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Cell Biology International (2009) 33, 10261031 (Printed in Great Britain)
Staurosporine-induced apoptosis in P388D1 macrophages involves both extrinsic and intrinsic pathways
Yuko Nakamura‑Lópeza1, Rosa Elena Sarmiento‑Silvaa, Julio Moran‑Andradeb and Beatriz Gómez‑Garcíaa*
aLaboratory of Virology, Department of Microbiology and Parasitology, Universidad Nacional Autónoma de México,Ciudad Universitaria, México DF 04510, Mexico
bDepartment of Neuroscience, Institute of Cell Physiology, Universidad Nacional Autónoma de México, Ciudad Universitaria,México DF 04510, Mexico
Treatment of P388D1, a macrophage-like cell line, with staurosporine triggered apoptosis through the activation of caspase-9 and caspase-3. Unexpected effects of staurosporine on the induction of apoptosis were the activation of caspase-8, and an increase of the levels of TNF-α. The increased TNF-α levels led to activation of caspase-8 by an autocrine effect via the TNF receptor expressed by the P388D1 macrophages. In contrast, P388D1 macrophages that either had been exposed to UV light or treated with dexamethasone did not undergo apoptosis.
Keywords: P388D1 macrophages, Apoptosis pathways, Staurosporine, UV light, Dexamethasone.
1Permanent address: Department of Molecular Biomedicine. Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional. Av Instituto Politécnico Nacional 2508, México DF 07360, Mexico.
*Corresponding author. Tel.: +52 5556232469; fax: +52 5556232386.
Apoptosis is a controlled process of cellular suicide that is important for development, tissue homeostasis, and the elimination of damaged or pathogen-infected cells. Apoptotic cells display a number of features such as DNA fragmentation, chromatin condensation, mitochondrial disruption, and alterations in the plasma membrane. Apoptosis is mediated by a family of intracellular caspases (Fuentes-Prior and Salvesen, 2004; Kerr et al., 1972), and can be induced by a wide variety of stimuli, such as ligands of death receptors (TNFR, Fas/CD95, TRAIL), chemotherapeutic drugs, and irradiation.
An agent frequently used for the induction of apoptosis is staurosporine. This alkaloid originally isolated from Streptomyces staurosporeus has been described as an inhibitor of protein kinases (Tamaoki and Nakano, 1990). Staurosporine-induced apoptosis involves the activation of mitochondrial caspase (Takahashi et al., 1997).
The P388D1 cell line, derived from a lymphoma induced with methylcholanthrene, has been characterized morphologically and functionally as macrophage-like cells. Although P388D1 cells have been a useful tool in virology, immunology, cardiology, and cancer research (Koren et al., 1975; Rauko et al., 2007; Rusiñol et al., 2004; Sarmiento et al., 2002), very little is known concerning apoptosis in this cell line. We show that P388D1 macrophages are resistant to apoptosis induced either by UV light or by dexamethasone. In addition, DNA fragmentation and caspase-8, -9, and -3 activity can be observed in P388D1 cells after treatment with staurosporine, indicating that both mitochondrial (intrinsic) and death receptors (extrinsic) apoptosis pathways had been activated.
2 Material and methods
2.1 Cell culture and induction of apoptosis
P388D1 cells (obtained from ATCC) were grown as reported previously (Sarmiento et al., 2002). To measure cell viability, cells were seeded at 1
2.2 Cell viability and morphology
Cell viability was evaluated by staining the cells with crystal violet as previously described (López-Marure et al., 2002). At the end of each incubation period, cells were fixed in 1.1% glutaraldehyde for 10
2.3 Apoptosis measurement 2.3.1 Detection of DNA fragmentation by agarose electrophoresis DNA in samples was obtained by using DNAzol Reagent (Invitrogen, Carlsbad, CA, USA), following the specifications of the manufacturer. After the extracted DNA was dissolved in 8
2.3.1 Detection of DNA fragmentation by agarose electrophoresis
DNA in samples was obtained by using DNAzol Reagent (Invitrogen, Carlsbad, CA, USA), following the specifications of the manufacturer. After the extracted DNA was dissolved in 8
3 Caspase activity
The cells were washed with cold PBS and homogenized in a caspase buffer containing 2
3.1 Measurement of TNFR1 expressionand of TNF-α production
TNFR1 expression was measured by flow cytometry. Cells (1
To determine the production of TNF-α, P388D1 cells were seeded at 1
3.2 Statistical analysis
Two-way ANOVA statistical tests were performed by using GraphPad Prism V 5.0 software (GraphPad Software, Inc, La Jolla, CA, USA). Results with p
4 Results and discussion
We found no morphological change or reduction in cell viability in the P388D1 macrophages at any time of exposure to UV light or treatment with dexamethasone (data not shown). Neither was DNA fragmentation observed at any time of UV light exposure and dexamethasone treatment. DNA fragmentation was not observed in cells that had been either exposed to UV light for 8
Agarose gel electrophoresis of DNA fragmentation in variously treated P388D1 macrophages. Extracted DNA from various samples was subjected to electrophoresis in agarose gel containing ethidium bromide. Lane 1: molecular weight marker; lane 2: untreated control cells; lane 3: cells exposed 8
Staurosporine, a broad-spectrum inhibitor of protein kinase, has been extensively used to induce apoptosis in a variety of cells. Treatment with 500
Morphology, cell viability, and DNA fragmentation in P388D1 cells after staurosporine-induced apoptosis. Panel A: Phase-contrast morphological analysis of cells after staurosporine treatment. Pictures were taken on an optical microscope at 40x (Nikon Diaphot 811454, Japan). Panel B: Cell viability was assessed by crystal violet assay; open circles, untreated cells (control); closed (black) circles, cells treated with 500
To elucidate the mechanism of action, we investigated the involvement of caspases in staurosporine-induced apoptosis. Staurosporine rapidly triggered the cleavage of LEHD-AMC peptide, a preferential substrate of caspase-9 (Fig. 3A). We also observed a stimulation of caspase-3 activity, as measured by the cleavage of DEVD-AMC (Fig. 3B). In accord with these findings, the results for P388D1 macrophages, which had first been incubated with the selective inhibitors for caspase-3 or -9 (DEVD-CHO or LEHD-CHO, respectively) and treated with 500
Caspase activity in P388D1 macrophages. Cells were treated for 0–12
Macrophages express death receptors (TNFR, Fas) and their ligands (TNF-α, FasL). We found that P388D1 macrophages expressed TNFR1 (Fig. 4A) and continuously produced TNF-α (2000
TNFR1 expression and TNF-α production in P388D1 cells. Panel A: Flow cytometry assay of the P388D1 cells expressing TNFR1 (dark line) and of negative control cells (light line); and Panel B: TNF-α levels in supernatant of P388D1 macrophages cultures at 8
Resistance to UV light-induced apoptosis has been described in different cell types, e.g. fibroblasts, Chinese-hamster ovary cells and keratinocytes (Carvalho et al., 2008; Chaturvedi et al., 2004; Rochette and Brash, 2008; Schenning et al., 2008; Yu et al., 2004). This resistance is related to the overexpression of phosphatidylserine synthases, phosphatidylinositol transfer protein alpha and anti-apoptotic protein BCL-xL (Chaturvedi et al., 2004; Rochette and Brash, 2008; Schenning et al., 2008; Yu et al., 2004). There are no reports of overexpression of these molecules in P388D1 macrophages. However, it has been reported that BCL-xL in P388D1 macrophages is downregulated by oxysterols and that these cells undergo apoptosis in response to these compounds (Rusiñol et al., 2004). There are no data showing that BCL-xL is overexpressed in P388D1 macrophages either after exposure to UV light or by treatment with dexamethasone. It has been suggested that both pro- and anti-apoptotic effects following the addition of glucocorticoids, and the up- or down-regulation of the bcl-X gene could occur in a cell-dependent context andin different cell types (Viegas et al., 2008).
Resistance to UV light-induce apoptosis is related to cell confluence and activation of p53 (Carvalho et al., 2008). For the assays, we used monolayers, but did not determine whether p53 was activated. Investigation of BCL-xL expression and p53 activation in P388D1macrophages is in progress. These further studies will delineate the molecular mechanisms that are responsible for the resistance to apoptosis by P388D1 macrophages treated with either dexamethasone or UV light. In this regard, it will be interesting to investigate the expression of pro-apoptotic and anti-apoptotic proteins in lysates of P388D1 macrophages after being treated with either UV light or dexamethasone.
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT; grant # 43944-M) and by the Dirección General de Asuntos del Personal Academico-Universidad Nacional Autonoma de Mexico (DGAPA-UNAM; grant # IN2004007). We thank Andi Espinoza-Sanchez and Berenice Hernández for the technical assistance, Rocio Tirado Mendoza, PhD, for helpful discussions, and Veronica Yakoleff for English revision and for editing of the manuscript. This work is part of the PhD dissertation submitted by Y. Nakamura-Lopez in partial fulfillment of the degree requirements.
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Received 16 February 2009/9 April 2009; accepted 3 June 2009doi:10.1016/j.cellbi.2009.06.010