PK15 48 (1) :58 63, (intermediate filament, IF) D ( Traub et al., 1994) IF P K15 D. ( Fuchs et al., 1994), , ( Fuchs et al., 1992) (apoptosis)

Transcription

1 48 (1) :58 63, 2002 A cta Zoologica S inica 3 PK15 (, ) D P K15 ( Porcrne Kidney215) DNA, DNA ladder ;,,,, P K15 D (intermediate filament, IF) D ( Traub et al., 1994) IF P K15 ( Porcine Kidney215),, DAPI, ( ) ( DNA ) 20,,, ; ( Fuchs et al., 1994), ( ), ( Fuchs et al., 1992) (apoptosis),,,, ( Wyllie et 111 al., 1980), P K15,?, 10 % DMEM IF Rao (p H 712, Gibico ) 37, (1996) Takahashi (1996) Hank s, D (10 g/ (lamin) ml, promaga ) DMEM 37, Giese ( 1997) TPA DMEM 24 h Lecai DM IL MPC211 (vimentin) Liao (1997) 112 DAPI D NA HT29 42 P K15 D, 8 ; Caulin (1997) 18 (etoposide), 1 (10 g/ ml) 48 h, PBS 3, DAPI 5 min, PBS, Lecai , (No1G ),, 28,, : E2mail :

2 1 : P K15 59 DMRB 4 5 min, CSK 0125 % P K15 24 h - CSK 60 min, PBS 3, PBS 3 (10 1 % 2PBS 30 min, PBS 3 mmol/ L Tris, 10 mmol/ L NaCl, 1 mmol/ L ED TA, 100 mg/ L proteinase K, 10 g/ L SDS, p H 810), 37, r/ min 5 min 115,, 2 P K15 D (10-20, r/ min, DNA TE 118 % 1 SDS, SDS ( %, 15 %) P K15 D, 3 %BSA, (10 g/ ml) 48 h, PBS 3, AE1 AE3 (, AE1 5 min, PBS 011 % Triton X2100 PBS 3 min, C1562 ( Sigma ) 37 1 h, PBS, FITC 1 h, BCIP NB T min, PBS DAPI 5 min,, Lecai DMRB P K15 ( 1 : a) Zhai (1988), P K15 D (10 g/ ml) 24 h, Formar, PBS, 015 % Triton X2100 CSK ( 10 mmol/ L PIPES, 100 mmol/ L mmol/ L MgCl 2, 1 mmol/ L KCl, 300 mmol/ L, 3 EGTA, 112 mmol/ L PMSF, 1 % TritonX2100, p H 618) 5 min, RSB2Magik ( 4215 mmol/ L NaCl, 216 mmol/ L Tris, 815 mmol/ L MgCl 2 6H 2 O, 112 mmol/ L PMSF, 1 % Tween240, 015 %, p H 813),,, CO 2 g/ ml) 96 h,, PBS 3, AE3 ) 37 2 h, 37 2, 48 h 72 h, 96 h, ( 1 : b), ( 1 : c),,, ( 1 : a) ;,, 1 PK15 Fig11 The phase contrast micrograph of induced PK15 cells 100 a. (Normal cells before induction) b. 96 h (Cells induced for 96 h) c. (Control cells),, ; 96 h,, ;, ( Normal cells were extended and closed, the outlines of nucleus and nucleolus were clear ; most cells dropped from walls after induction for 96 h, cells contracted and condensed ; no obvious changes in control cells)

3 ( 1 : b) ; ( 2 : ( 1 : c) DAPI, ( 2 : c) ; : ( 2 : a) ; ( 2 : d) b) ;, 2 PK15 DAPI Fig12 Cells stained with DAPI after induction a. (Nucleus of normal cell) b. ( Chomatin condensation of induced cell) c. ( Apoptotic bodies in induced cell) d. ( ) [ One apoptotic body (array) was released from cell ] 3 DNA Fig13 The electrophesis pattern of isolated DNAs from induced cells M : DNA (Molecular size marker) 1 : DNA, (Control) 2, 3, 4, 5, 6 : 12, 24, 48, h DNA, DNA ladder ( Induction for 12, 24, 48, 72 and 96 h, DNAs were digested to fragments) 4 Fig14 Indirect immunofluorescence of keratin in apoptotic cells a. (Normal cells) b, c, d. (Apoptotic cells) a, b, c, d. a, b, c, d DAPI (DAPI staining of the same cells as in a, b, c and d) ;,,,, (b),, (c d) ( The spreading keratin filament network was welldistributed in normal cells. After induction the antigen of keratin still existed in apoptotic cells, but its distribution was not even, keratin fluorescence was condensed in some cells as in b, and distributed around chomatin as in c and d)

4 1 : P K Fig15 Whole mount micrograph of keratin network in apoptotic cells a., N, (Normal cell, showing spreading keratin network. N : nuclei) b., ch,, ( ) (Apoptotic cell, keratin network was intact in some area of the cell, but the filaments were shortened, and the filaments around chomatin were condensed) P K15 12 h 24 h 48 h 72 h 96 h DNA,, : DNA ladder (DNA AE3 43 ku ), DNA ( 29 ku ; 29 ku 3) D P K15, 212 P K15 c1562, DAPI, ( 4 : a, a ) ;,, ( 4 : b, b ),, DNA, DNA ( 4 : c, c, d, d ), P K15 6 Fig16 Western blot of keratin protein in apoptotic cells,, [ 5 : a ( N) ], b. AE1, 45 ku, ( 5 : a) ;, ( Western blot with AE1, showed type keratin of 45 ku [ 5 : b (ch) ],, M : (Standard protein marker) [ 5 : b ( ) ] 96 h P K15 a. AE3, 43 ku 29 ku ( Western blot with AE3, showed type keratin of 43 ku and it s degraded product of 29 ku) which did not degrade) 1 : 96 h ( Proteins of induced cells for 96 h) 2 : ( Proteins of control cells)

5 ( 6 : a) ; AE1 45 ku,,, ( 6 : b) 3,,,,? ( Fuchs et al., 1994),,,, ;?, :,,,, (Liao et al., 1997) : :,,,,?,,? ( Wyllie et al.,, 1980) ( References) Caulin, C., G. S. Salvesen and R. G. Oshima 1997 Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J. Cell Biol. 138 : Giese, G., J. Schmidt, M. Gilbert, R. Albrecht and P. Traub 1997 TPA induces apoptosis in MPC211 cells grown in serum free medium. Biol. Fuchs, E. and K. Fuchs, E. and P. Liao, J., N. O. Cell 89 : Weber 1994 Intermediate filament : structure, dynamics, function and disease. A nnu. Rev. Biochem. 63 : Coulombe 1992 Of mice and men : Genetics skin diseases of keratin. Cell 69 : Ku and M. B. Omary 1997 Stress, apoptosis, and mitosis induce phosphorylation of human karetin 8 at ser273 in tissues and cultures cells. J. Biol. Chem. 272 : Rao, L., D. Perez and E. White 1996 Lamin proteolysis facilitates nuclear events during apoptosis. J. Cell Biol. 135 : Takahashi, A., E. S. Alnemri, Y. A. Lazebnik, T. F. Alnemri, G. Litwack and R. D. Moir 1996 Cleavage of lamin A by Mch2 but not cpp322 : multiple interleukin 1 2converting enzyme2related protease with distinct substrate recognition properties are active in apoptosis. N atl. Acad. Sci. USA 93 : Traub, P, R. and L. of Cytology 15 : Shoeman 1994 Intermediate filament proteins cytoskeletal elements with gene regulatory function? International Review Wyllie, A. H., J. F. R. Kerr and A. R. Currie 1980 Cell death : the significance of apoptosis. Int. Rev. Cytol. 68 : Zhai, Z. H., X. Wang and X. Y. Qian 1988 Alteration of intermediate filament2nuclear matrix system in Hela cells after adnovirus infection. Cell Biol. Int. Rep. 12 : Proc.

6 1 : P K15 63 ( Abstract) CHANGES IN KERATIN D URING APOPTOSIS OF PK15 ( PORCINE KID NEY215) CELLS 3 CHEN Dan2Ying GAO Yun2Fei L I Li2Xia ZHAI Zhong2He ( L if e Science College, Peki ng U niversity, Beiji ng , Chi na) Intermediate filament ( IF) is a major component of cytoskeleton. Recent advances suggest t hat IFs not only play a cytoskeletal role in cooperation with microfilaments and microtubules, but are also involved in cell differentiation, signal t ransduction and gene expression. Apoptosis is a p hysiological form of cell deat h, it describes t he orchest rated collapse of a cell, staging membrane blebbing, cell shrinkage, protein f ragmentation, chromatin condensation and DNA degradation. How do IFs change in apoptotic process and what kind of role do they play? The answers to these questions would be of great importance to further study of their functions. Epit helial cell P K15 ( Porcine Kidney215) was induced to apoptosis by actinomycin D. The cell shapes changed from spreading to contracting and rounding during the induction process, and most of the cells dropped f rom t he wall after induction for 96 h. DAPI staining showed t he chromatin condensation and apoptotic bodies formation characterized by apoptosis in this process. At the same time DNA ladder was detected by elect rop horesis. Indirect immunofluorescence result s showed t hat t he fluorescence of keratin dist ributed unevenly in t he apoptotic cells, and in some cells t he keratin fluorescence cont racted around t he condensed chromatin. Using selective ext raction combined wit h whole mount elect ron microscopy, we detected t he intact keratin filament network in some area of apoptotic cells, and t he filament s were obviously shortened. In consistent wit h t he result of immunofluorescence, t he keratin filament s around chromatin were condensed in comparison with filaments in other areas. Western blot analysis further showed that part of type keratin protein degraded only partially during apoptosis, whereas type keratin protein molecules were intact. These results suggested that in apoptotic processing the structure changes of keratin network might lead to the changes of cell shape, and might participate t he formation of apoptotic bodies. Key words Pig, Porcine Kidney cell, P K15, Keratin, Intermediate filament, Actinomycin D, Apoptosis 3 This work was supported by the Special Funds for Major State Basic Research of China (No. G )