39 2 2008 4 ACTA ANATOMICA SINICA Vol139,No12 Apr. 2008 207 B 1 3 2 (11, 361005 ; 21, 361004) [ ] (TPA) B (PKB) BrdU,DAPI,TPA BGC2823 ;TPA PKB ;,TPA PKB Ser 473,TPA PKB TPA BGC2823 ;TPA BGC2823 PKB,TPA TPA,PP2A ;TPA PKB Ser 473,Thr 308 ;TPA PKB Ser 473 ;TPA PKB PKB TPA ;TPA PKB Ser 473 [] ; ; B ;;;BGC2823 [] R3 [] A [ ] 052921356 (2008) 022207 THE ROLE OF PROTEIN KINASE B IN THE APOPTOSIS OF GASTRIC CANCER CELLS IND UCED BY 122O2TETRADECANOYLPHORBOL21,32ACETATE ZHANG Bing 1 3, XIA Chun 2 ( 11 Department of Histology and Embryology, Medical School of Xiamen University, Xiamen 361005, China ; 21 Zhongshan Hospital Xiamen University, Xiamen 361005, China) [ Abstract ] Objective To study the role of protein kinase B ( PKB) in gastric cancer BGC2823 cells treated by TPA. Methods Flow cytomety(fac) and fluorescence microscope were used to detect the effect of TPA on gastric cancer BGC2823 cells, after cells were treated by BrdU and stained by DAPI, respectively. The effect of TPA on the expression level of PKB protein and its phosphorylation of Ser 473 and Thr 308 were investigated by Western blotting. When nuclear and cytoplasmic protein fractions were prepared through lysis of cell and centrifugation, the effect of TPA on the expression level of PKB protein and its phosphorylation of Ser 473 in cytoplasm and nuclei were detected by Western blotting. Meanwhile, the effect of TPA on the distribution of PKB was observed under laser2scanning confocal microscope with immunofluorescence technique. Results TPA induced the apoptosis of gastric cancer BGC2823 cells. It also induced the inhibition of PKB protein expression in a TPA concentration2dependent and time2dependent manner in gastric cancer BGC2823 cells, regardless of dephosphorylation of PP2A. TPA inhibited the phosphorylation of PKB at Ser 473, but it did not affect its phosphorylation at Thr308. TPA only attenuated the expression level of PKB and its phosphorylation of Ser 473 in nuclei, whereas it did not change the distribution of PKB in BGC2823 cells. Conclusion These data suggest that the inhibition of PKB plays an important role in mediating the effect of TPA in gastric cell line. Apoptosis induced by TPA is partly due to the inhibition of PKB and its phosphorylation of Ser 473 in nuclei. [ Key words ] TPA ; Cell apoptosis ; Protein kinase B ( PKB) ; Western blotting ; Laser2scanning confocal microscope ; BGC2823 cells B (protein kinase B, PKB) [ ] 2007210209 [ ] 2007212220 [ ] (C0310001) [ ] (1966 ),( ),,, 3 (To whom correspondence should be addressed) E2mail :bingzhang1966 @yahoo. com. cn Tel : (0592) 2188672, ; 2A(proteinphosphatase 2A,PP2A) [1 ],,, [2 ],PKB,PKB
208 39,2,,PKB [3 ] ( 122O2tetradecanoylphorbol21, 32 acetate,tpa),,,, [4,5] TPA BGC2823, PKB,, TPA BGC2823 PKB 11 BGC2823 BGC2823 RPMI1640 ( 10 %,1mmolΠL 100mgΠL ), 37 5 % CO 2 21 5222 ( 52bromo22 2deoxyuridine, BrdU) [ 6] TPA 24h BrdU (20molΠL) 2h,, 4 % 4 30minPBS 3, 6 10-2 IUΠL DNA I (DNase I),25 30min,, 2PBS2T (0105 %Tween2202PBS ) 37 30min, BrdU,37 1hPBS 3 37 1h ( ) PBS,Beckman Coulter 31 [ 7] TPA (100gΠL) 24h, PBS, 4 % 4 30minDAPI, Olympus BX251 41 ( Western blotting) [ 8] PBS 23, 015ml 1mmolΠL PMSF PBS RIPA 50100 l, 30min,,SDS2PAGE, PVDF, ( enhanced chemiluminescence, ECL ) Tubulin actin LaminB 5, ( GraphPad Prism 4) B (20mmolΠL Hepes 719, 400mmolΠL NaCl, 1mmolΠL EDTA, 1mmolΠL EGTA,015 % NP240,1 % 1mmolΠL DTT,10 % ) 15min, 61 [ 7],, TPA PBS, 4 % 4 30min,37 3hPBS 3,,37 3hPBS, PI 10min MRC21024ES 11 TPA BGC2823 BrdU,TPA DNA 1, TPA (100gΠL) BGC2823 24h, ( ),BrdU ( ), TPA BGC2823 DNA,,TPA (100gΠL) BGC2823 24h,BGC2823 TPA 24h,,,,(2B, ) ; (2A),,TPA BGC2823, 1 TPA BGC2823 DNA Fig. 1 TPA decreases the rate of DNA synthesize in gastric cancer BGC2823 cells 51 [ 8] 21 TPA BGC2823 PKB A 100 l [ 10mmolΠL,BGC2823, Hepes 719,10mmolΠL KCl,011mmolΠL (EDTA) TPA(100gΠL) 12h,PKB,011mmolΠL 2, (22 ) ;TPA ( EGTA),0115 % NP240,011 % 1mmolΠL,PKB ;TPA (dithiothreitol, DTT),10 % ], 48h, PKB ( 5min,
Vol. 39,No. 2. B 209 3) TPA (4) 4,BGC2823, TPA(50150gΠL) 24h, 50gΠL TPA PKB,TPA BGC2823 PKB, TPA TPA PKB PP2A (okadaic acid, OA, 100nmolΠL) BGC2823 2h,TPA (100gΠL) 24h,,TPA BGC2823 PKB ; OA TPA 24h, PKB,TPA PKB (5) TPA PKB PKB PP2A,PP2A TPA PKB 31 BGC2823 TPA PKB PKB 473 (Ser 473) PKB 308 ( Thr 308) ( Phospho2 PKBΠSer 473 Phospho2PKBΠThr 308),, BGC2823 PKB,Ser 473 Thr 308 (6, 7) TPA(100gΠL) 6h,PKB Ser 473 ;TPA 24h, PKB Ser 473 ;TPA 48h, PKB Ser 473 3 TPA BGC2823 PKB A1 ; B1 Fig. 3 Effect of TPA on the expression of PKB in gastric cancer BGC2823 cells. A, Western blotting ; B, Statistical analysis of band intensity 4 TPA BGC2823 PKB A1 ; B1 Fig. 4 Effect of TPA in different concentration on the expression of PKB in gastric cancer BGC2823 cells. A, Western blotting ; B, Statistical analysis of band intensity 5 PP2A OA TPA BGC2823 PKB A1 ; B1 Fig. 5 Effect of OA (PP2A inhibitor) on the inhibition of PKB expression induced by TPA in gastric cancer BGC2823 cells. A, Western blotting ; B,Statistical analysis of band intensity
210 39,2 (6), TPA (100gΠL) BGC2823,PKB Thr 308 (7),TPA PKB Ser 473,, PKB Thr 308 41 TPA BGC2823 PKB Ser 473,,BGC2 823,PKB, TPA(100gΠL) 24h, PKB,TPA 48h,PKB, TPA 24h, PKB ; TPA 48h, PKB, (8), TPA PKB,BGC2823,PKB, 9, TPA(100gΠL) 6h, PKB ( ) ( ) ; TPA 24h, PKB,TPA 48h,PKB, (9), TPA PKB, TPA 6 TPA BGC2823 PKB Ser473 A1 ; B1 Fig. 6 Effect of TPA on the phosphorylation of PKBΠSer473 in gastric cancer BGC2823 cells. A, Western blotting ; B, Statistical analysis of band intensity 7 TPA BGC2823 PKB Thr473 A1 ; B1 Fig. 7 Effect of TPA on the phosphorylation of PKBΠThr308 in gastric cancer BGC2823 cells. A, Western blotting ; B, Statistical analysis of band intensity 8 TPA BGC2823 PKB A1 ; B1 Fig. 8 Effect of TPA on the expression of PKB in cytoplasm and nuclei in gastric cancer BGC2823 cells. A, Western blotting ; B, Statistical analysis of band intensity
Vol. 39,No. 2. B 211,PKB Ser 473,,BGC2823,PKB Ser 473, TPA(100gΠL) 24h, Ser 473,TPA 48h,Ser 473 TPA 24h, Ser 473 ;TPA 48h, Ser 473 (10) TPA PKB Ser 473, TPA 10 TPA BGC2823 PKB Ser473 A1 ; B1 Fig. 10 Effect of TPA on the phosphorylation of PKBΠSer473 in gastric cancer BGC2823 cells. A, Western blotting ; B, Statistical analysis of band intensity TPA [4,5 ] TPA BGC2823 [7 ], BrdU BGC2823, TPA BGC2823 DNA,BGC2823 (1), (2),TPA BGC2823,, B ( PKB), TPA, TPA TPA (3,4), TPA PKB, :102(2)22,102 232(2 )2 22 ( 172allylaminogeldanamycin, 172 AAG) ( gemcitabine ) (topotecan) PKB [911 ] ;PKB PP 2A OA ( TPA PKB PP 2A,5),, PKB TPA BGC2823 [8 ],PKB [12 ],PKB Thr 308 Ser 473 [13 ],,(72Hydroxystaurosporine, UCN201) (Ceramide) PKB [14,2 ] ;,PKBΠThr 308 PKB,PKB Ser 473 PKB,Thr 308 PKB [15 ], PKB,, TPA PKB Ser 473 [16 ],TPA PKB, Ser 473 (6), Thr 308 (7), TPA PKB Ser 473, PKB,PKB PKB ( gemcitabine ), (topotecan) PKB PKB [10,11 ], TPA PKB Ser473 (3,6), PKB Ser473 PKB, PKB, [17,18 ] (nerve growth factor, NGF) ( platelet derived growth factor receptor, PDGF) ( insulin2like growth factor21, IGF21) PKB, PKB
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