Removal of PSA from tumor cells inhibits proliferation.
Treatment of living cells with endoneuraminidase NE (endo-NE) (15
) degrades PSA with high specificity and leads to a better accessibility of NCAM on the cell surface (19
). In NCAM- and PSA-positive neuroblastoma (SH-SY5Y, Kelly, and LAN-5) and rhabdomyosarcoma (TE671) cells (44
), endo-NE induced a similar inhibition of cell growth, while growth of the NCAM- and PSA-negative neuroblastoma cell line LS was unaffected (Fig. ). Control incubations with heat-inactivated endo-NE, colominic acid (i.e., soluble PSA from bacteria), or N
-acetylneuraminic acid had no effect on cell growth. The endo-NE-induced reduction of cell growth was confirmed by counting the cells in some of the experiments. Detached, dead, or damaged cells were never observed, and DAPI staining revealed no abnormal condensation or fragmentation of nuclei. For SH-SY5Y cells, the absence of apoptosis after endo-NE treatment was confirmed by a cell death ELISA (see Fig. ), and the analysis of BrdU incorporation revealed that the endo-NE-induced reduction of cell growth was associated with a significant decline in proliferation (Fig. ). Thus, the observed growth inhibition was due to reduced rates of proliferation.
FIG. 1. PSA removal induces growth inhibition in PSA-NCAM-positive tumor cells. (a) LS, SH-SY5Y, Kelly, and LAN-5 neuroblastoma and TE671 rhabdomyosarcoma cells were grown for 2 days in control medium (ctrl., white bars) or in the presence of 6 ng/ml (80 pM) (more ...)
FIG. 6. PSA removal modulates MAPK-dependent survival and apoptosis. Serum-supplemented or serum-deprived SH-SY5Y cells were treated with the MEK inhibitor PD98059 (50 μM in dimethyl sulfoxide) or solvent (1 μl of dimethyl sulfoxide/ml) in the (more ...)
In primary cultures of cortical neurons, the expression of PSA has been shown to improve the sensitivity to BDNF, and in contrast to other neurotrophins, application of exogenous BDNF was able to rescue neurons from endo-NE-induced death (49
). Because SH-SY5Y cells express low levels of BDNF and TrkB (23
), we compared the influence of endo-NE on the mitogenic activities of BDNF, nerve growth factor, and fibroblast growth factor-2. The proliferative response to all three growth factors was drastically reduced in the absence of PSA (Fig. ). This pleiotropic effect argues against a BDNF-specific interaction with PSA but suggests that growth inhibition is a direct result of PSA failure.
FIG. 2. Growth inhibition after removal of PSA is mediated by NCAM interactions. (a) SH-SY5Y cells were treated for 2-day with 50 ng/ml BDNF, nerve growth factor or fibroblast growth factor-2 in the absence (ctrl., white bars) or presence of 6 ng of endo-NE per (more ...)
We then analyzed whether changes in NCAM binding abilities may be responsible for the effect of endo-NE. In a first experiment, the MAb 123C3 (29
) was used to interfere with NCAM binding. The PSA-specific MAb 735 and MAb 14G2a (31
), directed against the ganglioside GD2
, served as controls. All three reagents produced an 80% growth reduction, indicating a strong but nonspecific cytotoxic effect. Incubation with normal mouse IgG as a nonbinding control antibody had no effect on cell growth (data not shown). However, the endo-NE-mediated growth inhibition was completely prevented if cells were treated with the dendrimeric C3d peptide (39
), a synthetic ligand of the first immunoglobulinlike domain of NCAM (Fig. ). In contrast, an inactive variant of C3d, C3d2Ala (39
), was unable to protect the cells from the endo-NE-induced growth inhibition (not shown).
As controlled by cellular ELISA, incubation with the C3d peptide neither interfered with PSA degradation nor led to reduced cell surface expression of NCAM (cell surface immunoreactivities of untreated controls and of cultures treated with endo-NE in the presence and absence of C3d [arbitrary units ± standard error of the mean] were PSA, 0.77 ± 0.03, 0.02 ± 0.005, and 0.02 ± 0.002, respectively; NCAM, 0.54 ± 0.08, 0.76 ± 0.02, and 0.62 ± 0.05, respectively; n = 6). Also, C3d had no autonomous effect on proliferation (Fig. ). The protective effect of C3d therefore strongly suggests that the inhibition of proliferation involves interactions of NCAM. Moreover, because the C3d peptide binds to the first immunoglobulinlike domain of NCAM, this module appears to be involved in mediating this interaction.
The next question was if trans-interacting NCAM is also able to cause growth inhibition. In order to mimic the situation in the cell culture system, experiments were carried out with NCAM-positive and NCAM-negative cell membranes isolated from differentially transfected LS cells. The phenotypes of transfected LS cells are shown in Fig. . Parental LS are NCAM and PSA negative (Fig. ). The same is true for cells transfected with an empty vector (NCAM negative, PSA negative; Fig. , LSmock). Cells after transfection with a vector driving the expression of NCAM-140 stained positive in immunohistochemistry for NCAM but not for PSA (NCAM positive, PSA negative; Fig. , LSAM1), while double transfectants containing vectors driving NCAM-140 and polysialyltransferase ST8SiaIV expression, stained positive for both epitopes (PSA positive, NCAM positive; Fig. , LSAM1PST). Membranes from PSA-positive, NCAM-positive double transfectants were isolated before and after endo-NE digestion (compare Fig. , LSAM1PST, untreated versus LSAM1PST + endo-NE).
In the experiment, SH-SY5Y (PSA positive, NCAM positive) and the control cells (LS, PSA negative, NCAM negative) were overlaid with membrane preparations of all phenotypes. Microscopic control revealed that the membrane particles settled rapidly and dispersed uniformly over cells and cell-free areas (Fig. , lower panels for an example). After a coculture period of 2 days, the growth rates were compared (Fig. ). Membranes isolated from mock (PSA negative, NCAM negative) or double transfected LS cells (AM1PST; PSA positive, NCAM positive) did not noticeably affect cell growth in either of the cell systems. In contrast, the membrane fractions derived from LSAM1 or LSAM1PST after endo-NE treatment (both are PSA negative, NCAM positive) significantly inhibited the growth of SH-SY5Y cells (which are PSA positive, NCAM positive) and of LS cells (which are PSA negative, NCAM negative).
Although unexpected, this observation indicates that the growth inhibition induced by endo-NE or by NCAM itself results from trans binding of nonpolysialylated NCAM to heterophilic target structures. These findings received further support by the observation that growth of LSAM1 cells and LS AM1PST plus endo-NE (both PSA negative, NCAM positive, metabolic rates [day 2/day 0 ± standard error of the mean]: 1.62 ± 0.06 and 1.76 ± 0.09) was significantly reduced compared to nontransfected LS cells, LSmock (both PSA negative, NCAM negative), or LSAM1PST (PSA positive, NCAM positive; metabolic rates [day 2/day 0 ± standard error of the mean]: 2.21 ± 0.07, 2.19 ± 0.07, and 2.22 ± 0.08; n = 10 to 12; t test, P < 0.01).
The above data imply that PSA regulates NCAM-dependent cell-cell interactions, which requires its localization at cellular contact sites. To check this, the cell surface distribution of PSA and NCAM was studied. As visualized by immunogold detection with energy-filtering transmission electron microscopy (26
), PSA appears in clusters on SH-SY5Y cells and is preferably localized at sites of tight cell-cell contacts (Fig. ). Immunofluorescence staining and confocal microscopy clearly demonstrate that NCAM and PSA are mainly colocalized at cellular contact sites (Fig. , left panel). Removal of PSA did not change the strong NCAM-immunoreactivity at contact sites, however, the number of NCAM-positive contact zones increased significantly after 2-day of endo-NE treatment (Fig. , right panel). Even if seeded as single cell suspension at low densities, the tumor cells used in this study never grew without any contacts between each other. Time lapse observations performed with SH-SY5Y cells stably transfected to express the enhanced green fluorescent protein (EGFP) in the cytosol (SH-SY5YEGFP
) indicate that, besides the broad and rather stable contacts shown in Fig. , even those cells that have no contact at a given time point, readily will form new contacts or just broke up existing contacts due to extensive cellular motility (Fig. ).
FIG. 3. PSA-NCAM is localized at cell-cell contact sites. (a) Immunoelectron microscopic localization of cell-surface PSA by pre-embedding immunogold labeling and energy-filtering transmission electron microscopy of 500 nm thick sections. Antibodies were applied (more ...)
Taken together, these analyses of cell growth indicate that downregulation of PSA enables heterophilic NCAM interactions at contact sites between tumor cells leading to reduced proliferation in the recipient cell.
Endo-NE-induced NCAM signaling involves MAPK activation.
Because previous studies of NCAM signaling demonstrated the involvement of the p44/p42 MAPK ERK1/2 pathway (24
), this system was surveyed during endo-NE treatment of SH-SY5Y cells. Western blotting with anti-NCAM MAb 123C3 was used to monitor the conversion of the hardly detectable high-molecular weight smear typical for highly sialylated NCAM into PSA-free isoforms seen as sharp bands of 140 and 180-kDa (Fig. ). Already after 10 min of endo-NE digest, significant amounts of nonpolysialylated NCAM were generated, but only after 1 h the reaction was completed. Endo-NE was constantly present in the cultures and no PSA reappearance was detected during the 2 days of the experiment. Changes in ERK activity were examined by Western blotting with antibodies directed against dually phosphorylated ERK1/2, indicative for the activated MAP kinase (28
), and compared to the total amount of ERK protein (Fig. ).
FIG. 4. Removal of PSA leads to ERK activation. SH-SY5Y cells were supplied with fresh cell culture medium containing 60 ng of endo-NE per ml (Endo, Endo+) or solvent (ctrl., Endo−), incubated for the times indicated and analyzed by immunoblots (more ...)
To apply equal amounts of endo-NE, the enzyme was diluted in culture medium and medium were changed in all experimental groups at the beginning of the experiment. In the control group, the supply with fresh serum resulted in a moderate increase of ERK phosphorylation, relapsing to baseline within 16 h (Fig. ). In endo-NE treated cultures a strong increase of ERK phosphorylation was observed (Fig. ), while the total amount of ERK1/2 protein remained constant throughout the experiment (Fig. , lower panel). ERK phosphorylation was maximal as soon as 10 min after the beginning of the treatment and the peak level was maintained up to 1 h before it slowly returned to the level of the time-matched controls (Fig. ). In contrast to the effect of medium replacement, the strong MAPK activation during PSA digest was not caused by serum factors, because in the absence of serum endo-NE treatment carried out for 10, 30, or 60 min resulted in the same activation of MAPK as in serum-supplemented cultures (Table ). However, in contrast to the MAPK activation after supply with fresh serum, ERK phosphorylation was not increased by medium replacement and these experiments were limited to 1 h, because longer periods of serum withdrawal led to a marked increase of cell death. The parallel analysis of TE671 rhabdomyosarcoma cells produced an identical time course of ERK phosphorylation after endo-NE treatment (Table ).
Effect of endo-NE treatment on ERK phosphorylation
If the endo-NE-induced MAPK activation relates to signaling via the NCAM molecule, factors that prevented growth retardation as shown in Fig. , should be able to interfere with this induction. In fact, a complete inhibition of ERK phosphorylation was observed, if PSA removal was carried out in the presence of 1 μM C3d peptide (Fig. ). As shown in Fig. , similar results were obtained by antibody inhibition. Here it is important to note that, in contrast to the long-term incubations described above, short periods of antibody application had no apparent cytotoxic effect. After endo-NE treatment for 1 h, SH-SY5Y cells were incubated for 10 min with antibodies directed against NCAM (MAb 123C3), ganglioside GD2 (MAb 14G2a) or PSA (MAb 735). Although no complete reversal could be achieved by this specific protocol, application of MAb 123C3, but not 14G2a or 735, clearly reduced the ERK phosphorylation induced by endo-NE (Fig. , endo-NE-induced pERK intensities relative to the untreated control: 2.29 without additional antibody application, 1.38 after MAb 123C3, 1.96 and 2.53 after MAb 14G2a and 735, respectively). The same experiment performed with TE671 cells yielded identical results (endo-NE-induced pERK intensities relative to the untreated control: 1.77 without antibody, 1.30 after MAb 123C3, 2.23 and 1.95 after MAb 14G2a and 735, respectively).
FIG. 5. ERK activation by heterophilic NCAM interactions. ERK phosphorylation (pERK) and total ERK protein (ERK) were analyzed as in Fig. , with the ERK1/2-specific antibody at a dilution of 1:2,000. In all experiments shown, dual phosphorylation (more ...)
Next, NCAM-positive membranes were tested for their capability to activate MAPK. No MAP kinase activity could be detected in the membrane fractions themselves (not shown). NCAM-positive membranes (AM1, AM1PST +Endo) were able to induce ERK phosphorylation in PSA- and NCAM-positive SH-SY5Y cells, but membranes containing polysialylated NCAM (AM1PST) were ineffective (Fig. ). Likewise, ERK phosphorylation in TE671 cells was stimulated only by NCAM-positive, PSA-negative membranes (data not shown). Exposure of the PSA- and NCAM-negative LS cells to NCAM-positive membranes resulted in an ERK activation with a time-course similar to that, seen after removing PSA from the surface of the PSA-NCAM-positive neuroblastoma cells (Fig. ). As for the endo-NE treatment of the PSA- and NCAM-positive SH-SY5Y cells, this induction of MAPK signaling was clearly inhibited by concomitant application of C3d (Fig. , left panel) and completely blocked in the presence of PSA (Fig. , right panel, AM1PST). In accordance with the growth inhibition experiment (see Fig. ), these data are most consistent with the assumption that the MAPK activation after removal of PSA is induced by heterophilic NCAM interactions.
PSA removal promotes MAPK-dependent survival.
To elucidate the contribution of MAPK activity to changes of cell growth induced by endo-NE, the effects of MAPK inhibition were compared to those of PSA removal. In serum-supplemented cultures of SH-SY5Y cells, the inhibition of ERK phosphorylation by the MEK inhibitor PD98059 (Fig. ) reduced cell growth, and growth inhibition induced by endo-NE was significantly enhanced in the presence of PD98059 (Fig. , upper graph). The semiquantitative evaluation of intracellular mono- and oligonucleosomes by ELISA revealed that EndoNE treatment did not induce apoptotic cell death, whereas the inhibition of MAPK led to a small but clear-cut increase of apoptosis, which occurred independent of endo-NE treatment (Fig. , lower graph). As evident from the intracellular fragmentation of nuclei, a strong induction of apoptosis was observed after 2-day of serum withdrawal (Fig. ). Only 40% of the cells survived the 2-day starvation in otherwise untreated or in dimethyl sulfoxide-treated controls (Fig. , upper graph), and the high rate of apoptosis was confirmed by the semiquantitative evaluation of intracellular mono- and oligonucleosomes (Fig. , lower graph).
By endo-NE treatment, the survival rate of the serum-starved cells was significantly improved (Fig. , upper graph), while apoptosis was reduced (Fig. , lower graph). In the presence and in the absence of endo-NE, MAPK inhibition with PD98059 reduced the survival and increased apoptosis (Fig. ). In a second set of experiments, a clear increase of apoptosis was detected as soon as 4 h after the onset of serum withdrawal. In accordance with the results shown in Fig. for the 2-day incubation period, the incidence of apoptosis was enhanced by the inhibition of MAPK with PD98059, reduced by PSA removal with endo-NE and the anti-apoptotic effect of endo-NE was reversed by MAPK inhibition (apoptotic indices for the 4 h treatments: solvent-control [dimethyl sulfoxide]: 0.291, PD98059: 0.334, endo-NE: 0.213, endo-NE+PD98059: 0.302). Despite the significantly higher number of cells resulting from the endo-NE treatment, the percentage of BrdU-positive cells was decreased (Fig. ). Thus, removal of PSA from serum-deprived cells induced a similar inhibition of proliferation as under serum-supplemented conditions (see Fig. ). Together, these data indicate that the activation of MAPK after PSA removal or NCAM application exerts a survival promoting, anti-apoptotic effect but did not cause the growth inhibition observed after these treatments.
PSA removal induces neuronal differentiation.
The activation of MAPK observed in our experiments is highly reminiscent to the time course of ERK activity underlying the induction of neuronal differentiation in PC12 cells (28
). The induction of neuronal differentiation was therefore investigated by counting neuritic extensions in NCAM- and endo-NE treated SH-SY5Y cells. SH-SY5YEGFP
were used in these experiments because all processes can be reliably detected by fluorescence microscopy of living cells (Fig. ). In line with the well-characterized potential of SH-SY5Y cells to differentiate into a neuron-like phenotype (35
), process-bearing SH-SY5YEGFP
cells can be stained with an antibody against phosphorylated neurofilament, a marker of neuronal differentiation (46
) (Fig. , insets). Accordingly, long processes were referred to as neurites and cells with neurites longer than 20 μm were evaluated to assess the degree of neuronal differentiation. In untreated cultures grown for 48 h, an average of 21% of the SH-SY5YEGFP
cells developed processes longer than 20 μm, ranging up to 120 μm (mean length ± standard error of the mean, 36 ± 0.8 μm, n
FIG. 7. Effect of PSA removal, trans-interacting NCAM and MAPK on neuronal differentiation. (a to d) EGFP fluorescence of SH-SY5YEGFPcells grown for 48 h (a) in the absence or (b) in the presence of endo-NE (6 ng/ml) or together with crude membrane fractions (more ...)
Incubation with solvent (dimethyl sulfoxide) or MAPK inhibition by PD98059 caused no significant changes in neurite lengths or in the amount of neurite-bearing cells, while in the presence of endo-NE the neurites grew slightly longer and the number of neurite-bearing cells was clearly increased (Fig. ). This increase was completely prevented by the concomitant application of PD98059 (Fig. ). Similar to the effect of endo-NE, coculture of SH-SY5YEGFP with NCAM-positive membrane fractions from LSAM1 or endo-NE treated LSAM1PST induced a strong increase in the relative amounts of neurite-bearing cells together with a slight enhancement of neurite length (Fig. ). Consistent with the results on MAPK activation (see Fig. ), NCAM negative (mock) or PSA-NCAM-positive (AM1PST) membrane fractions had no effect (Fig. ). Despite the prolonged activation of MAPK after NCAM-exposure of untransfected LS cells (see Fig. ), no neurite outgrowth could be induced in this cell line (data not shown). Since protocols such as retinoic acid or growth factor treatment, which induce neuronal differentiation in other neuroblastoma cell lines, were also ineffective with LS cells (Seidenfaden, unpublished observation), these cells appear unable to differentiate morphologically. Thus, downregulation of PSA or exposure to nonpolysialylated NCAM induces a MAPK-dependent change towards a neuron-like phenotype in cells that exhibit the ability for such a differentiation.