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Regular Articles |
From the Cell and Molecular Biology Section,*
Pediatric
Oncology Branch, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland; the Department of
Pathology,
Seoul National University College
of Medicine, Seoul, Korea; and the Department of Nuclear
Medicine,
Samsung Medical Center,
Sungkyunkwan University, Seoul, Korea
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Abstract |
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Introduction |
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NTs exert profound effects on the biological behavior of neuronal cells during the processes of development, differentiation, and survival in the central and peripheral nervous systems, by binding to at least two types of receptor. The nerve growth factor receptor (LNGFR) is a member of the tumor necrosis factor family of receptors, and trk is a member of the tyrosine kinase family of receptors.6,7 The tyrosine kinase receptors selectively bind to their cognate ligands: trkA for nerve growth factor (NGF) and NT-3; trkB for BDNF, NT-4/5, and NT-3; and trkC for NT-3.8-10 Because NTs and trks are expressed in the central and peripheral nervous system tumors of childhood, there have been many studies regarding the role of these receptor-ligand interactions in these tumors.11,12
Many cell lines provide systems that have proved useful in the investigation of the major biological aspects of NB, because they retain the potential to respond to various biological response modifiers and growth factors.13 In our study, we used a tetracycline-regulatable vector system, in which two components of the tetracycline-controlled transactivator (tTA) promotor system have been organized in a single vector,14 to investigate the role of the activation of trkA and trkB in the SH-SY5Y (SY5Y) human neuroblastoma cell line. SY5Y is a neuronal subclone of the SK-N-SH cell line, which was established from a bone marrow aspirate of a thoracic NB.15 SY5Y expresses low levels of trkA and p75 LNGFR and lacks endogenous expression of trkB or BDNF.16
We examined how conditional activation of trkA and trkB affected the growth, survival, and differentiation of SY5Y cells. The effects of the activation of trkA and trkB signal transduction paths were quite similar for growth, survival, and differentiation; however, distinct differences were identified in the lineage-dependent markers. An increase in insulin-like growth factor-II (IGF-II) expression induced by trkA activation was reminiscent of the extra-adrenal NBs of infancy, which express high levels of trkA and show biochemical evidence of chromaffin differentiation.17 Furthermore, the inhibition of growth by blocking of the IGF-I receptor suggested that up-regulation of IGF-II expression is a component of the effector mechanism of trkA activation by NGF. Despite the different clinical implications, the biological effects mediated by trkA and trkB overlap in the case of SY5Y cells.
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Materials and Methods |
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Tetracycline-regulated vector, pBPSTR1,14
was
obtained as a gift from Dr. Steven Reeves. The vector contained the
puromycin resistance gene. Full-length human trkA cDNA (2.7 kb) was
subcloned into NotI, and a 3.1-kb fragment of rat trkB
spanning a full coding region, which was generated by
NotI-HpaI digestion of rat trkB cDNA (4.7 kb),
was subcloned into the NotI-PmeI site of
pBPSTR1. Stable transfectants were obtained from the human
neuroblastoma cell line SH-SY5Y by transfection using lipofectAMINE
(Life Technologies, Gaithersburg, MD). Subsequent selection was done
with 0.5 µg/ml puromycin (Sigma, St. Louis, MO). Cells were grown in
RPMI 1640 (Mediatech, Herndon, VA) containing 10% fetal bovine serum,
2 mmol/L glutamine, and antibiotics. The screening for
tetracycline-dependent regulation of transfected gene expression was
performed by Northern blot analysis. For the Northern blot analysis,
total RNA was extracted using a RNeasy kit (Quiagen, Santa Clarita, CA)
from cells grown in the presence (1 µg/ml) or absence of tetracycline
(Sigma) for at least 2 days. Ten micrograms of RNA was electrophoresed
in 1.2% formaldehyde agarose gel, transferred to a nitrocellulose
membrane (Nytran, Schleicher and Schuell, Keene, NH), and hybridized at
42°C with [
-32P]dCTP-labeled trkA (2.7 kb
EcoRI fragment) or trkB (3.1 kb) purified inserts in
solutions containing 50% formamide, 1 mol/L NaCl, 10% dextran
sulfate, 1% sodium dodecyl sulfate (SDS), and 250 µg/ml of salmon
sperm DNA. Membranes were washed twice with 2x standard saline citrate
(SSC), 0.1% SDS solution at room temperature for 15 minutes, and once
with 0.5x SSC, 0.1% SDS for 15 minutes at 50°C. They were then
exposed to Kodak XAR film at -70°C.
Functional Assessment of Signaling
To assess trkA and trkB autophosphorylation after NT stimulation, cells were plated in 100-mm tissue culture dishes (1 x 106 cells/dish) and cultured in the presence (1 µg/ml) and absence of tetracycline for 3 days. They were then harvested after stimulation with 100 ng/ml of 2.5S mouse NGF (Upstate Biotechnology, Lake Placid, NY) or recombinant human BDNF (Promega, Madison, WI) for 5 minutes. For the analysis of the early response gene profile after NGF and BDNF stimulation, 1 x 106 cells were plated in 100-mm tissue culture dishes in the presence or absence of tetracycline and were stimulated with 100 ng/ml of NTs on day 3 for the indicated periods of time. Cells were harvested, and RNA was extracted and analyzed for c-fos, c-jun, NGFI-A, NGFI-B/nur77, and NGFI-C by Northern blot analysis, according to the protocols described above. To assess the expression of endogenous NTs, selected clones were analyzed for the expression of NGF, BDNF, NT-4/5, and NT-3. NGF activity was assessed by application of the culture media to PC12 rat pheochromocytoma cells. Northern blot analysis was performed for BDNF, NT-4/5, and NT-3 expression. The level of LNGFR gene expression was also analyzed by Northern blot analysis.
Immunoblotting/Immunoprecipitation
For the protein analysis, cells were harvested in Tris-buffered saline (TBS) with cell scrapers and kept at -70°C until analysis. Cells were lysed in NP-40 lysis buffer containing 1% NP-40, 10% glycerol, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 500 µmol/L sodium orthovanadate in ice-cold TBS. Lysis was done on ice for 30 minutes, and the protein concentration was measured with the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA). For the immunoprecipitation of phosphorylated trkA and trkB receptors, 500 µg of protein from each lysate was immunoprecipitated with polyclonal rabbit anti-pan-trk antibody (C-14) (Santa Cruz Biotechnology, Santa Cruz, CA) and protein A Sepharose (Pharmacia Biotech, Piscataway, NJ). Immunoprecipitates were electrophoresed in an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and subsequently probed with an anti-phosphotyrosine antibody (4G10) (Upstate Biotechnology). Signals were detected using enhanced chemiluminescence reagents (Amersham Life Science, Arlington Heights, IL).
Analysis of Differentiation Lineage-Dependent Markers
Changes associated with differentiation in neural cell adhesion molecule (NCAM) and GAP-43 were analyzed by Western blot analysis, using 40 µg of protein with mouse monoclonal antibodies to NCAM (Zymed Laboratories, South San Francisco, CA) and GAP-43 (Oncogene Research Products, Cambridge, MA), respectively. Expression of bcl-2, IGF-II, and chromogranin A was analyzed by Northern blot analysis. The levels of norepinephrine in the media in which the cells were cultured were measured by high-pressure liquid chromatography (Beckmann Instruments, Fullerton, CA) at the Department of Laboratory Medicine, Seoul National University Hospital.
Radioimmunoassay for Insulin-Like Growth Factor-II
Cells were split in a six-well plate (1 x 105 cells/well) in the absence of tetracycline. NGF was added to individual wells the next day at a concentration of 100 ng/ml. The cells were then cultured for 5 days. The radioimmunoassay of culture supernatants for IGF-II was performed using a DSL-9100 ACTIVE IGF-II coated-tube immunoradiometric assay kit (Diagnostic Systems Laboratories, Webster, TX), according to the manufacturer’s instructions. The free IGF-II level was measured in samples not treated by acid/ethanol extraction, to enable the detection of only free IGF-II, which is not complexed with insulin-like growth factor-binding proteins (IGFBPs). Total IGF-II level was measured after an acid/ethanol extraction step to separate IGFBPs. Assay sensitivity was 0.13 ng/ml, and intra-assay and interassay coefficients of variation were 4.3–7.2% and 6.3–10.4%, respectively. The assay has no detectable cross-reactivity for IGF-I or insulin up to respective concentrations of 480 µg/ml and 4.3 µg/ml.
[3H]dThymidine Uptake Analysis
Cells were cultured in the presence of tetracycline, and 5 x 103 cells were split in a 96-well plate in different concentrations of tetracycline (1 µg/ml, 1 x 10-3 µg/ml, 5 x 10-4 µg/ml, 0 µg/ml) in quadruplicate. The next day (day 0), NGF and BDNF were added to individual wells at concentrations of 100 ng/ml. Cells were then cultured for 3 days (day 3). Twenty hours before harvesting, 1 µCi of [3H]dthymidine (ICN, Costa Mesa, CA) was applied to each well. Cells were harvested 20 hours after [3H]dthymidine application with a cell harvester (Inotech, Lansing, MI), and radioactivity was measured with a liquid scintillation counter (Beckmann Instruments).
Viable Cell Counting and Cell Cycle Analysis
To assess the changes in the number of cells and in the cell cycle distribution after NT treatment, 1 x 104 cells were split in 24-well plates in the presence or absence of tetracycline. NTs were applied at a concentration of 100 ng/ml the next day (day 0). Cells were harvested on day 5. The number of viable cells was then counted and subsequently subjected to cell cycle analysis. Cells were stained with propidium iodide (50 µg/ml) for cell cycle analysis, which was performed with a Becton-Dickinson FACScan.
Analysis of Cell Survival
Cells were split in 24-well plates at a density of 1 x 105 cells per well in triplicate, in the presence or absence of tetracycline. To assess the effects of NTs on the survival of cells after nutrient deprivation, cells were washed twice with serum-free RPMI 1640 on the following day (day 0). They were then cultured in RPMI 1640 containing 0.5% fetal bovine serum with and without tetracycline. NTs were applied at a concentration of 100 ng/ml. Cells were kept under these conditions, and the proportion of viable and nonviable cells was evaluated by trypan blue exclusion on day 5.
Assessment of the Effects of IGF-I Receptor Blocking
To analyze the effects of IGF-I receptor blocking on growth, the
cells were cultured in the absence of tetracycline, and mouse
anti-IGF-I receptor monoclonal antibody (IR-3; Oncogene Research
Products) was applied to individual wells on days 0 and 2.
Preincubation of SY5Y cells with 1 µg/ml of IR-3 prevents
autophosphorylation of the IGF-I receptor when stimulated with 100
ng/ml of IGF-II. To investigate whether the blocking of the IGF-I
receptor counteracts NGF-induced differentiation of trkA transfected
cells, 1 x 104
cells were split in a six-well plate
in triplicate. The cells were then treated with NGF for 4 days in the
presence and absence of IR-3 at a concentration of 2 µg/ml.
Neurite extension, which was defined as an elongation of the cell
processes to more than two times the cell body diameter, was analyzed
in at least 200 cells. To assess the effects of IGF-I receptor blocking
on growth, [3H]dthymidine uptake analysis was performed
as described above in the presence of different concentrations of
IR-3 (0, 0.5, 1, 2 µg/ml).
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Results |
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TrkA transfected clones SY5Y-TA25 (TA25) and SY5Y-TA88 (TA88),
trkB transfected clones SY5Y-TB3 (TB3) and SY5Y-TB8 (TB8), and a mock
transfected clone SY5Y-E2 (E2) were analyzed. The expression of trkA
from transfected genes differed from the expression of endogenous trkA
in terms of its regulatability and size. The expression of transfected
gene trkA or trkB mRNA was conditionally regulated by the application
of tetracycline in the range of 0–1 µg/ml (data not shown).
Experimental clones showed readily detectable levels of receptor
autophosphorylation when stimulated with cognate ligands, also in a
dose-dependent manner (Figure 1)
. E2 showed
detectable levels of autophosphorylation after NGF treatment but not
BDNF treatment. In the absence of tetracycline, trkA and trkB
autophosphorylation increased 1.44-fold for TA25, 12.18-fold for TA88,
2.33-fold for TB8, and 7.95-fold for TB3 when compared to that measured
in the presence of tetracycline, by quantitative densitometric
analysis. The intensities of trkB autophosphorylation in TB3 and TB8,
in the absence of tetracycline, were comparable to that of trkA
autophosphorylation in TA25 in the absence of tetracycline. In the
presence of tetracycline, TA25 and TA88 did not show detectable levels
of receptor autophosphorylation without NGF stimulation. TA25 and TA88
did not express NT-3 or NT-4/5 mRNA. In addition, they did not
produce a concentration of NGF in the conditioned media that would
induce neurites in PC12 cells (data not shown). TB3, TB8, and E2 showed
barely detectable levels of NT-4/5 on Northern blot analysis and did
not express BDNF or NT-3 (data not shown).
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. Induction of c-fos,
c-jun, and NGFI-A by NGF in E2 was presumed to be via the
activation of endogenous trkA. However, the response was transient when
compared to those of other experimental clones, and gave no detectable
message after 30 minutes. BDNF treatment of E2 did not induce
c-fos. Although the application of tetracycline
significantly repressed the expression of transfected genes, it was not
sufficient to completely block trkA and trkB expression in transfected
clones. Therefore, all of the experimental conditions were designated
as either low (in the presence of 1 µg/ml of tetracycline) or high
(in the absence of tetracycline) in terms of the expression of trkA and
trkB.
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Treatment with NTs induced differentiation, characterized by
neurite extension in both trkA and trkB transfected clones. The cells
began to show scattered neurite outgrowth 2 days after NT application,
and neuritogenesis was evident on day 3. With low trkA or trkB
expression, all of the experimental cells showed a flattening of the
soma with neurite formation. This was most evident in TA88 after NT
treatment. High trkA or trkB expression was associated with the most
differentiated features (Figure 3)
. E2 cells
did not show any significant alterations by NGF or BDNF.
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.
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. A detectable IGF-II mRNA
message was found after 2 days of NGF treatment, and the
radioimmunoassay results showed a chronological correlation. The
biggest difference in IGF-II concentration was reached on day 5, after
NGF treatment, increasing 3.3- and 6.2-fold in TA25 and TA88 cells,
respectively (Figure 4C)
. The highest concentrations of IGF-II in media
on day 5 were 77 ng/ml and 89 ng/ml for TA25 and TA88, respectively.
Up-regulation of IGF-II expression was not seen in BDNF-treated TB3 and
TB8, or in NT-treated E2 cells. Treatment of TA25 and TA88 with NGF
also resulted in a twofold increase in the secretion of norepinephrine
into the media (179% of control in TA25 and 214% of control in TA88),
whereas such changes were not seen in E2 cells and BDNF-treated TB3 and
TB8 cells (Figure 4D)
. The absolute concentration of norepinephrine in
media after NGF treatment was more than 3000 pg/ml in both of the
trkA-transfected cells on day 5. TB3 showed an increase in chromogranin
A expression due to BDNF treatment. All of the trkA and trkB
transfected clones showed increased expressions of GAP-43 and
bcl-2 after NGF and BDNF treatments, respectively (Figure 4, A and B)
. None of these significant biochemical changes were observed
in E2, suggesting that the intensity and/or duration of signaling by
endogenous trkA receptors may not be sufficient to elicit detectable
biological changes. Effects of TrkA and Trk B on Growth and Survival
When trkA was fully expressed and treated with NGF, TA25 and TA88
showed decreases in [3H]dthymidine uptake and cell
numbers. With low trkA expression, NGF treatment of TA88 did show
increases in [3H]dthymidine uptake and cell numbers
(Figure 5A)
. This suggests a differential
modulation of growth by the same receptor. Activation of trkB in TB3
and TB8 resulted in decreased [3H]dthymidine uptake in a
dose-dependent manner. The reduction of uptake ranged from 48% to 68%
of controls (Figure 5B)
. Cell numbers after 5 days of NT application
were consistent with trends in cell growth as determined by
[3H]dthymidine uptake analysis (Figure 5B)
. Although
activation of trkA and trkB significantly inhibited cell growth, flow
cytometric analysis did not show significant changes in the
distribution of the cell cycle in any of the clones. This suggests that
growth inhibition by NTs is not associated with major changes in the
cell cycle.
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. The percentage of viable cells increased
to 37% of controls in TA25 and to almost 45% in TA88 cells,
regardless of the levels of trkA. In TB3 and TB8, the addition of BDNF
rescued cells in a dose-dependent manner. BDNF stimulated a 15%
increase in the cell survival of TB8 and a 22% increase in the cell
survival of TB3 expressing low levels of trkB. This increased to 54%
in TB8 and 45% in TB3 expressing high levels of trkB (Figure 6B)
. The
survival of E2 was not significantly affected by NGF or BDNF.
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To assess whether up-regulation of IGF-II expression mediates
biological effects after trkA activation, TA25 and TA88 cells were
treated with NGF for 4 days in the presence and absence of IR-3 at a
concentration of 2 µg/ml. Treatment of the cells with IR-3 did not
counteract NGF-induced differentiation of the cells (Figure 7A)
. However, there was a persistent decrease
in [3H]dthymidine uptake up to 19% and 22% in TA25 and
TA88 cells, respectively, when these cells were treated with IR-3,
which suggested that up-regulation of IGF-II expression is a part of
the effector mechanism of trkA activation (Figure 7B)
. These changes
were not seen in BDNF-treated TB3, TB8, or NT-treated E2 cells.
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Discussion |
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Generally, the expression of trkA and that of trkB are mutually exclusive in primary NBs.2 Although a majority of neuroblastoma cell lines express low levels of trkA, few of them are responsive to NGF in terms of induction of differentiation or changes in proliferation and survival.22,23 Previous studies have reported the restoration of NGF responsiveness after trkA transfection in neuroblastoma cell lines and suggested that low levels of trkA, or defects in trkA signaling, may be responsible for the poor prognosis of these tumors.23,24 On the contrary, in our previous analysis of the effects of trkB induced by RA, BDNF treatment enhanced the invasiveness and survival of neuroblastoma cell lines without growth inhibition.25 Similar effects have been observed by others in the trkB-expressing SMS-KCN neuroblastoma cell line.5 These results may explain why the expression of trkA and the expression of trkB are associated with different prognoses.
In this study, we developed an experimental system in which we can differentially modulate the expression of trkA and trkB. Clonal variability, differences in the level of expression of receptors in individual clones, and the relatively high level of trkA expression, even in the presence of tetracycline in trkA-transfected cells, can be potential biases. However, we identified novel aspects of the effects of trkA and trkB in SY5Y cells. SY5Y expresses low levels of LNGFR, which binds all NTs, and may influence and elicit biological responses both dependent and independent of trk activation.26,27 Although it is possible that signaling via LNGFR may have influenced the effects by NGF and BDNF in our system, it is unlikely, as neither NGF nor BDNF significantly altered the expression level of LNGFR on Northern blot analysis (data not shown), and significant effects of NTs were not observed in E2.
Differentiation of adrenergic cells originating from the neural crest is characterized by its plasticity, because of the influence of environmental factors such as glucocorticoids and growth factors.28 Sympathetic neuroblasts can differentiate into both neuronal (sympathetic ganglionic) and neuroendocrine (adrenal or extra-adrenal chromaffin) lineages. A recent analysis of a series of primary NBs and the sympathetic nervous system showed an increased expression of trkA, trkC, IGF-II, and chromogranins and a decreased expression of bcl-2 and NCAM with ongoing morphological differentiation in extra-adrenal NBs.17 This pattern of gene expression corresponded to the antigenic profile of cells undergoing extra-adrenal chromaffin differentiation during human sympathetic nervous system development. A subsequent study demonstrated evidence of in vivo spontaneous neuronal to neuroendocrine lineage conversion in these NBs.29 The changes in the biochemical phenotype marked by NCAM down-regulation, increased catecholamine secretion, and a robust increase in IGF-II expression after NGF treatment of trkA-transfected clones in the present study suggest that in certain extra-adrenal NBs, activation of trkA may contribute in part to the differentiation of neuroendocrine phenotypes, although the increases in GAP-43 and bcl-2 are consistent with the development of neuronal phenotypes.
Increased expression of bcl-2 by NGF and BDNF in trkA and trkB transfected clones and enhanced cell survival after nutrient deprivation are consistent with the observations in other studies. The rescue of PC12 cells and BDNF-dependent neurons from apoptosis by NGF and BDNF was dependent on the up-regulation of bcl-2.30,31 It is interesting that trkA, a good prognostic marker in NB, is related to bcl-2 overexpression, although bcl-2 is not detected in tumors with a good prognosis. In contrast, bcl-2 expression is a major feature of NBs with unfavorable histologies and amplified N-myc. It also protects the NB cells from anticancer agents by producing a more drug-resistant phenotype.32,33 Therefore, increased bcl-2 expression would be more consistent in NBs with poor prognoses, which tend to express both BDNF and trkB, and in which chemosensitivity is altered by the activation of trkB by BDNF.34
The up-regulation of IGF-II expression, which can establish autocrine or paracrine loops of signaling in this IGF-I receptor-expressing cell line,35,36 was found to be the major feature that distinguished trkA from trkB. This was the first demonstration of the novel link between trkA activation by NGF and IGF-II expression in NB. The insulin-like growth factor system is important during the course of human development, and cell types with abundant IGF-II expression are strikingly correlated to the organomegaly and tumor predisposition of the Beckwith-Wiedemann syndrome, a syndrome characterized by an increased incidence of embryonal tumors such as Wilms’ tumor and NB.37 IGF-I and IGF-II have been reported to play an important role in the early development of chick sympathetic neurons. IGF-II has proven mitogenic effects, induces differentiation, and promotes the survival of cultured sympathetic neurons.38,39 IGF-II expression was also found in a significant proportion of primary NBs and cell lines, suggesting an important role in the tumorigenesis and biology of NB.40 However, NGF treatment of PC12 cells did not stimulate IGF-II expression (data not shown). Autocrine or paracrine loops between IGF-II and IGF-I receptors have been reported to induce proliferation and differentiation, prevent apoptosis, and enhance the tumorigenesis of SY5Y cells.35,36,41 They also mediate the autonomous growth of another NB cell line, SK-N-AS, which expresses IGF-II abundantly.42 Our findings suggest that up-regulation of bcl-2 and IGF-II expression are biologically relevant mechanisms that are coupled to trkA in NBs and possibly in the sympathetic nervous system. A recent report on the protease activity of 7S nerve growth factor for insulin-like growth factor-binding protein (IGFBP), which allows the level of free IGFs to increase, provides additional evidence that NGF can modulate the levels of IGF.43 In the context that IGF-II expression has been repeatedly advocated as a marker for extra-adrenal chromaffin differentiation,17,44 the findings from our study imply that up-regulation of IGF-II is not only a feature of the differentiated cells; it also has a role as a component of the effector mechanisms of trkA in these cells.
In contrast to previous studies, we found that the effects mediated by trkA and trkB receptors in SY5Y are overlapping. We showed that trkB activation is a growth-inhibiting signal for SY5Y, and that a particular level of trkA activation can be mitogenic. This strongly suggests that the effects of trkA and trkB activation can vary, depending on the biological phenotypes of individual NBs and the intensity of signaling in vivo. TrkA transfected SY5Y cells showed dissociation in the modulation of neuronal or neuroendocrine antigens and catecholamines. Moreover, the biochemical alterations seemed to be less dependent on the degree of morphological differentiation. These results are in line with those of previous observations of adrenal chromaffin cells, which demonstrated that distinct and independent modulations of peptidergic, catecholaminergic, and morphological properties of chromaffin cells were caused by different growth factors including NGF.45 It seems reasonable to speculate that no single growth factor could allow the SY5Y cells to differentiate into a specific biological phenotype. However, the relationship between trkA and IGF-II, along with increased catecholamine synthesis, implies that trkA-transfected SY5Y cells may allow us to study the major biological aspects of extra-adrenal NBs of infancy, which are characterized by a favorable prognosis and extra-adrenal chromaffin differentiation.
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Acknowledgements |
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Footnotes |
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Accepted for publication July 30, 1999.
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