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Cell Biology International (2009) 33, 1026–1031 (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


Abstract

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.


1 Introduction

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 1×104cells per well in 96-well plates (Nunclon, Roskilde, Denmark). To detect the DNA fragmentation, cells were cultured in 24-well plates (Costar, Cambridge, MA, USA) at 3×105 cells per well overnight before treatment. Caspase activity was measured in cells were cultured in 6-well plates (Nunclon, Roskilde, Denmark) at 3×106cells per well. To induce apoptosis, cells were washed with cold PBS (phosphate buffer saline), t incubated with RMPI 1640 medium supplemented with 1% (v/v) fetal bovine serum, under the following conditions: a) in the presence or absence of 500nM staurosporine (Sigma, St. Louis, MO, USA) for 8–24h; b) in the presence or absence of 75–200μM dexamethasone (Sigma) for 24–120h (Schmidt et al., 1999); or c) with or without exposure for a varied times (30s, 1min, 15min, 1h, 4h and 8h) to 254/365nm UV light from a lamp (UV Entela UVGL-25 mineral light lamp, Upland, CA, USA) placed 10cm above the cells; samples were taken at each time, or were cultured a further 24h before taking a sample for apoptosis assays. All incubations were done at 37°C.

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 10min. Cells containing crystal violet were solubilized in 10% acetic acid, and the absorbance at 590nm read in a Benchmark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Morphological analysis of cells was performed by using phase-contrast microscopy. Pictures were taken on an optical microscope at 40× (Nikon Diaphot 811454, Japan).

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 8mM NaOH, 10μl was mixed with 1μl loading buffer (Blue/Orange G; Promega, Madison, WI, USA). The samples were subjected to electrophoresis (1.8% agarose gel in 40mM Tris acetate, pH 8.5, containing 2mM EDTA and ethidium bromide (0.4mg/ml)) for 3h at 36volts. DNA was visualized by using a UV trans-illuminator (Universal Hood II, Bio-Rad Laboratories).

3 Caspase activity

The cells were washed with cold PBS and homogenized in a caspase buffer containing 2μl/ml protease inhibitor (Valencia and Moran, 2004). Caspase activities were assayed by a fluorometric method in a luminescence spectrometer (Spectronic Instruments, AMINCO-Bowman Series 2, Rochester, NY, USA) at an excitation wavelength of 360nm and an emission wavelength of 440nm. For this assay, the fluorogenic tetrapeptides, Ac-DEVD-AMC, Ac-IETD-AMC, and Ac-LEHD-AMC (Alexis Biochemicals, San Diego, CA, USA), were used as substrates to detect preferentially the activity of caspase-3, -8 and -9, respectively. The reactions were followed for 15min after the addition of substrate (10μM) using 30mg/ml of each cell homogenate. Values are expressed as relative fluorescence units (RFU) per h per mg of protein. To determine the effect of caspase inhibitors on cell viability, cells were pre-incubated (1h) with 20μM of either Ac-DEVD-CHO, Ac-IETD-CHO, or Ac-LEHD-CHO (Alexis Biochemicals), the preferential inhibitors of caspase-3, -8 and -9, respectively. Staurosporine was added and cell viability assayed after 12h incubation.

3.1 Measurement of TNFR1 expressionand of TNF-α production

TNFR1 expression was measured by flow cytometry. Cells (1×105) were detached by repeated pipetting with PBS, fixed with 4% paraformaldehyde (30min), and suspended in PBS containing 1% (w/v) BSA. The cells were incubated with fluorescently labelled antibody (anti-TNFR1-Alexa Fluor 647, AbD Serotec, Kidlington, OX, UK), diluted 1:10 in PBS containing 1% (w/v) BSA, for 1h in the dark, washed with cold PBS, and resuspended in 200μl PBS containing 1% (w/v) BSA. Cell labelling was analyzed by using FACScan (BD-Bioscience, San Jose, CA, USA) and WinMDI 2.9 software.

To determine the production of TNF-α, P388D1 cells were seeded at 1×104 cells per well in 96-well plates, washed several times with cold PBS, and incubated in the presence or absence of 500nM staurosporine for 8h. TNF-α was measured in the supernatants of these cells after staurosporine treatment using Mouse TNF ELISA Kit II (BD-Bioscience-Pharmingen, San Diego, CA, USA), according to the manufacturer's recommendations.

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<0.05 were considered as statistically significant.

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 8h and subsequently cultured for 24h (Fig. 1, lane 3), or treated with 200μM dexamethasone for 120h (Fig. 1, lane 4).


Fig. 1

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 8h to UV and subsequently cultured for 24h; lane 4: cells treated with 200μM dexamethasone for 120h; and lane 5: cells treated with 500nM staurosporine for 24h.


Staurosporine, a broad-spectrum inhibitor of protein kinase, has been extensively used to induce apoptosis in a variety of cells. Treatment with 500nM staurosporine-induced apoptosis in P388D1 macrophages. DNA fragmentation was observed in staurosporine-treated cells with specific DNA-laddering occurring after incubation lasting for >8h (Fig. 1, lane 5; Fig. 2C). After 8h of incubation with 500nM staurosporine, the cells showed morphological changes (Fig. 2A) and a decrease in cell viability; after 24h, no viable cells were found (Fig. 2B).


Fig. 2

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 500nM staurosporine. Data are expressed as mean±SD from three separate experiments performed in triplicate. Results showing statistically significant differences from control: **p<0.01 and ***p<0.001; and Panel C: Kinetics of DNA fragmentation of cells treated with 500nM staurosporine. Duration of incubation is indicated.


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 500nM staurosporine, showed no statistically significant differences in viability from control cells (Fig. 3D). When IETD-AMC was used, i.e. the substrate of caspase-8 as the most proximal regulatory caspase in receptor-mediated cell death, this caspase was activated (Fig. 3C). To confirm this observation, the effect of staurosporine on cell viability was blocked (Fig. 3D) when an inhibitor specific for caspase-8 (IETD-CHO) was used.


Fig. 3

Caspase activity in P388D1 macrophages. Cells were treated for 0–12h with 500nM staurosporine, then cell homogenates were incubated with caspase-9 substrate (LEHD-AMC), caspase-8 substrate (IETD-AMC), or caspase-3 substrate (DEVD-AMC). Panel A: caspase-9 activity; Panel B: caspase-3 activity; Panel C: caspase-8 activity; and Panel D: Viability of cells at 12h incubation: (medium), untreated control cells; (STS 500nM), cells treated with 500nM staurosporine (STS); or cells pre-treated with a caspase inhibitor (DEVD+STS: the caspase-3 inhibitor, DEVD-CHO; LEHD+STS: the caspase-9 inhibitor, LEHD-CHO; or IETD+STS: the caspase-8 inhibitor, IETD-CHO) before treatment with staurosporine. Data are expressed as mean±SD from three independent experiments performed in triplicate. Results showing statistically significant differences from control: *p<0.05 and ***p<0.001.


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 (2000pg/ml). After staurosporine treatment, the levels of this cytokine increased (2600pg/ml at 8h) (Fig. 4B), suggesting that the extrinsic pathway may have contributed to apoptotic death of P388D1 through activation of caspase-8.


Fig. 4

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 8h: medium, untreated control cells; and STS, cells treated with 500nM staurosporine. Data are expressed as the mean±SD from two separate experiments performed in duplicate.


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.

Acknowledgments

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 2009

doi:10.1016/j.cellbi.2009.06.010


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)