Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China.
J Exp Clin Cancer Res. 2010 Jun 17;29(1):74. doi: 10.1186/1756-9966-29-74.
The tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor. However, the inhibitory activities of these variants have not yet been compared in the same cells. Protein therapy is known to have several advantages over gene therapy. Thus, investigation of the exogenous protein molecule of the most effective suppressor may yield meaningful information regarding protein-based cancer therapy.
The inhibitory effects of p16INK4a, p14ARF and p12 were studied in the human lung cancer cell line A549 which lacks the CDKN2A locus. The eukaryotic expression plasmids of the three transcriptional variants were constructed and stably transfected into the cells. RNA and protein expression by the plasmids was confirmed using RT-PCR and fluorescence immunocytochemistry, respectively. Cell growth inhibition and cell-cycle redistribution after transfection were investigated based on growth curve and flow cytometry analyses. An exogenous His-tag fusion p16INK4a protein was obtained and purified by affinity chromatography. Cell growth inhibition and cell cycle arrest induced by the expression of p16INK4a protein were measured in A549 cells transduced with the exogenous protein.
While all three variants suppressed cell growth, p16INK4a had the strongest effect. Marked G1-phase accumulation and S-phase inhibition were induced by p16INK4a and p14ARF but not by p12. Exogenous p16INK4a protein was successfully expressed and purified and transduction of the fusion protein into A549 cells inhibited cell growth by G1-->S arrest.
Among the three transcript variants, p16INK4a has a greater inhibitory effect than p14ARF and p12; exogenous p16INK4a protein should be further investigated for use in cancer therapy as a protein agent.
肿瘤抑制基因 CDKN2A 至少产生三种不同的转录变体，每种转录变体都被认为编码一种肿瘤抑制蛋白。然而，这些变体在相同的细胞中的抑制活性尚未被比较过。与基因治疗相比，蛋白质治疗具有许多优势。因此，研究最有效的抑制因子的外源性蛋白质分子可能会为基于蛋白质的癌症治疗提供有意义的信息。
构建三种转录变体的真核表达质粒，并稳定转染缺乏 CDKN2A 基因座的人肺癌细胞系 A549。分别采用 RT-PCR 和荧光免疫细胞化学法检测质粒的 RNA 和蛋白表达。通过生长曲线和流式细胞术分析研究转染后细胞生长抑制和细胞周期重分布。通过亲和层析获得外源性 His 标记的 p16INK4a 融合蛋白，并进行纯化。用转导外源性蛋白的 A549 细胞测量 p16INK4a 蛋白表达诱导的细胞生长抑制和细胞周期阻滞。
虽然三种变体均抑制细胞生长，但 p16INK4a 的作用最强。p16INK4a 和 p14ARF 诱导明显的 G1 期积累和 S 期抑制，但 p12 则不然。成功表达和纯化了外源性 p16INK4a 蛋白，并将融合蛋白转导到 A549 细胞中，通过 G1→S 期阻滞抑制细胞生长。
在这三种转录变体中，p16INK4a 的抑制作用大于 p14ARF 和 p12；外源性 p16INK4a 蛋白作为蛋白质制剂应进一步用于癌症治疗的研究。
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