Inhibition Of Cell Proliferation And Induction Of P21～(WAF1) By Ectopic Expression Of Cyclin G2

Cell cycle regulation is the core event of cell proliferation regulation, which has a close relationship with cell carcinogenesis. Cyclin, cyclin -dependent ki-nase ( CDK) , and cyclin - dependent kinase inhibitor ( CKI) form a regulating network to coordinate normal cell cycle transition. The cyclins represent a group of closely related molecules which primarily function at specific stages of cell cycle as regulators of CDK activity by binding and forming active complexes with specific partner CDKs. This cyclin - CDK association is in part determined by the conserved cyclin region of -110 amino acid referred to as cyclin box, which is the symbol and regulating region of cyclin. The expression level of cyclins is obviously regulated by transcription and the rate of protein hydrolysis, which shows the phase speciality. Different cyclins bind to their corresponding CDK at different cell cycle stages, forming holoenzyme complex and acting as promoting molecules in cell cycle. Cyclin - CDK activity is also negatively controlled by CKI, thus achieving cell cycled dynamic balance. To date, at least 12 cyclins, from A to I, in mammalian cells are known, all cyclins display structural, functional, and stage specialities. For example, Gl cyclin are presented by cyclin C, D and E, mitotic cyclins by cyclin A and B, and cyclin A also functions at S stage.Currently, cyclin G family comprises 2 subtypes denoted cyclin Gl and cyclin G2. Cloning of cyclin G2 was first reported by M. C. Home in 1996, and only several papers about cyclin G2 have been published since then. Its function and regulation are still unclear. Classic view about cyclin function is its cell cycle promoting effect. Cyclins act as positive regulators of cell cycle progression and enhance cell proliferation. However, data accumulated so far have sugges-ted that cyclin G2 may not be a positive regulator like classic cyclins. Cyclin G2 has 60% nuleotide sequence identity and 53% protein identity with its family homologue, cyclin G1. Reports have shown that they are quite different in protein expression and regulation. And cyclin G2 may also be functionally quite different from cyclin G1. Their expression could be both induced by DNA damage agent while cyclin Gl s induction is p53 protein dependent and cyclin G2's induction is p53 independent. Their subcellular localization is also different, cyclin Gl usually accumulated in nuclear while cyclin G2 mainly located in cytoplasm. In B lymphocyte stimulated by growth inhibitory signal, cyclin G2 transcription level can be upregulated in a long duration, while the level of cyclin Gl remains unaffected. Cyclin Gl manifests a relatively constant expression level throughout the cell cycle while cyclin G2's expression shows contrasting fluctuation model and increases to a peak level in the late S - early G2 phase. Both of the two cyclins are frequently detectable in terminally differentiated tissues with cyclin G2 showing strong expression in cerebellum and cyclin Gl in skeletal muscle. Wykoff et. al. reintroduced the tumor supressor gene VHL into VHL deficient renal cancer cells and found that the transgene could induce a series of genes'expression including cyclin G2. Our recent work of gene expression profiling in oral squamous cell carcinomas revealed that 4 of 5 tumors showed decreased expression of cyclin G2. In order to make sure whether expression of the cyclin G2 gene promotes or inhibits the cell proliferation in vitro, we have cloned the whole length cyclin G2 c DNA and inserted it into an eukaryotic expression vector. The construct was then introduced into HeLa and CV -1 cells, the effect of cyclin G2 transgene expression on the colony forming capacity was observed, and protein expression of CKIs in transfectant cells was examined by immunocytochemical staining.1. Cloning of Cyclin G2 cDNACyclin G2 cDNA fragments covering the whole coding region was generated by RT - PCR using total RNA obtained from a premalignant keratinocyte cell line as template. RT - PCR product of 1070bp was digested...