Apoptosis triggered redistribution of caspase‐9 from cytoplasm to mitochondria

Potokar, Maja; Milisav, Irina; Kreft, Marko; Stenovec, Matjaž; Zorec, Robert

doi:10.1016/s0014-5793(03)00494-0

Cited by 27 publications

(21 citation statements)

References 44 publications

(68 reference statements)

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“…We used a relatively high concentration of rotenone (300 M) to trigger apoptosis. A 10-fold lower concentration of rotenone (30 M) was also effective in inducing apoptosis, consistent with the findings of a previous study (21). To verify that rotenone induced apoptosis and not necrosis, we labeled cells with annexin V. Figure 5 shows that annexin V-positive cells were Fig.…”

Section: Resultssupporting

confidence: 87%

“…The animals were euthanized in accordance with the following ethical codes and directives: International Guiding Principles for (21). The DNA was introduced into cells using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Apoptosis was triggered with rotenone, an inhibitor of complex I in the mitochondrial respiratory chain (10,16). Rotenone is widely used in studies of the activation of mitochondriadependent caspase pathways (20,21,29,30). Unless stated otherwise, cells were incubated with 300 M rotenone for the following periods (in min): 1, 5, 10, 15, 30 and 120.…”

Section: Methodsmentioning

confidence: 99%

“…Confocal images were analyzed using custom-made MatLab software as described previously (13,21). Briefly, the images obtained were exported as eight-bit tagged image file format, or TIFF, files and analyzed using the MatLab software (13) by counting the number of Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Binding of procaspase-9 to Apaf-1 leads to the cleavage of procaspase-9, converting it to an active caspase-9 (15). In pituitary somatotrophs, it appears that the caspase-9-proenzyme translocates to the proximity of mitochondria upon the activation of apoptosis (21), suggesting a mechanism of recruitment of apoptosome complex elements to the proximity of mitochondria, including Apaf-1.…”

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confidence: 99%

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Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

Potokar

Kreft²,

Chowdhury³

et al. 2006

American Journal of Physiology-Cell Physiology

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A key step in the intrinsic apoptotic pathway is the assembly of the apoptosome complex. The apoptosome components are well known; however, the physiology of the assembly of the apoptosome complex at the cellular level is still poorly defined. The aim of this work was to study the subcellular distribution of the apoptosome scaffold protein apoptotic protease-activating factor 1 (Apaf-1) before and after triggering apoptosis in single somatotrophs. Somatotrophs are the subject of extensive pituitary tissue remodeling in different physiological situations in which the quality and the number of pituitary cells are determined by cell proliferation and apoptosis. We show herein that 2 h after triggering apoptosis with rotenone, Apaf-1 redistributed to the proximity of mitochondria. In addition, the degree of colocalization between Apaf-1 and fluorescently labeled caspase-9 significantly increased during the same period. Furthermore, we show herein for the first time in single cells that the colocalization between Apaf-1 and cytochrome c increases only transiently, indicating a transient interaction between cytochrome c and Apaf-1 during the activation of apoptosis in these cells.

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Section: Resultssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

Potokar

Kreft²,

Chowdhury³

et al. 2006

American Journal of Physiology-Cell Physiology

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Preapoptotic cell stress response of primary hepatocytes

et al. 2010

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Primary hepatocytes are an important in vitro model for studying metabolism in man. Caspase-9 and Bcl-2-associated X protein (Bax) are regulators of the apoptotic pathway. Here we report on the translocation of procaspase-9 and Bax from cytoplasm to nuclei as well as on dispersion of mitochondria; these processes occur after isolation of primary hepatocytes. The observed changes appear similar to those at the beginning of apoptosis; however, the isolated hepatocytes are not apoptotic for the following reasons: (1) cells have a normal morphology and function; (2) the mitochondria are energized; (3) there is no apoptosis unless it is induced by, e.g., staurosporine or nodularin. Staurosporine does not trigger apoptosis through activation of caspase-9, as its activity is detected later than that of caspase-3. We propose that the translocation of procaspase-9 and Bax into the nuclei reduces the ability to trigger apoptosis through the intrinsic apoptotic pathway. The shifts of procaspase-9 and Bax are reversible in the absence of the apoptotic trigger; the spontaneous reversion was confirmed experimentally for procaspase-9, whereas Bax shifted from the nuclei to the cytosol and mitochondria after the initiation of apoptosis. To distinguish this process from apoptosis, we call it preapoptotic cell stress response. It shares some features with apoptosis; however, it is reversible and apoptosis has to be induced in addition to this process. Conclusion: Knowledge on preapoptotic cell stress response is important for assessing the quality of the cells used in cell therapies, in regenerative medicine, and of those used for modeling metabolic processes. (HEPATOLOGY 2010;51:2140-2151 C ell cultures, especially those of primary cells, are important models for studying biochemical and physiological phenomena; they are also used in cell therapies and in regenerative medicine. Ideally, the metabolism of isolated cells should not differ from the metabolism within the cells of intact tissues; therefore, the primary cell cultures are thought to be the closest models of in vivo processes. The primary cell cultures of isolated hepatocytes are being used as models to study drug metabolism or drug effects on the liver.1 Nevertheless, some metabolic changes occur upon culturing primary hepatocytes. Expression of cytochromes P450 (CYP) is especially well studied, because of their involvement in drug metabolism. The CYP messenger RNA (mRNA) levels of isolated hepatocytes are similar to those of liver 2 ; however, they decline progressively during the first days in culture.3 Also, there are significant perturbations of genes encoding for antioxidant enzymes, heat shock proteins, nitric oxide synthase, and methionine adenosyltransferase following the isolation and culture of hepatocytes. 2As a consequence, the use of primary cultured hepatocytes in drug metabolism studies is confined to the first days in culture. 4 Although the metabolic state of isolated hepatocytes is extensively studied, there are no data on modulation of apoptotic ma...

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Anticancer efficacy of 5F in NNK-induced lung cancer development of A/J mice and human lung cancer cells

Leung

Kong

et al. 2010

J Mol Med

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The mechanism responsible for the apoptotic effect induced by ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) is not fully understood and its in vivo effect has not been tested. In this study, the effect and mechanism of 5F was investigated in cigarette smoking carcinogen 4-methylnitrosamino-1-3-pyridyl-butanone (NNK)-induced mouse lung tumor model and in cultured lung cancer cells NCI-H23 and CRL-2066. 5F were given to mice after they were treated with NNK for 18 weeks. The effect of 5F on the lung tumor formation was examined, and its side effect was monitored. Cell proliferation and apoptosis were determined through expression of PCNA, Bcl-2, Bax, and TUNEL assay in in vivo animal model. 5F significantly inhibited the NNK-induced lung tumors by inducing apoptosis and suppressing cell proliferation in vivo with minimal side effects. Cell culture experiments showed that 5F translocated Bax into the mitochondria, downregulated Bcl-2, activated caspase-9 and caspase-3, released cytochrome c into the cytosol, and translocated AIF from the mitochondria to the nucleus, which leading to G2-M cell cycle arrest and cell apoptosis. 5F also activated ERK1/2 and the inhibition of ERK1/2 suppressed 5F-mediated changes in apoptotic molecules. In addition to ERK1/2, 5F activated Akt. The inhibition of Akt further facilitated the apoptosis induced, suggesting that Akt activation was anti-apoptotic rather than pro-apoptotic. Collectively, 5F is effective against lung cancer in vivo with minimal side effects. It induces apoptosis in lung cancer through the mitochondrial-mediated pathway, in which the activation of ERK is critical.

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Apoptosis triggered redistribution of caspase‐9 from cytoplasm to mitochondria

Cited by 27 publications

References 44 publications

Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

Preapoptotic cell stress response of primary hepatocytes

Anticancer efficacy of 5F in NNK-induced lung cancer development of A/J mice and human lung cancer cells

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