Study the effects of peripheral myelin protein 22 gene on cell growth in human lung cancer cell lines

• Main Authors: Kuo-Ru, 蔣濶如
• Other Authors: 蔡菁華
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/82973262992951265529

碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 95 === Peripheral myelin protein 22 (PMP22), also known as Growth arrest-specific-3 (GAS3) gene, is located at chromosome 17p11.2. PMP22 is composed of six exons and is a structural protein of the myelin sheath in the peripheral nervous system (PNS). The PMP22 gene plays an important role in cell proliferation, differentiate, cell-cell interactions and cell-matrix interaction. The mRNA level of PMP22 was 2 to 45 fold higher in normal lung than in lung cancer. The PMP22 expression level of CL1-0 is the lowest in CL1 series cell lines. We overexpressed PMP22 in CL1-0 to observe the effect of PMP22 on cell growth, apoptosis, colony formation, cell cycle distribution and expression of cell cycle regulators. When PMP22 is overexpressed in CL1-0 cells, 500μg/ml of G418 resistant cells developed mutinuclei, while this phenomenon disappeared in stable clones. MTT assay and cell counting data showed that overexpression of PMP22 decreased cell growth. Flow cytometry analysis showed that overexpression of PMP22 decreased the percentage of G1 and G2/M phase cells while increased S phase cells. We used Western blot analyses to examine the expression level of cell cycle regulators (CDKs). Overexpression of PMP22 decreased protein levels of CDKs. Among them, the expression of CDK1 decreased with the increasing expression level of exogenous PMP22, suggesting that PMP22 reduces CDK1 expression directly or indirectly. To assess whether PMP22 overexpression would decrease CDK1 by decreasing its mRNA level, RT-real time PCR was performed and showed that overexpression of PMP22 did not affect the mRNA level of CDK1. In addition, overexpression of PMP22 increased the protein level of p21, p53 and decreased the level of phosporylated-Rb. When PMP22 was overexpressed, CL1-0 cells formed losser colonies, and decreased the anchorage-independent growth. By Western blot analysis, the expression of β-catenin was decreased in cells overexpressed PMP22, suggesting that PMP22 may affect cell-cell interaction or cell-cell junction. Together, these results indicated that PMP22 might acts as a tumor suppressor in lung cancer. The detail mechanisms and pathways involved in tumor suppressor activity of PMP22 are required for further investigation.