| IntroductionProtein kinase B ( PKB) is a serine /threonine protein kinase with homology to protein kinase A and C , so it is also called RAG (related to A an C kinase). Three mammalian isoforms ( PKBα , PKBβ,PKB-r) ,have been cloned thus far. PKBa and PKBβ are ubiquitous , whereas PKB-r is expressed predominantly in brain, heart and kidney. Interest in PKB was piqued in 1995 when it was shown to be a direct downstream effector of phosphatidylinositol 3-kinase ( PI3-K).PKB has been shown to have a pivotal role in synthesis of protein and lipid, glucose metabolism and transportation, cell cycle progression and gene regulation, inhibition of apoptosis, angiogenesis and tumor transformation. PKB lies in the crosstalk of lots of signal pathways , and becomes a major participant in growth factor - mediated PI3 - Kinase signal transduction pathway. Tumor cells can secrete many growth factors, which bind to cell surface receptors by autocrine or paracrine production and activate a series of signal pathways, then induce cell proliferation. So study on growth factor - mediated signal transduction in tumor cells has some clinical values. Now it has been demonstrated that a variety of growth factors such as EGF,PDGF, VEGF,IGF -1 and bFGF can bind to tyrosine protein kinase receptors, activate PI3 -K, and generate Ins(3,4,5)P3 and Ins(3,4)P2. These PD-K products bind to the PH domain of PKB with high affinityand recruit it to the plasma membrane, where Ser473 and Thr308 are phosphorylated by PDK1 and PDK2. After activation, PKB is translocated to the nucleus by unknown mechanism and might be required for nucleus related gene transcription. Furthermore, activated PKB can escape cells from apoptosis through phosphorylation of BAD and activation of proapoptotic caspase-9. So we can block PD-K/PKB pathway, inhibit tumor cell growth and induce tumor cell apoptosis. This may be a novelty idea and method for cancer therapy.We haven 1 identified the mechanism by which bFGF regulates nasopharyngeal carcinoma cell proliferation through protein kinase signal pathways. In this article, we alter the time of bFGF stimulation on CNE -1, detect cytoplasm and membrane PKB activities and expression of c-fos in order to study the signal transduction mechanism by which bFGF regulates nasopharyngeal carcinoma cell proliferation through PI3-K/PKB pathway.Materials and Methods1. Materials: CNE -1 is a human nasopharyngeal carcinoma cell line. RPMI 1640 culture medium was purchased from Gibcol BRL. Inc; Histone 2B from Roche; cAMP - dependent protein kinase inhibitor (PKI) , PMSF, Wortmannin from Sigma; Polyclonal rabbit anti -c-fos from NeoMarkers; Secondary anti - rabbit horseradish peroxidase antibody were purchased from Promega. bFGF from Fujifu Bio. Tech. Inc; [r -32P] ATP from Beijing Yahui Bio. Tech. Inc. ; EGTA and DTT from Huamei Bio. Tech. Co.2. Methods:2. 1 Culture of CNE -1 cell line: CNE -1 cells were cultured inRPMI 1640 medium, supplemented with 10% fetal bovine serum at 37C in a 5% CO2 incubator.2. 2 Cell treatment and groups; the subconfluent cells were starved for 18h at 37C by adding 5ml of serum -free 1640 medium. At the end of the incubation period, cells were stimulated with bFGF described in following groups. The cells were randomly divided into different groups. For the time course experiment, the cells were stimulated with 50ng/ml bFGF for 0,15,30,45,60,90min. To block the PI3 - K signaling pathway, cells were pretreated with 100nM Wort-mannin for Ih prior to stimulation with bFGF.2. 3 Measurement of the PKB activity in CNE -1 cell line: cells were washed twice with ice -cold PBS, lysed in 150ul cell lysis buff-er(lmM EDTA, ImM EGTA, 10mM Tris, PH7.5, 100mM NaCl, 50mM sodium fluoride, InM sodium vanadate, 10u,g/ml leupeptin, 10uLg/ml protamin, 10ug/ml aprotinin, 10ug/ml pepstatin, ImM di-thiothreitol, ImM PMSF, 50mM p - glycerolphosphate, 0.09% Brij35) . The cell lysates were centrifuged for 1h at 100,000g at 4C. Supernatants were used for measurement of cyt...
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