CRE OLIGONUCLEOTIDE AS TRANSCRIPTION FACTOR DECOY/TUMOR GROWTH INHIBITOR

CRE 寡核苷酸作为转录因子诱饵/肿瘤生长抑制剂

• 批准号: 6101058
• 负责人: Y S CHO-CHUNG
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/6101058
• 关键词: DNA binding protein; athymic mouse; cell growth regulation; cyclic AMP; gene induction /repression; genetic regulatory element; genetic transcription; growth inhibitors; neoplastic growth; oligonucleotides; pleiotropism; transcription factor

Synthetic double-stranded DNA with high affinity for a target transcription factor can be introduced into target cells as decoy cis- elements to bind the factor and alter gene transcription. The CRE (cyclic AMP response element)-transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element TGACGTCA is palindromic, a synthetic single stranded oligonucleotide composed of the CRE sequence, which will self-hybridize to form either a duplex or hairpin, when introduced into a cell, can act as a decoy for the transcription factor. We have investigated the CRE-palindromic and - hairpin forming oligonucleotides as transcription factor decoys and the biological effects thereof. The 24 mer CRE palindrome oligonucleotide penetrated into the cell and competed with the cellular cis-element for the binding of sequence-specific CRE DNA-binding proteins, such as the 43 kDa CREB. The palindromic or hairpin-forming CRE oligonucleotide interfered with CRE-directed transcription in intact cells as determined by a transient transcription assay. The 24 mer CRE palindrome oligonucleotide produced potent growth inhibition in a variety of cancer cells including breast, prostate, lung, ovarian, colon, and epidermoid carcinomas, and multidrug-resistant cancer cell lines of MCF7-TH (MDR- breast cancer) and HCT-15 (MDR-colon carcinoma) (IC50, 100-150 nM). The growth of normal human mammary epithelial cell and lung epithelial cell lines was not affected by the CRE oligonucleotide. Treatment of nude mice bearing HCT-15 MDR colon carcinoma with 24 mer CRE oligonucleotide (0.1 mg/0.1 ml saline/mouse ip, 5x/week for 4 weeks) resulted in 85% inhibition of tumor growth. The CRE-oligonucleotide-induced growth inhibition accompanied changes in cell morphology and the appearance of apoptotic nuclei. Two base-mismatched control oligonucleotide or a palindromic oligonucleotide containing no CRE sequence had no effect on either CRE-directed transcription or cell growth. The mechanism by which the blockade of CRE-gene transcription brings about the selective inhibition of tumor cell growth but not normal cell growth is under investigation. Our data show that the CRE-transcription factor decoy can modulate in vivo gene transcription and restrain tumor growth in vivo. Thus, this technology offers great promise as a tool for defining cellular regulatory processes and treating diseased conditions.

对靶标具有高亲和力的合成双链 DNA 转录因子可以作为诱饵顺式引入靶细胞 结合因子并改变基因转录的元件。商业地产 （环磷酸腺苷反应元件）-转录因子复合物是 多效性激活剂，参与广泛的诱导 多种细胞和病毒基因。因为CRE顺式元素 TGACGTCA 是回文的，一种合成的单链寡核苷酸 由 CRE 序列组成，它将自杂交形成 当双链体或发夹引入细胞时，可以充当诱饵 转录因子。我们研究了 CRE 回文并且 - 作为转录因子诱饵的发夹形成寡核苷酸和 其生物学效应。 24 mer CRE 回文寡核苷酸 渗透到细胞内并与细胞顺式元件竞争 序列特异性 CRE DNA 结合蛋白的结合，例如 43 kDa CREB。回文或发夹形成的 CRE 寡核苷酸 干扰完整细胞中 CRE 指导的转录 通过瞬时转录测定。 24 mer CRE 回文 寡核苷酸对多种癌症产生有效的生长抑制 细胞包括乳腺、前列腺、肺、卵巢、结肠和表皮样细胞 癌和多药耐药癌细胞系 MCF7-TH (MDR- 乳腺癌）和 HCT-15（MDR 结肠癌）（IC50，100-150 nM）。这 正常人乳腺上皮细胞和肺上皮细胞的生长 细胞系不受 CRE 寡核苷酸的影响。裸体治疗 携带 24 mer CRE 寡核苷酸的 HCT-15 MDR 结肠癌小鼠 （0.1 mg/0.1 ml 生理盐水/小鼠腹腔注射，每周 5 次，持续 4 周）结果为 85% 抑制肿瘤生长。 CRE-寡核苷酸诱导的生长 抑制伴随着细胞形态和外观的变化 凋亡细胞核。两个碱基错配的对照寡核苷酸或 不含CRE序列的回文寡核苷酸对 CRE 指导的转录或细胞生长。其机制 CRE基因转录的阻断带来了选择性 抑制肿瘤细胞生长，但不抑制正常细胞生长 调查。我们的数据表明，CRE 转录因子诱饵可以 调节体内基因转录并抑制体内肿瘤生长。 因此，这项技术作为一种定义工具提供了巨大的前景。 细胞调节过程和治疗疾病。