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INTRODUCTION |
Neuroblastoma (NB)1 is a
pediatric solid tumor that arises most commonly from sympathetic
precursor cells in the adrenal medulla and, to a lesser extent, from
precursors of ganglion cells in the spinal cord (1). Examination of the
expression of enzymes involved in chromaffin cell differentiation
suggests that NB tumor cells arising from the adrenal medulla may be
derived from neuroblasts arrested during a stage of morphogenesis (2).
NB cells characteristically express neuronal markers, such as
neuron-specific enolase (3), tyrosine hydroxylase, and dopamine
-hydroxylase (4). This suggests that NB may arise from a neuroblast
that either fails to differentiate or is not eliminated by programmed
cell death at the appropriate stage of development. These hypotheses
encompass potentially inappropriate activation of signaling pathways
that could result in failure to properly execute differentiation or cell death during development. This could lead to a proliferative disease. If a subpopulation of NB cells is refractory to chemotherapy due to activation of signaling pathways required for survival in
sympathetic neurons, then an understanding these signaling pathways may
prove invaluable. Chemotherapeutic strategies for preventing the
arising of drug-resistant tumors or sensitizing tumors to existing
drugs can be envisioned.
Neurotrophin receptors, TRK, TRKB, and, TRKC are required for
development of the sympathetic nervous system (5). TRK
encodes the receptor for NGF (6-8), the archetypal member of the
neurotrophin family, which consists of NGF, BDNF, NT-3, and NT-4/5. The
TRK family (TRKs), which consists of TRK, TRKB, and TRKC, are receptor protein-tyrosine kinases. TRKB encodes a receptor for BDNF
(9-11) and NT-4/5 (12), although NT-3 also activates TRKB (9, 13). NT-3 is the ligand for TRKC receptor (14). Besides a full-length receptor protein-tyrosine kinase, TRKB expresses truncated
receptors containing the extracellular region and the transmembrane
domain but lacking the kinase domain (15, 16). The role that truncated receptors have is not known, although a potential role in signaling has
been proposed based on the finding that BDNF activation of either
TRKB.T1 or TRKB.T2 increases the rate of acidic metabolite release from
cells (17).
Genetic disruption of TRK expression in mice caused neuronal
cell loss in sympathetic ganglia and trigeminal and dorsal root ganglia
(5). Disruption of the TRKB locus in mice did not have a pronounced
effect on sympathetic neurons (18), although TRKB is expressed in
sympathetic neurons (19). However, TRKB is required for normal
development of neurons in the peripheral nervous system (trigeminal and
dorsal root ganglia) (20). In these TRKB (
/
) mice, there is an
increase in neuronal loss in the trigeminal ganglion earlier than
neuronal loss observed in mice homozygous for a null mutation of
TRK (21). This suggests that TRKB signaling is required for
survival of many trigeminal neurons before they become
NGF-dependent. In the central nervous system, additional studies on TRKB (
/
) mice indicate increased apoptotic cell death in
different regions of the brain, most significantly the dentate gyrus,
during early postnatal life (22). TRKB may have a neuroprotective function as well. Experiments that offer direct support of this concept
in vivo demonstrated lower rates of survival for axotomized hippocampal and motor neurons in TRKB (
/
) mice
contrasted to wild-type mice (22). So not only is TRKB important for
survival of neurons during postnatal development, but it may well
protect neurons from injury and axotomy-induced cell death.
With regard to NB, we wanted to test whether BDNF and its receptor,
TRKB, have a role in survival and, perhaps more importantly, to assess
whether TRKB may protect NB cells from chemotherapeutic drugs.
Neurotrophin receptor expression could, in theory, render a population
of NB cells refractory to chemotherapy and contribute to the
development of drug-resistant tumors. Indeed, there are data suggesting
that TRKB, and BDNF, are expressed in more aggressive tumors that have
N-MYC amplification (23). In vitro data with a NB
cell line indicate that BDNF may increase invasiveness of NB cells
(24). In this communication, we examine the role of BDNF in promoting
survival of NB cells with N-MYC amplification derived from a
stage D NB tumor and present the novel finding that BDNF protects NB
cells from cisplatin, a cytotoxic agent actually used in the treatment
of NB.
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EXPERIMENTAL PROCEDURES |
Reagents and Cell Culture--
5 mM ATRA (Sigma) and
9-cis-RA (Hoffman-La Roche) stocks were prepared by
dissolving retinoic acids in ethanol and stored for less than 3 months
at
80 °C. Human BDNF and NGF were generously supplied by AMGEN.
Unless otherwise stated, NB1643 cells were grown in 10% FBS/RPMI (RPMI
1640 medium containing 2 mM glutamine (BioWhittaker)
supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and
50 units/ml penicillin, 50 µg/ml streptomycin) (Life Technologies,
Inc.) at 37 °C with 5% CO2 in tissue culture plates
from Costar or Corning. Cells were trypsinized in trypsin versene
mixture (BioWhittaker) for routine counting and splitting. Nuclei were
prepared using the method of Butler (25) and counted using a Z2 counter
equipped with a 256 channelyzer (Coulter) for both cell plating and for
growth and survival assays monitored by nuclei counting. Poly-lysine
coating of tissue culture plates was accomplished by incubating plates
16 h with 1 ml of 50 µg/ml poly-lysine in H2O and
then washing once with H2O, air drying under UV light, and
subsequently cross-linking using UV light supplied by a Stratalinker
(Stratagene) using the "auto" setting.
Differentiation of NB1643 Cells--
NB1643 cells were plated at
a density of 2.5 × 105 per well in 6-well tissue
culture dishes (Costar) with or without 5 µM ATRA in 10%
FBS/RPMI medium. Final ethanol concentration was 0.1% in all wells.
Photographs at a magnification of × 225 were taken at 5 days
following ATRA treatment. To examine the effect of exogenous BDNF
treatment, NB1643 cells were plated at a density of 1 × 105 cells/well in 6-well dishes with 5 µM
ATRA. After 3 days, medium was changed to 5 µM ATRA with
or without 50 ng/ml BDNF. Final ethanol concentration was always 0.1%
in all wells. Photographs at a magnification of × 80 were taken
12 days after exogenous BDNF treatment.
Cell Viability Assays--
2.5 × 104 NB1643
cells/well were plated in medium (10% FBS/RPMI) containing 5 µM ATRA on poly-lysine-coated 96-well plates (Costar).
Final ethanol concentration was 0.1% through out the experiment.
48 h after ATRA treatment, BDNF (50 ng/ml) and varying concentrations of FBS (0, 0.1, 0.2, and 0.5%) were added in fresh medium containing 5 µM ATRA. After 4 days, wells were
aspirated, and 1 µM Calcein AM (Molecular Probes) was
added in Hanks' solution (Life Technologies, Inc.) for 1 h at
37 °C. Fluorescence was quantitated using a cytofluor fluorescent
plate reader (Millipore). Fluorescent signal from Calcein produced by
reactions catalyzed by cellular esterases is linear with respect to
viable cell number. In order to establish that detection of cells with
Calcein AM is linear, 5, 10, 25, 50, and 75 × 103
cells were plated in 96-well plates and cultured overnight. For Calcein
AM detection, quadruplicate wells were aspirated, and 1 µM Calcein AM (Molecular Probes) was added in Hanks'
solution (Life Technologies, Inc.) for 1 h at 37 °C.
Fluorescence was quantitated using a cytofluor fluorescent plate
reader. For direct assessment of cell number, triplicate wells were
trypsinized, and nuclei were prepared and counted as described above.
Survival Assays in Low Serum--
2.5 × 105
NB1643 cells were plated in 3.5-cm tissue culture wells (Costar) coated
with poly-lysine with 5 µM ATRA in 10% FBS/RPMI. Final
ethanol concentration was 0.1% in all wells. Medium was changed
48 h later to medium with or without 50 ng/ml BDNF and 5 µM ATRA in 0.1% FBS/RPMI. Triplicate wells were
trypsinized, and nuclei were counted as described above at various
times subsequent to BDNF addition.
Growth Assays--
5 × 105 NB1643 cells were
plated per well in 6-well tissue culture dishes (3.5-cm-diameter wells)
(Costar). Medium was changed 24 h later to medium with or without
5 µM ATRA. For the BDNF growth rate experiments, 2.5 × 105 NB1643 cells were plated per well in 6-well tissue
culture dishes (3.5-cm-diameter wells) (Costar). Medium was changed
24 h later to medium with or without 5 µM ATRA.
After 48 h, BDNF was added (100 ng/ml). Final ethanol
concentration was 0.1% in all wells. Triplicate wells were harvested
by trypsinization and nuclei were counted as described above. Linear
regression and statistical analysis to determine if the dependence of
growth rate (slopes) of NB1643 differ significantly were performed
using the method described by Zar (26) with Prism software (GraphPad).
Northern Analysis--
4 × 106 NB1643 cells
were plated in 10-cm dishes and treated 1 day later with or without
ATRA. Final ethanol concentration was 0.1% in both samples. After 5 days, total cellular RNA was isolated using RNAZol B using the method
provide by the manufacturer (Tel-Test) and quantitated and
characterized by using UV absorbance at 260 and 280 nM.
Total RNA preparations were examined by resolution on an agarose gel
with ethidium bromide staining to verify that the 18 S and 28 S
ribosomal RNA was not degraded. 20 µg of total RNA isolated from
NB1643 cells and 0.24-9.5-kilobase RNA ladder for size analysis (Life
Technologies, Inc.) was resolved on a agarose gel containing
formaldehyde (27). Northern transfer of the RNA to Hybond-N membrane
(Amersham Pharmacia Biotech) was accomplished using a Posiblot pressure
blotter (Stratagene) followed by UV cross-linking using a UV
Stratalinker (Stratagene). 32P-Labeled TRK
cDNA probe was synthesized using a random primed DNA labeling kit
(Roche Molecular Biochemicals) using the EcoRI restriction
fragment from pDM69 (28). Prehybridization was in 5× SSPE, 0.5% SDS,
5× Denhardt's solution, and 100 µg/ml salmon DNA prepared for
hybridization (Life Technologies, Inc.) for 2 h at 65 °C.
Probes were added for hybridization at 65 °C for 18 h at label
concentrations of 2.5-5 × 106 cpm/ml. Low stringency
washes were followed by two high stringency washes for 10 and 2 min,
respectively, in 0.2× SSPE and 0.1% SDS pre-equilibrated to 60 °C.
Images were visualized using a PhosphorImager (Molecular Dynamics).
RT-PCR of TRKB, BDNF, and NGF--
4 × 106
NB1643 cells were plated in 10-cm dishes (Corning) and treated with or
without ATRA 1 day later for analysis of TRKB, BDNF, and NGF mRNA. For analysis of
FOS induction, 2 × 106 NB1643 cells were
plated in 10-cm dishes and treated with or without ATRA 1 day later.
After 5 days, cells were treated with 100 ng/ml BDNF or NGF for 30 min.
Ethanol concentration was 0.1% in all dishes. Total RNA was prepared
and characterized as discussed above at 1, 2, and 5 days. cDNA was
made using the cDNA Cycle kit (Invitrogen) using 1 µg of total
RNA. One-tenth of the cDNA was used for PCR. 1 µM
primers, corresponding to the extracellular domain of TRKB
(5'-TCTCGAATCTCCAACCTCAG-3', 5'-TACTTCTGTTCGTGGTGTCC-3'), were used for
PCR using GeneAmp kit (Perkin-Elmer) using standard conditions with the
inclusion of 0.2 µl [33P]dATP per sample.
Taq was added after 5 min at 94 °C and 6 min at 60 °C,
and then 25 cycles with 2 min of extension steps at 72 °C, 1 min of
denaturation steps at 94 °C, and 1 min of annealing steps at
60 °C were performed, followed by a final 10-min extension at
72 °C. 1 µl of glyceraldehyde 3-phosphate dehydrogenase
(G3PDH) primers (CLONTECH) were used in
each reaction. Conditions for analysis of BDNF and
NGF were identical, except that 30 reaction cycles were
performed using 1 µl of transferrin primers
(CLONTECH). BDNF primers were
5'-GCAACGGCAACAAACCACAACATTATC-3' and
5'-GTCCCTGTATCAAAAGGCCAACTGAAG-3', and NGF primers were
5'-GCCCTTGATGTCTGTGGCGGTGGTC-3' and 5'-GCTTTTCTGATCGGCATACAGGCGG-3'. For FOS PCR, conditions were identical except that annealing
was at 57 °C. FOS primers (Continental Laboratory
Products) were 5'-CTACGAGGCGTCATCCT-3' and 5'-TCTGTCTCCGCTTGGAGTGTA-3',
and G3PDH primers were used for normalization. Samples,
along with 123-bp ladder DNA (Life Technologies, Inc.), were analyzed
on a 4% nondenaturing acrylamide:bisacrylamide (19:1) gel in TBE
buffer (29) and quantitated using a PhosphorImager. Levels of induction
were determined by normalizing to G3PDH or transferrin PCR products. PCRs were carried out with TRKB,
BDNF, NGF, and FOS primers and titrations of templates to ensure that the reactions were linear. G3PDH, transferrin,
and FOS PCR products exhibited log-linear relationships
between 15 and 30, 20 and 35, and 15 and 30 reaction cycles,
respectively. The lane containing the DNA ladder was cut from the gel
and stained in ethidium bromide for size analysis. Control cDNA
synthesis reactions without addition of reverse transcriptase to RNA
preparations, followed by PCR, did not result in detectable PCR
products. This verifies that the PCR products are derived from mRNA
as opposed to genomic DNA.
Immunoblotting Analysis of TRK and TRKB--
For analysis of TRK
and TRKB expression induced by ATRA, 4 × 106 NB1643
cells were plated in 10-cm dishes (Corning) and treated with or without
5 µM ATRA 2 days later. Ethanol concentration was 0.1%
in all samples. For analysis of 9-cis-RA induction, 4 × 106 NB1643 cells were plated with either 5 µM ATRA or 9-cis-RA for 5d. Cells in each dish
were lysed in 1 ml of RIPA (30) with fresh 1 mM
phenylmethylsulfonyl fluoride (diluted from 100× stock in methanol) at
4 °C, and repipetted four times through a 25 G needle. Samples were
either stored at
80 °C or used directly. Samples were thawed, and
lysates were cleared by centrifugation at 10,000 × g
for 30 min. To immunoprecipitate TRK and TRKB, supernatants were
incubated with 10 µl of TRK C-14 antisera (Santa Cruz) and then
incubated with rotation with 20 µl of protein A-Sepharose (Repligen)
at 4 °C. The immune complexes were pelleted in a microcentrifuge and
then resuspended in 1 ml of RIPA, vortexed, and pelleted in a
microcentrifuge. Washes were repeated three times, after which, the
remaining RIPA buffer was removed from the beads by aspiration through
a 26 gauge needle. The samples were then taken up in 30 µl of 2×
SDS-polyacrylamide gel electrophoresis sample buffer. After
SDS-polyacrylamide gel electrophoresis, the gels were transferred to
Immobilon-P (Millipore) using the Milliblot semidry apparatus (Millipore). Immunoblot analysis to detect TRK and TRKB was performed as follows at 22 °C. The membrane was blocked for 1 h in 5%
milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.05% Tween-20). The blot was washed once for 15 min and twice
for 5 min with TBST and then incubated with TRK C-14 (1:200) in 1%
milk/1% bovine serum albumin in TBST for 1 h. The membrane was
then washed as above and incubated with anti-rabbit horse radish
peroxidase in 1% milk/1% bovine serum albumin in TBST for 1 h.
The membrane was then washed for 15 min and then four times for 5 min
in TBST. TRK and TRKB bands were then visualized using chemiluminescent detection with the ECL kit (Amersham Pharmacia Biotech) and XAR 5 film
(Eastman Kodak Co.). For analysis of TRK- or TRKB-specific expression,
2 × 106 NB1643 cells were plated with either 0 or 5 µM ATRA for 4 days. TRK was immunoprecipitated using 1 µg of TRK-specific antibodies (catalog no. 9142, New England Biolabs)
followed by immunoblotting with TRK C-14 antisera using the methods
described above. For TRKB expression, TRKs were immunoprecipitated with
TRK C-14 antisera followed by immunoblotting with TRKB-specific
antiserum 5050 (1:400) (13, 31) and TRKB antibodies (1:200) (catalog
no. 794, Santa Cruz).
Kinase Activity of TRK and TRKB--
For analysis of NGF-induced
autophosphorylation, 4 × 106 NB1643 cells were plated
in 10-cm dishes (Corning) with or without 5 µM ATRA. 5 days later, cells were treated with 50 ng/ml of NGF for 5 min. For
analysis of BDNF-induced autophosphorylation, 5 × 106
cells were plated and treated with or without 5 µM ATRA 1 day later. 5 days later, cells were treated with 50 ng/ml BDNF for 5 min. Ethanol concentration was 0.1% in all dishes. Cells were lysed
immediately at 4 °C, and TRKs were immunoprecipitated, resolved using SDS-polyacrylamide gel electrophoresis, and transferred to
Immobilon-P as described above. Immunoblot analysis to detect TRKs was
performed as described above. Immunoblot analysis to detect
phosphotyrosine was performed as follows at 22 °C. The membrane was
blocked for 2 h in 5% bovine serum albumin (Sigma, catalog no.
A-6003) in TBST, and then fresh 4G10 anti-phosphotyrosine antibodies
(1:5000) (Upstate Biotechnology) were added for 1 h with rotation.
The blot was washed once for 15 min and twice for 5 min with TBST and
then incubated with anti-mouse horseradish peroxidase in 5% bovine
serum albumin (Sigma, catalog no. A-6003) in TBST. The membrane was
then washed for 15 min and then four times for 5 min in TBST.
Antiphosphotyrosine containing protein bands were then visualized using
chemiluminescent detection with the ECL kit (Amersham Pharmacia
Biotech) and XAR 5 film (Kodak).
Cisplatin Toxicity Assays--
2.5 × 104
NB1643 cells/well were plated in quadruplicate in medium (10%
FBS/RPMI) containing 5 µM ATRA on poly-lysine coated 96 well plates (Costar). Final ethanol concentration was 0.1% through out
the experiment. 48 h after ATRA treatment, BDNF (50 ng/ml) and
varying concentrations of cisplatin were added in fresh medium
containing 5 µM ATRA. Medium containing drug was removed 24 h later and replaced with fresh medium with BDNF (50 ng/ml) and
5 µM ATRA. After 5 days, wells were aspirated, and 1 µM Calcein AM (Molecular Probes) was added in Hanks'
solution (Life Technologies, Inc.) for 1 h at 37 °C.
Fluorescence was quantitated using a cytofluor fluorescent plate reader
(Millipore). Fluorescent signal resulting from Calcein AM product
produced by cellular esterases is linear with respect to viable cell
number. Data were analyzed with sigmoidal dose response equations using
Prism (GraphPad) to estimate EC50 values. Cell viability
assays with or without NGF (50 ng/ml) were done using the same
protocol, except that the assay was read with Calcein AM after 6 days
following the 24 h cisplatin pulse.
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RESULTS |
A Drug-sensitve Model of NB--
As there is evidence that BDNF
and its corresponding receptor, TRKB, may have a role in NB exhibiting
poor prognosis (23), we wanted to establish a model of NB using a cell
line to test the role of BDNF on growth and survival of NB cells. To
this end, we characterized a drug-sensitive cell line, NB1643, which
has N-MYC amplification and is derived from a stage D NB.
The cell line was established from a tumor isolated from a patient who had not yet been treated with chemotherapeutic drugs (32), which may
explain the unique drug-sensitive nature of this NB cell line. ATRA
induces a striking differentiated phenotype in the NB1643 cell line
that is most clearly characterized by extensive neurite outgrowth (Fig.
1). There is also a distinct
morphological change in the cell body, which can be described as
flattening and elongation. Neurite outgrowth started within a day of
ATRA addition and was extensive after 5 days. In addition, NB1643 cells
were less aggregated after differentiation induced by ATRA. As might
well be expected, differentiation in response to ATRA treatment was
accompanied by a reduced rate of cell growth (Fig.
2A). ATRA increased cell doubling time from 2.5 days to 3.5 days. However, there was still cell
proliferation, in the presence of serum, even 5 days after ATRA
addition. On the other hand, BDNF did not significantly alter the
growth rate observed in these differentiated cells in the presence of
serum (Fig. 2C). BDNF had no effect on the growth rate of
undifferentiated cells (Fig. 2B), which was expected because undifferentiated NB1643 cells do not express TRKB (see below). We next
wanted to test whether or not BDNF promoted survival in differentiated
NB1643 cells.

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Fig. 1.
ATRA induces a striking phenotypic
differentiation of NB1643 cells characterized by neurite
outgrowth. NB1643 cells were cultured in 0 or 5 µM
ATRA for 5 days. Photomicrograph magnification is × 225.
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Fig. 2.
Growth of NB1643 cells is decreased by ATRA
but unaffected by BDNF. NB1643 were grown in medium containing 0 or 5 µM ATRA and with or without 100 ng/ml BDNF. Cell
number was determined by harvesting and counting nuclei. A,
ATRA alone reduced the proliferation rate of NB1643 cells
(p = 0.002). B, BDNF (100 ng/ml) had no
significant effect on the growth rate of undifferentiated cells
(p = 0.66). C, BDNF had no significant
effect on the growth rate of differentiated cells (p = 0.35), which were cultured for 2 days in 5 µM ATRA and
then grown in medium containing 10% FBS and with or without 100 ng/ml
BDNF.
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BDNF Promotes Survival of Differentiated Human NB Cells--
BDNF
promotes survival of cells under stress resulting from varying levels
serum (Fig. 3A). It is
interesting that increasing levels of serum in the presence of a
constant concentration of BDNF results in increased survival. Perhaps
there is synergism between BDNF and a growth factor in serum that
promotes survival. Alternatively, perhaps pretreatment with BDNF is
required for the effects of a growth factor in serum in these NB cells.
Cellular viability was assessed using Calcein AM, which is an
established method for measuring cell viability. This assay requires
both cellular esterases for conversion of cell-permeable,
nonfluorescent, Calcein AM to fluorescent Calcein and intact plasma
membranes for retention of the cell impermeant, highly charged, Calcein (33-36). The production of Calcein from Calcein AM by cellular esterases in intact cells was linear with respect to cell number (Fig.
3B). In an independent assay, in which cell number was
counted directly, it was also demonstrated that BDNF induced increased survival of NB1643 in low serum (Fig. 3C). Because exogenous
BDNF increased cell survival in NB1643 cells, we next wanted to assess the expression of TRKB, the receptor for BDNF.

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Fig. 3.
BDNF promotes survival of NB1643 cells in low
serum conditions. A, BDNF increases cellular viability
in low serum conditions. NB1643 cells were cultured for 2 days in 5 µM ATRA. Cells were then cultured in medium containing
varying levels of serum (0, 0.1, 0.2, or 0.5% FBS) with or without
BDNF (50 ng/ml) for 4 days, after which, cellular viability was
assessed by using Calcein AM. B, Calcein AM detection of
cells was linear with respect to cell number. NB1643 cells were plated
at varying densities and grown overnight. Replicate wells were either
assayed with Calcein AM or nuclei were harvested for direct counting.
C, BDNF increased survival of NB1643 in low serum
conditions. NB 1643 cells were cultured 2 days in 5 µM
ATRA and complete medium and then cultured in 5 µM ATRA
in 0.1% FBS/RPMI medium with or without 50 ng/ml BDNF. Cell number was
quantitated by preparing and counting nuclei.
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TRKB and TRK Are Induced by ATRA--
It seemed likely that NB
cell differentiation may be comparable to NGF-induced differentiation
of rat PC12 cells, because the origins of these cell lines are similar.
We examined the expression of both TRK and TRKB and their corresponding
ligands, NGF and BDNF. ATRA induced expression of TRK
3.2-kilobase mRNA in NB1643 cells (Fig.
4). Although TRK mRNA was
not detected in the cell prior to ATRA treatment, TRK protein was
expressed in NB1643 cells (see Fig. 6A, below). On the other
hand, TRKB protein was not detected in these cells prior to ATRA
treatment (see Fig. 6A, below). TRKB mRNA
expression was induced about 7-, 10-, and 17-fold after 1, 2, and 5 days of ATRA treatment, respectively (Fig.
5A). Clearly, regulation of
TRK and TRKB by ATRA occurs at the transcriptional level. However,
these experiments do not rule out the possibility of a translational
contribution to the regulation of TRK and TRKB levels. The time course
of expression of both TRK and TRKB proteins following ATRA treatment
was determined using antibodies raised against the C-terminal 14 amino
acids of TRK, which recognize TRK and TRKB (Fig.
6A). Interesting, TRK was
induced in 1 day, whereas TRKB was induced after 2 days and TRKB
expression was further increased at 5 days following ATRA treatment.
Although TRK was expressed before ATRA treatment, ATRA treatment
further increased the levels of TRK. On the other hand, TRKB protein
was not detected before ATRA treatment in NB1643 cells. Because the antibody raised to the C-terminal 14 amino acids recognizes both TRK
and TRKB, the expression of these proteins was examined using specific
antibodies. Immunoprecipitation with a TRK-specific antibody followed
by immunoblotting with the C-14 TRK antibodies confirmed that TRK was
expressed and induced by ATRA in these cells (Fig. 6C).
Immunoprecipitation with a TRK C-14 antibodies followed by immunoblotting with TRKB-specific antibodies (13, 31) confirmed that
TRKB was expressed and induced by ATRA in these cells (Fig. 6D). In summary, ATRA induces full-length TRKB, which is a
receptor for BDNF and a protein-tyrosine kinase.

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Fig. 4.
ATRA induces expression of TRK
mRNA. Northern analysis on 20 µg of total RNA isolated
from NB1643 cell lines grown in 0 or 5 µM ATRA for 5 days. The top panel is hybridized with a human TRK
32P-labeled probe, whereas the bottom panel is hybridized
with a human 32P-labeled G3PDH probe to verify
equivalent loading of RNA samples. The 3.2-kilobase TRK
mRNA and G3PDH mRNA are indicated with
arrows.
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Fig. 5.
ATRA induces expression of TRKB,
and BDNF and NGF are
expressed in NB1643 cells. Total RNA was isolated from NB1643
cells grown in 0 or 5 µM ATRA for 1, 2, and 5 days.
A, RT-PCR analysis for expression of TRKB and
G3PDH are shown. Arrows indicate the predicted
371-bp TRKB and 983-bp G3PDH PCR products.
B, RT-PCR analysis for expression of BDNF,
NGF, and G3PDH are shown. NB1643 cells were
treated with 0 or 5 µM ATRA. Arrows indicate
the predicted 492-bp BDNF, 432-bp NGF, and
1347-bp transferrin receptor PCR products. 33P-Labeled PCR
products were analyzed using a PhosphorImager.
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Fig. 6.
ATRA and 9-cis-RA induce
expression of TRKB and TRK. NB1643 cells were grown in 0 or 5 µM ATRA or 9-cis-RA. gp140TRK and
gp145 TRKB bands are indicated with arrows.
A, immunoprecipitation of TRKs followed by immunoblotting
analysis is depicted at 1, 2, and 5 days after ATRA treatment.
B, immunoprecipitation of TRKs followed by immunoblotting
analysis is depicted after cells were grown in either ATRA or
9-cis-RA. C, TRK was immunoprecipitated with
TRK-specific antiserum followed by immunoblotting with TRK C-14
antisera. D, TRKs were immunoprecipitated with C-14 TRK
antiserum followed by immunoblotting using TRKB-specific antibodies
(794 and 5050) and generic TRK C-14 antibodies.
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There is considerable clinical interest in both ATRA and the recently
discovered hormone, 9-cis-RA, as biological modifiers (37).
Therefore, experiments on the effects of the latter hormone on NB1643
cells were also carried out. 9-cis-RA also causes
differentiation and expression of TRK and TRKB in NB1643 cells,
although it is less potent than ATRA (Fig. 6B).
BDNF and NGF Are Expressed in NB1643 Cells--
Next, we
determined whether BDNF or NGF is expressed in these cells.
BDNF and, to a lesser extent, NGF, are expressed
in NB1643 cells (Fig. 5B). ATRA treatment results in a
decrease in BDNF mRNA levels. It seems likely that
differentiation of NB1643 cells induced by ATRA or 9-cis-RA
may be due to an autocrine loop involving BDNF and TRKB, respectively,
and perhaps to a lesser extent, NGF and TRK. The time course of
induction of TRKB, as opposed to TRK, is more closely correlated with
the time course of phenotypic differentiation. This supports the notion
that differentiation may be the result of an autocrine mechanism
involving BDNF and TRKB.
TRKB and TRK Are Functional in NB1643 Cells--
The initial step
in signal transduction involving TRKs is ligand-induced dimerization
(38) and autophosphorylation of the receptors, which results in
increased protein-tyrosine kinase activity. In order to determine
whether TRKB and TRK are functional, experiments to investigate ligand
stimulated kinase activity were undertaken. In NB1643 cells, ATRA
induced TRK and TRKB expression; however, only TRK was
autophosphorylated in response to NGF (Fig. 7A). TRK was not
phosphorylated on tyrosine in the absence of NGF (data not shown).
Likewise, BDNF definitively stimulated only autophosphorylation of TRKB
receptor in ATRA-treated NB1643 cells (Fig. 7B). Both BDNF
and NGF definitively increased protein-tyrosine kinase activity of TRKB
and TRK, respectively, in differentiated NB1643 cells. This
demonstrates that, at a minimum, TRKB and TRK are functional
ligand-dependent kinases in NB1643 cells.

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Fig. 7.
ATRA induces TRK and TRKB, which is competent
for NGF- and BDNF-induced autophosphorylation. A,
NB1643 cells grown for 5 days in 0 or 5 µM ATRA were
treated with 50 ng/ml NGF for 5 min. Immunoprecipitation of TRKs,
followed by anti-phosphotyrosine and anti-TRKs immunoblotting indicates
that gp140TRK, but not gp145 TRKB, is
phosphorylated on tyrosine in response to NGF. B, NB1643
cells grown for 5 days in 0 or 5 µM ATRA were treated
with 0 or 50 ng/ml BDNF for 5 min. Immunoprecipitation of TRKs,
followed by antiphosphotyrosine immunoblotting indicates that
gp145TRKB, but not gp140TRK, is phosphorylated
on tyrosine in response to BDNF.
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BDNF and NGF Induce FOS--
One of the earliest detectable events
in growth factor-induced mitogenesis or NGF-induced differentiation of
PC12 cells is induction of the immediate early gene, FOS. In
NB1643 cells differentiated by ATRA treatment, BDNF and, to a lesser
extent, NGF induced FOS expression 19.5- and 2.5-fold,
respectively (Fig. 8). This experimental result, coupled with evidence that both TRKB and TRK have kinase activity, indicates that, at a minimum, some of the neurotrophin signaling pathways resulting in gene expression are competent in NB1643
cells. Because BDNF induces FOS expression more effectively than NGF, it seems likely that an autocrine loop involving BDNF and
TRKB may contribute more to the differentiated phenotype of the cells
than NGF and TRK. To more directly test whether BDNF is involved in
differentiation of these cells, we investigated whether exogenous BDNF
treatment resulted in phenotypic changes.

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Fig. 8.
BDNF and NGF induce FOS
expression. NB1643 cells were treated with 5 µM ATRA. After 5 days, cells were treated with either
BDNF or NGF (100 ng/ml) for 30 min and then analyzed for FOS
and G3PDH expression using RT-PCR. 33P-Labeled
PCR products were analyzed using a PhosphorImager. Arrows
indicate the predicted 482-bp FOS and 983-bp
G3PDH PCR products.
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Exogenous BDNF Causes an Increase in Neurite Outgrowth--
NB1643
cells were pretreated with ATRA for 2 days and then cultured with or
without exogenous BDNF for 12 days (Fig.
9). BDNF causes increased neurite
outgrowth, which suggests that ATRA induces differentiation by
up-regulating TRKB expression. This in turn could form an autocrine
loop involving BDNF and TRKB resulting in differentiation of NB1643
cells. Because exogenous BDNF increased the survival of NB1643 cells in
low serum, we next addressed whether neurotrophins might protect NB
cells from chemotherapeutic drugs.

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Fig. 9.
Exogenous BDNF treatment causes increased
neurite outgrowth in NB1643 cells. NB1643 cells were cultured in 5 µM ATRA for 2 days and then grown with or without
exogenous BDNF (50 ng/ml) for 12 days. Rows A and
B depict different fields in the same sample.
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BDNF and NGF Protect NB Cells from Cisplatin--
As cisplatin is
one of the drugs used in chemotherapeutic treatment of NB, we tested
whether or not BDNF afforded protection from this drug. BDNF does
indeed protect NB1643 cells from cisplatin (Fig.
10A). The viability of
NB1643 cells in varying concentrations of cisplatin with and without
BDNF shows that BDNF induces almost a 2-fold shift in cisplatin
toxicity. The EC50 values were estimated to be 2.7 and 5 µg/ml without or with BDNF treatment, respectively. Although
resistance, determined by these assays, was modest, it could well
represent a clinically significant level (see discussion below). The
protection afforded by BDNF was reproduced in two additional
experiments. This protection is unlikely to be afforded by a withdrawal
from cell cycle, because, as shown in Fig. 2C, BDNF did not
significantly alter growth of NB1643 cells. NGF also protects NB1643
cells from cisplatin. The viability of NB1643 cells in varying
concentrations of cisplatin with and without NGF shows that NGF also
induced a similar shift in cisplatin toxicity (Fig. 10B).
BDNF and NGF, therefore, have a similar protective effect against
cisplatin treatment. This is not surprising considering the sequence
homology and functional similarities between the TRK family
members.

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Fig. 10.
BDNF and NGF protect NB1643 cells from
cisplatin. NB1643 cells cultured for 2 days in ATRA were treated
simultaneously with and without 50 ng/ml BDNF and cisplatin for 24 h (A) or with or without 50 ng/ml NGF and cisplatin for
24 h (B). After 24 h, medium was replaced with
fresh medium containing BDNF or NGF. Cell viability was assayed 5 (A) or 6 (B) days later using Calcein AM.
Error bars represent S.E.
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DISCUSSION |
The data presented here indicate that BDNF is a trophic factor for
NB cells, at least in this model of NB. Perhaps more significantly, BDNF protects these NB cells from cisplatin, which is a drug that damages DNA. We tested this hypothesis, in part, because of the extensive data for the function of neurotrophins in models addressing neurodegenerative and other trauma-induced diseases of the nervous system. These studies range from findings that BDNF and TRKB are induced in models of stress to data from an in vivo model
suggesting that TRKB is required for survival of damaged neurons. In
models of neuronal damage in the hippocampus, TRKB and
BDNF expression is increased following cellular insult (39).
Hippocampal kindling, which causes seizures, leads to a rapid transient
increase of TRKB mRNA and protein in the hippocampus.
Levels of TRK or TRKC were not altered by
kindling. Other conditions, such as ischemia and hypoglycemic coma,
resulted in a similar increase in TRKB mRNA in the
dentate gyrus. These treatments also left TRK and TRKC mRNAs levels unaltered. Following exposure to
kainate, which also induces seizures, TrkB mRNA
increased in dentate granule cell and CA1 pyramidal cell layers of the
adult hippocampus (40). A similar increase in BDNF mRNA was also
observed following kainate treatment in the pyramidal and granule cell
regions. Spinal cord lesions in rats, which do allow some axonal
regrowth, lead to increased levels of TRKB, suggesting a role for TRKB
in axonal sprouting in injured spinal cords (41, 42). These results suggest that up-regulation of TRKB and BDNF may form an autocrine loop
promoting cell survival. This may be a protective response to neuronal insult.
There are data that support a target-derived mechanism for trophic
support for neurons provided by neurotrophins. However, in experiments
designed to test an autocrine role for BDNF using BDNF and TRKB
antisense oligonucleotides resulted in increased death of sensory
neurons (43). This provides direct evidence that BDNF may function in
an autocrine fashion in some neuronal populations. BDNF protects rat
hippocampal, septal, and cortical cultured neurons against metabolic
and excitotoxic insults (44). Activation of voltage-sensitive calcium
channels results in increased survival of cultured rat embryonic
cortical neurons (45). It was shown, using antibodies to BDNF, that
BDNF is required for this observed increase in survival. Taken
together, these studies suggest that BDNF may have a role in protection
of mature neurons from cellular insults. It seemed plausible, based on
these studies, that neurotrophins may protect cells from a plethora of
stressful conditions, which led us to investigate and demonstrate that
BDNF induces drug resistance in a NB cell line.
BDNF and NGF induce about a 2-fold shift in the EC50 values
for cisplatin toxicity in NB1643 cells, indicating that the factors do
induce drug resistance. The complete survival observed in the presence
of neurotrophins is even greater, as it seems to derive from a combined
effect on survival and drug resistance by these factors. To address the
clinical significance of these data, we have contrasted them with
clinical pharmacokinetic data on serum cisplatin levels. Delivery of
cisplatin by infusion of 90 mg/m2 over 6 h resulted in
serum levels between 1-4 µg/ml that declined rapidly (46). In
another study contrasting infusion of 100 mg/m2 cisplatin
over varying periods, 2-7 h, peak cisplatin serum levels were in the
1-5 µg/ml range (47). Shorter infusion periods resulted in
predictably higher peak serum concentrations of cisplatin. Infusion
over the longest period, 7 h, resulted in serum cisplatin concentrations of 1 µg/ml with a several-hour plateau. Cisplatin levels declined rapidly following infusion in every case. This concentration range (1-5 µg/ml) is comparable to the effective concentration range of cisplatin presented in Fig. 10. If the
sensitivity of NB cells to cisplatin is altered in this concentration
range, it may allow survival of some NB cells. Clinically relevant drug resistance may not require order of magnitude shifts, but rather just
severalfold shifts in the concentrations of drugs required to induce
cell death. Cells need only survive the achievable therapeutic dose to
have effective drug resistance.
In vitro selection of two different NB cell lines using
escalating doses of cisplatin resulted in resistant cell lines (48). The resistant cell lines, IMR and SK-N-SH, were 6.6- and 3.8-fold, respectively, more cisplatin-resistant than the parental NB lines. The
parent SK-N-SH line was more resistant originally than the parent IMR
line. Mechanisms of resistance in these cell lines were not
established, although the involvement of enhanced DNA repair is
discussed. These data also underscore that shifts in the cisplatin
concentrations required to induce cell death resulting in drug
resistance may be small.
The findings presented here and previously (49, 50) underscore the
importance of an autocrine loop involving TRKB and BDNF in
differentiation of NB cells. In SH-SY5Y NB cells, TRKB is induced by
ATRA and, here, treatment with exogenous BDNF causes differentiation.
ATRA treatment of KCNR cells, which already express BDNF, induces TRKB
expression and subsequent differentiation. However, in 15N NB cells,
ATRA induces only a truncated TRKB receptor and although these cells
express BDNF, ATRA does not induce differentiation. These reports, and
the data reported here, provide evidence that ATRA induces
differentiation by creating an autocrine loop with BDNF and TRKB.
It seems plausible that ATRA, by regulating expression of TRK and TRKB,
may be required for normal development or maintenance of sympathetic
neurons. ATRA induces NGF-dependent survival in embryonic
day 7 sympathetic neurons from chick (51). ATRA treatment of embryonic
chick sympathetic neurons leads to increased levels of TRK, resulting
in NGF-dependent survival (52). This response is mediated
by the
-retinoic acid receptor. Although not conclusive, these
studies implicate ATRA in a developmental role during sympathetic neuronal development. A reasonable hypothesis is that ATRA, or other
retinoids, may be required for induction of TRK family members during
development of sympathetic neurons. Experiments with a panel of
retinoic acid receptor selective retinoids has provided evidence that 3 distinct retinoic acid receptor/retinoid X receptor heterodimers may be
involved in mediating the effects of ATRA and 9-cis-RA in NB
cells (53).
In contrast to TRKB expression in NB, there are findings that
TRK expression is correlated with a favorable prognosis in
NB (54-56). In addition to a correlation between TRK
expression and stage, there is an inverse relationship between
TRK and N-MYC expression in NB (56-58).
N-MYC amplification is a well established negative
prognostic indicator for NB (59). Although the evidence is compelling
that TRK expression is a positive prognostic indicator for
NB, the reason TRK expression is correlated with prognosis and stage is
unknown. One explanation for this correlation between TRK
expression and a favorable outcome in NB is that TRK might cause
differentiation and regression of low stage NB. Alternatively, low
stage NB with favorable prognosis may require TRK for survival, whereas
high stage NB may not. In contrast, in this study, we have examined
neurotrophin function in a cell line derived from a high stage NB tumor.
Investigations of some NB cell lines have suggested that restoration of
TRK expression, either by transfection or up-regulation, leads to differentiation in response to NGF. Transfection of
TRK cDNA into HTLA230 cells followed by NGF treatment
resulted in growth arrest and differentiation, as well as
N-MYC down-regulation (60). This cell line, HTLA230, was
isolated from a patient with stage IV disease and notably lacks
expression of TRK. In SH-SY5Y, NGF in conjunction with aphidicolin,
an inhibitor of DNA polymerase
, induce differentiation
(61).
Differentiation is accompanied by up-regulation of TRK and
down-regulation of c-MYC. In another report on the same cell
line, the role of TRK was tested directly by exogenous expression of TRK (62). SH-SY5Y cells engineered to express TRK differentiate in
response to exogenous NGF treatment. In contrast to the previous report
(61), treatment of these cells with
12-O-tetradecanoylphorbol-13-acetate induced endogenous TRK
expression, but the cells did not differentiate in response to NGF
treatment. It is possible that TRK expression induced by
12-O-tetradecanoylphorbol-13-acetate is not sufficient for
the sustained response necessary for NB differentiation. Alternatively, there may be defects in the endogenous TRK receptor in these cells. Regardless, these data do suggest that TRK receptor causes
differentiation and growth arrest of NB. This could explain TRK
expression in NB with favorable prognosis and, likewise, how decreased
or loss of TRK expression or defects in signaling events
downstream of TRK could cause NB with poor prognosis.
Whether BDNF requires common or independent signaling pathways for
promoting survival or chemoresistance remains unknown. Neurotrophins,
via TRKs, activate at least three signaling pathways (RAS, PI3K, and
PLC-
1), and perhaps a fourth pathway requiring SNT, which may be
identical to FRS2 (63, 80). Ligand-induced dimerization (38) and
autophosphorylation (38) forms binding sites for protein substrates
containing SH2 (13, 64) and PTB (65) domains. RAS is activated in a
pathway requiring SHC association with TRK (66). Neurotrophins induce
PLC-
1 association with TrkB (13), and PLC-
1 is phosphorylated and
activated (13, 64, 67). PI3K is definitively activated by TRK. However,
it is likely that PI3K does not form a ligand-dependent
association complex with TRK (68). There is, however, a report that
PI3K associates with phosphorylated tyrosine 751 of TRK (69). However, it is more probable that PI3K is activated indirectly by TRK through another mechanism, such as another adapter phosphoprotein or RAS. RAS
can activate PI3K (70). There are also recent data indicating that BDNF
may activate other signaling pathways involving rAPS and SH2-B
(40).
The question of which signaling pathways activated by TRKs are required
for NB cell differentiation and survival is not completely resolved.
PC12 cells differentiate in response to NGF (71). RAS activation is
required for PC12 cell differentiation. SHC, and to some extent
PLC-
1, may be required for differentiation of PC12 cells (66),
although other data indicate redundant roles for PLC-
1 and RAS in
ERK1 activation (72). PC12 cells expressing a point mutant of TRK,
which cannot activate PLC-
1, still extend neurites in response to
NGF (73). There are data suggesting that a fourth pathway may be
activated by TRK involving SNT (74), which may be required for
neuritogenesis, but not survival (75). The role that SNT, which may be
identical to FRS2 (63, 80), has in both differentiation and survival in
response to neurotrophins is not understood. However, FRS2 has been
shown to be required for differentiation of PC12 cells in response to
FGF (76). A role for PI3K in differentiation has also been suggested.
Wortmannin, an inhibitor of PI3K, has been reported to block
NGF-induced neurite outgrowth in PC12 cells (77). It is likely that the
primary role for neurotrophins during development and, perhaps, in the mature adult is as trophic or survival factors. In PC12 cells, NGF
prevents cell death in serum-free medium (71). PI3K signaling may be
required for NGF-induced survival of PC12 cells (78). It will prove
interesting to examine the role that PI3K may have in chemoprotection.
It is now apparent that investigations into the role that SNT or FRS2
has in neurons will also be required to resolve the signaling pathways
required for the phenotypic effects of neurotrophins.
In conclusion, BDNF promotes survival and chemoprotection against
cisplatin in differentiated NB1643 cells. Experiments assessing whether
BDNF affords protection from other DNA-damaging agents, such as
topoisomerase inhibitors and ionizing radiation, are underway. This
will suggest whether BDNF antagonizes a general apoptotic response to
DNA damage or affects just specific classes of DNA-damaging agents.
BDNF has been shown to protect another NB cell line, 15N, which was
engineered to express TRKB, from vinblastine (79). Vinblastine promotes
tubulin depolymerization. In NB1643 cells, ATRA induces a phenotypic
differentiation that is accompanied by increased expression of
functional TRKB receptor protein-tyrosine kinase. Because BDNF is
expressed in these cells, an autocrine loop is formed involving BDNF
and TRKB that is likely involved in differentiation of these cells.
Treatment with exogenous BDNF promotes even greater neurite extension.
It will be of great interest to delineate the signaling pathways
required for survival and drug resistance in NB wells, both at the
membrane receptor level and further downstream in other pathways
controlling cell growth and apoptosis.