Regulation Of MTOR And MPF In Growth And Proliferation Of Cell And Oral Tumor

1. In order to investigate the relation of mTOR and cell cycle phases, the expression of mTOR(mammalian target of rapamycin) and it' s a1 , a2, B1, B2 isoforms of p70 S6 kinase(p70 S6K)and 4EBP1 in different cell cycle phases of synchronized Hela cells were studied.2. To observe the expression of mTOR and its substrates in oral squamous cell carcinoma.3. Studying the expression of mTOR/ p70 S6K signal pathway in oral acin-ic cell carcinoma and parotid adenocarcinoma.4. Studying the expression of mTOR/ eIF-4EBP1 signal pathway in muco-epidermoid carcinoma .5. To investigate the content and activity of M-phase promoting factor (MPF) in pleomorphic adenoma, mucoepidermoid carcinoma, buccal carcinoma and normal tissue, in order to evaluate the role of MPF in the development of tumor and relationship between MPF and malignant degree.Methods1. Hela cell culture and synchronization of cell cycleHela cell line was from Biosignal research center of Kobe university of Japan.Reagents: 250mM TdR in PBS, 100mM nocodazol in DMSO.Procedure: Inoculate cells at 2 x 104 cells / 35 mm dish (3.2 x 105 cells / 100mm dish) in Dulbecco' s modified Eagle' s medium ( DMEM) , 10% FBS 10ml; 37℃ overnight, add TdR to a final cone, of 2mM, 37℃ for 17h, wash the cell layer twice with PBS prewarmed at 37℃ , add fresh medium prewarmed at 37℃ , 37℃ for 9h,add TdR to final cone, of 2mM, 37℃ 15h.Wash the cell layer twice with PBS prewarmed at 37 ℃, add fresh medium prewarmed at 37 ℃, 37℃ for Xh : 4h S phase 8h G2 phase 10h Ml phase 12h M2 phase 16h Gl phase2. RT-PCRPCR was performed in 50ul reactions containing 10 mM Tris HC1 ( pH 9.2), 1.5 mM MgCl2, 75 mM KC1, 0.4 uM of each 3' primer, 0. 8 uM 5' primer, 160 uM each dNTP, and 2. 5 units of Taq polymerase (GIBCO). Thirty-five cycles of PCR were performed; each cycle consisted of 30 s at 94℃ , 30 s at 56℃ , and 1 min at 68℃ followed by a single 7-min extension at 68℃.3. Western BlottingCells (5 x 106) were washed with PBS and lysed at 4℃ with 25 mM Tris HC1, pH 7. 4/50 mM NaCl/0. 5% sodium deoxycholate/2% Nonidet P40 (NP40)/0. 2% SDS/1uM phenylmethylsulfonyl fluoride (PMSF)/50 ug/ml aprotinin/50 uM leupeptin. Lysates were resolved by SDS/6% (for mTOR) or 10% (for p70 S6K) or 15% (for 4EBP1) polyacrylamide gels and transferred to nitrocellulose filters. After blocking of the filters with a solution containing 1% BSA, the filters were incubated with either a mouse polyclonal antibody raised against the common carboxyl-terminal sequence ( NSGPYKKQAF-PMISKRPEHLRMNL) of the p70 S6K isoforms, or a mouse polyclonal antibody raised against a recombinant His-tagged mouse PHAS1 (4EBP1) , Specific reactive proteins were detected by an enhanced chemiluminescence ( ECL) method, employing a sheep anti-mouse IgG antibody linked to horseradish peroxidase.4. p70 S6 Kinase ActivityThe specific activities of p70 S6K were determined by[32P] incorporation into S6 peptide in the immune complex as described previously. Briefly, cells (5 x 106) were washed with PBS and lysed at 4℃ in 500 ul of lysis buffer (10 mM potassium phosphate/1 mM EDTA/5 mM EGTA/10 mM MgCl2/50 mM B-glycerophosphate/1 mM Na3VO4/2 mM DTT/40 ug/ml PMSF/0. 1% NP40).The extract was incubated for 30 min at 4℃ with the C18 antibody. The immune complex was absorbed to Protein G-coupled beads ( Zymed) for 30 min and washed twice with the lysis buffer and once with kinase buffer (20 mM Tris ?HCl, pH 7.5/10 mM MgCl2/1 ug/ml IP-20/0.1 mg/ml BSA/0.4 mM DTT). After the final wash, the immune complexes were suspended in 50 ui of the kinase buffer containing 100 uM unlabeled ATP, 200 uCi/ml [ r-32P]-ATP (1 Ci = 37 GBq), and 125 uM S6 peptide (RRRLSSLRA, Upstate Biotechnology). The reaction was allowed to proceed for 15 min at 301 and terminated by the addition of 20 ul of 250 mM EDTA and boiling for 5 min. After a brief centrifu-gation, the supernatant (25 ul) was applied to phosphocellulose paper and radioactivity was determined by using a liquid scintillation co...