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1.
Detection of DNA fragmentation and endonucleases in apoptosis 总被引:3,自引:0,他引:3
DNA degradation during apoptosis is endonuclease mediated and proceeds through an ordered series of stages commencing with the production of large DNA pieces of 300 kb which are then degraded to fragments of 50 kb. The 50-kb fragments are further degraded, in some but not all cells, to smaller pieces (10-40 kb) releasing the small oligonucleosome fragments that are detected as a characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of both DNA ladders and the initial stages of DNA fragmentation using pulsed-field gel electrophoresis. We have developed electrophoresis conditions that resolve large fragments of DNA and also retain the smaller fragments on the same gel. Methods for the detection of endonuclease activities responsible for the cleavage of DNA during apoptosis are also presented. 相似文献
2.
Transfected human apoptosis signal-regulating kinase 1 (ASK1) produces a 150 kDa protein. However, we have detected endogenous ASK1 predominantly as 39 and 50 kDa C-terminal and 75 and 110 kDa N-terminal fragments in a panel of nontransfected cancer cell lines and HUVEC endothelial cells. This suggests that in nonapoptotic cells, endogenous ASK1 protein is normally cleaved at a number of specific sites, some of which are in the kinase domain. Transfected ASK1 protein is known to be degraded by the proteasome. In contrast, the cleavage of endogenous ASK1 is independent of the proteasome as treatment with the proteasome inhibitor, lactacystin did not inhibit cleavage. Cisplatin treatment decreased the amount of 39 kDa C-terminal ASK1 fragments in mutant p53 cell lines suggesting a decrease in cleavage associated with apoptosis. Transfected ASK1 may, therefore, not accurately reflect the role of endogenous ASK1. 相似文献
3.
Muthukalingan Krishnan 《Insect Science》2015,22(5):629-638
The family of 30 kDa lipoproteins (LP1–5) is abundant in silkworm pupa fat body (FB) and hemolymph. One of its members, the 29 kDa protein decreased in concentration from peripheral (PP) FB tissue but was sustained in perivisceral (PV) FB tissue at the time of apoptosis. This study investigated the correlation of the 30 kDa proteins with FB apoptosis. Two protein fractions were purified, a 29 and a 30/31 kDa protein fraction, and they were used to test for activity against actinomycin D‐induced apoptosis in the FB tissues. Concentrations as little as 50 μg/mL of the 29 kDa protein fraction efficiently inhibited apoptosis. Less antiapoptotic activity was detected for the higher MW fraction; DNA fragmentation was observed in FB tissue treated with 50 μg/mL of the 30/31 kDa fraction. The viability of the cells in the 29 kDa protein‐supplemented culture was 40% higher than in the 31 kDa protein‐supplemented culture. However, the 30 kDa lipoproteins were not able to prevent scheduled FB degeneration during silkworm metamorphosis. Thus, it is hypothesized that the antiapoptotic 29 kDa protein needs to be proteolytically degraded by a regulatory mechanism to allow programmed cell death of FB tissue. 相似文献
4.
BAPTA blocks DNA fragmentation and chromatin condensation downstream of caspase-3 and DFF activation in HT-induced apoptosis in HL-60 cells 总被引:2,自引:0,他引:2
Min Zhang Yanping Li Hongqing Zhang Shaobai Xue 《Apoptosis : an international journal on programmed cell death》2001,6(4):291-297
DFF ((DNA Fragmentation Factor) is a heterodimer composed of 40 kDa (DFF40, CAD) and 45 kDa (DFF45, ICAD) subunits. During apoptosis, activated caspase-3 cleaves DFF45 and activates DFF40, a DNase that targets nucleosomal linker region and cleaves chromatin DNA into nucleosomal fragments. We have previously reported that HT induced apoptosis in HL-60 cells, and intracellular Ca2+ chelator BAPTA blocked apoptosis-associated DNA fragmentation induced by HT. We report here that HT also induced activation of caspase-3 and cleavage of DFF45. BAPTA prevented neither the caspase-3 activation nor the cleavage of DFF45. Mitochondrial membrane potential was disrupted in BAPTA-AM treated cells. However, BAPTA did prevent DNA fragmentation and chromatin condensation in HT-treated cells. These data suggest a novel role for intracellular calcium in regulating apoptotic nuclease that causes DNA fragmentation and chromatin condensation. 相似文献
5.
Apoptosis-associated cleavage of beta-catenin in human colon cancer and rat hepatoma cells 总被引:2,自引:0,他引:2
Fukuda K 《The international journal of biochemistry & cell biology》1999,31(3-4):519-529
Proteases belonging to the caspase family play a crucial role in apoptotic processes. Identification of protein cleavage specific to apoptosis may therefore provide further information about the mechanisms of apoptosis. In this study, apoptosis and necrosis were induced in cells of the human colon cancer cell lines, WiDr and DLD-1, and the resulting protein cleavage patterns investigated for beta-catenin. beta-Catenin was detected as a 92 kDa protein in control viable cells, while 65-72 kDa beta-catenin cleavage fragments were characteristically observed in apoptotic cells. These fragments were not observed in necrotic cell death. Similar apoptosis-specific beta-catenin cleavage was also demonstrated in the rat hepatoma cell line McA-RH7777, suggesting that the beta-catenin cleavage is a common event in apoptosis in various cell types. The formation of 65-72 kDa beta-catenin cleavage fragments was completely prevented by a caspase-1 inhibitor Z-VAD-CH2F and a caspase-3 inhibitor Z-DEVD-CH2F, indicating that the cleavage is associated with caspase-dependent process. Since beta-catenin is implicated in cell adhesion and signal transduction, these findings may suggest various possible roles of beta-catenin degradation in the dramatic cytoskeletal and morphological changes, as well as signaling events that accompany apoptosis. 相似文献
6.
Schiavone N Papucci L Luciani P Lapucci A Donnini M Capaccioli S 《Biochemical and biophysical research communications》2000,270(2):406-414
Genotoxic damage induces cell cycle arrest and/or apoptosis by activation of p53 oncosuppressor protein. A number of anticancer drugs are genotoxic and their damaging effect upon cells is mediated by this mechanism. Microinjection of defined DNA species directly into nucleus has been reported previously to activate p53 and inhibit cell cycle. Here, we demonstrate that simple addition of heterogeneous degraded DNA to cultured cells (Rat-1 fibroblasts) in combination with lipotransfecting agent DOTAP leads to apoptosis induction and mitosis inhibition by a molecular mechanism which mimics that of the cellular response to genotoxic anticancer agents. Indeed, both cellular effects induced by lipotransfected degraded DNA (essentially, heterogeneous small DNA fragments) are associated to p53 activation and modulated by two apoptosis-related genes, such as bcl-2 and c-myc, which also modulate the apoptotic threshold to anticancer agents. Here we raise the hypothesis of exogenous DNA segment lipotransfection as possible new tool for anticancer therapy. 相似文献
7.
The review summarizes the authors’ and literature data on accumulation of DNA breaks in differentiating cells. Large 50-kb free DNA fragments were observed by several research teams in non-apoptotic insect, mammal, and plant cells. More intense DNA breakage was observed during maturation of spermatides, embryo development, and differentiation of myotubes, epidermal cells, lymphocytes, and neutrophils. In general, accumulation of DNA breaks in differentiating cells cannot be attributed to a decrease in the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. We hypothesize that DNA fragmentation is an epigenetic tool for regulating the differentiation process. Scarce data on localization of the differentiation-associated DNA breaks indicate their preferable accumulation in specific DNA sequences including the nuclear matrix attachment sites. The same sites are degraded at early stages of apoptosis. Recent data on non-apoptotic function of caspases provide more evidence for possible existence of a DNA breakage mechanism in differentiating cells, resembling the initial stage of apoptosis. Excision of methylated cytosine and recombination are other possible explanations of the phenomenon. Elucidation of mechanisms of differentiation-induced DNA breaks appears to be a prospective research direction. 相似文献
8.
The review summarizes the authors' and literature data on accumulation of DNA breaks in differentiating cells. Large 50-kb free DNA fragments were observed by several research teams in non-apoptotic insect, mammal, and plant cells. More intense DNA breakage was observed during maturation of spermatides, embryo development, and differentiation of myotubes, epidermal cells, lymphocytes, and neutrophils. In general, accumulation of DNA breaks in differentiating cells cannot be attributed to a decrease in the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. We hypothesize that DNA fragmentation is an epigenetic tool for regulating the differentiation process. Scarce data on localization of the differentiation-associated DNA breaks indicate their preferable accumulation in specific DNA sequences including the nuclear matrix attachment sites. he same sites are degraded at early stages of apoptosis. Recent data on non-apoptotic function of caspases provide more evidence for possible existence of a DNA breakage mechanism in differentiating cells, resembling the initial stage of apoptosis. Excision of methylated cytosine and recombination are other possible explanations of the phenomenon. Elucidation of mechanisms of differentiation-induced DNA breaks appears to be a prospective research direction. 相似文献
9.
Ferraris J Radl DB Zárate S Jaita G Eijo G Zaldivar V Clapp C Seilicovich A Pisera D 《PloS one》2011,6(7):e21806
The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal. 相似文献
10.
Umeda T Kouchi Z Kawahara H Tomioka S Sasagawa N Maeda T Sorimachi H Ishiura S Suzuki K 《Journal of biochemistry》2001,130(4):535-542
Members of the caspase family have been implicated as key mediators of apoptosis in mammalian cells. However, few of their substrates are known to have physiological significance in the apoptotic process. We focused our screening for caspase substrates on cytoskeletal proteins. We found that an actin binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135, 120, and 110 kDa major C-terminal fragments when apoptosis was induced by etoposide in both the human monoblastic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat. The cleavage of filamin was blocked by a cell permeable inhibitor of caspase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ac-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examine whether caspase-3 cleaves filamin in vitro, we prepared a recombinant active form of caspase-3 directly using a Pichia pastoris overexpression system. When we applied recombinant active caspase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved into the same fragments seen in apoptosis-induced cells in vivo. Platelet filamin was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-terminal fragments, and the cleavage pattern was the same as observed in apoptotic human cells in vivo. These results suggest that filamin is an in vivo substrate of caspase-3. 相似文献
11.
Solovyan VT Bezvenyuk ZA Salminen A Austin CA Courtney MJ 《The Journal of biological chemistry》2002,277(24):21458-21467
Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50-100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage. 相似文献
12.
Maria Grazia Bottone Cristiana Soldani Annunzia Fraschini Anna Cleta Croce Giovanni Bottiroli Tania Camboni Anna Ivana Scovassi Carlo Pellicciari 《Histochemistry and cell biology》2009,131(3):391-399
Photosensitization of tumor cells after incubation with Rose Bengal acetate (RB-Ac) induces multiple organelle photodamage
followed by apoptotic cell death. We used immunocytochemical techniques in multicolor fluorescence microscopy to elucidate
whether this occurs through the simultaneous activation of different apoptotic pathways, in HeLa cells. We detected in situ
the activated forms of caspases 9 and 3, and the translocation from the mitochondria to the nucleus of the apoptosis inducing
factor; DNA electrophoretic techniques were also used to assess the occurrence of nuclear DNA cleavage into either high- or
low-molecular-weight fragments. Both the caspase-dependent and caspase-independent apoptotic pathways are activated. The genomic
DNA is degraded into high molecular weight molecules only, without the formation of oligonucleosome-sized fragments. The ability
of RB-Ac to induce the simultaneous release of apoptogenic signals from different photodamaged organelles makes it an especially
powerful cytotoxic agent. 相似文献
13.
The Escherichia coli CT596 prophage exclusion genes gmrS and gmrD were found to encode a novel type IV modification-dependent restriction nuclease that targets and digests glucosylated (glc)-hydroxymethylcytosine (HMC) DNAs. The protein products GmrS (36 kDa) and GmrD (27 kDa) were purified and found to be inactive separately, but together degraded several different glc-HMC modified DNAs (T4, T2 and T6). The GMR enzyme is able to degrade both alpha-glucosy-HMC T4 DNA and beta-glucosyl-HMC T4 DNA, whereas no activity was observed against non-modified DNAs including unmodified T4 cytosine (C) DNA or non-glucosylated T4 HMC DNA. Enzyme activity requires NTP, favors UTP, is stimulated by calcium, and initially produces 4 kb DNA fragments that are further degraded to low molecular mass products. The enzyme is inhibited by the T4 phage internal protein I* (IPI*) to which it was found to bind. Overall activities of the purified GmrSD enzyme are in good agreement with the properties of the cloned gmr genes in vivo and suggest a restriction enzyme specific for sugar modified HMC DNAs. IPI* thus represents a third generation bacteriophage defense against restriction nucleases of the Gmr type. 相似文献
14.
Yamaguchi K Uzzo R Dulin N Finke JH Kolenko V 《Biochemical and biophysical research communications》2004,318(3):710-713
Apoptotic DNA fragmentation minimizes the risk of transferring genetic information from apoptotic cancer cells to the neighboring cells. We have reported previously that caspase-deficient human renal cell carcinoma (RCC) lines were almost completely resistant to apoptosis in response to cytotoxic agents. In the present report we examined apoptotic process in caspase competent RCC-91 cells. Apoptosis in RCC-91 cells was accompanied by activation of caspases-3 and -9; cleavage of PARP and DFF45 proteins; typical apoptotic nuclei fragmentation and mitochondrial collapse. Nevertheless, DNA in these cells was not degraded into oligonucleosomal fragments compared to control Jurkat cells. Expression of caspase-activated DNase, DFF40 accountable for characteristic ladder pattern was easily detectable in Jurkat but not renal cancer cells, providing one possible explanation for the lack of oligonucleosomal DNA fragmentation in apoptotic RCC cells. Lack of typical DNA fragmentation indicates a potential threat of transferring genetic information from one tumor cell to another or to the neighboring healthy cells. 相似文献
15.
Semple JI Smits VA Fernaud JR Mamely I Freire R 《Cell death and differentiation》2007,14(8):1433-1442
Apoptosis plays a crucial role in development and tissue homeostasis. Some key survival pathways, such as DNA damage checkpoints and DNA repair, have been described to be inactivated during apoptosis. Here, we describe the processing of the human checkpoint protein Claspin during apoptosis. We observed cleavage of Claspin into multiple fragments in vivo. In vitro cleavage with caspases 3 and 7 of various fragments of the protein, revealed cut sites near the N- and C-termini of the protein. Using mass spectrometry, we identified a novel caspase cleavage site in Claspin at Asp25. Importantly, in addition to cleavage by caspases, we observed a proteasome-dependent degradation of Claspin under apoptotic conditions, resulting in a reduction of the levels of both full-length Claspin and its cleavage products. This degradation was not dependent upon the DSGxxS phosphodegron motif required for SCF(beta-TrCP)-mediated ubiquitination of Claspin. Finally, downregulation of Claspin protein levels by short interfering RNA resulted in an increase in apoptotic induction both in the presence and absence of DNA damage. We conclude that Claspin has antiapoptotic activity and is degraded by two different pathways during apoptosis. The resulting disappearance of Claspin from the cells further promotes apoptosis. 相似文献
16.
JIAN-GUO REN RONG-LIANG ZHENG YI-MING SHI BO GONG JIAN-FENG LI 《Cell biology international》1998,22(1):41-49
The effects of oxidative stress (ascorbic acid—ferrous system) on the proliferation, differentiation and apoptosis of the human hepatoma cell SMMC-7721 were studied. Oxidative stress significantly inhibited cell proliferation and induced morphological differentiation. Whatever the indices related with cell malignancy, such as α-fetoprotein and c-glutamyltranspeptidase or the index related with cell differentiation, such as tyrosine-α-ketoglutarate transaminase, all inclined evidently to normalization. The tumour's clonogenic potential decreased significantly. Moreover, together with differentiation, the phenomenon of apoptosis was found by the appearance of apoptotic bodies, detached cells, and apoptotic morphological feature. Although, their DNA was not degraded into oligonucleosomal fragmentation, the DNA was cut into larger fragments (about 21.2kbp) of a size associated with chromatin loops. These findings indicated that oxidative stress can induce both differentiation and apoptosis simultaneously in tumour cells. All the results showed that oxidative stress may initiate the tumour cells reverse transformation. The possible mechanism of the differentiation and apoptosis induced by oxidative stress may be related to the lipid peroxidation of cell membrane. 相似文献
17.
Immunochemical characterization of mucins. Polypeptide (M1) and polysaccharide (A and Leb) antigens. 总被引:3,自引:0,他引:3
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Seven monoclonal antibodies (MAbs) reacting with high-molecular-mass components (greater than 20,000 kDa) isolated from an ovarian mucinous cyst of an A Le(a-b+) patient are described. By the use of immunoradiometric methods, these MAbs characterized seven different epitopes associated with components having a density of 1.45 g/ml by CsCl-density-gradient ultracentrifugation, like mucins. Two MAbs reacted with A and Lewis blood-group antigens respectively (polysaccharide epitopes). The five other MAbs characterized five M1 epitopes (called a, b, c, d and e), mainly associated with components of more than 20,000 kDa and 2000 kDa. They were completely destroyed by papain and 2-mercaptoethanol treatment (polypeptide epitopes). Moreover, timed trypsin digestion of native mucin resulted in a progressive loss of M1 activity and degraded these mucins into smaller M1-positive fragments. The a and c epitopes were partially degraded from relatively high-molecular-mass fragments (2000 kDa to 500 kDa) into a 100 kDa fragment. The b and d epitopes were completely degraded into smaller fragments ranging from 100 kDa to 40 kDa. The e epitope was completely destroyed by trypsin. These different pathways of M1 antigen degradation suggest the occurrence of different epitopes located in separate regions of the mucin molecules. 相似文献
18.
Hashimoto S Ishisaki A Yamato K Aiko K Amagasa T Nishihara T 《Journal of cellular physiology》2001,186(1):65-72
We previously reported that concanamycin A, a specific inhibitor of vacuolar type H+-ATPases, induces DNA fragmentation in B cell hybridoma HS-72 cells. In the present study, we found that the cytosol from concanamycin A-treated HS-72 cells had a cytotoxic effect on intact cells in a cell viability assay. While activin A also induced apoptosis in HS-72 cells, the cytosol from activin A-treated HS-72 cells had no effect on cell viability. We purified the cytosol from concanamycin A-treated HS-72 cells by a four-step procedure: ultracentrifugation; HiTrap heparin column chromatography; HiTrap Q column chromatography; and reverse-phase high performance liquid chromatography on a C18 hydrophobic support. The biologically active fraction, which was used as partially purified cytosol, gave a specific band of protein with a molecular mass of 33 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis in cells cultured with the partially purified cytosol. The overexpression of human Bcl-2 suppressed apoptosis, indicating that the cytosol from concanamycin A-treated HS-72 cells induces apoptosis by a Bcl-2-inhibiting mechanism. These findings suggest that concanamycin A, a vacuolar type H+-ATPase inhibitor, produces intracellular apoptosis-inducing factor in B cell hybridoma. 相似文献
19.
Bcl-2 cleavages at two adjacent sites by different caspases promote cisplatin-induced apoptosis 总被引:1,自引:0,他引:1
The protein encoded by bcl-2 proto-oncogene plays an important role in the mitochondria-mediated apoptotic pathway. Although the general role of Bcl-2 is anti-apoptotic, previous work showed that Bcl-2 fragments cleaved by caspases could promote apoptotic process. We report herein that Bcl-2 protein was cleaved to produce two fragments of around 23 kDa in human hepatocarcinoma BEL-7404 cells or in Bcl-2 overexpressing CHO cells induced by cisplatin. Treating cells with the general caspase inhibitor z-VAD-fmk blocked the induced cleavage of Bcl-2. Mutagenesis analyses showed that Bcl-2 was cleaved by caspases at two adjacent recognition sites in the loop domain (YEWD31↓AGD34↓V), which could be inhibited by caspase-8 and -3 inhibitors, respectively. Overexpression of the carboxyl terminal 23 kDa fragments increased the sensitivity of CHO cells to cisplatin-induced apoptosis. These results indicate that Bcl-2 can be cleaved into two close fragments by different caspases during cisplatin-induced apoptosis, both of which contribute to the acceleration ofapoptotic process. 相似文献
20.
Q Song S P Lees-Miller S Kumar Z Zhang D W Chan G C Smith S P Jackson E S Alnemri G Litwack K K Khanna M F Lavin 《The EMBO journal》1996,15(13):3238-3246
Radiosensitive cell lines derived from X-ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA-dependent protein kinase, DNA-PK, suggesting that DNA-PK plays an important role in DNA double-strand break repair. Another enzyme implicated in DNA repair, poly(ADP-ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA-PKcs, the catalytic subunit of DNA-PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA-binding component of the enzyme, remains intact. Degradation of DNA-PKcs was accompanied by loss of DNA-PK activity. One cell line resistant to etoposide-induced apoptosis failed to show degradation of DNA-PKcs. Protease inhibitor data implicated an ICE-like protease in the cleavage of DNA-PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA-PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA-PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA-PKcs is a critical target for proteolysis by an ICE-like protease during apoptosis. 相似文献