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Implication of p16 inactivation in tumorigenic activity of respiratory epithelial cell lines and adenocarcinoma cell line established from plutonium-induced lung tumor in rat

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Abstract

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.

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Acknowledgments

The authors are grateful to Dr. Evelyne May of the National Center for Scientific Research, France for providing the BP cell lines and to Dr. Sei-ichi Kohno, Toho University and Dr. Masako Minamihisamatsu, NIRS for their help in the chromosome analysis. The authors also thank Mr. Isao Takahashi, Ms. Miyuki Inoue, and Ms. Nozomi Noro for their technical assistance.

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Authors and Affiliations

  1. 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

    Yutaka Yamada, Akifumi Nakata & Yoshiya Shimada

  2. Department of Radiation Dosimetry, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba, 263-8555, Japan

    Mitsuaki A. Yoshida

  3. Department of Radiobiology, Institute for Environmental Sciences, 1-7, Ienomae, Obuchi, Rokkasho-mura, Kamikita-gun, Aomori, 039-3212, Japan

    Yoichi Oghiso

  4. Départment de Radiobiologie et de Radiopathologie, Laboratoire de Radiotoxicologie, CEA-Direction des Sciences du Vivant, BP 12 91680, Bruyères-le-Châtel, France

    Jean-Luc Poncy

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  1. Yutaka Yamada
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  2. Akifumi Nakata
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  3. Mitsuaki A. Yoshida
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  4. Yoshiya Shimada
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  5. Yoichi Oghiso
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  6. Jean-Luc Poncy
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Correspondence to Yutaka Yamada.

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Editor: J. Denry Sato

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Yamada, Y., Nakata, A., Yoshida, M.A. et al. Implication of p16 inactivation in tumorigenic activity of respiratory epithelial cell lines and adenocarcinoma cell line established from plutonium-induced lung tumor in rat. In Vitro Cell.Dev.Biol.-Animal 46, 477–486 (2010). https://doi.org/10.1007/s11626-009-9269-3

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