CRE OLIGONUCLEOTIDE AS TRANSCRIPTION FACTOR DECOY/TUMOR GROWTH INHIBITOR

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导入靶细胞， 结合因子并改变基因转录的元件。综合招聘考试 （环AMP反应元件）-转录因子复合物是一种 一种多效性激活剂，参与广泛的 各种细胞和病毒基因。因为CRE顺式元件 TGACGTCA是回文的，是一种合成的单链寡核苷酸， 由CRE序列组成，其将自杂交以形成 当双链体或发夹被引入细胞时，可以作为诱饵， 转录因子我们研究了CRE回文和- 作为转录因子诱饵的发夹形成寡核苷酸， 其生物学效应。24 mer CRE回文寡核苷酸 渗透到细胞中，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CRE寡核苷酸 干扰完整细胞中的CRE定向转录， 通过瞬时转录测定。24 mer CRE回文 寡核苷酸在多种癌症中产生有效的生长抑制 包括乳腺、前列腺、肺、卵巢、结肠和表皮样细胞 癌和MCF 7-TH（MDR-1）的多药耐药癌细胞系。 乳腺癌）和HCT-15（MDR-结肠癌）（IC 50，100-150 nM）。的 正常人乳腺上皮细胞和肺上皮细胞的生长 品系不受CRE寡核苷酸的影响。裸体治疗 用24 mer CRE寡核苷酸携带HCT-15 MDR结肠癌的小鼠 (0.1 1 mg/0.1ml盐水/小鼠ip，5x/周，持续4周）导致85%的 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。的机制 CRE基因转录的阻断导致选择性的 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 调节体内基因转录和抑制体内肿瘤生长。 因此，这项技术提供了很大的希望，作为一种工具， 细胞调节过程和治疗疾病。

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.