Vol. 51 No. 2/2004 539–544 QUARTERLY Communication

In this study we have investigated the impact of differentiation of neuronal cells on their sensitivity to microbial toxins. We used the human neural crest-derived tumor cell line Paju, which can be induced to differentiation in vitro by treatment with phorbol 12-myristate 13-acetate. Addition of the highly toxic potassium ionophores cereulide (4.5 and 9.0 ng/ml) or valinomycin (20 ng/ml), to cultures of undifferentiated Paju cells caused collapse of the mitochondrial membrane potential - measured with the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazole carbocyanine iodide (JC-1) followed by detachment of the cells and their apoptotic death. After induced differentiation of the Paju cells, their mitochondria retained the membrane potential upon exposure to the toxins and the cells displayed increased resistance to apoptosis as compared with undifferentiated cells. This effect may be caused by an elevated expression of the anti-apoptotic protein Bcl-2 and of the neuroprotective factor, stanniocalcin, in differentiated cells.

caused by an elevated expression of the anti-apoptotic protein Bcl-2 and of the neuroprotective factor, stanniocalcin, in differentiated cells.
When neural Paju cells are induced to differentiation in culture, the expression of Bcl-2 rises (Zhang et al., 1996).Bcl-2 is known to protect against the mitochondrial permeability transition induced by Ca 2+ and/or prooxidants (Shimizu et al., 1998;Murphy et al., 2001).Neuronal differentiation, both in vivo and in vitro, also leads to a strongly upregulated expression of stanniocalcin (Zhang et al., 1998).Stanniocalcin confers a protective effect on neurons during cerebral ischemia (Zhang et al., 2000).Mitochondria have a central role in the signal transduction of apoptosis and necrosis (Gottlieb, 2000;Lemasters et al., 2002).Valinomycin, a potassium ionophore, facilitates the selective transport of K + ions across the inner membrane of mitochondria (Duax et al., 1996) and induces apoptosis in ascites hepatoma cells (Inai et al., 1997) by disrupting the mitochondrial membrane potential.Valinomycin has been reported to cause apoptosis in other mammalian cell lines as well (Penning et al., 2000;Li & El-Mallahk, 2000;Krick et al., 2001).Andersson et al. (1998a) isolated from an indoor environment Streptomyces griseus strains that produce valinomycin.It inhibited human NK cell activity and induced apoptosis as did commercially available valinomycin (Paananen et al., 2000).Cereulide is a toxin, which may cause serious food poisoning in man.We have characterised cereulide isolated from Bacillus cereus and found that it acts as a potassium ionophore with valinomycin-like effects on mitochondrial functions (Mikkola et al., 1999).It induced loss of mitochondrial membrane potential (DY m ) in human cells (HeLa, Caco-2 and Calu-3) (Jääskeläinen et al., 2003) and initiated apoptosis of human NK cells as a consequence of mitochondrial damage (Paananen et al., 2002).
Here we report that the mitochondria in Paju cells that have been induced to neural differentiation are more resistant to the toxic effects of cereulide and valinomycin than the mitochondria in undifferentiated Paju cells.It is suggested that this could be due to higher expression of Bcl-2 and stanniocalcin in differentiated Paju cells.
The Paju cell line was established in the Department of Pathology, Haartman Institute (Zhang et al., 1996) from the pleural fluid of a girl who had a widespread metastatic neural crest-derived tumour.The cells grow surface-adherent in RPMI 1640 medium, supplemented with 10% FCS (fetal calf serum), penicillin G (6 mg/ml), streptomycin sulfate (10 mg/ml), and 1 mM glutamine.For subculturing, the cells were detached by treatment with 0.5 M EDTA.After detachment the cells were seeded (40 000 cells/chamber) onto 8-well chambered cover glasses (Naperville, U.S.A.), grown to 20% confluence whereafter 10 nM PMA was added for 4 days to induce terminal neural differentiation.
Bacterial toxin from S. griseus was purified by HPLC and shown to be valinomycin as previously described (Andersson et al., 1998a).The toxin is hydrophobic and therefore it was diluted in methanol and applied to cells in 8-well chambers at a concentration of 5 ng/ml (isolated from S. griseus strain 10/A1) or 20 ng/ml (from the same strain or commercial from Sigma).In controls, methanol alone was used as a solvent.Cereulide, the emetic toxin, was isolated from B. cereus strain F4810/72 as described (Andersson et al., 1998b) and applied to the cells at a concentration of 4.5 and 9 ng/ml.
Changes in DY m in intact cells were examined with fluorescence microscope after staining with the fluorescent probe JC-1 (Reers et al., 1995).The plasma membrane integrity was assessed by PI, which enters into dead cells only (Yeh et al., 1981).These dyes were used in combination as a cell viability test.Staining of the slide cultures was done at 3 mM JC-1 and 5 mM PI for 20 min at 37°C in the dark in culture medium under humidified atmosphere containing 5% CO 2 .Free dye was removed by washing with RPMI-1640 medium and the slide was analysed immediately with a Leitz photomicroscope with epifluorescence optics and a 100-W mercury lamp.To visualize green and red fluorescence simultaneously, a long-pass filter system LP 520 nm, dichromic mirror RKP 510, and exciter (450-490 nm) were used.

RESULTS
The morphology of Paju cells before (WT) and after induced differentiation is shown in Fig. 1.Treatment with PMA provoked neural sprouting of dendrites carrying growth-cone like structures at their tips (Fig. 1B).The PMA-treated cells also largely exit the cell cycle and accumulate in G2 (not shown).
A collapse of DY m in connection with the mitochondrial permeability transition often is an early, irreversible event in cell death.Therefore we followed DY m by staining with the JC-1 fluorescence probe.The integrity of the plasma membrane was probed by staining with PI. Figure 2 shows epifluorescence micrographs of WT (left panels) and differentiated (right panels) Paju cells exposed to the microbial toxins.In the control, the cells were exposed to the solvent (methanol) only.The mitochondria display yellow-orange fluorescence, indicating a high DY m , both in WT (panel A) and differentiated (panel B) cells.WT cells lost their DY m after exposure to 4.5 ng cereulide/ml for 20 min and only a few mitochondria emitted yellow fluorescence while the cytoplasms emitted green (panel C).Treatment of WT cells with 9 ng cereulide/ml for 20 min caused an almost complete loss of the DY m (green colour), rounding up and detachment of the cells (panel E).Treatment of differentiated Paju cells with the same concentrations of cereulide induced only small if any changes (panels D and F).In the case of valinomycin, the effects were analogous.Normal cells lost their DY m after exposure for 20 min to 5 ng/ml valinomycin isolated from S. griseus.In contrast, the differentiated cells were unaffected (not shown).Even 20 ng/ml valinomycin isolated from S. griseus (panel H) or commercial valinomycin (panel J) caused no visible damage in differentiated cells.Normal (WT Paju) cells treated under identical conditions showed green fluorescence with JC-1, corresponding to a decreased DY m .The cells were, however, still not permeable to PI (panels G and I), indicating an intact plasma membrane.
Taken together, these results show that, under conditions where all three tested micro- bial toxins caused loss of DY m in WT Paju cells, the mitochondria in differentiated Paju cells displayed resistance to the toxins.

DISCUSSION
In this study Paju cells were used to monitor for neurotoxicity of cereulide from B. cereus (Mikkola et al., 1999) and of valinomycin, both commercial and isolated from S. griseus (Andersson et al., 1998a).We examined the effect of neuronal cell differentiation on the sensitivity to these microbial toxins.Profound effects of the toxins (all potassium ionophores) on undifferentiated Paju cells were observed (Fig. 2).JC-1 staining showed a loss of DY m in normal cells within 20 min of exposure to the toxins, the cells rounded and detached from the monolayer.The cells lost viability without obvious damage to the plasma membrane.In contrast, in differentiated cells, mitochondria were largely intact after similar exposure to the same toxins, indicating an increased resistance.
Mitochondria may loose their DY m by three main mechanisms: 1) DY m -driven uptake of K + as a positively charged ionophore complex; 2) Opening of the permeability transition pore; 3) Increased transmembrane transport of protons, i.e. protonophorous uncoupling.Since we deal here with K + ionophores, mechanism 1) is mainly responsible for the depolarisation.Given that K + is the main cation in the cytosol, the uptake of K + could be considerable, resulting in extensive swelling of mitochondria.We might in addition have stimulation of the mitochondrial permeability transition.This and swelling will cause some secondary increase in H + flux, i.e. uncoupling with decreased synthesis of ATP, and hydrolysis of ATP by the F o F 1 -ATPase.Mitochondrial volume is regulated by the combined activities of the ATP-sensitive K + channel and the K + /H + antiporter (Garlid & Paucek, 2003).In the presence of potassium ionophores it is to be expected, that the stimulated influx of K + and swelling results in activation of the antiporter, which in turn causes K + efflux and influx of H + .This cycling mechanism may contribute to an uncoupling.
The toxicity of the studied potassium ionophores is mainly due to their effects on mitochondria, i.e. loss of DY m and swelling.This generally occurs in the mitochondrial permeability transition, however, valinomycin was found also to cause release of cytochrome c without inducing permeability transition (Shinohara et al., 2002).Swelling itself would rupture the outer membrane and release proapoptotic factors like cytochrome c (Cai et al., 1998).The associated decrease in ATP would be expected to result in cell necrosis (Lemasters et al., 2002), but the cell death seen in this study rather corresponds to apoptosis.
The molecular mechanism(s), conferring higher resistance to the toxins in differentiated Paju cells, is not clear.The expression of several proteins is increased during induced neural differentiation, among which Bcl-2 is known to suppress the mitochondrial permeability transition (Shimizu et al., 1998;Murphy et al., 2001).This property of Bcl-2 is not of interest since valinomycin was found to release cytochrome c and induce apoptosis without permeability transition (Shinohara et al., 2002).Stanniocalcin has a protective effect on neurons during cerebral ischemia (Zhang et al., 2000).Moreover, binding of stanniocalcin to the inner membrane of mitochondria was recently reported and stanniocalcin was found to stimulate electron transport in submitochondrial particles in vitro (McCudden et al., 2002).These observations suggest that stanniocalcin may contribute to the maintenance of the energy state even under stressful conditions.Studies are in progress to elucidate whether stanniocalcin or/and some other factors are instrumental for the increased resistance to bacterial toxins in differentiated neuronal cells.

Figure 1 .
Figure 1.The morphology of Paju cells before (WT) (panel A) and after induced differentiation (panel B).

Figure 2 .
Figure 2. JC-1 and PI-stained human neural undifferentiated (WT) (left panel) and differentiated (right panel) Paju cells exposed to the microbial toxins.JC-1 fluoresces yellow-orange in cell areas with high DY m and green in areas of lower potential.PI fluoresces red in cells with damaged cell membrane, whereas there is no fluorescence in intact cells.Paju cells were exposed (20 min, 37°C) to the solvent only (panels A and B), to 4.5 ng/ml (C and D), or to 9 ng /ml of cereulide (panels E and F), to 20 ng/ml of valinomycin isolated from S. griseus (panels G and H), and to 20 ng/ml of valinomycin from Sigma (panels I and J).