Role of p53 family genes in acquiring drug resistance of neuroblastoma.

我们研究了p53家族基因在人类神经母细胞瘤多药耐药的发生和获得中的作用。我们得到的结果如下。(1)在初次手术获得的原发性神经母细胞瘤中p53基因突变非常罕见。然而，它在复发性肿瘤和许多神经母细胞瘤细胞系中被诱导。在30株神经母细胞瘤细胞系中，我们通过酵母功能测定和DNA测序发现6个p53基因功能缺失突变。此外，我们发现高水平的p53 mRNA表达与神经母细胞瘤的不良预后显著相关，通过使用我们内部的cDNA微阵列，该芯片携带了从我们生成的原代神经母细胞瘤cDNA文库中获得的5300个cDNA。此外，我们已经知道p53通常局限于晚期神经母细胞瘤的细胞质中。这些提示p53可能在原发性神经母细胞瘤中起调节肿瘤生长和凋亡的作用。(2)寻找p53家族基因在维甲酸诱导的神经母细胞瘤细胞凋亡中的作用。然而，p53家族分子的水平太低而无法检测到。在蛋白水平上仅诱导出p73蛋白。(3)在包括神经母细胞瘤在内的人类肿瘤细胞系中，p73和p63在顺铂耐药细胞系中表达水平较高。我们进一步的分析表明，Mdm2在这些顺铂耐药细胞系中表达高，进而诱导p53蛋白降解，导致获得耐药。目前，我们正在进一步寻找针对侵袭性神经母细胞瘤的精确机制，以开发新的治疗策略。

We have examined the role of p53 family genes in the genesis and acquisition of multi-drug resistance in human neuroblastoma. The results we obtained are as follows. (1)Mutation of the p53 gene was very rare in primary neuroblastomas obtained at initial surgery. However, it was induced in recurrent tumors as well as in many neuroblastoma cell lines. Among 30 neuroblastoma cell lines, we found 6 loss of function mutations in the p53 gene by yeast functional assay and DNA sequencing. Furthermore, we found that high levels of p53 mRNA expression were significantly associated with poor prognosis of neuroblastoma by using our in-house cDNA microarray which carried 5,300 cDNAs obtained from primary neuroblastoma cDNA libraries we generated. In addition, it is already known that p53 is often localized in cellular cytoplasm in advanced stages neuroblastomas. These suggest that p53 may function in primary neuroblastomas to regulate the tumor growth and apoptosis. (2)We searched for the role of p53 family genes in retinoic acid-induced apoptosis of neuroblastoma cells. However, the levels of p53 family molecules were too low to be detected. Only p73 protein was induced at the protein level. (3)In the panel of human cancer cell lines including neuroblastoma, the levels of p73 and p63 expression were high in the lines with resistance to cisplatin. Our further analysis revealed that Mdm2 expression was high in those cisplatin-resistant cell lines, that in turn induced degradation of p53 protein, leading to acquiring the resistance. Currently, we are further searching for the precise mechanism to develop new therapeutic strategies against aggressive neuroblastomas.