Re Richard N, Zhang Zhuo, Cook Julia L
Department of Research, Ochsner Clinic Foundation, New Orleans, LA.
Ochsner J. 2007 Summer;7(2):58-60.
We have previously reported that oligodeoxyribonucleotides, designed to bind in a triplex fashion to a specific p53 binding site homology, inhibit the proliferation of colon cancer cells in vitro and in vivo. The present study was designed to extend these observations and to determine whether ribonucleic acid (RNA) generated from a retroviral vector (RVV) and possessing a corresponding triplex forming site can, in a similar fashion, inhibit proliferation of p53-null K-562 leukemia cells. Viral vectors may offer advantages over oligonucleotides for tumor treatment. RVVs have the potential to be taken up more efficiently than oligonucleotides and to be expressed continuously and long-term, circumventing the need for repeated and frequent oligomer administration.
The p53-null human erythroleukemia cell line, K-562, was stably transfected with a tetracycline-repressible p53 expression construct (p53/pUHD10-3). p53 protein in these cells is expressed in the absence of tetracycline but down-regulated upon tetracycline treatment. Triplex-forming oligonucleotides [Hoog 1 (experimental) and Hoog 3 (control)] were cloned into RVVs in order to generate triplex-forming fusion mRNAs. Naive K-562 cells and p53/pUHD10-3-transfected K-562 cells (with and without tetracycline treatment) were infected with viruses that express the triplex-forming RNAs. Cell growth was measured by BrdU incorporation into DNA.
RVVs encoding Hoog 1, in both orientations, inhibit the growth of naive K-562 cells and p53-transfected, tet-repressed K-562 cells. p53 expression in K-562 cells decreases growth to the same extent as Hoog 1 RVV treatment. However, Hoog 1-RVV does not further inhibit growth of p53-expressing K-562 cells. Treatment with an RVV encoding the control, Hoog 3, has no growth inhibitory effect.
Triple helix-forming RNAs directed to a p53 consensus sequence homology reduce leukemia cell proliferation, suggesting a novel method of treatment.
我们之前曾报道，设计以三链体形式与特定p53结合位点同源序列结合的寡脱氧核糖核苷酸，在体外和体内均可抑制结肠癌细胞的增殖。本研究旨在拓展这些观察结果，并确定由逆转录病毒载体（RVV）产生的、具有相应三链体形成位点的核糖核酸（RNA）是否能以类似方式抑制p53基因缺失的K-562白血病细胞的增殖。病毒载体在肿瘤治疗方面可能比寡核苷酸具有优势。RVV有可能比寡核苷酸更有效地被摄取，并能持续长期表达，从而无需反复频繁地给予寡聚物。
将四环素可抑制的p53表达构建体（p53/pUHD10-3）稳定转染至p53基因缺失的人红白血病细胞系K-562。这些细胞中的p53蛋白在无四环素时表达，但在四环素处理后下调。将形成三链体的寡核苷酸[Hoog 1（实验性）和Hoog 3（对照）]克隆到RVV中，以产生形成三链体的融合mRNA。用表达形成三链体RNA的病毒感染未处理的K-562细胞和p53/pUHD10-3转染的K-562细胞（有或无四环素处理）。通过将BrdU掺入DNA来测量细胞生长。
编码Hoog 1的RVV，无论何种方向，均能抑制未处理的K-562细胞以及p53转染、四环素抑制的K-562细胞的生长。K-562细胞中p53的表达降低生长的程度与Hoog 1 RVV处理相同。然而，Hoog 1-RVV不会进一步抑制表达p53的K-562细胞的生长。用编码对照Hoog 3的RVV处理没有生长抑制作用。
靶向p53共有序列同源性的三链螺旋形成RNA可降低白血病细胞增殖，提示一种新的治疗方法。
