Experimental Radiobiology for Children's Health Research Group, Research Center for Radiation Protection, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba, 263-8555, Japan.
In Vitro Cell Dev Biol Anim. 2010 May;46(5):477-86. doi: 10.1007/s11626-009-9269-3. Epub 2010 Feb 5.
To investigate whether p16 inactivation is involved in the development of rat pulmonary tumors, we compared the p16 status and tumorigenicity of cell lines which indicated different p16 status. The tumor cell line (PuD2) was established from lung adenocarcinoma induced in plutonium dioxide-inhaled rat in this study. The virus-immortalized SV40T2 cells, benzo[a]pyrene-induced BP cells, BP-derived BP(P)Tu cells, and gamma ray-transformed RTiv3 cells were utilized as the respiratory epithelial cell lines. A tumorigenicity assay-inoculating cells into nude mice revealed that PuD2, BP, and BP(P)Tu cells were tumorigenic, but SV40T2 and RTiv3 cells were not. Methylation-specific PCR of the p16 promoter region revealed that SV40T2 cells were unmethylated, BP cells displayed heterogeneous methylation, and BP(P)Tu and RTiv3 cells were completely methylated. Methylation-specific PCR and PCR of genomic DNA in the p16 region did not amplify product in PuD2 cells, indicating deletion of p16. Banded karyotypes prepared from PuD2 cells exhibited trisomy of chromosome 4, inversion in chromosome 11, and partial deletion of chromosomes 4 and 5. The demethylating agent 5Aza2dC partially demethylated the p16 promoter region of BP(P)Tu, BP and RTiv3 cells, increasing expression of the p16 transcript and decreasing growth of the cells. These results indicate that hypermethylation of the p16 promoter region occurs early in neoplastic transformation before acquisition of tumorigenicity in rat respiratory epithelium. Loss of genes located on chromosomes 4 and 5 may be important for tumor progression and acquisition of high tumorigenic activity in the Pu-induced rat lung tumor.
为了研究 p16 失活是否参与大鼠肺部肿瘤的发生，我们比较了具有不同 p16 状态的细胞系的 p16 状态和致瘤性。在本研究中，我们从钚二氧化物吸入大鼠诱导的肺腺癌中建立了肿瘤细胞系（PuD2）。我们利用 SV40T2 细胞、苯并[a]芘诱导的 BP 细胞、BP 衍生的 BP（P）Tu 细胞和γ射线转化的 RTiv3 细胞作为呼吸上皮细胞系。将细胞接种到裸鼠中的肿瘤发生测定表明，PuD2、BP 和 BP（P）Tu 细胞具有致瘤性，但 SV40T2 和 RTiv3 细胞没有。p16 启动子区的甲基化特异性 PCR 显示 SV40T2 细胞是非甲基化的，BP 细胞显示异质性甲基化，而 BP（P）Tu 和 RTiv3 细胞则完全甲基化。甲基化特异性 PCR 和基因组 DNA 的 p16 区 PCR 未能扩增 PuD2 细胞中的产物，表明 p16 缺失。从 PuD2 细胞制备的带型核型显示染色体 4 的三体性、染色体 11 的倒位和染色体 4 和 5 的部分缺失。去甲基化剂 5Aza2dC 部分去甲基化 BP（P）Tu、BP 和 RTiv3 细胞的 p16 启动子区，增加 p16 转录本的表达并降低细胞的生长。这些结果表明，在大鼠呼吸上皮细胞获得致瘤性之前，p16 启动子区的高甲基化发生在肿瘤转化的早期。位于染色体 4 和 5 上的基因的缺失可能对肿瘤的进展和 Pu 诱导的大鼠肺癌中获得高致瘤活性很重要。
