Download Deletion Map of Chromosome 9 and p16 (CDKN2A) Gene Alterations

ICANCER RESEARCH57. 907-912. March I. 19971
Deletion Map of Chromosome 9 and p16 (CDKN2A) Gene Alterations
in Neuroblastoma1
Junko Takita, Yasuhide Hayashi, Takashi Kohno, Naohito Yamaguchi, Ryoji Hanada, Keiko Yamamoto,
and Jun Yokota2
Biology Division If. T., T. K., J. Y.J and fancer
Information and Epidemiology Division fN. Y.J, National Cancer Center Research Institute, 1.1. Tsukifi 5-chome, Chuo-ku, Tokyo
104: Department of Pediatrics. Faculty of Medicine. Unis'ersitv of Tokyo. 3-I, Hongo 7-chome. Bunkyo-ku, Tokyo I 13 Ii. T., Y. HI; and Division of Hematology/Oncology.
Saitama Children ‘s
Medical Center, 2100, Magome. Iwatsuki 339 (R. H., K. Y.J. Japan
ABSTRACT
We reported previously that loss of heterozygosity (LOH) on chromo
somes
2q, 9p and
18q frequently
occurs
in neuroblastoma
and that
patients with 9p LOH in the tumors showed statistically significant asso
ciation with an advanced stage of the disease and poor prognosis. To
determine the role of chromosome 9 loss in neuroblastoma, we performed
deletion mapping of chromosome 9 in 80 cases of neuroblastoma using 11
polymorphic microsatellite markers and a restriction fragment length
porymorphism marker. LOll at one or more loci on chromosome 9 was
detected in 33 ofSO cases (41%). Chromosome 9p was lost in 24 of8O cases
(32%), whereas chromosome 9q was lost in 18 of 80 cases (23%). There
were two commonly deleted regions mapped to 9p2l between the D9S171
marker and the IFNBJ marker and 9q34—qterdistal to the D9S176
marker. In addition, patients with LOH at 9p21 but not at 9q34—qterin
the tumors showed statistically significant association with poor prognosis
(P = 0.023). Because the commonly deleted regions at 9p21 includes the
p16 (CDKN2A) gene, the status of thepl6 gene was further examined in 80
fresh tumors and 19 cell lines of neuroblastoma.
A missense mutation
was
detected at codon 52 in a fresh tumor. ThepI6 gene was not expressed in
13 of 19 cell lines (72%), and 5 of the 13 cell lines displayed methylation
ofthe CpG island surrounding the first exon of thepl6 gene. These results
suggest
that
the p16
gene
is a candidate
tumor
suppressor
gene
for
neuroblastoma, and its inactivation may contribute to the progression of
neuroblastoma.
INTRODUCTION
Recent molecular studies have revealed that the genesis and pro
gression of human cancer is largely attributed to accumulation of a
series of genetic events that culminate in the transformation of a cell
into a malignant clone (1). Central to this theory are the roles of
oncogenes and tumor suppressor genes, the activation and inactivation
of which, respectively, cause disruption of critical events in cell
division and differentiation (1). The paradigm of alterations in the
tumor suppressor gene is a mutation of one allele and a loss of the
other allele. Reduction to homozygosity of the tumor suppressor gene
can be detected as LOH3 of informative polymorphic markers in the
region of the tumor suppressor gene. Thus, allelic losses are hallmarks
of chromosomal regions harboring tumor suppressor genes (2).
Although NB is one of the most common childhood tumors, little is
known about the genetic changes that contribute to the development
of tumor. It has been reported that LOH occurs frequently on at least
three chromosome arms, ip, 1lq, and 14q, in NB (3—10).In addition,
we demonstrated recently that three additional loci on chromosomes
MATERIALS
as stage
whom
requests
for
reprints
1 year. Patients
or surgery
3 The
abbreviations
used
are:
IVS. In our 80 NB patients,
74
multidrug
with stage I, II, or IVS were treated
radiotherapy.
chemotherapy
consisting
Patients
consisting
with either
of vincristine
surgery
alone
and cyclophosphamide
with stage III or IV were treated
of cyclophosphamide,
Adriamycin,
with
cispla
tin, and etoposide with or without surgery and radiotherapy. In 53 cases,
histological data was available; thus, tumors were histologically classified as
described by Ota and Shimizu (19). There were 4 cases of GNB classified as
well differentiated, 8 cases of GNB classified as composite, 9 cases of GNB
classified as poorly differentiated, 27 cases of NB classified as rosette-fibril
lary, and 5 cases of NB classified as round cell. We also used 19 NB cell lines,
should
LOH,
IV, and 8 as stage
plus chemotherapy
with or without
NB1, NB9, NB16, NB19, NB39, NB69, LAN1, LAN2, LAN5, KP-N-NS,
GOTO, CHP-134, IMR-32, TNB-l, TGW, SCMCN2, SCMCN3, SCMCN4,
be addressed.
Phone:
81-3-3542-251
and SCMCN5, for the analysis of the pitS gene alterations in NB.
DNA, RNA, and Protein Extraction. DNA was isolated from tumors,
normal tissues, and cell lines by proteinase K digestion and phenol/chloroform
isoamyl alcohol (24:1) extraction as described previously (20). mRNA was
1, Ext.
extracted from cells growing in culture using the FastTrack 2.0 mRNA
4650: Fax: 8 1-3-3542-0807.
toma; NB, neuroblastoma;
III, 16 as stage
patients were infants under I year of age at diagnosis, and 6 patients were over
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported in part by a Grant-in-Aid from the Ministry of Health and
Welfare for the second term Comprehensive 10-Year Strategy for Cancer Control and
Grants-in-Aid from the Ministry of Health and Welfare and from the Ministry of
Education, Science, Sports and Culture of Japan.
2 To
AND METHODS
Primary Tumors and Cell Lines. Tumor samples were randomly obtained
from 80 patients admitted to various institutions between May 1987 and July
I993 at surgery or at autopsy. Corresponding normal tissues were available in
all cases. The patients were staged according to the classification of staging in
NB (18). Of the 80 cases, 21 were classified as being stage I, 27 as stage II, 8
Received 8/5/96; accepted 1/6/97.
@
2q, 9p, and l8q were deleted with high frequency in NB. Moreover,
several studies have shown the correlation of genetic changes with
prognosis of the patients with NB (1 1). N-myc (NMYC) oncogene
amplification has been known to be an appreciable prognostic factor
in an advanced stage of the disease. It is also indicated that chromo
some Ip deletion frequently occurs in an advanced stage of the
disease, and there may be two tumor suppressor genes on chromo
some Ip associated with progression of the disease (9, 10). In addi
tion, we also reported that 9p LOH was significantly associated with
advanced stages of the disease and with poor prognosis (12).
To determine the locus on chromosome 9 that may harbor putative
tumor suppressor genes involved in the progression of NB, we per
formed deletion mapping of chromosome 9 in 80 cases of NB using
11 microsatellite markers and a RFLP marker. The result indicated
that there were two commonly deleted regions on chromosome 9,
9p2l and 9q34—qter, in NB, and that LOH at 9p2l was significantly
associated with poor prognosis. The p16 (CDKN2A) tumor suppressor
gene has been mapped to 9p2l and is inactivated in a variety of
malignancies by various mechanisms, including deletion, point mu
tation, and methylation of the CpG island in the 5' end of the p16 gene
(13—17).Therefore, we further examined the alterations of the pitS
gene in NB. Although deletions and mutations of the pi6 gene are
infrequent, transcriptional silencing and DNA methylation were fre
quently detected in NB cell lines. Thus, it was indicated that the p/tS
gene is a candidate tumor suppressor gene involved in the progression
of NB.
loss
of heterozygosity;
SSCP, single-strand conformational
GNB,
isolation kit according to the manufacturer's
ganglioneuroblas
polymorphism.
protein
was extracted
by lysing
instructions (lnvitrogen). Cellular
1 X 106 cells with 40 p1 of lysis buffer
907
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1997 American Association for Cancer Research.
[50 mM
CHROMOSOME9 DELETIONSAND p16 IN NEUROBLASTOMA
HEPES-NaOH (pH 7.0), 1% NP4O, 1% sodium deoxycholate, 0. 1% SDS, 250
mM NaCl, 5 mM EDTA,
50 msi NaF, 1 mi@iDTT,
1 mist phenylmethylsulfonyl
fluoride, and 50 @g/ml
aprotinini.
Sequencing. PCR productsselected for sequencingwere ligated with the
pCRIITA-cloning vector (Invitrogen) and transformed into competent DH5a
cells (Life Technologies,
Inc.). Both strands were sequenced for each PCR
PCR-LOHAnalysis.DNAfromtumorsandcorresponding
normaltissues product from at least three independent clones.
were analyzed for LOH by PCR amplification of microsatellite sequences.
Microsatellite
reaction
markers for PCR-LOH
volumes
were 10
analysis were listed in Fig. I . Total
@l
containing
50—100 ng DNA,
10 msi Tris-HCI
(pH
8.3), 50 mM KCI, 1.5 mM MgC12,250 @.LM
of each deoxynucleotide triphos
phate, 0.01% gelatin, 125 ng of each primer, 1.14 @sCi
of [a-32P)dCTP,and 1
40%, as described
unit of Taq
trisomy, besides LOH by this definition. Therefore, it is possible that certain
DNA
polymerase
(Pharmacia
Biotech,
Inc.).
Because
unequal
previously
(23). We could score allelic
imbalance,
such as
amplification of alleles occurs with 35 cycles of PCR (21), PCR amplifications
were performed for 35 cycles consisting of denaturation at 94°Cfor 40 s,
chromosome
annealing
and clinical features of the disease among the patient group was examined by
Fisher' s exact test. The vital status of the patients was observed until December
system
at 55°C for 40 s, and extension
9600 (Perkin-Elmer)
as described
at 72°C for 90 s in a Gene Amp PCR
(12).
Southern and Northern Blot Analyses. Approximately10 @.tg
of purified
DNA were digested with appropriate restriction enzymes and separated by
electrophoresis on 0.8% agarose gel. DNA was transferred from the gel to
nylon membranes ( 12). The membranes were hybridized with a PCR-generated
fragment corresponding to exon 1 of the p16 gene (p16-i), a full-length pitS
eDNA fragment, JNFBi, and pNB-l labeled with [a-32P]dCTP. LOH at the
IFNB1 locus was examined
by Southern
blot analysis
l.Lgof mRNA was denatured with 40% formamide/32%
(12). Approximately
loci scoring
Statistical
Analyses.
3 1, 1995. The survival
LOH are trisomic
rather
than monosomic.
Significance of the differences in various biological
curves
for each group of the patients
were estimated
by
the Kaplan-Meier method, and the resulting curves were compared using the
log-rank
test for univariate
analysis.
Multivariate
analysis
was performed
using
the Cox proportional hazards model.
Western Blot Analysis. Fifty jsg of protein were separatedin a 4—20%
gradient SDS/polyacrylamide gel and electroblotted to Hybond-Enhanced
Chemiluminescence
3
lington
formaldehyde and was
Heights,
(ECL)
nitrocellulose
membrane
IL). After being blocked
(Amersham
with 5% nonfat
Corp.,
Ar
dry milk and 0.1%
electrophoresed on a 1.2% agarose gel containing 25% formaldehyde. Then
mRNA was transferred to nylon filters. The filters were hybridized with p16-I
and full-length eDNA probes labeled with [a-32P] dCTP and were exposed to
Kodak XAR-5 film at —80°C.
Prehybridization, hybridization, and posthy
Tween 20 in Tris-buffered saline, membranes were incubated at 37°Cfor 2 h
bridization
Biotechnology). The blot was subsequently probed by the ECL Western
washes
were performed
basically
as described
(20).
PCR-SSCPAnalysis.All sampleswerescreenedformutationsinexons1
with the 1:400 dilution
amino
acids
blotting
137—156 at the COOH
detection
confirmed
analysis.
RESULTS
The primer
sets for the pi6
gene were: exon
1, PQIS,
5'-TCTGCG
were separated by electrophoresis
on 6% polyacrylamide
gel with 5% glycerol
and without glycerol. Moreover, to better assure detection of mutations by
PCR-SSCP, we used two cases of lung carcinomas with p16 mutations as
positive
controls.
9
4
9
16
18 23
24 28
of a rabbit polyclonal
anti-p16
antibody
(PharMingen),
the epitope of which is unknown, and a rabbit polyclonal anti-p16 antibody for
and 2 of the pi6 gene by PCR-SSCP analysis (22). Exon 1 was amplified as
one fragment, whereas exon 2 was split into two fragments for PCR-SSCP
GAGAGGGGGAGAGCAGGCA (sense) and PQ1A, 5'-TCTGCGGAGA
GAGGGGGAGAGCAGGCA (antisense); first exon 2, PQ2AS, 5'ACAAGCTFCCTITFCCGTCATGCCG (sense) and PQ2AA, 5'-CCAG
GCATCGCGCACGTCCA (antisense); and second exon 2, PQ2BS, 5'-TFC
CTGGACACGCTGGTGGT (sense) and PQ2BA, 5'-TCTGAGClTFGGA
AGCTCTCAG (antisense).
PCR conditions for exon I of the p16 gene were 35 cycles of 50 s at 94°C,
50 5at 65°C,
and 50 s at 72°C.
PCR conditions for exon 2 of the pi6 gene were
35 cycles of 50 5 at 94°C,50 s at 64°C,and 50 s at 72°C.
The PCR products
system
by staining
terminus
(Amersham
the membrane
Corp.).
of the p16 protein
Equal
loading
(Santa
of protein
Cruz
was
after detection.
Frequency and Common Regions of LOH on Chromosome 9 in
NB. Eighty cases of NB were examined for LOH on chromosome 9
using 11 microsatellite polymorphic markers and a RFLP DNA
marker. The incidence of LOH at each locus is summarized in Fig. 1.
All cases showed heterozygous genotypes in their normal tissue at one
or more loci on chromosome 9, and LOH at one or more loci was
detected in 33 of 80 cases (41%). LOH on chromosome 9p was
detected in 24 of 80 cases (30%), whereas LOH on chromosome 9q
was detected in 18 of 80 cases (23%). Five of 33 cases (15%) showed
LOH at all informative loci, whereas the other 28 tumors showed
partial deletions of chromosome 9 (Fig. I). Case 23 showed LOH at
29 30 39 42 60 61 79 80
8
15 33 34
58 59
70
76
7722
40
67
75 27 31 373872
I—
D9S281
2135(6%)
24
23
22
L
21
21
@\D9S162 6/41(15%)
13
I\\\FNBI9/27(33%)
12
k\tNA@ 6/41(20%)
11
11
13
21.1
212
21.3
22.1
222
22.3
A
I
I
I
I
\@D9s@5
2135(9%)
\\
12
@
Definition of LOH. The signal intensity of the polymorphic alleles was
quantified and calculated by the scanning densitometer and data analysis
system (The Discovery Series; Quantity One, pdi, NY). LOH was considered
to be present if reduction rates of signal intensities in tumors were more than
I
I
S1712137(8%)
09S186
20
1/32(9%)
l—D9S152 4/37(11%)
I
27
D9S195 1127(4%)
V26
D9S176 9/44(20%)
/34
D9S158 8/339(21%)
31
32
33
34.1
342
34.3
Fig. I . Schematic representation of deletion map of chromosome 9 in NB. The approximate locations of markers used are shown on the left and tumor numbers are shown above.
., LOH; 0, heterozygosity; and no symbol, not informative. cM distances between the markers are indicated in italics. and the number of LOH/informative cases is shown on the right
of each locus name.
908
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CHROMOSOME 9 DELETIONS AND p16 IN NEUROBLASTOMA
the D9Si62, IFNB1, and IFNA loci, but heterozygosity was retained
at the D9Si 7i locus (Fig. 2). In case 60, LOH was detected at the
D9Si7i locus, but heterozygosity was retained at all loci distal to the
IFNB1 locus (Fig. 2). Cases 33, 75, and 76 showed LOH at D9S/58,
but heterozygosity was retained at all informative loci proximal to the
D9Si76 locus (Fig. 2). The result from these five patients implicates
the presence of two commonly deleted regions that are mapped
between the IFNBi and D9S17J loci at chromosome 9p2l and distal
to the D9S176 locus at chromosome 9q34—qter (Fig. 1). The size of
genetic distance of a commonly deleted region on chromosome 9p2l
is 5 cM and that on 9q34—qter is more than 34 cM.
23603376TNTNTNTN
TL
D9S1 62 D9SI 62 D9S1 52 D9S1 52
a
IFNBI
IFNB1 D9S195 D9S195
IFNA
INFA
D9S176 D9S176
I
D9S1 71
D9S1 71 D9S1 58
D9S1 58
Fig. 2. LOH in cases showing partial or interstitial deletions of chromosomes 9. DNA
was isolated from tumors (Lanes 7) and corresponding normal tissues (Lanes N) from
patient 23 (Lane I), patient 60 (Lane 2), patient 33 (Lane 3), and patient 76 (Lane 4).
Allelic fragments that showed LOH are indicated by arrowheads.
Table I Correlation
Relationship between LOH on Chromosome 9 and Clinicopath
ological Findings of NB. Because age, stage, and N-myc amplifica
tion are known to be associated with prognosis of patients with NB,
the relationship between LOH on chromosome 9 and these clinico
pathological findings was examined. There were 56 patients with
stage I+II+IVS, 8 patients with stage III, and 16 patients with stage
Iv. N-myc amplificationwas detected in 10 of 80 cases. Because 62
of 80 (78%) were found by a mass screening program, the ratio of
infantile and early-stage patients in this study was higher than that in
previous studies (5, 10). However, age, stage, and genotype of N-myc
were also significantly associated with short survival of the patients in
our 80 cases (P < 0.001). Therefore, clinical outcome of patients in
this study was similar to that of patients in previous studies (5, 10). All
patients were followed-up for at least 36 months and up to 82 months.
The mean follow-up periods of patients with LOH on chromosomes 9,
9p, and 9q were 44, 46, and 47 months, respectively. The medium
ranges of survival of the patients with LOH on chromosomes 9, 9p,
and 9q were 50, 42, and 57 months, respectively. LOH on chromo
some 9p was significantly associated with the stage of the disease
(P = 0.029) and the survival of the patients (P
0.023). However,
neither LOH on chromosome 9q nor LOH on chromosome 9 showed
association with any of these parameters (Table 1). Ten of 24 patients
with 9p LOH died within 24 months, whereas only 9 of 56 patients
without 9p LOH died within this period (Fig. 3). However, significant
association was not observed between 9q LOH and survival of the
patients (P = 0.551; Table 2). Accordingly, LOH on chromosome 9
was not significantly associated with survival (P —0.387; Table 2).
There was no correlation between 9p LOH and other clinicopatho
logical findings, such as age, course of diagnosis (found by mass
screening or clinical symptoms), histological types, and genotype of
N-myc and lp LOH. When multivariate analysis of survival was
performed by age (under 1 year versus over 1 year) and genotype of
N-myc amplification (present or absent) as covariates, no significant
of LOH on chromsome 9 with biological and clinical variables in neurblastoma
LOH
99p21—229q34—qterAge<
I yr
(0.566)Stage―I,
(0.478)Result
of screening+
3/18(0.871)N-myc
amplification+
(0.438)Histological
typeGNB
1 yr29/74
II, IVS
III
IV20/55
4/6 (0.293)―22/74
3/9
10/16 (0.185)12/55
—28/62
5/18(0.565)18/62
—5/10
28/70 (0.349)4/1
Well differentiated
Composite
Poorly differentiated
NB Rosette fibrillary
Round cell3/4
3/8
4/9
12/27
2/5 (0.793)
0.3872/4
Survivalb
a Fisher's
exact
b P-value,
log-rank
2/6 (0.339)14/74
2/6
3/9
9/16 (0.029)13/55
1/9
2/16
6/18(0.807)13/62
0
20/70 (0.580)1/10
1/8
4/9
7/27
3/5 (0.584)
0.0232/4
15/70
3/8
3/9
6/27
0/5 (0.412)
0.551
test.
test.
909
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CHROMOSOME9 DELETIONSAND p16 IN NEUROBLASTOMA
mRNA was not detected in 13 of 19 cell lines (72%). The p16 protein
was undetectable by Western blot analysis in the corresponding 13
cell lines (Fig. 5).
% Survival
100
Methylation of the p16 Gene in NB Cell Lines. We next exam
90
80
70
60@
50@
40@
9p LOH-(n=56)
30@
20@
10@
0
—.------
9p
LOH+
(n=24)
med whether the pi6 gene was transcriptionally silenced in NB cell
lines by methylation of a CpG island in the pitS gene. DNA was
digested with EC0RI and SinaI and analyzed by Southern blot hybrid
ization. Unmethylated fragments of 650 and 350 bp were not detected
in five cell lines that did not express piO mRNA, indicating that the
critical SmaI site within exon I of all alleles was methylated in these
cell lines. However, this site was not methylated in eight other cell
lines in which pitS mRNA was not detected. The remaining six cell
lines that express pi6 mRNA showed unmethylated 650- and 350-bp
bands. There were no cell lines in which p/tS mRNA was expressed in
spite of having the methylated SmaI site in exon I.
P0.023
DISCUSSION
12
24
36
Monthes after diagnosis
Fig. 3. Kaplan-Meier survival curves for patients with NB classified according to
patients with LOH on chromosome 9p. The difference in survival times between the
patients with 9p LOH and without 9p LOH was significant by log-rank test.
We present here the deletion map of chromosome 9 and alterations
of the pi6 gene in NB. In this study, we detected two commonly
deleted regions: one was between the IFNB1 and D9Si 7/ loci at
chromosome 9p2l and the other was distal to the D9Si76 locus at
chromosome 9q34—qter. Recently, it has been demonstrated that both
short and long arms of chromosome 9 are frequently deleted in many
types of human cancers. Loss of the short arm of chromosome 9, in
association was found, but patients with 9p LOH showed poorer
survival than patients without 9p LOH (P
0.0486).
Mutation of the p16 Gene in NB. Because the pitS gene has been
mapped to 9p2l between the JFNB/ and D9S171 markers, PCR-SSCP
analysis was performed to screen for p/iS mutations in 19 cell lines
and 80 fresh tumors using three sets of primers flanking two exons of
the pitS gene. Although SSCP variants were not detected in the 19 cell
lines, a tumor-specific SSCP pattern was observed in one of 80 fresh
tumors. Sequence analysis of the variant SSCP band revealed a
missense mutation at codon 52 from ATG (Met) to AAG (Lys; Fig. 4).
Because these patients did not show heterozygous genotypes at 9p2l,
we could not determine whether LOH had occurred on chromosome
9p2l. Although several polymorphisms were reported (15), we did not
detect such polymorphisms in this study. Previously, we detected
polymorphisms only in 3 of 71 Japanese patients (4%) with lung
cancers (15). Thus, the population with polymorphic alleles would be
rare in Japanese.
Expression of the p16 Gene in NB Cell Lines. To investigate the
expression pattern of the pitS gene in NB, we performed Northern and
Western blot analyses in 19 NB cell lines (Table 2; Fig. 5). pitS
linesCell Table 2Status
Fig. 4. PCR-SSCP (A) and sequence (B) analy
TN
19 cases
of NB
ceIl
geneDNAmRNAProteinMethylationTGW
linep'6
IMR-32
WT
—
-
-
LAN1
LAN2
LANS
NBI
NB9
NBI6
NBI9
NB39
NB69
SCMCN2
SCMCN3
SCMCN4
SCMCN5
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
WT
+
+
-
-
-
+
CHP-l34
TNBI
GOTO
-(,
KP-N-NSWV'
WT,
wild
-
-
-
-
-
-
—
-
+
-
-
-
-
-
+
-
-
-
-
-
-
+
+
-
+
+
-
-
-
+
-
-
+
WT
+
+
—
WT
WT
WT+
+
+
-
-
-
-
-+
--
type.
B
A
ses of the p!6 gene in a case of NB. DNA was
of the pitS gene iii
Tumor
ACGT
Normal
ACGT
isolated from a tumor (7) and corresponding normal
JrG
T 52Met
tissue (N) of patient 4. A. abnormal conformers
were detected in the 5-region of the p16 gene exon
2. B, point mutation resulting in amino acid change
from methionine to lysine was detected at codon 52.
_J
,@
I
52
A
—Ic
IT
LG
•@•1
T
LG
910
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1997 American Association for Cancer Research.
Lys*
CHROMOSOME9 DELETIONSAND p16 IN NEUROBLASTOMA
123456789
A
4.3 kb —
.
0.65kb—
•.
0.35kb—
•
B
1
*
2 3
4
56789
0.8 kb
C
123456789
p16 —@
Fig. 5. Methylation ofthe 5' CpG island in thepl6 gene and expression ofthepió gene
in NB cell lines. N4l7 small cell lung carcinoma cell line and A549 non-small cell lung
carcinoma cell line are used as positive and negative controls, respectively. The pitS gene
is expressed in N417, whereas it is homozygously deleted in A549. Lane I, N417; Lane
2, A549; Lanes 3—9,NB cell lines, TOW, IMR-32, LAN 1, NB 16, NB69, LAN2, and
NB19. A, Southern blot analysis of the 5-region of exon 1 of the p16 gene. Digestion with
SmaI plus EcoRI yields two small fragments (0.65 and 0.35 kb) in six cell lines (Lanes 1,
N-myc amplification. Although the age of children at diagnosis is also
a factor to predict the outcome of patients, 9p LOH was not correlated
with the age of patients. This might be due to the small number of
patients over 1 year in this study. Therefore, further studies with a
large population of children over 1 year may lead to conclusive data
for the correlation between age of patients and 9p LOH. Although lp
LOH is also considered to correlate with poor survival, we found no
association between lp LOH and 9p LOH. Because we used only two
markers at lp32 for detection of Ip LOH (12), it could influence the
statistical analysis.
The pi6 gene, a candidate tumor suppressor gene involved in many
types of human cancers (13—15),have been mapped to chromosome
9p2l between the IFNBi and D9Si7i loci, which is one of the
commonly deleted regions on chromosome 9 in NB. However, no
homozygous deletions have been reported in NB (32). It is also
reported that there were no pitS gene mutations and no LOH at the
JFNA locus close to the pi6 gene locus (32). In this study, we found
no homozygous deletions in both primary tumors and cell lines, and
a missense mutation was detected only in a primary tumor. Because
this type of mutation has not been reported previously, we do not
know if it is functionally significant. These data suggest that the pitS
gene is not a target tumor suppressor gene inactivated in NB. How
ever, it is possible that the p16 gene is inactivated by alternative
mechanisms in most tumors, such as intronic deletions and mutations
not detected by sequence analysis of exons or Southern blot analysis.
Moreover, recent evidence indicated that transcriptional repression by
DNA methylation of promoter and 5' regulatory sequences may be a
pathway for inactivation of the pitS gene in several types of human
cancers (16—17).To clarify whether the p/tS gene is inactivated in NB,
we examined the status of the pi6 gene using Southern, Northern, and
Western blot analyses. The pi6 gene was not expressed in most NB
cell lines. Absence of the pi6 mRNA in the samples lacking the p16
protein suggests that pitS expression is likely to be regulated at the
transcriptional level. Moreover, we found that hypermethylation of
the 5' CpG island in the pitS gene is frequent in cell lines lacking p16
expression. Thus, it is likely that the pitS gene is inactivated mostly by
5' CpG island methylations rather than DNA alterations in NB.
Similar results were also reported in several other types of human
cancers (16—17).However, the mechanisms for the absence of the pi6
mRNA in the remaining cell lines is not clear. We cannot role out the
3.4. 5. 6,and7),indicating
thattheSmaIsiteisunmethylated,
andonelargefragment(4.3 possibility that mutation harbors in the promoter region of the pi6
kb) in two cell lines (Lanes 8 and 9), indicating that the SmaI site is methylated. B,
gene with the consequence of gene inactivation. Recent studies mdi
Northern blot analysis of the p16 gene. The pitS transcripts of 0.8 kb were detected in two
cated that expression of not only the pi6 gene but also the pi5 gene
of NB cell lines (Lanes 3 and 5). C, Western blot analysis of the p16 protein. p16 protein
was detected in two of NB cell lines (Lanes 3 and 5).
is suppressed by homozygous deletion, point mutation, and hyperm
ethylation of the 5' CpG island of this gene in several human cancers
(33, 34). Particularly, in leukemia, the methylation of the 5' CpG
particular 9p2l—22, occurs in a variety of human cancers, including
island
in the first exon of the p/S gene is more frequent than that in
melanoma (24), renal cell carcinoma (25), lung cancer (26), bladder
the pi6 gene (17). Therefore, to determine the pathways to inactivate
cancer (27), head and neck cancer (28), and ovarian cancer (29).
Chromosome 9q, in particular 9q34—qter, is also frequently deleted in these genes and whether the p15 and/or pi6 genes are involved in the
progression of NB, more detailed analysis of these genes will be
several human cancers (27, 29—31).Interestingly, both of the short
necessary.
and long arms are deleted in some human cancers, such as bladder
In conclusion, it was demonstrated here that at least two tumor
cancer, ovarian cancer, esophageal carcinoma, and renal cell carci
suppressor genes on chromosome 9 are involved in the genesis
noma (27, 29 —3
1). Therefore, as in the case of several other types of
and/or progression of NB. Particularly, the gene on chromosome
cancers, at least two tumor suppressor genes on both short and long
9p is likely to be associated with progression of NB, and the pi6
arms of chromosome 9 may contribute to genesis and progression of
gene is a candidate target tumor suppressor gene involved in the
NB. Furthermore, we found that 9p LOH significantly correlates with
progression of NB. However, because we have not examined for
an advanced stage of the disease and poor prognosis of the patient, and
9q LOH did not correlate with these clinical parameters. In the present p16 methylation in primary tumors, the biological significance of
p16 inactivation in the genesis and progression of NB is still
study, 9p LOH was significantly associated with poor prognosis
independently of N-myc amplification. Thus, it is possible that a unclear. For this reason, we are currently investigating the asso
ciation ofpl6 expression and methylation in tumors with prognosis
tumor suppressor gene located on chromosome 9p2l plays an impor
(ant role in the progression of NB through a different pathway from
of patients with NB.
911
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CHROMOSOME9 DELETIONSAND p16 IN NEUROBLASTOMA
ACKNOWLEDGMENTS
silencing of the tumor suppressor p16/CDKN2JMTSI in human cancers. Nat. Med., 1:
686—692, 1995.
17. Herman, J. 0., Merlo, A., Mao, L., Lapidus, R. G., Issa. J-P. J., Davidson, N. E.,
We thank Dr. S. Nagata for providing the IFNBI cDNA probe.
Sidransky, D., and Baylin. S. B. Inactivation of the CDKN2JpI6JMTSI gene is
frequently associated with aberrant DNA methylation in all common human cancers.
Cancer Res., 55: 4525—4530, 1995.
18. Evans, A. E., D'Angio, G. J., and Randolph. J. A proposed staging for children with
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Deletion Map of Chromosome 9 and p16 (CDKN2A) Gene
Alterations in Neuroblastoma
Junko Takita, Yasuhide Hayashi, Takashi Kohno, et al.
Cancer Res 1997;57:907-912.
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