Differentiation-associated apoptosis of neural stem cells is effected by Bcl-2 overexpression: impact on cell lineage determination

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European Journal of Cell Biology 80, 539 ± 553 (2001, August) ´  Urban & Fischer Verlag ´ Jena http://www.urbanfischer.de/journals/ejcb

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Differentiation-associated apoptosis of neural stem cells is effected by Bcl-2 overexpression: impact on cell lineage determination Christina Esdara, Sandra Milastab, Alfred Maelickeb, Thomas Herget1) a, b a b

Axxima Pharmaceuticals AG, Martinsried/Germany Laboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University, Mainz/Germany

Received January 2, 2001 Received in revised version April 12, 2001 Accepted April 29, 2001

Neural differentiation ± neural determination ± apoptosis ± retinoic acid ± PCC7-Mz1 ± mouse Apoptosis is an integral part of neural development. To elucidate the importance of programmed cell death on cell lineage determination we utilized murine PCC7-Mz1 cells, a model system for neural differentiation. Treatment of pluripotent PCC7-Mz1 stem cells with 0.1 mM all-trans retinoic acid (RA) causes a cease of proliferation and an initiation of differentiation into neurons, glial cells and fibroblasts. Simultaneously, a fraction of the cell culture (ca. 25%) dies within 24 h by apoptosis. We transfected PCC7-Mz1 cells with the human bcl-2 cDNA and generated PCC7-Mz-Bcl-2 cell lines expressing two- to tenfold higher levels of Bcl-2 than parental cells. Overexpression of Bcl-2 resulted in hypophosphorylation of the retinoblastoma (Rb) protein and consequently prolonged the doubling time of the culture from 18 h to 23 h. RAinduced apoptosis was drastically reduced to 3 to 15% depending on the level of Bcl-2 expression. RA-induced caspase activation, cytochrome c release from the mitochondria to the cytosol and DNA fragmentation was completely blocked. Furthermore, treating Bcl-2 cultures with ceramide (10 mM), a second messenger mediating the RA-initiated death signal in parental cells, no longer caused DNA laddering. Bcl-2 overexpression did not interfere with the potential of PCC7-Mz cells to develop into neurons, glial cells and fibroblasts. However, the relative distribution of cell types in the culture was shifted such that the fraction of neurons was reduced to half (from 60 to 30%) with a concomitant increase in the number of glial and fibroblastoid cells. Furthermore, Bcl-2-overexpressing neurons, but not neurons of parental or mock-transfected PCC7-Mz1 cultures, were able to grow as single cells. PD Dr. Thomas Herget, Axxima Pharmaceuticals AG, Am Klopferspitz 19, D-82152 Martinsried/Germany, e-mail: [email protected], Fax: ‡ 49 89 7401 6520.

1)

Abbreviations. Caspases Aspartate-specific cystein proteases. ± d1 ± d6 Day 1 to 6 of differentiation. ± GAP-43 Growth-associated protein of 43 kDa. ± PARP Poly(ADP)ribose polymerase. ± RA All-trans retinoic acid. ± Rb protein Retinoblastoma protein. ± SDS-PAGE Sodium dodecyl sulfatepolyacrylamide gel electrophoresis. ± z-IETD-fmk Benzyloxycarbonylisoleucyl-glutamyl-threonyl-aspartyl fluoromethyl ketone. ± z-VAD-fmk Benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone.

Introduction During brain development, up to 80% of all neurons are eliminated by apoptosis (Oppenheim, 1991). Initially neurons seem to be produced in excess in order to allow for the maximal probability of forming contacts with their cellular partners. This process, called neurotrophic strategy (reviewed in (Pettmann and Henderson, 1998)), is followed by the elimination of excess cells through apoptosis. While initiation of apoptosis depends on cell type and stimulus, the execution phase of apoptosis is often independent of the apoptotic trigger and strikingly similar in various tissues. In recent years, three distinct pathways leading to apoptosis have been identified (Mehmet, 2000). The first one (type I) is activated by oligomerization of death receptors i. e. CD95, TRAIL- and tumor-necrosis factor receptors, which recruit adaptor molecules involved in activation of caspase-8 and -10 (Scaffidi et al., 1999). The second pathway (type II) depends on the release of pro-apoptotic molecules, such as cytochrome c, from the mitochondria into the cytosol. Together with ATP, cytochrome c forms a complex (apoptosome) with Apaf-1 (apoptotic protease activating factor) and pro-caspase-9, which is released in an active form (Villa et al., 1997) (reviewed in (Green and Reed, 1998)). Since activation of caspase-9 initiates a caspase cascade irreversibly resulting in apoptosis (Li et al., 1997; Liu et al., 1997) this process is regulated in a complex manner. A class of proteins called IAPs (inhibitors of apoptosis

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proteins) blocks caspase activity and thereby prevents apoptosis. On the other hand, a protein called Smac (second mitochondria-derived activator of caspase) (Du et al., 2000) or DIABLO (direct IAP binding protein with low pI) (Verhagen et al., 2000) was recently identified that promotes cytochrome c/Apaf-1-dependent caspase activation by opposing the inhibitory activity of IAPs on apoptosis. Lately, a novel, third apoptotic pathway (type III) was identified. After stress to the endoplasmatic reticulum, including the release of Ca2 ‡ from intracellular stores, caspase-12 is activated (Nakagawa et al., 2000). It was suggested that the Bcl-2 gene family, which is the homologue of the C. elegans CED-9, is involved in regulation of the mitochondria-dependent pathway (Green and Reed, 1998). Several Bcl-2 family proteins reside in the outer membrane of the mitochondrium, nucleus and endoplasmatic reticulum. Bcl-2, Bcl-xL and Bax share striking similarity to the poreforming domain of some types of bacterial toxins, e. g. diphteria toxin and colicins (Kroemer et al., 1998) and may form ion channels. Furthermore, Bcl-2 may act to target the protein kinase Raf-1 to the mitochondrial membrane (Wang et al., 1996). Bcl-2-related proteins have been demonstrated to regulate cell survival in response to a variety of apoptotic stimuli, including growth factor withdrawal and genotoxic damage, in a number of cell types (White, 1996). The homodimeric form of Bcl-2 has been shown to increase the survival of both central and peripheral neurons in culture and has been demonstrated to counter the effects of a variety of apoptotic and necrotic stimuli including ischemia (Martinou et al., 1994), traumatic brain injury (Clark et al., 1997), neurotrophic factor removal (Allsopp et al., 1993), growth factor deprivation and generation of free radicals (Merry and Korsmeyer, 1997). Evidence suggests that the susceptibility of cells to programmed cell death in neuronal cells can be affected by the ratio of the levels of the negative regulators of apoptosis to proapoptotic members of the Bcl-2 family (Middleton et al., 1996) and is, in the case of neuronal cells, regulated by the levels of various neurotrophic factors (Middleton et al., 1996; Allsopp et al., 1995). In vivo, Bcl-2 expression has been demonstrated in the murine nervous system to occur as early as embryonic day 10, with bcl-2 mRNA expression peaking on day 15 i. e. during the periods in which remodeling of the nervous system occurs (Abe-Dohmae et al., 1993). Although the expression is downregulated at the time of birth, the continued expression into adulthood suggests a role for Bcl-2 also in the maintenance of the nervous system (Farlie et al., 1995; Bernier and Parent, 1998). In this context, Bcl-2 has been discussed among the factors that induce and maintain axonal growth (reviewed in (Holm and Isacson, 1999)). Although the protective effect of Bcl-2 toward developmental and induced neuronal cell death is well documented, the role of Bcl-2 in neurogenesis and neural cell lineage determination remains elusive. Rat pheochromocytoma PC12 cell lines stably transfected with Bcl-2 cDNAwere protected from death caused by removal of trophic factors. However, Bcl-2 in these cells neither mimicked nor interfered with promotion of neurite outgrowth by nerve growth factor (NGF) (Batistatou et al., 1993). Sato and colleagues reported that overexpression of Bcl-2 in PC12 cells caused survival and neuritogenesis in serumfree, but not in serum-containing medium (Sato et al., 1994). Neurite outgrowth was also observed in a dopaminergic cell line overexpressing Bcl-2 (Oh et al., 1996). Furthermore, when

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Bcl-2 was overexpressed in the human neural crest-derived tumor cell line, Paju, which undergoes spontaneous neural differentiation, accelerated and extensive neural sprouting was observed (Zhang et al., 1996). In the present study, we investigated whether Bcl-2 has novel functions during neural differentiation either in addition to providing protection against neural cell death or as a consequence of blocking cell death. To address this question we studied the expression of Bcl-2 in the embryonal carcinoma cell line PCC7-Mz1, which, upon incubation with all-trans retinoic acid (RA), differentiates into neurons, astroglia cells and fibroblasts, with the neurons forming a fully functional neuronal network (Lang et al., 1989; Berger et al., 1997). The PCC7Mz1 model of neural development allows access to the full course of neural differentiation, beginning with the stem-cell stage and leading via neural precursor stages to the terminally differentiated phenotypes. Cell lineage determination starts within hours after exposure to RA and is essentially completed within one day (Jostock et al., 1998; Bauer et al., 1997). At day three of RA treatment, when the neuronal derivatives have formed long extensions and begin to establish a neuronal network, the first fibroblasts can be detected by their marker expression, followed by phenotypic astroglial cells at around day 6. From day 6, neuronal derivatives begin to develop polarity which is completed in the second week of differentiation (Berger et al. 1997). The embryonic carcinoma cell line PCC7-Mz1 may therefore be considered to be a clone of pluripotent cells of neuroectodermal origin and serve as a model of neural development. We recently reported that apoptosis is part of early neurogenesis i. e. when PCC7-Mz1 cells become committed to their specific cell lineages (Herget et al., 1998). Within 24 hours of RA treatment, a considerable fraction (ca. 23%) of the PCC7Mz1 culture detaches and dies, whereas the remaining cells of the culture begin to differentiate along their specific lineages (neuronal, astroglial and fibroblastoid). Neurotrophic factors were ineffective in preventing this cell death as was also shown for early cell death in the avian cervical cord (Yaginuma et al., 1996), implying that cell-autonomous mechanisms are responsible. Therefore, apoptosis may be instructed by the gene regulatory program that controls cell lineage determination and the formation of patterns of neuroectodermal derivatives. Bcl-2 transcription becomes up-regulated in PCC7-Mz1 cells within a few days of RA treatment and is conversely related to the extent of apoptosis, which rapidly declines once the neuronal network starts to emerge (Herget et al., 1998). Hence, expression of Bcl-2 seems to provide protection from apoptosis for PCC7-Mz1 cells that have already achieved a certain level of differentiation. In the present study, we transfected PCC7-Mz1 cells with the human Bcl-2-coding cDNA and generated stable cell lines. These cells showed resistance towards RA- and ceramideinduced apoptosis. Bcl-2 overexpression reduced RA-caused programmed cell death to 3 to 15% and influenced cell lineages determination in favor of cells with flat morphology (i. e. glial cells and fibroblasts). Furthermore, cell cycle analysis revealed an accumulation of the Bcl-2-overexpressing cells in the G0/G1 phase of the cell cycle accompanied by activation of the retinoblastoma protein (Rb). Bcl-2-overexpressing neurons showed a different morphology compared to mock- or untransfected PCC7-Mz1 cells.

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Materials and methods Cell culture

The mouse embryonic carcinoma cell line PCC7-Mz1 is a subclone of the PCC7-S-AzaR1 (clone 1009) cell line (Lang et al., 1989). Culture conditions, growth characteristics and the RA-induced differentiation pattern have been described previously (Lang et al., 1989; Berger et al., 1997; Herget et al., 1998). PCC7-Mz1 cells were grown in plastic tissue culture flasks in Dulbeccos modified Eagles medium (DMEM) (Life Technologies, Karlsruhe, Germany), supplemented with 12.5% fetal bovine serum (FCS) (batch 148, Roche Molecular Biochemicals, Germany), at 37 8C in humidified air/10% CO2. Prior to induction of differentiation, PCC7-Mz cells were seeded at a density of 1.75  104 cells/cm2 in plastic tissue culture dishes. For differentiation, cultures were treated with 0.1 mM (final concentration) all-trans retinoic acid (RA) (Sigma, Germany) one day after plating. 24 hours later, the culture medium was replaced by DMEM, supplemented with 12.5% FCS, 0.1 mM RA and 1 mM dibutyryl cAMP (dbcAMP; Roche Molecular Biochemicals, Germany).

Cell viability assay

For quantification of the degree of cell death in cell culture, we employed the viability assay based on the reduction of tetrazolium salt to formazan by mitochondrial dehydrogenase activity. The assay was performed in 96-well microtiter plates (Greiner, Frickenhausen, Germany) as described previously (Herget et al., 1998) but WST-1 (Roche Molecular Biochemicals, Germany) was used instead of MTT. The light absorbance at 405 nm of the medium including all factors but without cells was determined and subtracted from the absorption readings with cells. Eight wells per sample point were analyzed and each experiment was repeated independently at least three times. Caspase inhibitors used were z-VAD-fmk (Alexis, Grunberg, Germany) and z-IETD-fmk (Calbiochem, Bad Soden, Germany).

Preparation of cell lysates and Western blot analysis

Western blot analyses were performed as described previously (Esdar et al., 1999; Oehrlein et al., 1998; Herget et al., 1998). To prepare extracts of apoptotic cells, detached PCC7-Mz1 cells in the supernatant were collected by centrifugation (10 min at 2 000g) and resuspended in 150 ml lysis buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 1% Triton X-100, 0.4 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT, 100 mg/ml leupeptin, 100 mg/ml aprotinin, 10 mM benzamidine, 2 mM phenylmethylsulphonyl fluoride (PMSF), 20 mM b-glycerophosphate and 0.1 mM sodium-ortho-vanadate). Adherent cells were washed twice with ice-cold PBS and scraped off the dish with 200 ml lysis buffer using a rubber policeman. The lysed cells were centrifuged for 10 min at 20 000g at 4 8C. Protein concentrations of the supernatants were determined using BCA reagent (Pierce, Bruchsal, Germany). For preparation of mitochondrial and S100 fractions (supernatant of 100 000g), cells grown on 15-cm culture dishes were harvested in 200 ml of buffer A (10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 50 mM sucrose, 0.1 mM PMSF, 0.5 mM DTT) and homogenized by thirty strokes in a Dounce homogeniser with an S-pistle. After centrifugation at 750g for 10 min at 4 8C, the pellet was resuspended in 200 ml buffer A and centrifuged again at 750g. The supernatants were pooled and centrifuged for 30min at 10 000g. Then, the pellets containing the mitochondria were resuspended in 100 ml reducing Laemmli sample buffer. The supernatant was spun for 30 min at 100 000g in a Beckman TL100 centrifuge (TL100.2 rotor) as described (Li et al., 1997). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore, Bedford, MA) by semi-dry blotting. The membranes were blocked with PBS/Triton (0.1% Triton X-100 in phosphate-buffered saline, pH 7.2), supplemented with 5% low-fat milk powder (w/v) for 1 h and then incubated overnight at 4 8C with a monoclonal mouse antibody recognizing Rb (PharMingen, San Diego), diluted 1 : 500 in PBS/Triton with 1% low-fat milk powder (w/v), a

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monoclonal n-clathrin antibody (Berger et al., 1997) (kindly provided by R. Jahn; Göttingen) diluted 1 : 1 500, a monoclonal cytochrome c-specific antibody (PharMingen, San Diego), diluted 1 : 500, or with an affinity-purified rabbit GAP-43 antiserum, diluted 1 : 4 000 (Esdar et al., 1999). After washing, membranes were incubated with horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit antibody (1 : 2 000 in PBS/Triton with 1% low fat milk powder; w/v) (DAKO, Hamburg, Germany) for 1 h at room temperature to detect bound antibodies. Membranes were washed again with PBS/Triton and secondary antibodies were visualized by the enhanced chemiluminescence (ECL) detection system according to the manufacturers instructions (Amersham, Braunschweig, Germany) using medical X-ray films (Fuji).

Genomic DNA analysis

Cells were cultivated in 6-cm dishes and genomic DNA was isolated as described previously (Herget et al., 1998). Briefly, cells of the medium were collected by centrifugation (2 000g, 10 min, 4 8C) and the pellet was resuspended in 20 ml cell lysis buffer (0.5% Triton X-100, 20 mM EDTA, 5 mM Tris-HCl, pH 8). Adherent cells were washed with ice-cold phosphate-buffered saline (PBS) once, detached with a rubber policeman and centrifuged. This cell pellet was also resuspended in 20 ml cell lysis buffer. The lysed cells were immediately incubated with 0.5 mg/ml proteinase K (Sigma) and then with 0.5 mg/ml RNase A (Roche Molecular Biochemicals, Germany) at 50 8C for 1 h each. The reactions were stopped by heat treatment at 70 8C for 10 min. The samples were kept at 56 8C, and 15 ml of pre-warmed sample buffer (1% low-melt agarose, 10 mM EDTA, 0.25% bromphenol blue, 40% sucrose) was added. The samples were then loaded onto a 1.4% agarose gel. After electrophoresis, the DNA banding pattern was visualized under UV light using ethidium bromide.

Flow cytometric analysis

The amount of cells in G0/G1-, M- or S-phase of the cell cycle was determined by fluorescence-activated cell sorting (FACS) analysis. Approximately 1  106 cells, untreated (d0) or treated with 0.1 mM RA for 24 h (d1), were washed twice with ice-cold PBS and collected in 1 ml PBS. Then, 10 ml of lysis buffer (0.5% BSA, 0.1% Triton X-100 in PBS) were added, cells were centrifuged at 800g for 10 min and resuspended in 10 ml of lysis buffer. After another centrifugation step (800g, 10 min), chromosomal DNA was stained by adding propidium iodide to a final concentration of 5 mg/ml. Following 15 min on ice, flow cytometry analysis of 105 cells per sample was performed on a FACScan (Becton Dickinson). Cell cycle distribution was calculated using FACScan software and manual gating. The average value of at least three independent experiments is shown.

Estimation of population doubling times and growth rates

Under the assumption that cells in cultures undergo sequential symmetric divisions leading to an exponential increase in cell number, we determined the population doubling time of PCC7-Mz1 cells and PCC7-Mz-Bcl-2 clones using the following term: population doubling level (pdl) ˆ 3.32  (log Nh ± log Ni) whereby Nh is the number of cells harvested after a period of cultivation and Ni is the number of cells inoculated (McAteer and Davies, 1994). After division of the time of cultivation by pdl the population doubling time was calculated. The mean value of 20 passages of the clones analyzed was determined. To analyze the growth rates of PCC7-Mz1 cells and PCC7-Mz-Bcl-2 clones, cells were plated on 24-well plates with a density of 2  104 cells per cm2 and induced to differentiate as described. At the indicated time points (4 hours after plating (d-1), d0, d1 and d3) all cells of one well were harvested, viable cells identified by trypan blue exclusion and counted with a Neubauer ruled hemocytometer. The experiment was repeated three times.

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Immunofluorescence studies

PCC7-Mz cells were grown and differentiated on ethanol-cleaned glass coverslips at a density of 1  104 cells/cm2. At the differentiation stage to be studied, cells were washed and fixed in 4% PFA in PBS supplemented with 4% sucrose for 20 min at room temperature. Fixed cells were stored in 120 mM sodium phosphate buffer (pH 7.4) at 4 8C until use. Cells were washed as follows: three times in 120 mM sodium phosphate buffer (pH 7.4), twice in low-salt-PBS (LS-PBS: 150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4) and twice in high-salt-PBS (HS-PBS: 500 mM NaCl, 20 mM sodium phosphate buffer, pH 7.4). Washed cells were incubated with blocking buffer (450 mM NaCl, 0.3% Triton X-100, 20 mM sodium phosphate buffer, pH 7.4, supplemented with 16.5% FCS) for 30 min at room temperature. The coverslip was incubated with 50 ml blocking buffer containing the primary antibody in a humidity chamber for one hour. After three washes with HS-PBS, each coverslip was incubated with the appropriate secondary antibody diluted in blocking buffer. This incubation, and all subsequent ones, were performed in the dark. Prior to mounting the cells, they were washed twice with HS-PBS and twice with 120 mM sodium phosphate buffer. The coverslips were mounted in PBS, supplemented with 1 mg/ml phenylenediamine, 50% glycerol. They were kept in the dark at ÿ 20 8C for storage. Marker proteins were Thy1.2 for fibroblasts; GAP-43, synaptophysin, TAU and Ex-1 for neurons; and GFAP for glial cells. The antisera were diluted and detected as described previously (Müller-Husmann et al., 1994; Berger et al., 1997; Oehrlein et al., 1998; Esdar et al., 1999).

Results Establishing Bcl 2-overexpressing PCC7-Mz1 cell lines

We showed previously that bcl-2 mRNA levels were nearly undetectable during early stages of differentiation, but were up-

Fig. 1. PCC7-Mz-Bcl-2 cell lines overexpressing the Bcl-2 protein. PCC7-Mz1 cells and four Bcl-2-transfected cell lines (B12, F3, H5, D2) were treated on d0 for the indicated periods (d1 to d10) with RA to induce neural differentiation. Cells were harvested, proteins were extracted and 15 mg per lane were separated by 15% SDS-PAGE. Bcl-2 protein was detected by Western blotting and visualized by the enhanced chemiluminescence method. Exposure to Fuji Medical Xray film was for 10 min. Relative expression levels were estimated by densitometric scanning and depicted in comparison to PCC7-Mz1 stem

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regulated in PCC7-Mz1 cultures within a few days of RA treatment i. e. when a stable neuronal network was formed (Herget et al., 1998). The human bcl-2 cDNA (kindly provided by Dr. J. Boise; University of Lleida) was cloned into the pcDNA3 vector with CMV promoter (Stratagene) and into a modified version of this vector which contains a SV40 promoter instead of the original CMV promoter (Jostock et al., 1998). To investigate a potential link between Bcl-2 expression and viability, PCC7-Mz1 cells were plated at 1.5  104 cells/cm2 in 3-cm culture dishes coated with laminin and were transfected with the two bcl-2 constructs the following day using EscortTM (Sigma, Munich, Germany). This treatment (lipofection) itself did not cause any cell death in contrast to the Ca2 ‡-phosphate co-precipitation method (data not shown). G-418 (600 mg/ml; LifeTechnologies-GIBCO-BRL) resistant colonies were propagated, cloned by limited dilution and analyzed by Western blotting. Six cell lines obtained from the pcDNA3-Bcl-2 transfection and 15 cell lines obtained from the pcDNA3SV40-Bcl-2 transfection were identified overexpressing the Bcl-2 protein at least twofold. The expression of Bcl-2 was evaluated in PCC7-Mz1 stem cells and 1, 3, 6 and 10 days after treating with RA. In clones B12, F3 and H5 the bcl-2 cDNA was driven by the SV40 promoter, while it was driven by the CMV promoter in clone D2. The clone F3 turned out to be an N-type clone developing only neurons (Lang et al., 1989; Jostock et al., 1998) which died within three days of RA treatment since the supporting glial cells were missing. The intensities of the Bcl-2 bands detected by Western blotting were densitometrically quantified and compared with the Bcl-2 expression of untransfected PCC7-Mz1 stem cells (day 0) (Fig. 1).

cells (ˆ 1). Expression of the transgenic bcl-2 cDNA was driven by the SV40 promoter in cell lines B12, F3 and H5, while it was controlled by the CMV promoter in D2 cells. Expression of Bcl-2 was two- to eightfold higher in transfected than in parental PCC7-Mz1 cells. The CMV promoter (cell line D2) was activated by the change of medium containing RA and dbcAMP on d1, while expression of the SV40 promoter constructs (cell lines B12, F3 and H5) remained unaffected by this treatment.

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Effect of Bcl-2 on early neural differentiation 543

The data show that in the stem cells of the transfectants the Bcl-2 levels were already at least as high as on d3 in the parental PCC7-Mz1 cells. During the course of differentiation, the Bcl-2 protein levels of the transfectants were three- to eightfold higher than in the parental cells. Noteworthily, the level of Bcl-2 in stem cells of clone D2 was only two-fold higher than in PCC7Mz1 stem cells but increased dramatically from d3, i. e. two days after change of medium containing RA and 1 mM dbcAMP (Fig. 1). This sixfold upregulation from d1 to d3 can be attributed to the activation of the CMV promoter by dbcAMP as was already observed previously in PCC7 cells (unpublished data) (Neuman et al., 1995). In conclusion we generated stable cell lines overexpressing the Bcl-2 protein severalfold in both undifferentiated and differentiated PCC7-Mz-Bcl-2 cells.

Bcl-2 overexpression inhibits RA-induced cell death

We showed previously, that 24 hours after incubation with 0.1 mM RA the number of viable PCC7-Mz1 cells in the culture was reduced by about 25% (Herget et al., 1998). To quantify viability, we used the WST-1 assay to monitor the mitochondrial dehydrogenase activity. Bcl-2-transfected cultures treated with RA for 24 hours showed little cell death. This effect correlated nicely with the degree of Bcl-2 overexpression. While PCC7Mz1 and mock-transfected cultures had a viability of 75  6.5%, a twofold overexpression (clone D2) resulted in a viability of 83  7.4%, and a sevenfold overexpression was necessary to increase viability to 97  8.2% (clone H5) (n ˆ 8; Fig. 2A). Since the WST-1 test does not discriminate between apoptotic and necrotic cell death, we examined whether the genomic DNA of PCC7-Mz-Bcl-2 cells was protected against intranucleosomal cleavage, a hallmark of apoptotic cell death. PCC7Mz1 and PCC7-Mz-Bcl-2 cells were treated with RA for 24 hours, all detached cells of a culture were harvested, genomic DNA isolated and fractionated on an agarose gel. Treating PCC7-Mz1 cells with RA caused significant DNA cleavage. A two- to fourfold overexpression of Bcl-2 (clone D2 and B12) was sufficient to efficiently suppress DNA degradation while a higher overexpression (> 5-fold, cell line F3) completely prevented DNA laddering (Fig. 2B). Without RA treatment, no DNA laddering was detectable, neither in PCC7-Mz1 nor in PCC7-Bcl-2 cultures. Next, the effect of Bcl-2 on caspase activity was assayed. A major substrate for these cysteine-specific proteases is the poly(ADP)ribose polymerase (PARP). Upon induction of apoptosis, the 120-kDa PARP protein is cleaved into a 85kDa N-terminal and a 25-kDa C-terminal fragment. Western blot analyses revealed that adherent PCC7-Mz1 cells and adherent bcl-2-transfected cells contained only the intact PARP protein, whereas detached PCC7-Mz1 cells displayed only the 85-kDa cleavage product (Fig. 3A). However, in Bcl-2overexpressing cell lines (B12, E10, F3, H5, and D2) PARP protein in detached cells was not completely cut, but a sizable fraction of it (between 30% and 60%) remained intact. These data demonstrate that Bcl-2 expression blocks the RA-triggered apoptotic signal transduction pathway upstream of caspase activation. Depending on cell type and stimulus the caspase-cascade is activated by a cytoplasmic complex, called the apoptosome, consisting of cytochrome c, Apaf-1 and the pro-caspase 9. We addressed the question of whether RA causes release of cytochrome c from the mitochondria and whether this process is inhibited by Bcl-2 overexpression. PCC7-Mz1 and PCC7-Mz-

Fig. 2. Protection of PCC7-Mz-Bcl-2 cells against RA-induced apoptosis. A) PCC7-Mz1 cells and six cell lines transfected with the SV40bcl-2 construct and two transfected with the CMV-bcl-2 construct were seeded in 96-well microtiter plates and treated with 0.1 mM RA for 24 hours the following day. Then, cells were incubated with the WST-1 reagent for two hours. Light absorbance reflecting the viability of the cultures is shown as percentage of the signal obtained with parallel cultures having received solvent only (ˆ 100%, upper dashed line). Bars depict means  SEM of eight independent experiments with eight samples per condition each. The statistical significance in comparison with PCC7-Mz1 cells was calculated according to the students t-test. * p < 0.05 and ** p < 0.2. While PCC7-Mz1 and mock-transfected cells showed a loss of viability by 25% (lower dashed line), the viability of SV40-bcl-2-transfected cells dropped on average by 5% and that of CMV-bcl-2-transfected cells by about 15%. B) PCC7-Mz1 stem cells and stem cells of the Bcl-2-overexpressing cell lines B12, F3 and D2 were plated in 6-cm culture dishes and 0.1 mM RA was added the following day (‡ RA). After incubation for 24 hours, genomic DNA of cells of the supernatant was isolated and separated on a 1.4% agarose gel as described in Materials and methods. After staining with ethidium bromide, DNA laddering typical of apoptotic cells became visible only when PCC7-Mz1 cells, but not when PCC7-Mz-Bcl-2 cultures were treated with RA. Incubation with the solvent (0.05% DMSO) did not cause any DNA laddering (-RA). The marker (lane M) was a mixture of l phage DNA and pYH48 DNA digested with the restriction enzymes HindIII and AluI, respectively

Bcl-2 stem cells (B12, F3 and H5) remained untreated or were incubated with RA for 24 hours. Then, cells were harvested, lysed and the cell extracts separated into a mitochondrial and a cytosolic S100 (supernatant of 100 000g) fraction by centrifugation. Western blot analysis utilizing cytochrome c-specific

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Fig. 3. Bcl-2 inhibits RA-induced caspase activation and cytochrome c release. A) PCC7-Mz1 and Bcl-2 stem cells (B12, E10, F3, H5 and D2) were plated and incubated with 0.1 mM RA for 24 hours. Then, proteins were extracted from cells attached to the substratum (adh) and from detached cells (SN). 10 mg protein were separated per lane by 10% SDS-PAGE, transferred onto a PVDF membrane and full-length (120 kDa) and clipped (85 kDa) PARP was detected by an antibody. All adherent cells contained only intact PARP, while detached PCC7Mz1 cells contained only cleaved PARP. The detached cells of Bcl-2overexpressing cultures had both intact and digested PARP protein. B) PCC7-Mz1 cells and Bcl-2 cells (B12, F3 and H5) were grown in 15-cm

culture dishes and incubated without (ÿ) or with (‡) RA for another 24 hours the following day. Then cells were collected, lysed and the extracts were separated into a mitochondrial (Mito.) and a cytosolic S100 (S100) fraction by centrifugation. 20 mg protein each were loaded and separated by 15% SDS-PAGE, transferred onto a PVDF membrane and cytochrome c was detected by an antibody. Bound antibody was visualized by enhanced chemiluminescence and exposure to Fuji X-ray film for 10 min. Cytochrome c was present in all mitochondrial fractions, and RA caused a release into the cytosol in PCC7-Mz1, but not in the Bcl-2-overexpressing cells.

antibodies revealed that cytochrome c was present in all mitochondrial fractions. In PCC7-Mz1 cells, RA caused a release of cytochrome c from the mitochondria and a concomitant increase in the cytoplasm (Fig. 3B, left panel). However, cytochrome c was not detectable in the cytoplasm of the RA-treated Bcl-2-overexpressing cell lines. These data demonstrate that RA causes release of cytochrome c from the mitochondria to the cytoplasm in PCC7-Mz1 cells. This translocation is inhibited by Bcl-2 overexpression, and explains the drastically reduced caspase activity in the PCC7-Mz-Bcl-2 cells (Fig. 3A).

efficiently blocked apoptosis of PCC7-Mz1 cells, as described previously (Herget et al., 1998), and of Bcl-2-overexpressing cells (Fig. 4). Taken together, RA activates a death pathway of type II, which is completely independent of caspase-8, involves the mitochondria and is blocked by Bcl-2.

Effect of caspase inhibition on RA-induced apoptosis

Since overexpression of Bcl-2 did not completely block RAmediated cell death (Fig. 1 and 2A), we investigated whether there exists an additional, mitochondria-independent pathway in PCC7-Mz1 cells. By using specific caspase-8 inhibitors, pathways of type I, initiated by death receptors, can efficiently be stopped (Rytömaa et al., 1999). However, 10 mM of the potent caspase-8 inhibitor z-IETD-fmk did not reduce the degree of cell death in RA-treated parental PCC7-Mz1 cultures nor in bcl-2-transfected cultures (B12 and H5; Fig. 4). Therefore, caspase-8 does not seem to be involved in the RA-induced cell death. As a control, we treated the same cell lines in parallel with RA and z-VAD-fmk. This general caspase inhibitor

Bcl-2 inhibits cell death caused by ceramide

After having demonstrated that Bcl-2 hindered RA-induced apoptosis, we studied the effect of Bcl-2 overexpression on ceramide-induced apoptosis. Ceramides were shown to be synthesized upon RA treatment and responsible for cells undergoing apoptosis (Herget et al., 2000). Treating mock transfectants or PCC7-Mz1 stem cells for 24 hours with increasing concentrations of membrane-permeable C6-ceramide caused a dose-dependent loss of viability (continuous line, Fig. 5A). The Bcl-2-overexpressing cells, however, showed a significantly reduced loss of viability (dashed lines, Fig. 5A). The ceramide-induced cell death was due to apoptotic and not necrotic processes, as shown by DNA analysis. While detached cells of C6-ceramide (10 mM)-treated PCC7-Mz1 and mocktransfected PCC7-Mz1 cultures displayed a striking DNA laddering, Bcl-2-overexpressing cells (clones B12, F3 and D2) did not show any signs of this type of DNA degradation (Fig. 5B). These results demonstrate that Bcl-2 is involved in both, RA- and ceramide-induced apoptosis. Furthermore, they

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Fig. 4. Effect of caspase inhibitors on RA-induced apoptosis. Stem cells of PCC7-Mz1 and of the bcl-2-transfected clones B12 and H5 were treated with 10ÿ 7 M RA in the absence or presence of the caspase-8 inhibitor z-IETD-fmk (10 mM) and the pan-caspase inhibitor z-VADfmk (50 mM), as indicated. After 24 hours, a viability assay was performed as described in Materials and methods (untreated controls ˆ 100% viability). A representative experiment (n ˆ 3) is depicted. While inhibition of caspase-8 had no effect on RA-induced apoptosis ( ‡ RA, ‡ z-IETD-fmk), general inhibition of caspases efficiently prevented cell death ( ‡ RA, ‡ z-VAD-fmk) in all cell lines. The inhibitors z-IETD-fmk and z-VAD-fmk themselves had no effect on viability of cultures (data not shown).

support the notion that apoptosis caused by RA and ceramide occur via the same pathways, with Bcl-2 controlling apoptosis downstream of ceramide production. Observations obtained with R-clones, which are PCC7-Mz cells resistant to RA-induced differentiation and apoptosis (Lang et al., 1989; Oehrlein et al., 1998; Herget et al., 2000), are in agreement with this conclusion. These RA-resistant clones also responded to ceramide by increased apoptosis but did not express higher levels of Bcl-2 (Oehrlein and Herget; unpublished data). This indicates that in R-clones the RA-initiated apoptotic signal is obviously blocked at the level of retinoic acid receptors and not at a downstream step, e. g. by an up-regulation of Bcl-2. The Bcl-2 protection of PCC7-Mz1 stem cells is not limited to blocking the apoptotic effects of retinoic acid and ceramide (Figs. 2 and 4). The degree of apoptosis induced by treatment with staurosporine (5 nM) or by serum deprivation for 24 hours, by UV light illumination (10 min, 50 cm distance, 30 W) (Herget et al., 1998), chemical hypoxia (iodoacetic acid and cyanide) were also reduced in Bcl-2-overexpressing cell lines (data not shown). Furthermore, the cytotoxic effect of 10 mM glutamate causing necrotic cell death in PCC7-Mz1 was nearly completely abolished in Bcl-2-overexpressing derivatives (Düwelhenke and Herget, unpublished results).

Proliferation rate of Bcl-2-expressing cultures

Since Bcl-2 overexpression blocked efficiently RA-induced apoptosis we speculated that after RA treatment the number of cells of PCC7-Mz-Bcl-2 cultures should be larger than in PCC7Mz1 cultures. Parental PCC7-Mz1 and bcl-2-transfected clones (B12, H5 and D2) were plated and treated with RA the following day. Cell numbers were monitored until day 3 of

Fig. 5. Bcl-2 overexpression inhibits ceramide-induced apoptosis. A) PCC7-Mz1 and Bcl-2-overexpressing cultures (B12, F3, H5 and D2) were seeded in 96-well microtiter plates and treated with increasing concentrations of synthetic C6-ceramide for 24 hours. Viability was measured and compared with solvent-treated (0.05% DMSO) cultures (100% viability). The diagram depicts the means of three independent experiments performed with eight samples per condition each. The table summarizes the EC50 values of C6-ceramide for the PCC7-Mz1 and the Bcl-2-overexpressing cells. B) PCC7-Mz1 and the Bcl-2 clones B12, F3 and D2 were plated in 6-cm culture dishes and incubated with 10 mM C6-ceramide or with solvent (Co) for 24 hours the following day, as indicated. Genomic DNA of the detached cells was isolated and analyzed on a 1.4% agarose gel. C6-ceramide induced DNA laddering only in PCC7-Mz1 stem cells but not in the Bcl-2-overexpressing cell lines.

differentiation. Surprisingly, none of the growth curves of the cultures investigated differed significantly and was apparently independent of Bcl-2 expression (Fig. 6). Cell proliferation was detected for up to two days after RA treatment before cultures ceased to proliferate, as described previously for PCC7-Mz1 cells (Esdar et al., 1999). A theoretical growth curve for Bcl-2overexpressing cultures was calculated with the program Microcal origin 5.0 (OriginLab, Coop.) on the basis of the growth behavior of PCC7-Mz1 cultures described previously (Herget et al., 1998; Esdar et al., 1999) except that cell death was assumed to be zero. Figure 6 shows that growth curves of PCC7-Mz-Bcl-2 cultures (B12, H5 and D2) differed significantly from this theoretical evaluation. Thus, Bcl-2 expression is not simply blocking cell death.

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546 C. Esdar, S. Milasta et al.

Fig. 6. Growth curves of PCC7-Mz1 and Bcl-2 cultures. PCC7-Mz1 and Bcl-2 cells (clones B12, H5 and D2) were seeded in 24-well plates (4  104 cells per well). Four hours after plating, cells were harvested, stained with trypan blue, and numbers of living cells were calculated (ÿ 1). After one day (d0), RA (0.1 mM) was added and the incubation was prolonged for three additional days. Cell numbers were determined at the indicated time points. The growth rate of the bcl-2-transfected cell lines B12, H5 and D2 do not significantly differ from the one of the parental PCC7-Mz1 cells. The theoretical growth curve (red) for the Bcl-2-overexpressing cultures, calculated with the program Microcal origin 5.0, predicted a higher growth rate than actually measured.

To assess whether a prolonged cell cycle of the PCC7-MzBcl-2 cells was responsible for the finding that these cultures did not show a higher cell number despite the reduced apoptosis rate, we investigated the generation time. Growth of the respective cultures in the log phase was monitored over 20 passages and the population doubling time (PDT, expressed in hours) was calculated. Although PDT is not the same as cell generation time (cell cycle time), which is the interval between successive divisions (mitosis to mitosis) for an individual cell, in practice, PDT is used as an estimate of cell cycle time and to determine the length of the phase of the cell cycle (McAteer and Davies, 1994). While the PDT of PCC7-Mz1 cultures was 17.5  2.25 hours, the PCC7-Mz-Bcl-2 cells had a PDT which was on average about 6 hours longer (Table I). The prolongation of the generation time can be attributed to the overexpression of Bcl-2 rather than to clonal variation since analysis of two mock-transfected cell lines in parallel revealed similar PDTs (16.9  1.5 h and 17.3  2.2 h) as the PCC7-Mz1 cultures. To further evaluate the effect of Bcl-2 overexpression on the generation time, we monitored the distribution of cells in the various phases of the cell cycle. The nuclei of three bcl-2transfected cell lines and the untransfected PCC7-Mz1 cells Tab. I. Population doubling times. Cell line

Population doubling time ( SEM )

PCC7-Mz1 PCC7-Mz-Bcl-2-B12 PCC7-Mz-Bcl-2-F3 PCC7-Mz-Bcl-2-H5 PCC7-Mz-A3 (mock) PCC7-Mz-B9 (mock)

17.5  2.25 h 25.6  2.5 h 23.0  2.5 h 23.2  2.45 h 16.9  1.5 h 17.3  2.2 h

The population doubling times, which are similar to the generation times, of PCC7-Mz1, bcl-2- and mock-transfected cell lines were estimated over a growth period of 20 passages. The figures were calculated as described in Materials and methods.

were stained by propidium iodide and analyzed by flow cytometry (n ˆ 3). In PCC7-Mz1 stem cell cultures 31  5% of the cells were in the G0/G1-phase, while in Bcl-2-overexpressing cultures about 45  9% of the cells were in the G0/G1phase. This difference in cell cycle distribution was even more significant in cultures treated for one day with RA. 38  6% of the PCC7-Mz1 cells were in the G0/G1-phase, which is in good agreement with previously published data (Esdar et al., 1999), however, already 54 to 70% of the cells of the PCC7-Mz-Bcl-2 cultures had entered the G0/G1-phase. These experiments show, that the overexpression of Bcl-2 results indeed in a higher degree of cells in the G0/G1-phase in both, untreated and RAtreated (1 day) cultures. The length of a cell cycle depends mainly on the duration of the G0/G1-phase. The entry into the cell cycle is determined by the restriction point in the G1-phase, which is controlled e. g. by the retinoblastoma protein (Rb). The active form of Rb in the G0/G1-phase and in terminally differentiated cells is hypophosphorylated and binds transcription factors like E2F thus inhibiting entry into S-phase. Therefore, hyperphosphorylated, inactive ppRb is mainly found in cycling cells. Consequently, we investigated the effect of Bcl-2 expression on the phosphorylation status of Rb. Proteins of stem cells and of differentiating PCC7-Mz1 and PCC7-Bcl-2 cells (H5, B12 and F3) were extracted and Rb was analyzed by Western blotting (Fig. 7). PCC7-Mz1 stem cells (d0) expressed only hyperphosphorylated, inactive Rb (ppRb). On d1 additionally the faster migrating hypophosphorylated, active Rb (pRb) became detectable. During the course of differentiation the ratio of ppRb to pRb changed in favor of pRb. The Bcl-2-overexpressing stem cells (d0) (H5, B12 and F3) contained already the hypophosphorylated, active Rb-protein in contrast to PCC7Mz1 stem cells (Fig. 7). Hence, overexpression of Bcl-2 seems to induce an activation of the Rb protein and thus delays the transition from G1-phase to S-phase or causes a faster exit of the cell cycle into G0. At present the reason is not clear for the extended cell cycle and the higher degree of cells in the G0/G1phase. Finally, six days after induction of neural differentiation, Rb was down-regulated in all cell lines (Fig. 7).

Effect of Bcl-2 expression on neural differentiation

The contribution of Bcl-2 to neural differentiation was so far difficult to assess. The PCC7-Mz1 cell line is one of the few systems which allow an analysis of the effects of the expression of a particular gene on the process of neural determination. Accordingly, we evaluated the potential of the 21 generated PCC7-Mz-Bcl-2 cell lines to differentiate along the neuronal, astroglial and fibroblastoid lineage. Three types of PCC7-Mz1 subclones (P-, O- and N-type) with distinct differentiation potential have been previously classified (Lang et al., 1989). The cell lines were seeded on glass coverslips and induced to differentiate with RA. The differentiating cultures were observed microscopically every day and the developing cell types were identified phenotypically (Table II). Twelve of the 21 bcl-2 cDNA-transfected cell lines showed the full differentiation potential and developed neurons, astroglial cells and fibroblasts. These cultures revealed the same differentiation pattern as the parental PCC7-Mz1 cells and were classified as Psubtype. Six of the 21 transfectants had the differentiation potential of the N-type. Stem cells of these clones differentiated exclusively into neurons and less than 1% of all cells had a flat

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Effect of Bcl-2 on early neural differentiation 547

Fig. 7. Expression of the retinoblastoma protein. The parental PCC7Mz1 cells and the bcl-2-transfected cell lines (H5, B12 and F3) were plated in 10-cm tissue culture dishes and induced for differentiation. Proteins were extracted from stem cells (d0) and at the indicated days after addition of RA. 50 mg detergent-soluble protein were separated by 7.5% SDS-PAGE, blotted onto a PVDF membrane and probed with a retinoblastoma protein (Rb)-specific antibody. The antibody recognizes the active hypophosphorylated (pRb) as well as the inactive hyperphosporylated (ppRb) form of Rb. Detection of bound antibody

was performed by enhanced chemiluminescence and exposure to Fuji medical X-ray film for 5 min The position and size of protein markers are shown on the left. An example of three independent experiments is depicted. In PCC7-Mz1 stem cells (d0) only hyperphosphorylated, inactive ppRb was detectable, while stem cells of the bcl-2-transfected cell lines (H5, B12 and F3) expressed already hypophosphorylated, active pRb. Rb expression ceases in all cultures during the course of differentiation.

Tab. II. Influence of Bcl-2 on cell lineage determination.

with the empty pcDNA-3-SV40 vector in parallel and generated stable clones (mock transfections). Thirteen of the 21 mock transfectants turned out to be of the P-type, three of the N-type and one of the O-type; four of them did not differentiate but died upon incubation with RA. Therefore, the potential of the mock transfectants to develop the various cell types was similar to PCC7-Mz-1 cells (Lang et al., 1989) and Bcl-2overexpressing cells (Table II). Accordingly, Bcl-2 does not seem to affect lineage decision on the stem cell level.

Transfectants

Type of differentiation P-Type O-Type N-Type No differentiation

PCC7-Mz-Bcl-2 (n ˆ 21) 12 PCC7-Mz-mock (n ˆ 21) 13

2 1

6 3

1 4

Cell lines transfected with the bcl-2 expression vector or with the empty pcDNA3 vector (mock transfection) were generated and their potential to differentiate into neurons, glial cells and fibroblasts was estimated. Cell lines, which (comparably to the parental PCC7-Mz1 cells) produced all three types of cells, were of the P-type (about 40% of cells had a flat morphology at d6). Cultures showing a strongly reduced percentage of non-neuronal cells are of the O-type (1 to 10% of cells had a flat morphology at d6), and those cell lines differentiating into neurons only are called N-type (less than 1% of cells had a flat morphology at d6). There were a few cell lines unable to differentiate but dying upon incubation with RA ( No differentiation). Each cell line was differentiated six times for six days each, and the cell types were identified by immunofluorescence with antibodies recognizing the marker proteins Thy1.2 for fibroblasts, GAP-43 and Ex-1 for neurons, and GFAP for glial cells. The differentiation potential of each cell line was stable for over 20 passages.

morphology. Because of the absence of the supporting astroglial cells and fibroblasts, the neurons detached and died around day 4. Two cell lines turned out to exhibit a differentiation potential intermediate to the P- and the N-type. These clones are of the O-type and developed mainly neurons, but much less derivatives with a flat morphology (around 1% to 10% of the cells on d6) than the parental P-type. These Oclones also died between d4 and d6, because neurons were not sufficiently sustained by cells with flat morphology. There was one PCC7-Mz-Bcl-2 cell line, which could not be induced to differentiate into defined cell types, because these cells died within a few days of RA treatment. The differentiation potential of all 21 cell lines were monitored in three independent sets of experiments. The differentiation pattern (P-, O- and N-type) of each single cell line was stable and did not change with number of passages. To determine the significance of these figures (summarized in Table II), we performed transfections

Expression of neuronal marker proteins

We studied the influence of high levels of Bcl-2 on maturation of the neuronal phenotype. Therefore, three Bcl-2 cell lines (B12, H5 and D2), which had proven to be of the P-type (Table II), were incubated with RA and the expression of two neuronal marker proteins was monitored. Expression of the neuron-specific PKC substrate GAP-43 was up-regulated within 24 hours of RA treatment (Esdar et al., 1999). The light chain of the neuronal n-clathrin is a marker for the late neuronal development and expressed in PCC7-Mz1 cells from d2 (Herget et al., 2000). Proteins from PCC7-Mz1 and Bcl-2 cell lines (B12, H5 and D2) were extracted from untreated cells and 1, 3 and 6 days after RA treatment. The Western blot analysis show that in PCC7-Mz1 cells, expression of the GAP-43 protein is up-regulated one day after induction of differentiation and that levels remained high till day 6 (Fig. 8) as described before (Esdar et al., 1999). The PCC7-Mz-Bcl-2-D2 cell line, which was transfected with the bcl-2 cDNA under the control of the weaker CMV promoter (Fig. 1), showed the identical GAP-43 expression pattern as the PCC7-Mz1 cells. The cell lines PCC7Mz-Bcl-2-B12 and -H5, which due to the stronger SV40 promoter expressed higher levels of Bcl-2 (Fig. 1), displayed a different picture. GAP-43 was weakly detectable in untreated stem cells (d0) and was slightly up-regulated at day 1. However, this increase was, in contrast to the parental PCC7-Mz1 cells, only transient, i. e. GAP-43 signals on day 3 and 6 were low again.

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Fig. 8. Expression of neuronal marker proteins. Cultures of PCC7Mz1 and of bcl-2 transfectants (B12, H5 and D2) were plated in 10-cm culture dishes and incubated with RA the following day (d0). Proteins were extracted from stem cells (d0) and 1, 3 and 6 days after induction of differentiation. For GAP-43 detection (upper panels), 5 mg protein were loaded on a 10% polyacrylamide gel, and for detection of the light chain of the neuronal n-clathrin (lower panels) 15 mg were separated by 12.5% SDS-PAGE. Bound antibody was detected by chemiluminescence and exposure to Fuji X-ray films for 1 min and 15 min for GAP-

43 and n-clathrin, respectively. Equal loading of all lanes of a gel and transfer onto PVDF membranes was examined by staining the membrane with Coomassie brilliant blue (data not shown). In PCC7Mz1 cells, expression of GAP-43 and n-clathrin is up-regulated from day 1 and day 3, respectively. In the Bcl-2 cell lines (B12 and H5) upregulation of these marker proteins was less pronounced and only transient. The Bcl-2 clone D2 expressed comparable levels of Bcl-2 in stem cells as PCC7-Mz1 cells (Fig. 1) and showed an expression pattern of GAP-43 and n-clathrin as PCC7-Mz1 cells do.

The expression pattern of n-clathrin verified the findings of the GAP-43 analysis. Both, PCC7-Mz1 and Bcl-2 cells (D2) strongly expressed n-clathrin on d3 (Fig. 8). The signals were slightly reduced on day 6, probably due to the increasing fraction of flat cells, which do not express n-clathrin. The Bcl-2overexpressing cell lines B12 and H5 weakly induced n-clathrin synthesis on day 3 and at day 6 n-clathrin levels were barely detectable. The levels of the two neuronal marker proteins imply that the Bcl-2 cell lines (B12 and H5) developed less neurons than the parental PCC7-Mz1 cultures. This observation was confirmed when we estimated the fraction of neuronal cells in differentiating cultures. PCC7-Mz1 as well as Bcl-2 cells (B12 and H5) were differentiated for three and six days. Then all cells of a dish were carefully removed and placed as individual cells on cell adhesion slides (BioRad, Munich, Germany). Expression of the neuron-specific markers GAP-43 (Esdar et al., 1999) and Ex-1 (Müller-Husmann et al., 1994) was studied by immunofluorescence. In PCC7-Mz1 cultures about 70% of the cells expressed these neuronal markers and thus were identified as neurons. However, less than half of this value, namely about 30%, of the Bcl-2 cultures (B12 and H5) showed expression of these two marker proteins (Fig. 9). At day 6 about 60% of the cells in PCC7-Mz1 cultures were neurons. This number was slightly reduced in comparison to d3 because of the ongoing proliferation of cells with flat morphology. In Bcl-2-overexpressing cultures only 30 to 40% of the cells were GAP-43- and Ex-1-positive at d6. Taken together, these data (Figs. 8 and 9) demonstrate that Bcl-2 expression supports the survival of cells with flat morphology, which in turn results in cultures with less cells developing a neuronal phenotype.

having the potential to develop all three cell types (P-type clone, Table II), were treated with RA. While all stem cells and cultures during the initial phase of differentiation (d3) looked very similar (Fig. 10a ± f), striking differences became obvious at day 6 and day 10 of differentiation. At that time, the neurons of PCC7-Mz1 cultures form aggregates consisting of 20 to 50 neurons (Fig. 10g, k). These cell clusters, which contain also some dead cells, are interconnected via bundles of neurites. Single neurons are hardly detectable. The neurons of the MzBcl-2 cultures, however, did not form these clusters but laid as single cells on cells with flat morphology. Not more than 2 ± 3 neurons were attached to each other (Fig. 10h, i, l, m). These individual neurons were connected via a network of branched neurites, which was similar to the one in primary neuronal cell culture systems (Oehrlein et al., 1998). A second method was employed to verify the morphological differences. We performed immunostaining of d8 cultures with the neuron-specific GAP-43 antiserum (Esdar et al., 1999) to visualize neuronal aggregates and the processes in PCC7-Mz1 cultures (Fig. 11). This staining shows, that neurons of Bcl-2 clones B12 and H5 laid as single neurons on a confluent layer of cells with flat morphology. Overall, it became obvious that the Bcl-2 cultures looked robuster than PCC7-Mz1 cells especially during the late phase of differentiation (e. g. on d12) in that they contained less dead and membrane-damaged cells. Since cultured neurons from Bcl-2ÿ/ÿ embryos showed a slower maturation program (Middleton et al., 1998) we investigated the expression of the neuronal marker GAP-43, TAU, MAP2a/b and synaptophysin by immunocytochemistry (d0 to d9). We never observed a significant difference in the spatial or temporal expression of these proteins in parental and Bcl-2 transfected (B12, H5) PCC7 cells. Figure 11 depicts the analysis of the TAU protein and synaptophysin at day 8 of differentiation. In summary, Bcl-2 does not seem to influence neural cell lineage determination or the kinetics of maturation, but supports survival of cells developing a flat morphology and allows neurons to persist as single cells.

Morphology of Bcl-2-overexpressing neurons

The finding that Bcl-2 reduces the number of developing neurons (Figs. 8 and 9) prompted us to question whether overexpression of this protein also alters the morphology of cells. PCC7-Mz1 cells and Bcl-2 cell lines (B12 and H5), both

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Effect of Bcl-2 on RA-induced apoptosis

Fig. 9. Fraction of neuronal derivatives in PCC7-Mz cultures. PCC7Mz1 cells (black bar) and Bcl-2 clones B12 (light gray) and H5 (dark gray) were differentiated on glass coverslips for three (A) and six (B) days, detached and transferred onto cell adhesion slides as single cells. Neuronal derivatives were identified by reacting with GAP-43- (left bars) and with Ex-1-specific (right bars) antibodies. The means ( SEM) of three independent experiments with about 300 counted cells each are depicted. The fraction of neuronal derivatives is drastically reduced in Bcl-2 cell lines (B12, H5) compared to untransfected PCC7-Mz1 cells.

Discussion During recent years it has been repeatedly shown that the Bcl-2 protein, originally identified as an oncogene in human lymphomas (Tsujimoto et al., 1984; Bakhshi et al., 1985), displays anti-apoptotic effects in various cell systems (Farrow and Brown, 1996; White, 1996; Reed, 1997). In the present study, we investigated the role of Bcl-2 on all-trans retinoic-acid (RA)induced neural differentiation and apoptosis. Retinoids exert their biological effects by altering gene expression and were shown to regulate the level of signal transduction elements such as p21WAF (Lin et al., 1996), Fas ligand (Okamura et al., 1998) and Bcl-2 (Nagy et al., 1996) as well as effector enzymes such as transglutaminase (Chiocca et al., 1988) and sphingomyelinase (Riboni et al., 1995). All these molecules have been implicated in the induction and execution of the cell death program.

We show here that overexpression of Bcl-2 in PCC7-Mz1 cells, a model system of neural differentiation, efficiently blocks RAinduced apoptosis, but not RA-induced differentiation. While parental and mock-transfected cultures exhibit a loss of viability by about 25% in response to RA treatment, Bcl-2overexpressing cultures have a reduced viability of 3 to 15% depending on the actual level of Bcl-2 overexpression. Furthermore, the present findings establish for the first time that the RA-induced apoptotic signal is transduced via the mitochondria: Thus: (i) Treating PCC7-Mz1 cells with RA causes a translocation of cytochrome c from the mitochondria to the cytosol. (ii) Overexpression of Bcl-2 prevents release of cytochrome c. Concomitantly, caspase activity and DNA laddering is inhibited by Bcl-2. Only in type II cells, apoptosis can be blocked by overexpressed Bcl-2 or Bcl-xL (Scaffidi et al., 1999). (iii) Additionally, RA induces de novo synthesis of ceramides (Herget et al., 2000), which are known to activate the type II death pathway (Scaffidi et al., 1999). Remarkably, even an eightfold overexpression of Bcl-2 did not prevent RA-mediated apoptosis completely. Since the relative amounts of death agonists and antagonists rather than their individual levels seem to define the sensitivity of cells to apoptosis, it can be suggested that Bcl-2 levels were still too low to neutralize the proapoptotic partners by heterodimerization. Recent results based on studies on heterodimerization-deficient mutant proteins argue, however, for the existence of both heterodimerization-dependent and -independent mechanisms of action (Minn et al., 1999). We excluded the possibility that in PCC7-Mz1 cells an RA-induced mitochondria- and Bcl-2independent pathway contributes to apoptosis. Inhibition of the initiator caspases, which are activated by stimulation of death receptors, did not diminish the degree of RA-induced apoptosis neither in parental PCC7-Mz1 nor in Bcl-2-overexpressing cells (data not shown). Therefore, we conclude that RA activates solely a death pathway of type II in PCC7-Mz1 cells.

Bcl-2 influences neural differentiation

The effect of Bcl-2 on neural development has been investigated in several studies. Elevated expression of Bcl-2 was found in neuroblastoma cells concomitantly with in vitro differentiation (Hanada et al., 1993). Overexpression of Bcl-2 in a spontaneously differentiating neural crest-derived tumor cell line (Paju) induced extensive neurite outgrowth (Zhang et al., 1996). Additionally, Bcl-2 promotes neuronal differentiation of PC12 cells (Batistatou et al., 1993; Sato et al. 1994; Suzuki and Tsutomi, 1998). However, the cell lines tested were of neuronal origin and therefore determined to become neurons. The observed enhanced neuronal differentiation of these cell lines is, therefore, due to an anti-apoptotic effect rather than a neurogenic result of Bcl-2. A survival effect of Bcl-2 was also found during neural differentiation of RA-treated P19 cells (Okazawa et al., 1996). Since in this cell system RA-induced apoptosis occurs earlier than differentiation, the effect of Bcl-2 on differentiation and lineage determination was not analyzed. Yet, the crucial, so far only inadequately answered question concerns the role of Bcl-2 in neurogenesis and neural cell lineage determination. We show, that overexpression of Bcl-2 did not block the capacity of the polypotent PCC7-Mz1 stem cells to develop into neurons, astroglial cells and fibroblasts. This result is surprising on the background that many cell types, including stem cell lines like F9, P19 and myeloid precursor

550 C. Esdar, S. Milasta et al.

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Fig. 10. Morphology of differentiating PCC7-Mz1 and PCC7-Mz-Bcl2 cultures. PCC7-Mz1 cells (a, d, g, k) as well as Bcl-2 cells (B12: b, e, h, l and H5: c, f, i, m) were seeded on glass coverslips and differentiated with RA. Photographs of living stem cells (a ± c) and of cultures at d3 (d ± f), d6 (g ± i) and d10 (k ± m) were taken. There are no obvious

differences between these cultures at d0 and d3. At d6 and d10 the neuronal PCC7-Mz1 cells form aggregates (*), interconnected via neurites. The neurons of the Bcl-2 clones (arrows) remain as single cells and communicate by branched neurites. Bar ˆ 62 mm.

cells (Okazawa et al., 1996; Kohzaki et al., 1999), show downregulation of Bcl-2 upon induction of differentiation implying that low levels of Bcl-2 are a prerequisite for differentiation. Differentiating PCC7 cultures overexpressing Bcl-2 synthesize less of the neuronal proteins GAP-43 and n-clathrin and these cultures produce only half of the numbers of neurons than the parental cells do. Those effects of Bcl-2 vanish when Bcl-2 overexpression is induced after one day of induction of differentiation. This result corroborates the hypothesis that processes deciding cell survival and cell lineage are accomplished within the first 24 hours of neural differentiation. Also factors like RA, bFGF, laminin and ceramide, capable of influencing the fate of stem cells, are incompetent to do so at later phases of development (Jostock et al., 1998; Herget et al., 1998, 2000). It seems that a high level of Bcl-2 in PCC7-Mz1 stem cells has two consequences: (i) It dramatically reduces differentiation-

induced cell death and (ii) Bcl-2 promotes the non-neuronal phenotype. While the first effect can be attributed to the blockage of the mitochondria-dependent death pathway (Fig. 3), the second one may be due to modulating the gene regulatory program of stem cells. Recently, it was indeed suggested that Bcl-2 instructs gene expression independently of its control of apoptosis. Constitutive expression of Bcl-2 maintained the levels of an extracellular matrix protein, the proteoglycan aggrecan, in a chondrocyte cell line in response to serum withdrawal, a treatment which causes normally downregulation of both, Bcl-2 and aggrecan mRNA, in parental cells (Feng et al., 1999). Accordingly, Bcl-2 overexpression in PCC7 cells may enhance the production of extracellular proteins or other compounds acting in an autocrine or paracrine fashion, which might be responsible for promoting the non-neuronal phenotype. In fact, we could show that the fate of PCC7-MzN cells, which purely differentiate to neuronal derivatives, is

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Fig. 11. Comparison of differentiated PCC7-Mz1 and Bcl-2 neurons by GAP-43, TAU and synaptophysin immunostaining. PCC7-Mz1 cells and Bcl-2 cells (B12, H5) were plated on glass coverslips, treated for 8 days with RA and then fixed. Neurons were visualized by GAP-43-, TAU- and synaptophysin-specific antibodies (as indicated) which stain soma and neurites (Berger et al., 1997, Esdar et al., 1999). The

Effect of Bcl-2 on early neural differentiation 551

corresponding phase-contrast images are depicted underneath the immunofluorescence images. The neurons of PCC7-Mz1 cultures form aggregates (*) while neurons of the Bcl-2 transfectants remain as single cells (arrows) on a monolayer of cells with a flat morphology. Bar ˆ 15.6 mm

552 C. Esdar, S. Milasta et al.

shifted when cells are grown on laminin-coated surfaces (Jostock et al., 1998). Altered expression of proteins involved in cell : cell and cell : matrix interaction were shown to be important for the survival of neurons (Frisch and Ruoslahti, 1997; Chen et al., 1999) and may also be responsible for allowing PCC7-Mz-Bcl-2 neurons to survive as single cells (Fig. 11). Our result that Bcl-2-overexpressing cultures contain twice as many non-neuronal cells than parental PCC7-Mz1 cultures can be explained not only by a changed gene expression program: It is likely that during early neural differentiation of PCC7-Mz1 cultures the precursors of non-neuronal cell types are more vulnerable to apoptosis than the neuronal precursor cells. Therefore, blockage of cell death by Bcl-2 overexpression results in an altered ratio in favor of non-neuronal cells. At the moment we cannot discriminate between these two possibilities, i. e. whether Bcl-2 modulates expression of differentiationassociated proteins or whether it reduces apoptosis in nonneuronal precursor cells. The phenomenon described here, namely that Bcl-2 favors quiescence over the cycling state, may also be attributed to an altered gene expression. Overexpression of Bcl-2 is associated with a cessation of PCC7-Mz1 stem cell proliferation, which may be due to a slower entry of the cell cycle as described previously (OReilly et al., 1996; Lind et al., 1999). Furthermore, the exit into G0 in response to RA-induced differentiation is accelerated. We show that these changes correlate with a decrease in pRb phosphorylation (Fig. 7). Cycling PCC7-Mz1 cells treated with RA encounter a crossroad of mitosis, differentiation and cell death. The decision of the subsequent fate may partly be made by the actual phase of the cell cycle and the gene regulatory program. In the present study we show that Bcl-2 is able to exert various functions during early neural differentiation. In addition to its antiapoptotic properties, Bcl-2 controls directly or indirectly entry and exit of the cell cycle and determination of cell fate. Using mutagenesis of the Bcl-2 protein will be interesting to elucidate whether these tasks are mechanistically distinct or connected. Acknowledgement. We would like to thank I. Koziollek, H. Taschner and N. Düwelhenke (all Laboratory of Molecular Neurobiology, University of Mainz) for experimental assistance; Dr. R. Jahn (MPI Göttingen) for providing the monoclonal antibody against neuronal clathrin; Dr. D. Vaux (Walter and Eliza Hall Institute, Melbourne, Australia), Dr. J. Boise (University Lleida, Spain) for pcDNA3-Bcl-2 vector and Dr. M. Cotten (Axxima AG, Munich) for critical reading of the manuscript. The work was financially supported by the Deutsche Forschungsgemeinschaft (He 2557.1 ± 2) and the NMFZ of the University of Mainz (T. Herget). The award of a post-graduate fellowship from the Fonds of the Chemical Industry to C. Esdar is gratefully acknowledged.

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