The nerve growth factor receptor, TrkA, has a
critical role in the survival, differentiation, and function of neurons
in the peripheral and central nervous systems. Recent studies have
demonstrated a strong correlation between abundant expression of TrkA
and a favorable prognosis of the pediatric tumor, neuroblastoma. This correlation suggests that TrkA may actively promote growth arrest and
differentiation of neuroblastoma tumor cells and may be an important
therapeutic target in the treatment of this disease. In the present
study, we have examined the mechanistic basis for TrkA gene expression
in human neuroblastoma cells. Northern blotting and nuclear run-on
analyses demonstrated that transcription is a primary determinant of
both cell-specific and variable expression of the TrkA gene in
neuroblastoma cell lines that express it to different degrees.
Cell-specific and variable transcription in neuroblastoma cells was
recapitulated by transient transfection of TrkA promoter-luciferase
reporter constructs, and regulatory sequences mediating these processes
were localized to a 138-base pair region lying just upstream of the
transcription initiation region. This neuroblastoma regulatory region
formed multiple DNA-protein complexes in gel shift assays that were
highly enriched in neuroblastoma cells exhibiting abundant TrkA
expression. Thus, TrkA-positive neuroblastoma cells are distinguished
by differential expression of putative transcription factors that
ultimately may serve as targets for up-regulating TrkA expression in
tumors with poor prognosis.
 |
INTRODUCTION |
Neuroblastoma is a predominantly pediatric neoplasia with nearly
all cases occurring in children younger than 10 years of age (1). It is
the major form of extracranial solid tumor in children and accounts for
10-15% of childhood cancer-related deaths (2). Based on the early
onset of this disease and the expression of specific cellular and
developmental markers, neuroblastoma appears to arise from
sympathoadrenal precursors that fail to undergo terminal
differentiation and/or cell death during fetal development (2-4).
Significant advances have been made in prognostic markers for this
disease (5-8). In particular, diploid DNA content, N-MYC
amplification and chromosome 1p deletion are highly associated with
rapid tumor progression and poor outcome. Conversely, near triploidy
and absence of 1p deletions are linked to a favorable outcome in
infants and greater responsiveness to chemotherapy (2, 9). However, the
outcome for neuroblastoma patients remains generally poor despite
multitherapy strategies (2, 10). An interesting and characteristic
feature of neuroblastoma is the occurrence of spontaneous regression or
differentiation into benign ganglioneuromas in a minority of patients,
independent of treatment (2, 11). A critical question is what
regulatory mechanisms characterize these spontaneously regressing
neuroblastomas and distinguish them from the more aggressive,
unfavorable forms?
TrkA is a member of the neurotrophin tyrosine kinase receptor family
that also includes TrkB and TrkC. TrkA specifically mediates signaling
for nerve growth factor
(NGF)1 (although it can also
be activated by NT-3) (12) and is critical for both survival and
terminal differentiation of sympathetic and a subset of sensory neurons
(12-14). Within the central nervous system, TrkA signaling is
important for basal forebrain cholinergic neurons, among other
functions (14). Recent findings have demonstrated that abundant
expression of TrkA is strongly correlated with favorable prognosis for
neuroblastoma, while low or absent expression is linked to a poor
outcome (15-19). TrkA expression within neuroblastomas occurs
specifically in neuroblasts and differentiated ganglion cells, with the
highest levels occurring in the latter cells (20). In addition, forced
expression of TrkA in neuroblastoma cells lacking this receptor
converts them into NGF-responsive cells that undergo growth arrest and
terminal differentiation in the presence of NGF (17, 21-23). These
findings have led to the suggestion that TrkA expression in
neuroblastoma tumors actively promotes their growth arrest and
differentiation into a regressed or benign state (16, 24).
Alternatively, TrkA expression may be associated with a generally more
differentiated state in neuroblastoma cells that are predisposed to
growth arrest and further differentiation.
Whether an active participant in neuroblastoma tumor arrest or simply a
marker for a more differentiated state, elucidating the mechanisms
responsible for TrkA expression (and its absence) in neuroblastoma
cells is potentially of great importance for understanding and treating
this disease. In particular, it may ultimately reveal regulatory
mechanisms that distinguish favorable from poor prognosis tumors that
could serve as future therapeutic targets. The existence of human
neuroblastoma cell lines that express TrkA at low or moderate to high
levels provides an opportunity to explore the determinants of its
variable expression in this tumor cell type. To this end, we have
examined the role of gene transcription in the differential expression
of TrkA in various human neuroblastoma cell lines and have
characterized the human TrkA promoter in these cells using transient
transfection. Our findings indicate that proximal regulatory sequences
play a critical role in both cell-specific and variable TrkA promoter
activity in neuroblastoma cells.
 |
MATERIALS AND METHODS |
Cell Culture--
The human neuroblastoma cell lines SH-SY5Y,
IMR32, LA-N-6, SK-N-SH, SMS-KCN (25-29) and kidney K293 cells were
cultured in complete RPMI 1640 medium (Life Technologies, Inc.)
supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 50 units/ml sodium penicillin, and 50 units/ml streptomycin sulfate at
37 °C in humidified 5% CO2.
Preparation of Total RNA and Northern Blotting--
Total RNA
was extracted from fresh or frozen cell pellets using the guanidinium
isothiocyanate/CsCl2 method (30). Twenty µg of total RNA
were separated on formaldehyde gels and electrophoretically transferred
to GeneScreen Plus membranes (NEN Life Science Products). Membranes
were hybridized with a 1.5-kb BamHI-KpnI fragment
from the human TrkA cDNA pLM6 (31) that was labeled using random primers. Variation in the loading of total RNA was normalized by
hybridization with a 1.2-kb PstI fragment derived from a
human glyceraldehyde-3-phosphate dehydrogenase cDNA (pHcGAP)
(32).
Isolation of Nuclei and Nuclear Run-on Analysis--
Cell nuclei
were isolated using a modification of the method of Greenberg and Ziff
(33). Approximately 2-3 × 107 cells were lysed and
homogenized in hypotonic buffer (10 mM Tris-HCl, pH 7.4, 70 mM NaCl, 3 mM MgCl2, 1% Nonidet
P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 3 mM dithiothreitol) at 4 °C. The nuclei were pelleted and
resuspended in 200 µl of storage buffer (25% glycerol, 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
3 mM dithiothreitol, and 1 µg/ml each of the proteinase inhibitors pepstatin A, bestatin, aprotinin, and leupeptin) at a
concentration of 5-7.5 × 107 nuclei/ml.
The run-on reaction was performed with fresh nuclei as described
previously (34). The reaction was run at 37 °C for 30 min, and RNA
was extracted by the acid-phenol method (35). Various human
complementary DNAs (cDNAs) used were as follows: TrkA,
BamHI- and KpnI-digested pLM6;
glyceraldehyde-3-phosphate dehydrogenase, PstI-digested
pHcGAP (32); and
-actin, XhoI-digested pHFbeta-A-1 (36)
and EcoRI-digested pBR322. 1.5 µg of each DNA fragment was
denatured and blotted onto GeneScreen Plus membranes and hybridized to
the 32P-labeled RNAs. Data were quantified using a PDI DNA
ImageWare System (Huntington Station, NY).
Isolation of Human TrkA Genomic Sequences--
A 3.1-kb
HindIII fragment was isolated from an EMBL-3 genomic library
derived from human placental DNA using a 236-bp
EcoRI-SmaI fragment of the human TrkA cDNA as
well as an oligonucleotide complementary to sequences 22-48 of the
TrkA cDNA (5
-TCTGTGCGCTCCCAGCTGCAGCTGCCA-3
) as probes (31). This
genomic sequence contained the 5
-flanking region as well as exon I and
a portion of the first intron of the human TrkA gene based on dideoxy
sequencing and comparison with the human TrkA cDNA sequence (31).
It was cloned into the HindIII site of pBluescript (KS) to
generate pJD1.
Primer Extension and RNase Protection Assays--
Primer
extension was performed essentially as described elsewhere (37).
An end-labeled primer
(5
-CGATGTAGAGCTCAGTCAGGTTCTCTGCGCCGGGCAGGTGGTGGAGG-3
corresponding to sequences +174 to +221 relative to the main
translation start codon for human TrkA) (31) was hybridized to total
RNA in hybridization buffer (150 mM KCl, 10 mM
Tris-HCl, pH 8.3, and 1 mM EDTA) at 65 °C for 4 h.
Reverse transcription was carried out with SuperScript II RNase
H-reverse transcriptase (Life Technologies, Inc.) at 55 °C for 40 min. Reactions were phenol/chloroform-extracted, precipitated, and
analyzed on 8% polyacrylamide-8 M urea sequencing gels.
The plasmid pBS-N2.8 was generated by isolating a
HindIII-NarI fragment from pJD1 and ligation into
the HindIII and XhoI sites of pBluescript (KS).
pTrkA-NarI was generated by BamHI digestion of pBS-N2.8 and
self-ligation of the vector fragment. Antisense riboprobe was
synthesized from BamHI-digested pTrkA-NarI using phage T3
RNA polymerase. Twenty µg of total RNA from various cell lines were
hybridized and processed as described previously (38).
Generation of TrkA Promoter-Luciferase Contructs--
The 3.1-kb
HindIII fragment from pJD1 was cloned into the
HindIII site of pGL3 to generate pTrkLuc3.1 and pTrkLuc3.1AS
(antisense). The pGL3basic vector was modified prior to this insertion
by digestion with MluI, treatment with T4 DNA polymerase,
and religation to remove the MluI site. pTrkLuc2.8 and
pTrkLuc2.7 were made by partial digestion of pJD1 with NarI
and treatment with Klenow fragment, followed by complete digestion with
SpeI. The 2.7- and 2.8-kb fragments were then inserted into
the HindIII and NheI sites of pGL3. pTrkLuc2.6
was produced by isolating a 2.6-kb KpnI-AflIII fragment from pTrkLuc3.1 and inserting it into pGL3 at the
KpnI and HindIII sites. pTrkLuc1.2 was generated
from pTrkLuc2.6 by digestion with NheI and SacII
and religation of the 6.0-kb plasmid fragment. pTrkLuc1.0 and
pTrkLuc0.7 were produced by digestion of pTrkLuc2.6 with
BglII and SacI, respectively, followed by
religation of the vector fragments. pTrkLuc0.2 was made from pTrkLuc2.6
by digestion with BamHI and NheI and
self-ligation of a 5.1-kb vector fragment, and pTrkLuc0.14 was
generated from pTrkLuc0.2 by digestion with SacI and
ApaI and self-ligation of the 5.0-kb vector fragment.
Constructs were verified by DNA sequencing using the dideoxy method.
The ApaI-AflIII region of the human TrkA promoter
was sequenced completely on both strands using internal and external primers and analyzed for transcription factor binding elements using
the TFsites program (Genetics Computer Group, Madison, WI).
Transient Transfection and Reporter Gene Assays--
Supercoiled
plasmid DNAs were prepared on Qiagen columns (Qiagen, CA), and
constructs were transfected in duplicate. Co-transfected pCMV-
-galactosidase DNA was used for normalizing transfection efficiency and each construct was tested a minimum of three times. Nonspecific promoter activity was determined in each experiment using
the antisense construct pTrkLuc3.1AS and its value was subtracted from
the activity of each sense promoter construct after normalization. Nonspecific activities were approximately 10, 22, and 27% of total promoter activities for SMS-KCN, SH-SY5Y, and K293 cells, respectively. Cells were seeded 12-24 h prior to transfection in 35-mm culture dishes at 0.3-0.8 × 106 cells/dish. 1.5 µg of
pTrkLuc construct, and 0.75 µg of pCMV-
-galactosidase were
combined with 6 µl of LipofectAMINE (Life Technologies, Inc.) in 200 µl of RPMI 1640 medium (antibiotic- and serum-free) and incubated for
30 min at room temperature. The DNA mixture was diluted with 800 µl
of RPMI 1640 medium and then added to cells for 5 h at 37 °C.
Cells were then washed with culture medium containing serum and
antibiotics and cultured for an additional 43 h. Transfected cells
were washed with phosphate-buffered saline, lysed with 200 µl 1 × lysis buffer (Promega, Madison, WI), and supernatants were stored at
80 °C.
Luciferase reporter activities were assayed with a luciferase assay
system (Promega) using a Packard Pico-Lite 6100 luminometer. Relative
light units were normalized using the
-galactosidase activity
determined in the same extract.
-Galactosidase activities were
assayed using the fluorescence method as described by Stuart et
al. (39). Reactions were assayed in a fluorescence
spectrophotometer (Perkin-Elmer, model LS-3), by excitation at 365 nm
and measurement at 445 nm. Purified
-galactosidase enzyme (Promega)
was used as a standard.
Gel Shift Analysis--
Protein extracts were prepared from
freshly isolated nuclei in 20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, and 1 µg/ml each of the proteinase inhibitors pepstatin A,
bestatin, aprotinin, and leupeptin. Protein was determined using a
Bio-Rad assay kit, and relative concentrations in different extracts
were verified on SDS-polyacrylamide gels and Coomassie Blue
staining.
Gel shift assays were performed as described previously by
Galcheva-Gargova et al. (40). The probe was prepared by
isolating an ApaI-AflIII fragment from pTrkLuc2.7
and labeling by Klenow fill-in using [32P]dCTP. Binding
reactions contained 1.0 ng of 32P-labeled probe and 3 µg
of nuclear extract. Competition assays were performed with a 50-fold
mass excess of unlabeled ApaI-AflIII genomic
fragment or a double-stranded TATA box oligonucleotide: 5
-GCAGAGCATATAAGGTGAGGTAGGA-3
. DNA-protein complexes were resolved on
4% nondenaturing polyacrylamide gels.
 |
RESULTS |
Relative Transcription Rates of the TrkA Gene in Human
Neuroblastoma Cell Lines--
Neuroblastoma cell lines exhibit varying
degrees of TrkA expression (41-43) and thus can be used to examine the
basis for both its cell-specific and variable expression in this tumor
cell type. Gene transcription often plays a primary role in
cell-specific gene expression (44). To determine whether this was the
case for TrkA in human neuroblastoma cells, Northern blots were
initially performed using different cell lines (Fig.
1). SK-N-SH, IMR32, and SMS-KCN cells
contained relatively high levels of a 2.9-kb TrkA mRNA, with IMR32
being the most enriched. In contrast, SH-SY5Y cells contained low
levels of the 2.9-kb transcript, and LA-N-6 cells expressed
intermediate levels. TrkA mRNA was undetectable in human kidney
K293 cells (data not shown).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 1.
Northern blot analysis of TrkA mRNA
levels in different neuroblastoma cell lines. Total RNA (20 µg)
was examined from the following cell lines: SK-N-SH (lane
1), SMS-KCN (lane 2), SH-SY5Y (lane 3)
LA-N-6 (lane 4), SMS-KCN (lane 5), IMR32
(lane 6), and SH-SY5Y (lane 7). The size of the
human TrkA mRNA is indicated in kilobases. Membranes were
subsequently stripped and rehybridized with a probe to human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (shown
below).
|
|
Nuclear run-on studies were performed to determine the degree to which
Northern blotting data reflected differences in the rate of TrkA gene
transcription between various cell lines. In agreement with the above
results, TrkA transcription was low but detectable in SH-SY5Y cells
and ~ 4-5-fold higher in SMS-KCN and IMR32 cells (Fig.
2). No transcription was measurable in K293 cells.
Thus, gene transcription appears to be a major determinant of both
cell-specific and degree of TrkA expression in neuroblastoma cells.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 2.
Transcriptional analysis of the human TrkA
gene in different cell lines. Equivalent amounts of
32P-labeled RNAs were generated from K293, SH-SY5Y,
SMS-KCN, and IMR32 cell nuclei and then hybridized to the indicated
plasmid DNAs.
|
|
Identification of the Transcription Initiation Region for the Human
TrkA Gene in Neuroblastoma Cells--
A combination of RNase
protection and primer extension analysis was used to accurately
localize the TrkA promoter region employed in human neuroblastoma
cells. RNase protection was performed with a riboprobe
(pTrk-NarI) complementary to sequences spanning the 5
-end
of the human TrkA cDNA originally isolated from K562
erythroleukemia cells (31) (Fig.
3A). Analysis of total RNA
from SH-SY5Y, SMS-KCN, and IMR32 cells revealed two prominent
protection products of ~110 and 125 nucleotides (nt) in each case
(Fig. 3B). Additional products ~150-160 nt in length
sometimes were observed upon longer exposure (e.g. in IMR32
cells, Fig. 3B). No specific products were detected for
TrkA-negative K293 kidney cells. The two major start sites predicted by
these results lie ~60-70 bp downstream of the 5
-end for the TrkA
cDNA originally isolated from K562 cells (Fig. 3A).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 3.
Mapping the transcription start sites for the
human TrkA gene. A, schematic representation of the human
TrkA genomic fragment showing the transcription start sites determined
by RNase protection (asterisks) and primer extension
(square). An additional start site sometimes observed by
RNase protection is shown in parentheses. Locations of the
primer used for extension analysis (a and arrow),
translation initiation site (ATG and arrow), and 5 -end of the TrkA cDNA from K562 cells (inverted
arrowhead) are also shown. The pTrkA-NarI riboprobe
used in RNase protection assays is indicated by the bar below.
B, mapping of start sites by RNase protection. Total RNA (20 µg)
was analyzed from SH-SY5Y (lane 1), SMS-KCN (lane
2), IMR32 (lane 3), and K293 (lane 5) cell
lines. Yeast tRNA (lane 4) served as a negative control. C, mapping of start sites by primer extension. Total RNA (50 µg each except lane 1, 80 µg) was examined from SH-SY5Y
(lane 1), SMS-KCN (lane 2), IMR32 (lane
3), and K293 cells (lane 4). The specific extension
product is indicated by an asterisk. In both A
and B, the sizes of DNA markers are shown in nucleotides
(nt).
|
|
Primer extension analysis was performed to further examine this
question using a 47-bp oligonucleotide complementary to sequences between +174 and +221 relative to the TrkA translation start codon (Fig. 3A). A single major extension product ~281 nt in
length was observed in SH-SY5Y, SMS-KCN, and IMR32 cells (Fig.
3C). The predicted initiation site based on this result
occurs ~32 bp upstream of the more 5
-major start site identified by
RNase protection (Fig. 3A). Together these findings indicate
that transcription of the TrkA gene generally initiates in
neuroblastoma cells from one or more sites within a 30-50-bp region
lying just downstream of the 5
-end for the TrkA cDNA originally
isolated from K562 erythroleukemia cells.
Localization of Neuroblastoma Cell-specific Elements within the
Human TrkA Promoter--
To identify regions within the TrkA promoter
that are important for expression in neuroblastoma cells, a series of
promoter constructs were generated in the luciferase-containing
plasmid, pGL3basic (Fig. 4). The largest
of these, pTrkLuc2.8, consisted of a 2.8-kb
HindIII-NarI fragment containing ~2.6 kb of
5
-flanking sequence and a portion of exon I. Additional versions
consisted of 5
- and/or 3
-deletions of pTrk2.8 as well as an antisense construct containing a 3.1-kb genomic fragment that served as a control
for nonspecific activity. The low TrkA-expressing SH-SY5Y line and
TrkA-negative K293 cells were transiently transfected with these
various constructs to determine the presence of neuroblastoma cell-specific regulatory sequences within the human TrkA promoter. Transfection of SH-SY5Y cells yielded comparable promoter activity for
all constructs tested (Fig.
5A). The smallest of these,
pTrkLuc0.14, consisted of a 138-bp proximal 5
-flanking sequence
defined by ApaI and AflIII sites lying just
upstream of the transcription initiation region (see Fig. 4). While the
activities of various constructs were generally constant in K293 cells
as well, their absolute levels were ~10-fold lower than in SH-SY5Y
cells (Fig. 5B). Thus, the 138-bp
ApaI-AflIII region is sufficient to mediate neuroblastoma cell-specific regulation of the TrkA promoter. Based on
the various constructs tested, no significant enhancer or repressor sequences are apparent lying upstream or downstream of this region.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Schematic diagram of human TrkA genomic
sequences used to generate pTrkLuc promoter constructs. The 3.1-kb
HindIII fragment derived from the human TrkA gene is shown
at the top along with relevant restriction sites. The 5 -end of exon I
(shaded box) is based on primer extension analysis. Genomic
fragments used in various pTrkLuc constructs are indicated below and
were cloned into the pGL3basic reporter plasmid.
|
|

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 5.
Cell specificity of TrkA promoter activity in
transiently transfected cells. A, luciferase activities for
different pTrkLuc constructs in SH-SY5Y neuroblastoma cells.
B, reporter activities of the same constructs in
TrkA-negative human kidney K293 cells. For comparison, the activity of
the pTrkLuc2.6 construct in SH-SY5Y cells is also shown.
Bars indicate the standard errors of duplicate
samples.
|
|
Promoter Sequences Mediating Variable TrkA Expression in
Neuroblastoma Cells--
Northern and nuclear run-on analyses
demonstrated that TrkA transcription occurs to varying degrees in human
neuroblastoma cell lines, similar to what is observed in primary
tumors. To explore the nature of the regulatory elements responsible
for elevated transcription of the TrkA gene in neuroblastoma cells, promoter constructs also were transiently transfected into SMS-KCN cells. This neuroblastoma cell line exhibits a severalfold higher level
of TrkA mRNA and transcription than occurs in SH-SY5Y cells (see
Figs. 1 and 2). As with the other two cell lines examined, the activity
of the pTrkLuc0.14 promoter construct was very similar to the longer
versions tested (Fig. 6). In addition,
absolute promoter activity was ~5-fold greater than observed in
SH-SY5Y cells. This indicates that enhanced TrkA promoter activity is recapitulated in transiently transfected SMS-KCN cells and that the
relevant cis-elements are contained within a 138-bp proximal 5
-flanking region. It should be noted that further deletion of 102 bp
from the 5
-end of the ApaI-Af1III sequence resulted in >93% loss of luciferase activity in all three cell lines (data not
shown), confirming the critical importance of this region for TrkA
promoter activity.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 6.
Promoter activity in neuroblastoma SMS-KCN
cells which express TrkA at relatively high levels. Reporter gene
activities were determined as outlined in Fig. 5. The activity of
pTrkLuc2.6 in transfected SH-SY5Y (low TrkA-expressing) cells is shown
for comparison.
|
|
Cell-specific Binding of Nuclear Factors to the Neuroblastoma
Regulatory Region of the TrkA Promoter--
The above findings
indicated that nuclear factor interactions within the 138-bp
ApaI-AflIII region mediated both cell-specific and variable expression of the TrkA promoter in neuroblastoma cells.
Gel shift experiments were performed to examine these interactions directly and compare them in different cell lines using the
ApaI-AflIII sequence as a probe. Four major
complexes were detected in nuclear extracts prepared from SMS-KCN cells
(Fig. 7, a-d), which express the TrkA gene at relatively high levels. All four complexes were specifically competed by a 50-fold excess of unlabeled homologous DNA
but not by an equivalent amount of an unrelated competitor DNA. In
SH-SY5Y cells, which express TrkA at low levels, complex a
was present in concentrations similar to SMS-KCN cells but
complex b was markedly reduced, and complexes c
and d were extremely low (Fig. 7). The complexes detected in
SH-SY5Y cells also were specifically competed by homologous unlabeled
competitor. Further, all four complexes were essentially undetectable
in TrkA-negative K293 cells. Thus, multiple DNA binding proteins
interact with the proximal TrkA regulatory region mediating
cell-specific and variable expression in TrkA-expressing neuroblastoma
cells. These factors are extremely low in TrkA-negative cells and at
least three of them (complexes b, c, and d) are specifically elevated
in neuroblastoma cells exhibiting enhanced TrkA promoter activity.

View larger version (77K):
[in this window]
[in a new window]
|
Fig. 7.
Nuclear factor binding to the neuroblastoma
regulatory region of the TrkA promoter. Gel shift analyses were
performed with an end-labeled ApaI-AflIII genomic
fragment. Lane 1, free probe; lanes 2-4, nuclear
extracts from SMS-KCN cells; lanes 5-7, extracts from
SH-SY5Y cells; lanes 8-10, extracts from K293 cells. Three
µg of nuclear protein were used per lane. The presence or absence of
a 50-fold excess of homologous or unrelated competitor DNA is indicated
above each lane. The positions of the four DNA-protein complexes
observed in SMS-KCN cells (a-d) are also indicated.
|
|
Sequence analysis of the ApaI-AflIII 5
-flanking
region revealed the presence of multiple recognition sites for known
transcription factors, including ATF, Sp1, ets factors,
Egr-1, and AP2 (Fig. 8). In
some cases, multiple consensus sequences are present and some sites are
overlapping, as for Sp1, AP2, and Egr-1 elements. Certain of
these sites, and/or yet to be defined regulatory elements, may be bound
by factors selectively present in TrkA-expressing neuroblastoma cells
and mediate cell-specific and variable TrkA promoter activity.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 8.
Sequence of the human TrkA 5 -proximal
promoter region. Potential binding sites for known DNA binding
proteins are shown by boxes. Sequences that were missing in
the human TrkA gene sequence reported by Greco et al. (62)
are indicated by a dash (-), and two bases that were inverted are
shown in lowercase letters above the corresponding
sequences.
|
|
 |
DISCUSSION |
While TrkA expression and/or signaling is often deficient in
neuroblastoma cells derived from advanced stage tumors (45, 46), its
signal transduction pathway was shown to be intact in cells obtained
from a favorable primary tumor (16). Forced expression of TrkA in
receptor-deficient neuroblastoma cells also leads to growth arrest and
differentiation of tumors in nude mice following in vivo
treatment with NGF (21). Recent studies have further suggested that
Schwann cells are capable of infiltrating favorable neuroblastomas and
may provide a local source of NGF (47). These and other studies have
led to the hypothesis that TrkA has an active role in the spontaneous
maturation/regression of favorable neuroblastoma tumors (16, 24).
However, a direct role for NGF and TrkA signaling in this process
remains to be established.
The strong correlation between high TrkA expression and a favorable
outcome for neuroblastoma (16, 21-23) indicates that understanding the
mechanisms responsible for TrkA expression in neuroblastoma cells is
likely to provide important insight into this disease and may assist in
the development of potentially novel treatments. For example, it could
lead to therapies focused on induction of TrkA that, in combination
with neurotrophin treatment, could promote tumor differentiation. Even
if TrkA expression is simply a marker for a more differentiated tumor
state, such analyses will reveal fundamental regulatory pathways that
distinguish TrkA-expressing neuroblastoma cells from their
TrkA-negative counterparts.
The present studies form a strong basis for this approach by
demonstrating a major role for gene transcription in the elevated expression of TrkA in neuroblastoma cells. In contrast, enhanced expression of N-MYC in human neuroblastoma cells is
regulated largely at the level of mRNA stability (48). Distinct
mechanisms thus determine differential expression of these markers for
neuroblastomas having favorable and poor prognoses. Cis
elements mediating both cell-specific and elevated TrkA transcription
in neuroblastoma cells have been localized to a 138-bp proximal
promoter sequence. Differential gene transcription can occur by various
mechanisms, including expression of unique transcription factors,
elevated levels, and/or novel combinations of more generally expressed factors and epigenetic processes such as
chromatin-dependent transcriptional effects or DNA
methylation (49-51). The present studies indicate that differential
expression of DNA-binding proteins plays an important role in TrkA
promoter regulation in neuroblastoma cells. That is, several
DNA-protein complexes were identified involving the proximal regulatory
promoter region that were common to TrkA-expressing neuroblastoma cells
and extremely low or undetectable in TrkA-negative cells. Further,
certain complexes were markedly elevated in a neuroblastoma cell line
that transcribes the TrkA gene at relatively high levels. Although
these findings do not rule out a role for chromatin structure or DNA
methylation in TrkA promoter regulation, such mechanisms are not
required for differential activation within the proximal 5
-flanking
region defined here since it is unlikely they contribute to promoter
activity determined in transient transfection assays.
Several candidate elements were identified within the proximal
regulatory region that may be bound by transcription factors specifically elevated in TrkA-expressing neuroblastoma cells. These
include several GC-box sites such as for Sp1, AP2, and
Egr-1, as well as sites for ATF and ets-related
factors. Sp1 elements have been implicated in cell-specific or
differentiation-associated regulation of several promoters, including
those for p21/WAF1, hepatocyte growth factor and human
KDR/flk-II (52-54). Further, Egr-1 sites are
involved in the regulation of certain neuronally expressed promoters,
and this factor is expressed and inducible in neuroblastoma cells (55,
56). ATF and AP2 family members also have an important role in neuronal
gene expression (55, 57-59). Finally, ets domain proteins
have been implicated in cell-specific gene expression and at least some
members of this family are restricted to or enriched in neuronal cells,
including PEA3 and NERF (55, 60, 61). Thus, the present findings define
a region critical for TrkA promoter expression in neuroblastoma cells
which contains several candidate regulatory elements. It is also
possible that additional, novel regulatory elements exist within the
proximal 5
-flanking region that remain to be identified. Their
definition should provide important insight into the mechanistic basis
for TrkA gene regulation in neuroblastoma.
We thank Cathy Warren for her excellent
assistance in preparing the manuscript and Dr. Alonzo Ross for his
encouragement and advice.