Analysis of IGF-I receptor transcriptional regulation by mutant p53 and its clinical implication

研究了野生型P53对HPV感染的宫颈癌来源细胞株SIHA和HeLa S3中IGF-I受体表达的影响。瞬时将E6基因导入SIHA细胞后，P53蛋白表达下调，IGF-I受体表达上调。相反，瞬时将E2导入HeLa S3细胞后，P53表达上调，IGF-I受体表达下调。我们可以通过建立这些基因的稳定转化体来重新确认这些现象。IGF-I受体启动子驱动的荧光素酶分析表明，IGF-I受体表达的这些变化是基于转录调控。这些稳定的转化子中IGF-I受体表达的变化对它们在软琼脂中的非锚定生长和裸鼠体内的致瘤性产生了影响。然后，用免疫组织化学方法检测其在人宫颈标本中的表达水平及其酪氨酸磷酸化状态。IGF-I受体表达水平在CIN III及以上病变中显著升高。在所有的CIN和浸润性癌标本中都观察到了磷酸化的IGF-I受体，并且它的促进作用与疾病的进展有关，然后评估突变的P53对IGF-I受体的转录调控。我们构建了编码野生型和各种突变体的表达载体，尤其是在热点突变区域。瞬时转染野生型和突变型P53基因后，荧光素酶活性检测显示IGF-I受体的转录调控不同。野生型以及P72R和V143A突变体下调IGF-I受体的转录，而其他突变体T123A、R175H、R248Q、R273H则没有影响。

The role of wild type p53 on IGF-I receptor expression was studied in cervical cancer derived cell lines SiHa and HeLa S3 harboring HPV. Transient transfection of E6 into SiHa cells downregulated p53, and upregulated IGF-I receptor. In contrast, transient transfection of E2 into HeLa S3 cells upregulated p53, and downregulated IGF-I receptor. We could reconfirm these phenomena by establishing stable transformants of these genes. Luciferase assay driven by IGF-I receptor promoter revealed that these alternation of IGF-I receptor expression was based on transcriptional regulation. Alternation of IGF-I receptor expression within these stable transformants did effect on their anchorage-independent growth in soft agar, and tumorigenesity in nude mice.Then, expression levels and their tyrosil phosphorylated status in human cervical specimens were evaluated by immunohistochemistry. IGF-I receptor expression levels were significantly promoted in CIN III lesions and more. Phosphorylated IGF-I receptor was observed in all CIN and invasive cancer specimens, and its promotion was related to the progression of the disease.Then, transcriptional regulation on IGF-I receptor by mutant p53 was evaluated. We constructed expression vectors encoding wild type and various p53 mutants especially within hot spot mutation areas. Luciferase assay after transient transfection of these wild type and mutant p53 genes showed different transcriptional regulations of IGF-I receptor. Wild type as well as P72R and V143A mutants downregulated IGF-I receptor transcription, whereas other mutants T123A, R175H, R248Q, R273H had no effect.