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1.
G418 is used extensively in transfection experiments to select eukaryotic cells that have acquired neomycin
resistance genes, but the mechanism is still elusive. To investigate this, we treated normal rat kidney cells with
G418 for 3 days and found that the cells presented typical apoptotic features such as cell shrinkage, nuclear
fragmentation, and caspase-3 activation. However, there was no low-molecular DNA ladder. The pan caspase inhibitor
z-VAD-fmk completely inhibited this type of apoptosis, suggesting a caspase-dependent mechanism. Caspase cascades
in apoptosis induced by G418 were initiated by at least two pathways: the release of cytochrome c from
mitochondria, which was observed under confocal microscopy, and endoplasmic reticulum stress, demonstrated by the
increase in Ca2+ concentration and the cleavage of m-calpain and procaspase-12. Both
pathways activated caspase-9. Inhibition of caspase-9 activity by z-LEHD-fmk prevented most of the cells from
apoptosis, and E-64d, an inhibitor of calpain accentuated this block. The cleavage of casapse-9 and caspase-12 was
blocked only by simultaneous application of z-VAD-fmk and E-64d, but not by either alone. E-64d did not prevent
the release of cytochrome c. These results indicated that these two pathways were independent of each other. Received 1 April 2004; received after revision 21 April 2004; accepted 26 May 2004 相似文献
2.
Analysis of nuclear apoptotic process in a cell-free system 总被引:2,自引:0,他引:2
We report an analysis of the apoptotic process of mouse liver nuclei induced in a cell-free carrot cytosol system by cytochrome
c. Typical characteristics of apoptosis were observed, such as chromatin condensation, margination, apoptotic bodies and DNA
ladders. Furthermore, transmission and scanning electron microscope analysis of the apoptotic nuclei detected chromatin-free
nuclear vesicles before apoptotic bodies appeared at a comparatively late phase. When AC-YVAD-CHO, an inhibitor of caspase
6, was introduced into the system, these vesicles and apoptotic bodies disappeared completely within our study sections. We
confirmed the results using whole-mount electron microscopy, and found that although the nuclear lamina was destroyed early,
the nuclear matrix largely remained intact during the course of apoptosis. The nuclear matrix played an important role in
maintaining the integrity of apoptotic cells and connecting the apoptotic bodies and apoptotic nucleus.
Received 29 September 2000; revised 10 December 2000; accepted 13 December 2000 相似文献
3.
Mitochondria and calpains mediate caspase-dependent apoptosis induced by doxycycline in HeLa cells 总被引:2,自引:0,他引:2
Doxycycline (Dc) has been demonstrated to inhibit cell growth and induce apoptosis in tumor cells, although its mechanism
of action is not fully understood. The present study demonstrates that apoptosis can be induced in HeLa cells. Western blot
data demonstrated that cytochrome c (Cyt c), Smac (the second mitochondria-derived activator of caspase), calpain I, caspase-9,
−3 and −8 were involved in the apoptotic process, while the pan caspase inhibitor zVAD-fmk almost completely inhibited Dc-induced
apoptosis. We further demonstrated that the release of mitochondrial proteins and the activation of calpains occurred upstream
of the caspase cascade, in which caspase-9 was activated in response to the release of Cyt c, that caspase-8 activation was
caspase and calpain dependent, and that caspase-3 was activated mainly by caspase-8 and −9. Caspase-8 played important roles
in the activation of caspase-3 and induction of apoptosis, whereas the role of the caspase-9 was limited.
Received 26 November 2005; received after revision 14 February 2006; accepted 1 March 2006 相似文献
4.
Kulikov AV Shilov ES Mufazalov IA Gogvadze V Nedospasov SA Zhivotovsky B 《Cellular and molecular life sciences : CMLS》2012,69(11):1787-1797
Cytochrome c is a well-known mitochondrial protein that fulfills life-supporting functions by transferring electrons to the respiratory chain to maintain ATP production. However, during the activation of apoptotic machinery, it is released from mitochondria and, being in the cytosol, it either triggers the activation of the caspase cascade in intrinsic apoptotic pathway, or it is involved in the amplification of extrinsic apoptotic signaling. Accumulating evidence suggests that only unmodified holocytochrome c is efficient in the stimulation of apoptosis. Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the complete or partial cytochrome c deficiency in vivo. Here, we discuss the importance of distinct amino acid residues for various functions of cytochrome c in cells and mice with targeted cytochrome c mutations. 相似文献
5.
Kim DS Jeong YM Moon SI Kim SY Kwon SB Park ES Youn SW Park KC 《Cellular and molecular life sciences : CMLS》2006,63(22):2661-2668
Indole-3-carbinol (I3C) has been found to act against several types of cancer, while ultraviolet B (UVB) is known to induce
the apoptosis of human melanoma cells. Here, we investigated whether I3C can sensitize G361 human melanoma cells to UVB-induced
apoptosis. We examined the effects of combined I3C and UVB (I3C/UVB) at various dosages. I3C (200 μM)/UVB (50 mJ/cm2) synergistically reduced melanoma cell viability, whereas I3C (200 μM) or UVB (50 mJ/cm2), separately, had little effect on cell viability. DNA fragmentation assays indicated that I3C/UVB induced apoptosis. Further
results show that I3C/UVB activates caspase-8, −3, and Bid and causes the cleavage of poly(ADP-ribose) polymerase. Moreover,
I3C decreased the expression of the anti-apoptotic protein, Bcl-2, whereas UVB increased the translocation of Bax to mitochondria.
Thus, an increased Bax/Bcl-2 ratio by I3C/UVB may result in melanoma apoptosis. In conclusion, our study demonstrated that
I3C sensitizes human melanoma cells by down-regulating Bcl-2.
Received 5 July 2006; received after revision 25 August 2006; accepted 11 September 2006 相似文献
6.
Control of respiration by nitric oxide in Keilin-Hartree particles,mitochondria and SH-SY5Y neuroblastoma cells 总被引:1,自引:0,他引:1
Mastronicola D Genova ML Arese M Barone MC Giuffrè A Bianchi C Brunori M Lenaz G Sarti P 《Cellular and molecular life sciences : CMLS》2003,60(8):1752-1759
The pattern of cytochrome c oxidase inhibition by nitric oxide (NO) was investigated polarographically using Keilin-Hartree particles, mitochondria and human neuroblastoma cells. NO reacts with purified cytochrome c oxidase forming either a nitrosyl- or a nitrite-inhibited derivative, displaying distinct kinetics and light sensitivity of respiration recovery in the absence of free NO. Keilin-Hartree particles or cells, respiring either on endogenous substrates alone or in the presence of ascorbate, as well as state 3and state 4mitochondria respiring on glutamate and malate, displayed the rapid recovery characteristic of the nitrite derivative. All systems, when respiring in the presence of tetramethyl-p-phenylenediamine, were characterised by the slower, light-sensitive recovery typical of the nitrosyl derivative. Together the results suggest that the reaction of NO with cytochrome c oxidase in situ follows two alternative inhibition pathways, depending on the electron flux through the respiratory chain.Received 1 April 2003; received after revision 22 May 2003; accepted 3 June 2003 相似文献
7.
Nitric oxide can inhibit apoptosis or switch it into necrosis 总被引:4,自引:0,他引:4
Melino G Catani MV Corazzari M Guerrieri P Bernassola F 《Cellular and molecular life sciences : CMLS》2000,57(4):612-622
Nitric oxide (NO) and its related molecules are important messengers that play central roles in pathophysiology. Redox modulation of thiol groups on protein cysteine residues by S-nitrosylation can modulate protein function. NO has emerged as a potent regulator of apoptosis in many cell types, either preventing cell death or driving an apoptotic response into a necrotic one. NO protects neuroblastoma cells from retinoid- and cisplatin-induced apoptosis, without significantly increasing necrotic cell damage. Nitrosylation of thiol groups of several critical factors may be important for cell survival. Indeed, S-nitrosylation of the active-site cysteine residue of apoptotic molecules, such as caspases and tissue transglutaminase, results in the inhibition of their catalytic activities and has important implications for the regulation of apoptosis by NO. On the other hand, NO is able to shift the anti-CD95- and ceramide-triggered apoptotic response of Jurkat T cells into necrotic cell death. In these apoptotic models, NO is therefore unable to solely inhibit cell death, indicating that it may act below the point of no return elicited by CD95-ligation and ceramide stimulation. 相似文献
8.
Cancer cell metabolism is characterized by limited oxidative phosphorylation in order to minimize oxidative stress. We have
previously shown that the flavonoid flavone in HT-29 colon cancer cells increases the uptake of pyruvate or lactate into mitochondria,
which is followed by an increase in O2−.. production that finally leads to apoptosis. Similarly, a supply of palmitoylcarnitine in combination with carnitine induces
apoptosis in HT-29 cells by increasing the mitochondrial respiration rate. Here we show that flavone-induced apoptosis is
increased more than twofold in the presence of palmitoylcarnitine due to increased mitochondrial fatty acid transport and
the subsequent metabolic generation of O2−. in mitochondria is the initiating factor for the execution of apoptosis.
Received 12 August 2005; received after revision 12 October 2005; accepted 14 October 2005 相似文献
9.
Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB 总被引:2,自引:0,他引:2
The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus
N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by western blot analysis. The role of the
kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a
specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase).
The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death.
Moreover, NO-induced apoptosis involves the release of cytochrome c.
Received 15 March 2002; accepted 25 March 2002 相似文献
10.
DNA replication is essential for accurate transmission of genomic information from parental to daughter cells. DNA replication
is licensed once per cell division cycle. This process is highly regulated by both positive and negative regulators. Over-replication,
under-replication, as well as DNA damage in a cell all induce the activation of checkpoint control pathways such as ATM/ATR,
CHK kinases, and the tumor suppressor protein p53, which provide “damage controls” via either DNA repairs or apoptosis. This
review focuses on accumulating evidence, with the emphasis on recently discovered Killin, that S-phase checkpoint control
is crucial for a mammalian cell to make a life and death decision in order to safeguard genome integrity. 相似文献
11.
R. T. Allen M. W. Cluck D. K. Agrawal 《Cellular and molecular life sciences : CMLS》1998,54(5):427-445
Apoptosis is an essential and highly conserved mode of cell death that is important for normal development, host defense
and suppression of oncogenesis. Faulty regulation of apoptosis has been implicated in degenerative conditions, vascular diseases,
AIDS and cancer. Among the numerous proteins and genes involved, members of the Bcl-2 family play a central role to inhibit
or promote apoptosis. In this article, we present up-to-date information and recent discoveries regarding biochemical functions
of Bcl-2 family proteins, positive and negative interactions between these proteins, and their modification and regulation
by either proteolytic cleavage or by cytosolic kinases, such as Raf-1 and stress-activated protein kinases. We have critically
reviewed the functional role of caspases and the consequences of cleaving key substrates, including lamins, poly(ADP ribose)
polymerase and the Rb protein. In addition, we have presented the latest Fas-induced signalling mechanism as a model for receptor-linked
caspase regulation. Finally, the structural and functional interactions of Ced-4 and its partial mam malian homologue, apoptosis
protease activating factor-1 (Apaf-1), are presented in a model which includes other Apafs. This model culminates in a caspase/Apaf
regulatory cascade to activate the executioners of programmed cell death following cytochrome c release from the mitochondria
of mammalian cells. The importance of these pathways in the treatment of disease is highly dependent on further characterization
of genes and other regulatory molecules in mammals.
Received 18 February 1998; accepted February 1998 相似文献
12.
Kysenius K Muggalla P Mätlik K Arumäe U Huttunen HJ 《Cellular and molecular life sciences : CMLS》2012,69(11):1903-1916
The secreted protease proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipid (LDL) receptor family members LDLR, very low density lipoprotein receptor (VLDLR) and apolipoprotein receptor 2 (ApoER2), and promotes their degradation in intracellular acidic compartments. In the liver, LDLR is a major controller of blood LDL levels, whereas VLDLR and ApoER2 in the brain mediate Reelin signaling, a critical pathway for proper development of the nervous system. Expression level of PCSK9 in the brain is highest in the cerebellum during perinatal development, but is also increased in the adult brain after ischemia. The mechanism of PCSK9 function and its involvement in neuronal apoptosis is poorly understood. We show here that RNAi-mediated knockdown of PCSK9 significantly reduced the death of potassium-deprived cerebellar granule neurons (CGN), as shown by reduced levels of nuclear phosphorylated c-Jun and activated caspase-3, as well as condensed apoptotic nuclei. ApoER2 protein levels were increased in PCSK9 RNAi cells. Knockdown of ApoER2 but not of VLDLR was sufficient to reverse the protection provided by PCSK9 RNAi, suggesting that proapoptotic signaling of PCSK9 is mediated by altered ApoER2 function. Pharmacological inhibition of signaling pathways associated with lipoprotein receptors suggested that PCSK9 regulates neuronal apoptosis independently of NMDA receptor function but in concert with ERK and JNK signaling pathways. PCSK9 RNAi also reduced staurosporine-induced CGN apoptosis and axonal degeneration in the nerve growth factor-deprived dorsal root ganglion neurons. We conclude that PCSK9 potentiates neuronal apoptosis via modulation of ApoER2 levels and related anti-apoptotic signaling pathways. 相似文献
13.
Hydrogen peroxide and hydroxyl radical involvement in the activation of caspase-3 in chemically induced apoptosis of HL-60 cells 总被引:3,自引:0,他引:3
Kajiwara K Ikeda K Kuroi R Hashimoto R Tokumaru S Kojo S 《Cellular and molecular life sciences : CMLS》2001,58(3):485-491
Apoptosis of HL-60 cells induced by actinomycin D, H7, or daunorubicin was shown to involve the activation of caspase-3-like
protease, 2 h after the addition of these drugs, based on microassay of enzyme activity by high-performance liquid chromatography.
Catalase and a spin trap, N-t-butyl--phenylnitrone, which effectively inhibited the apoptosis induced by these drugs, also inhibited the activation of caspase-3-like
protease. These results suggest that hydrogen peroxide and the hydroxyl radical are common mediators of caspase-3 activation
caused by these chemicals, with apparently different functional mechanisms. Based on mitochondrial activity determined by
oxygen consumption, complexes I, II, and IV were inhibited by actinomycin D. H7 inhibited complexes I and IV, 1 and 1.5 h
respectively, after the addition of the drug to HL-60 cells. Daunorubicin inhibited complex IV, 1.5 h after the addition of
the drug to HL-60 cells. Inhibition of complex IV by actinomycin D, H7, and daunorubicin were almost fully restored by the
addition of cytochrome c. The release to the cytosol of cytochrome c by these drugs was also demonstrated by Western blot
analysis. Addition of catalase inhibited the depression of complex IV activity induced by actinomycin D and H7. These observations
indicate a direct relationship between hydrogen peroxide and the release of cytochrome c during apoptosis caused by actinomycin
D, H7, and daunorubicin.
Received 24 November 2000; received after revision 2 January 2001; accepted 30 January 2001 相似文献
14.
Glucocorticoids in T cell apoptosis and function 总被引:5,自引:0,他引:5
Glucocorticoids (GCs) are a class of steroid hormones which regulate a variety of essential biological functions. The profound
anti-inflammatory and immunosuppressive activity of synthetic GCs, combined with their power to induce lymphocyte apoptosis
place them among the most commonly prescribed drugs worldwide. Endogenous GCs also exert a wide range of immunomodulatory
activities, including the control of T cell homeostasis. Most, if not all of these effects are mediated through the glucocorticoid
receptor, a member of the nuclear receptor superfamily. However, the signaling pathways and their cell type specificity remain
poorly defined. In this review, we summarize our present knowledge on GC action, the mechanisms employed to induce apoptosis
and the currently discussed models of how they may participate in thymocyte development. Although our knowledge in this field
has substantially increased during recent years, we are still far from a comprehensive picture of the role that GCs play in
T lymphocytes.
Received 20 August 2005; received after revision 27 September 2005; accepted 10 October 2005 相似文献
15.
Dynamics of the nuclear envelope at mitosis and during apoptosis 总被引:4,自引:0,他引:4
B. Buendia J.-C. Courvalin P. Collas 《Cellular and molecular life sciences : CMLS》2001,58(12-13):1781-1789
The nuclear envelope is a highly dynamic structure that reversibly disassembles and reforms at mitosis. The nuclear envelope also breaks down--irreversibly--during apoptosis, a process essential for development and tissue homeostasis. Analyses of fixed cells, time-lapse, imaging studies of live cells and the development of powerful cell-free extracts derived from gametes or mammalian somatic cells have provided insights on the fate of nuclear envelope proteins during mitosis and apoptosis, and on the mechanisms behind nuclear envelope modifications in these processes. In this review, we discuss evidence leading to our understanding of the dynamics of the nuclear envelope alterations at mitosis and during apoptosis. We also present novel imaging and genetic approaches to the study of nuclear envelope dynamics and function. 相似文献
16.
Ludivine A. Pradelli Marie Bénéteau Jean-Ehrland Ricci 《Cellular and molecular life sciences : CMLS》2010,67(10):1589-1597
Mitochondria control whether a cell lives or dies. The role mitochondria play in deciding the fate of a cell was first identified
in the mid-1990s, because mitochondria-enriched fractions were found to be necessary for activation of death proteases, the
caspases, in a cell-free model of apoptotic cell death. Mitochondrial involvement in apoptosis was subsequently shown to be
regulated by Bcl-2, a protein that was known to contribute to cancer in specific circumstances. The important role of mitochondria
in promoting caspase activation has therefore been a major focus of apoptosis research; however, it is also clear that mitochondria
contribute to cell death by caspase-independent mechanisms. In this review, we will highlight recent findings and discuss
the mechanism underlying the mitochondrial control of apoptosis and caspase-independent cell death. 相似文献
17.
18.
The interaction between BW755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline), a potent inhibitor of both lipoxygenase and cyclo-oxygenase, and respiratory chain in mitochondria and electron transport particles (ETP) from rat livers was examined. BW755C accelerated the oxygen uptake by mitochondria without the addition of substrate for the respiratory chain. Spectrophotometric study revealed that BW755C was quickly oxidized by cytochrome oxidase in mitochondria to a compound possessing an absorption maximum at 524 nm. p-Phenylenediamine (p-diaminobenzene, PPDA), which, like BW755C, serves as an electron donor to cytochrome oxidase, was shown to inhibit the generation of active oxygen in macrophages; the inhibition was stronger than that of BW755C. These results strongly suggest that the oxidative conversion of BW755C by mitochondrial cytochrome oxidase is associated with its potentially inhibitory action on the active oxygen-generating system in phagocytes. 相似文献
19.
Lombardo MF Ciriolo MR Rotilio G Rossi L 《Cellular and molecular life sciences : CMLS》2003,60(8):1733-1743
SH-SY5Y neuroblastoma cells were cultured for up to three serial passages in the presence of the copper chelator triethylene tetramine (Trien). The copper-depleted neuroblastoma cell line obtained showed decreased activities of the copper enzymes Cu, Zn superoxide dismutase and cytochrome c oxidase with concomitant increases in reactive oxygen species. Mitochondrial antioxidants (Mn superoxide dismutase and Bcl-2) were up-regulated. Overexpression and activation of p53 were early responses, leading to an increase in p21. Eventually, copper-depleted cells detached from the monolayer and underwent apoptosis. Activation of up-stream caspase-9, but not caspase-8, suggested that apoptosis proceeds via a mitochondrial pathway, followed by caspase-3 activation. The addition of copper sulfate to the copper-depleted cells restored copper enzymes, normalized antioxidant levels and improved cell viability. We conclude that prolonged copper starvation in these replicating cells leads to mitochondrial damage and oxidative stress and ultimately, apoptosis.Received 24 April 2003; accepted 23 May 2003 相似文献
20.
Bryja V Pacherník J Soucek K Horvath V Dvorák P Hampl A 《Cellular and molecular life sciences : CMLS》2004,61(11):1384-1400
In mouse embryonic stem (mES) cells, the expression of p27 is elevated when differentiation is induced. Using mES cells lacking p27 we tested the importance of p27 for the regulation of three critical cellular processes: proliferation, differentiation, and apoptosis. Although cell cycle distribution, DNA synthesis, and the activity of key G1/S-regulating cyclin-dependent kinases remained unaltered in p27-deficient ES cells during retinoic acid-induced differentiation, the amounts of cyclin D2 and D3 in such cells were much lower compared with normal mES cells. The onset of differentiation induces apoptosis in p27-deficient cells, the extent of which can be reduced by artificially increasing the level of cyclin D3. We suggest that the role of p27 in at least some differentiation pathways of mES cells is to prevent apoptosis, and that it is not involved in slowing cell cycle progression. We also propose that the pro-survival function of p27 is realized via regulation of metabolism of D-type cyclin(s).Received 25 February 2004; received after revision 5 April 2004; accepted 15 April 2004 相似文献