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Molecular Biology and Genetics |
Division of Oncology [P. M. T., J. M. M., M. D. H., G. M. B., P. S. W.], The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104; Department of Pediatrics [J. M. M., M. D. H., G. M. B., P. S. W.], University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; Division of Hematology-Oncology, Children’s Hospital Los Angeles, Los Angeles, California 90027 [R. C. S., C. P. R.]; and Department of Pediatrics, University of Southern California School of Medicine, Los Angeles, California 90027 [R. C. S., C. P. R.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Neuroblastoma is a common pediatric tumor of the peripheral nervous
system and is often incurable when diagnosed after 1 year of age.
Neuroblastoma displays remarkable clinical heterogeneity, ranging from
spontaneous regression and/or differentiation to rapidly progressive
and metastatic disease, and considerable genetic heterogeneity is also
apparent. Cytogenetic, molecular genetic, and functional analyses of
primary tumors and cell lines have identified a number of genomic
regions frequently exhibiting hemizygous deletion (4)
.
Deletion of 1p36 correlates strongly with advanced disease and is the
most well-characterized region of deletion in neuroblastoma
(5, 6, 7)
. LOH studies have narrowed the region of consistent
overlapping deletion to 1p36.3, and this region is deleted in 
35%
of primary neuroblastomas (5
, 8
, 9)
. Several groups have
also hypothesized the existence of one or more additional TSGs located
elsewhere within 1p36 (10, 11, 12)
. Despite the
characterization of numerous candidates, no 1p36 TSG has yet been
identified, largely because of the paucity of tumors with localized
1p36 rearrangements (4)
.
Besides 1p36, several additional regions of the genome are frequently deleted in primary neuroblastomas, including 3p, 4p, 11q23, and 14q32 (4) . However, no TSG consistently mutated or rearranged in neuroblastoma tumors has yet been identified, and analyses of several known TSGs, including TP53, RET, CDKN2A, and MADH4, have detected few if any mutations in these genes (4) . Furthermore, HD of known tumor suppressor loci has been reported only rarely in neuroblastoma (13, 14, 15, 16) . The lack of evidence for genetic alterations in the genes encoding the DNA damage sensor p53 and the cyclin-dependent kinase inhibitory protein p16INK4a is notable, as these two genes are commonly disrupted in most malignancies (17 , 18) .
In the present study, we investigated whether HD occurs with significant frequency in neuroblastoma by systematically screening a large panel of cell lines with markers mapping to 1p36 and also with markers representing known TSGs. We report evidence for HD at CDKN2A but not within distal 1p or at other known TSGs in our cell line panel.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cell lines CHLA-10, CHLA-101, CHLA-103, CHLA-108, CHLA-124, CHLA-132,
CHLA-136, CHLA-138, CHLA-140, CHLA-143, CHLA-152, CHLA-153, CHLA-171,
CHLA-174, CHLA-178, CHLA-179, CHLA-185, CHLA-52, CHLA-54, CHLA-60,
CHLA-95, and CHLA-98 were established as described from patients
treated with myeloablative chemoradiotherapy (19)
. Cell
lines CHP-901 and CHP-902R were established at the Children’s Hospital
of Philadelphia from a bone marrow biopsy and a relapsed tumor mass,
respectively, and cultured in RPMI 1640 (Life Technologies, Inc.,
Gaithersburg, MD) with 10% fetal bovine serum, 25 µg/ml gentamicin,
and 1% oxaloacetate/pyruvate/bovine insulin-media supplement
(OPI; Life Technologies, Inc.). MYCN amplification
and 1p allelic loss determinations were performed as described
previously (7)
. Frozen tumor and corresponding normal
marrow samples of patients from which cell lines CHLA-101, CHLA-174,
and CHLA-179 were derived were obtained from the Children’s Cancer
Group Neuroblastoma Biology Resource Laboratory at Children’s Hospital
Los Angeles. To obtain primary tumor DNA from the patient who gave rise
to the LA-N-6 cell line, cryopreserved marrow was thawed, washed to
remove DMSO in Iscove’s DMEM with 10% fetal bovine serum, and treated
with 10 units/ml of DNase for 1 h at 37°C in a 5%
CO2 incubator. The mononuclear cells were
separated using a Ficoll density centrifugation. Mononuclear cells were
then treated with magnetic immunobeads (one bead/total cells) and tumor
cells removed as described previously (20)
. The resulting
mononuclear cells, which contained <0.1% tumor, were centrifuged to a
pellet, medium-aspirated, and flash-frozen for future DNA extraction.
The Jurkat T-cell line was kindly provided by S. Lessin (University of
Pennsylvania). DNA was isolated from cell line pellets or frozen
tissue using anion-exchange chromatography (Qiagen, Valencia, CA). DNA
from Centre d’Etude du Polymorphisme Humain reference family
member 1331-01 (Coriell Cell Repositories, Camden, NJ) was used as a
normal control.
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and 3
, respectively. Primers used for detailed mapping of HD around
CDKN2A are included in Table 3
. Primer sequences were
obtained from the Genome
Database4
and from literature reports of specific TSG characterizations or were
generated from a representative sequence using Primer
3.5
If possible, TSG primers targeted gene regions commonly disrupted in
other tumors. Markers for 1p36 were mapped using a distal 1p-specific
mapping
panel6
as described previously. Markers mapping to 9p were ordered using map
information from the HUGO chromosome 9 integrated map (Genome
Database accession no. 6276683) and genomic sequence tracts from
GenBank.7
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. A pooled DNA template containing human RH cell lines
21-30 from the Stanford G3 panel (Research Genetics, Huntsville, AL),
each of which had no human DNA fragment retention for markers within
1p36, was used as a control for detecting 1p36 HD. The 46 cell lines, a
normal DNA control (1331-01), and a negative control (PCR reaction with
no added template or the RH control) were amplified by PCR in 20 µl
volumes containing 4 µl of 10x PCR buffer II (Perkin-Elmer, Norwalk,
CT), 0.4 µM of each primer, 0.2
mM of each deoxynucleotide triphosphate, 0.2
units AmpliTaq Gold DNA polymerase (Perkin-Elmer), 1.5
mM MgCl2, 0.5%
Ficoll, 0.00625% bromphenol blue, and 14.4 ng template DNA.
Reactions were amplified for 1 cycle at 95°C (3 min); 15 cycles at
95°C (45 s) with the annealing/extension temperature starting at
70°C and decreasing by 0.7°C each cycle (1 min); 35 cycles at
95°C (45 s), 55°C (30 s), and 72°C (1 min); and 1 cycle at 72°C
(10 min). Twenty µl of each reaction solution was analyzed by
electrophoresis on an enhanced sensitivity gel system (Visigel;
Stratagene, La Jolla, CA), with products detected by ethidium bromide
staining. PCR reactions yielding no visible signal for a specific
marker were repeated, followed by radiolabeled amplification, PAGE, and
autoradiographic detection as described above. DNA sequences for
CDKN2A exon 2 were obtained on an Applied Biosystems Model
377 DNA sequencer using the ABI Taq DyeDeoxy Terminator Cycle
Sequencing kit (Perkin-Elmer).
Southern Analysis.
Ten µg of each genomic DNA sample was restriction digested for
16 h with PstI (Promega, Madison, WI) and
electrophoresed on a 0.6% agarose gel in 1x TBE. Gels were
sequentially immersed in 0.25 M HCl for 30 min,
1.5 M NaCl/0.5 M NaOH for
30 min, and 0.5 M Tris (pH 7.4)/1.5
M NaCl for 30 min. Electrophoresed DNA was then
transferred onto Hybond N+ membranes (Amersham
Pharmacia, Uppsala, Sweden), washed in 6x SSC, UV cross-linked, and
hybridized to either a 1.1-kb CDKN2A DNA probe amplified
from genomic DNA using primers CDKN2ex2-1 and CDKN2ex2-3 (Table 3)
or
to a probe spanning exons 3–5 of the MLL gene at 11q23
(kindly provided by Maureen Megonigal (Children’s Hospital of
Philadelphia, PA). Probe radiolabeling was performed with the
Rediprime Random Labeling Kit and Redivue
[
-32P]dCTP (Amersham Pharmacia). Membranes
were prehybridized for 3 h in 20 ml of prehybridization solution
(10% dextran sulfate, 0.75 M NaCl, 0.04
NaPO4 (pH 7.2), 4 mM EDTA
(pH 8), 0.5% N-lauryl sarcosine, 5x Denhardt’s solution, and 125
µg/ml sheared herring sperm DNA), hybridized for 16 h at 65°C
after the addition of labeled probe, and washed with 2x SSC and then
in decreasing concentrations of SSC-0.2% N-lauryl sarcosine at 65°C,
followed by autoradiography.
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RESULTS |
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. Thirty-nine of
the 46 cell lines (85%) had evidence of a 1p hemizygous deletion.
Previously, we identified a smallest overlapping region of consistent
deletion (SRO) spanning 1 Mb within 1p36.3 by LOH analyses of
primary neuroblastomas (5)
. This region has been further
characterized by the construction and analysis of a distal 1p-specific
mapping panel, which subdivides 1p35–p36 into 50 distinct genomic
intervals (21)
. The 1p36.3 SRO is entirely contained
within mapping intervals 6–11 (Table 2
; Ref. 5
).
Seventy-six markers within the SRO, providing an average HD detection
resolution of approximately 13 kb, were used to survey the 46
neuroblastoma cell lines by PCR. Included in this set of markers were
primers representing each gene or expressed sequence tag cluster known
to map within or near the SRO, including the proposed candidate
neuroblastoma TSGs TP73, TNFRSF12, and
HKR3 (22, 23, 24)
. To our knowledge, HD for 1p36
has never been reported; therefore, we created a HD control by pooling
DNA from 10 RH cell lines known to contain several human DNA fragments
but no fragments within 1p36. A product of the predicted size was
detected with all 76 markers for every neuroblastoma cell line but not
for the HD control or a negative control (no template; Fig. 1A
).
|
. Markers mapping within each of the 50
intervals defined by the distal 1p mapping panel were included to
assure that each portion of 1p36 was well represented. We also designed
markers for the proposed 1p36 TSGs (TNFRSF1B,
RIZ, PAX7, NBL1, TCEB3,
E2F2, C1ORF4, LAP18, and
ID3; Ref. 4
), as well as for twelve
markers surrounding a reported 1p36.2 translocation breakpoint in the
neuroblastoma cell line NGP (9
, 25)
. The 86 markers, which
provide an average 1p36 resolution of 350 kb, were used to survey the
46 neuroblastoma cell lines. A product of the predicted size was
visible for each marker in every neuroblastoma cell line and in a
normal control but not in a negative control.
HD Screen of Known TSGs.
Several known TSGs have been characterized for mutations in
neuroblastomas, but no abnormalities have been detected with
significant frequency. However, most of these analyses have included
only small cohorts of tumors and/or cell lines, and many identified
TSGs have not been investigated. Therefore, we extended our HD search
to include 21 known TSGs throughout the genome (Table 3)
. Intragenic
primers suitable for genomic PCR were designed for each TSG. Whenever
possible, primers were selected to span intragenic regions previously
demonstrated to be the most frequently deleted and/or mutated in
malignant cells. The 21 TSG markers were each used to survey the
neuroblastoma cell line panel and the Jurkat T-cell line, which is
homozygously deleted for CDKN2A (26)
. Twenty of
the 21 TSG markers yielded an amplification product in every cell line.
However, the CDKN2A primers, which span exon 2 of the gene,
did not amplify a product in three neuroblastoma cell lines (CHLA-174,
CHLA-179, and LA-N-6), nor in the Jurkat control, in repeated trials
using both ethidium staining and radioisotopic detection (Fig. 1B)
. In addition, a CDKN2A product 100 bp
shorter than the predicted length was consistently generated in cell
line CHLA-101 (Fig. 1B)
and was the only product generated
in this cell line.
Analysis of CDKN2A Deletions.
The CDKN2A HDs detected in the four neuroblastoma cell lines
were investigated further with 28 additional markers located within or
flanking CDKN2A to map the proximal and distal HD
boundaries. Included were markers spanning exons 1, 1ß, and 3 and
introns 1, 1ß, and 2 of CDKN2A; exons 1 and 2 of
CDKN2B; and several proximal and distal loci (Fig. 2)
. LA-N-6, CHLA-174, and the control Jurkat line demonstrated HD for the
entire CDKN2A gene including exon 1ß, which encodes the 5'
portion of p14ARF, and both exons of
CDKN2B. CHLA-179 showed HD for all exons of
CKDN2A but not for CDKN2B. No additional
alterations were detected for CHLA-101. A survey of the remaining 42
neuroblastoma cell lines with the four CDKN2A and two
CDKN2B exon-specific markers detected products of the
expected size in all cases. The LA-N-6, CHLA-174, and CHLA-179 HDs were
confined to a region of 9p21 flanked by IFNA and
D9S171 (Fig. 2)
, and each deletion would be predicted to
completely abolish production of both the p14ARF
and p16INK4a protein products.
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. Corresponding primary tumor and constitutional DNAs for LA-N-6,
CHLA-174, and CHLA-179 were analyzed in parallel to determine whether
HD occurred in vivo or only after cell-culture
establishment. A CDKN2A exon 2 probe hybridized to the
requisite 3.4-kb PstI fragment in constitutional DNA samples
from the three cell lines showing HD by PCR, as well as in primary
tumor DNA from CHLA-179. However, in agreement with the PCR results, no
hybridization was observed for the three cell lines with
CKDN2A HD. Furthermore, primary tumor DNA for CHLA-174 also
failed to hybridize to the CDKN2A probe, suggesting that HD
occurred in vivo in this tumor (no LA-N-6 tumor sample was
available for analysis). Hybridization with a control probe from a
different chromosome to the same filter yielded a band of identical
size and approximately the same intensity for each sample (not shown).
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. Both techniques
demonstrated an 100-bp deletion within exon 2. DNA sequencing of the
truncated exon 2 PCR product showed a deletion of 104 bp (bp 246–349
and residues 69–103 of p16INK4a; and bp 390–493
and residues 83–118 of p14ARF) entirely
contained within exon 2. PCR analysis of primary tumor DNA for the
patient from which CHLA-101 was derived showed only the truncated exon
2 fragment, suggesting that the primary tumor contained an exon 2 HD
identical to the cell line (not shown). Sequencing of CDKN2A
exon 2 from the primary tumor DNA confirmed these results. No
corresponding constitutional DNA for CHLA-101 was available for
analysis. The 104-bp deletion of CDKN2A exon 2 in CHLA-101 is predicted to disrupt both p16INK4a and p14ARF, which use alternate reading frames within exon 2. For p16INK4a, the COOH-terminal 87 residues of the wild-type protein would be replaced with the 15 COOH-terminal residues of p14ARF because of a frameshift from the p16INK4a reading frame to the p14ARF reading frame. It is unlikely that such a fusion protein, if expressed in CHLA-101, would be functional, as the deleted region of p16INK4a contains the Cdk4/Cdk6 binding site where most loss-of-function point mutations localize (27) . For p14ARF, the final 50 residues would be replaced by 42 frameshifted residues.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our findings of no HD within 1p36 are consistent with the possibility that disruption of only a single 1p36 homologue can facilitate neuroblastoma tumorigenesis. Although the marker density we used to survey HD within the 1p36.3 SRO was high (13 kb), very small regions of HD may have gone undetected. In most cases, biallelic inactivation of a TSG occurs by intragenic deletion or bp mutation of one allele, and then by a second, larger deletion event in the remaining allele, as is common with TP53 (31) . Because we surveyed only cultured cell lines for HD at 1p36.3, it is formally possible that HD occurs more frequently in primary tumors. However, this seems unlikely, given that many of the cell lines in our study were derived from advanced neuroblastomas passaged repeatedly in vitro, and that most cell lines had hemizygous deletion of distal 1p.
With the exception of CDKN2A, HD was not detected for any of the 21 TSGs surveyed. Our inclusion of positive and negative controls for HD and the confirmation of CDKN2A HD by Southern analysis for all four identified HD cases suggests that our PCR-based HD assay is both sensitive and specific. It remains possible that HD of one or more TSGs went undetected with the exon primers used for TSG surveying. However, because we targeted the most commonly disrupted gene region of each TSG, it is likely that the majority of HD events would have been identified. HDs and/or inactivating mutations in neuroblastoma are known to be rare or absent in the TP53, DCC, MADH4, and RET tumor suppressor genes (4) . Previously, four cases of HD have been reported for neuroblastoma: HD of several exons of the NF1 gene at 17q11.2 in a primary tumor from a patient with neurofibromatosis type 1 (13) and in a neuroblastoma cell line (32) ; HD of the CASP8 gene at 2q33 in one cell line (16) ; and HD of CDKN2A in a single cell line (see below; Ref. 14 ). Whereas we did not detect HD of NF1, the NF1 locus has not yet been well characterized in neuroblastoma, although there appears to be no increased incidence of neuroblastoma in patients with neurofibromatosis or vice versa (32) . The present study does not address whether HD occurs at other chromosomal loci than those included here. However, experiments using representational differential analysis did not detect HD in several neuroblastoma cell lines.8 Furthermore, a lack of HD at a tumor suppressor locus does not preclude inactivation of the gene by other genetic or epigenetic events, such as gene mutation or methylation-mediated gene silencing.
The region of 9p21 is frequently deleted in a wide range of
malignancies (33)
. Three loci in 9p21 have been implicated
as TSGs: CDKN2A/p16INK4a and
CDKN2A/p14ARF, which partially share a
coding exon but are encoded by two distinct reading frames; and the
highly homologous CDKN2B/p15INK4b
(Fig. 2
; Refs. 3
, 34
). Substantial genetic evidence
suggests that disruption of both p16INK4a and
p14ARF, but not p15INK4b,
is crucial for tumor development (18)
. Almost all
tumor-specific rearrangements of 9p21 alter exon 2, which is shared by
p16INK4a and p14ARF, and
only a few mutations affecting just one of the three implicated
proteins have been reported (18
, 35)
. Targeted deletion
experiments of the three loci in mice also suggest a causative role for
CDKN2A but not CDKN2B, as mice with germ-line
disruptions of CDKN2A are cancer-prone (36)
.
p16INK4a acts as an inhibitor of the cell cycle
activators cdk4 and cdk6, which in turn inactivate the pRB tumor
suppressor protein, whereas p14ARF is thought to
derepress p53 by binding to and inactivating mdm2 (18)
.
Disruptions of the CDKN2A locus that concomitantly eliminate
functional p16INK4a and
p14ARF are thus believed to inactivate both the
p53 and Rb tumor suppression pathways.
Inactivation of both the Rb and p53 pathways are key events for tumorigenicity in almost all neoplasms (31 , 37) . Because mutations and gene rearrangements of TP53 and RB1 are rare in neuroblastoma, these pathways might instead be disrupted by other mechanisms. One possible alternative is a CDKN2A gene rearrangement that simultaneously inactivates both p16INK4a and p14ARF. The four neuroblastoma HDs described in the present study all target CDKN2A exon 2 and disrupt both the p16INK4a and p14ARF coding regions, consistent with the type of CDKN2A rearrangements seen in other malignancies. The exon 2-specific 104-bp CDKN2A deletion observed in cell line CHLA-101 supports this hypothesis. Also, because the CHLA-101 and CHLA-179 HDs do not extend to CDKN2B, it is unlikely that biallelic disruption of CDKN2B plays a role in neuroblastoma tumorigenesis, although monoallelic deletion of this gene could conceivably have an ancillary effect. However, mutations or deletions limited to CDKN2B have not been detected in neuroblastomas (38) .
Previous genetic analyses have reported evidence for infrequent disruption of CDKN2A in neuroblastoma. Several LOH studies cumulatively found 9p21 allelic loss in 29 of 131 (22%) primary neuroblastomas (14 , 38, 39, 40, 41) , with LOH most frequently observed in tumors identified by mass screening of urinary catecholamine metabolites. However, HD of CDKN2A has not been found in primary tumors (3 , 14 , 38 , 39 , 41, 42, 43) , although Dicicianni et al. (14) reported HD for an adriamycin-resistant subclone of the Be2C cell line that was not observed in the parent cell line. Comparative genomic hybridization studies of primary neuroblastomas have also reported only a low frequency of 9p deletions (4) . Furthermore, only a single CDKN2A mutation, a missense mutation at residue 52 of p16INK4a in exon 2 has been identified out of 178 primary tumors and 28 cell lines screened (14 , 38 , 39 , 41, 42, 43) . Notably, exon 1ß (encoding p14ARF) has not been included in any of the mutation screens, but these results together suggest that a purely genetic disruption of the CDKN2A locus is uncommon. Our results agree with these findings in that only 4 of 46 (9%) cell lines demonstrate gross biallelic inactivation. Nevertheless, our identification of HD in two of three corresponding primary tumors suggests that CDKN2A inactivation is a contributing in vivo genetic event for a subset of neuroblastomas, rather than a strictly in vitro phenomenon.
It is also conceivable that p16INK4a and/or
p14ARF are inactivated by alternative mechanisms.
A correlation between promoter hypermethylation and decreased
expression of the p16INK4a transcript, presumably
providing an epigenetic mechanism for CDKN2A suppression,
has been noted in other malignancies (44, 45, 46)
. Three
studies of CDKN2A methylation in neuroblastoma cumulatively
found exon 1 methylation in 35% of tumors and cell lines, but no
correlations between methylation status and expression levels were
apparent (14
, 38 , 41)
. The methylation status and
mutational analysis of CDKN2A exon 1ß in neuroblastoma has
not been reported. The expression pattern of CDKN2A in
primary tumors is varied and exhibits no obvious correlations with
other parameters (38
, 41 , 47)
. Likewise, analyses of other
genes in the CDKN2A pathway have found few abnormalities in
neuroblastoma (14
, 38
, 42
, 47, 48, 49)
.
Mutation of the TP53 gene is very rare in primary neuroblastomas, and deletion or LOH of 17p is uncommon (4) . There is, however, evidence that inactivation of the p53 pathway is important in some neuroblastomas. Amplification of the p53-inhibitory MDM2 locus has been identified in several neuroblastoma cell lines and in a single tumor (50, 51, 52) . Furthermore, immunohistochemistry studies suggest that p53 is sequestered in the cytoplasm in neuroblastomas, which has been postulated as a posttranslational mechanism for functional repression of p53 (53 , 54) . The CDKN2A deletions observed in the present study, which would be predicted to affect p53 function through p14ARF, are consistent with alterations in p53 signaling playing a role in the tumorigenesis of some neuroblastomas. More detailed analysis of p14ARF expression and function in neuroblastoma will be needed to fully understand the contribution that this protein plays in neuroblastoma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants CA39771 (to
G. M. B.), CA78966 (to J. M. M.), CA60104 (to R. C. S.), and
CA82830 (to C. P. R.) from the NIH, and the Audrey E. Evans Chair in
Molecular Oncology (to G. M. B.). 
2 To whom requests for reprints should be
addressed, at Division of Oncology, Room 902, 3516 Civic Center
Boulevard, Philadelphia, PA 19104-4318. Phone: (215) 590-5241; Fax:
(215) 590-3770; E-mail: white{at}genome.chop.edu
3 The abbreviations used are: TSG, tumor
suppressor gene; HD, homozygous deletion; LOH, loss of heterozygosity;
SRO, smallest region of overlap; RH, radiation hybrid.
4 Internet address: www.gdb.org.
5 Internet address:
www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi.
6 Internet address:
compgen.rutgers.edu/chr1/data/1p36map/index.shtml.
7 Internet address: www.ncbi.nlm.nih.gov/.
8 J. M. Maris and N. Lisitsyn, unpublished
results.
Received 6/19/00. Accepted 11/13/00.
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