Change of Cell-cycle Related Proteins and Tumor Suppressive Effect in Non-small Cell Lung Cancer Cell Line after Transfection of p16(MTS1) Gene

Kim, Young Whan; Kim, Jae Yeo; Yoo, Chul Gyu; Han, Sung Koo; Shim, Young Soo; Lee, Kye Young

Abstract

BACKGROUND: It is clear that deregulation of coil cycle progression is a hallmark of neoplastic transformation and genes involved in the G1/S transition of the coil cycle are especially frequent targets for mutations in human cancers, including lung cancer. P16 gene product, one of the G1 cell-cycle related proteins, that is recently identified plays an important role in the negative regulation of the kinase activity of the cyclin dependent kinase (cdk) enzymes. Therefore p16 gene is known, to be an important tumor suppressor gene and is also called MTS1 (multiple tumor suppressor 1). No more oncogenes have ken reported to be frequently related to multiple different malignancies than the alterations of p16 gene. Especially when it comes to non-small cell lung cancer, there was no expression of p16 in more than 70% of cell lines examined. Ann also it is speculated that p16 gene could exert a key role in the development of non-small cell lung cancer. This study was designed to evaluate whether p16 gene ould be used as a candidate for gene therapy of non-small cell lung cancer.
METHODS
After the extraction of total RNA from normal fibroblast cell line and subsequent reverse transcriptase reaction and polymerase chain reaction, the amplified P16 cDNA was sukloned into eukaryotic expression plasmid vector, pRC-CMV. The constructed pRC-CMV-p16 was transfected into the NCI-H441 NSCLC cell line using lipofectin. The changes of U I cell-cycle related proteins were investigated with Western blot analysis and immunoprecipitation after extraction of proteins from cell lysates and tumor suppressive effect was observed by clonogenic assay.
RESULTS
(1) p16(-) NCI-H441 cell line transfected with pRC-CMV-p16 showed The formation of p16 : cdk 4 complex and decreased phosphorylated Rb protein, while control cell line did not. (2) Clonogenic assay demonstrated that the number of colony formation was markedly decreased in p16(-) NCI-H441 cell line transfected with pRC-CMV-p16 than the control cell line.
CONCLUSION
It is confirmed that the expression of p16 protein in p16 absent NSCLC cell line with the gene transfection leads to p16 cdk4 complex formation, subsequent decrease of phosphorylated pRb protein and ultimately tumor suppressive effects. And also it provides the foundation for the application of P16 gone as a important candidate for the gene therapy of NSCLC.