nonsense nature of the target sequences of the CRIPTO gene. We studied the effect on three colon carcinoma cell lines [viz NTERA-2, NOG-8, and GEO. which have been analyzed for the cripto gene] for their inhibition in cell growth. Preliminary results showed a 90% inhibition of NOG-8 colon cancer celline in soft agar growth.

Cripto是最近从人类胚胎中鉴定和克隆的基因。 癌细胞系 人cripto cDNA长2020个碱基对， 564个碱基对的开放阅读框。 开放阅读帧编码 一种188个氨基酸的蛋白质，含有37个氨基酸的控制部分， 与其它氨基酸共享结构上富含半胱氨酸基序的氨基酸 EGF超基因家族的成员，如人EGF、人TGF α和人TGF β 1。 人双调蛋白 以前已经证明， EGF或TGF α cDNA在成纤维细胞和上皮细胞中的表达可 导致体外转化和在某些情况下导致体内致瘤性 vivo. 初步实验表明，转染人cripto cDNA表达载体，其中转录在RSV-1的控制下。 LTR进入小鼠NIH-3 T3成纤维细胞诱导转化的 焦点 由于cripto和TGF α在未分化的人类中表达， 胚胎癌细胞系，其中它们可能作为自分泌细胞发挥作用， 生长因子，并且由于“cripto”蛋白质的一部分共享 与EGF和TGF α同源，我们决定制备针对EGF和TGF α的反义寡核苷酸。 CRIPTO cDNA序列，以显示关闭 这个因素的作用。 作为该项目的第一步，我们 筛选3种不同的硫代磷酸寡核苷酸的反义、有义和 CRIPTO基因的靶序列的无义性质。 我们研究了 对三种结肠癌细胞系[即NTERA-2、NOG-8和GEO. 已经分析了cripto基因]在细胞中的抑制作用， 增长 初步结果显示对NOG-8结肠癌的抑制率为90% 细胞在软琼脂中生长。