Mutational Analysis of the CDKN2 (MTSI/p16i”k4A

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Mutational Analysis of the CDKN2 (MTSI/p16i”k4A)Gene
in Primary B-Cell Lymphomas
By Toshiki Uchida, Takashi Watanabe, Tomohiro Kinoshita, Takashi Murate, Hidehiko Saito, and Tomomitsu Hotta
The CDKNZ gene located on chromosome 9p21 encodes
the
cyclin-dependentkinase4 inhibitor p16. This gene isputaa
tive tumor-suppressor gene because of its frequent alterations in many kinds of tumor cell lines. We analyzed the
CDKNZ gene to evaluate its alterations in 52 primary specimens of non-Hodgkin’s lymphoma (NHL) or chronic lymphocytic leukemia (CLL) of B-cell origin by Southernblot analysis, polymerase
chain
reaction-mediated
single-strand
conformationpolymorphism (PCR-SSCP)analysis,anddirect sequencing.By Southern blot analysis, we showed homozygous deletion of the CDKNZ gene in 3 of 42 patients
with B-NHL (7.1%). After screening by PCR-SSCP analysis,
direct sequencing identified one missense mutation at codon 72 (nucleotide 233) and two frameshifts due to a 35-bp
deletion arising at codon 3 (nucleotides 27 to 61) and a 13bp deletion arising at codon 49 (nucleotides 163 to 175) in
patients with B-NHL (3 of 42, 7.1%). In the patient carrying
the missense mutation, hemizygous deletion of the CDKNZ
gene was also suspected. In this study, we detected alterations in CDKNZ in 6 of 42 patients (14.3%) with B-NHL and
in none of 10 patients with B-CLL. Our results suggest that
the CDKNZ alterations contribute in tumorigenesisin some
patients with B-NHL.
0 1995 by The American Societyof Hematology.
T
fragment 1, also known as CIPI, SDII, CAPI, and CDKNI)
were absent in
many
primary
tumors.” Alterations in
CDKN2 have been reported inmanyprimaryhuman
cancers?O-29
following their demonstration in cell lines. In contrast to WAF1 mutations, the CDKN2 gene was found to be
homozygously deleted or mutated at a high frequency in
some types of primary tumors, showing thatthe CDKN2
gene is a tumor suppressor.20”2However, because there was
a discrepancy between the rate of the CDKN2 mutations and
that of loss of heterozygosity at 9p21 in primary tumors”
and also between the rate of CDKN2 alterations in primary
tumors and that of cell line^,*^-*^ itis still unclear whether
the CDKN2 gene also plays an important role in tumorigenesis of other primary tumors. The extent to which the CDKN2
gene is involved in tumorigenesis is currently under intensive
investigation.
In hematologic malignancies, the CDKN2 alteration has
beenwell
investigated in acute lymphocytic leukemias
(ALL),’4.’o.’I which frequently show chromosome 9 abnormalities.17 However, detailed investigations of this gene in
other hematologic malignancies have not yet been reported.
In this study, to evaluate the alterations in the CDKN2 gene
and to determine whether this gene plays an important role
in tumorigenesis or disease progression of primary B-cell
lymphomas, we performed Southern blotting, polymerase
chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP), and direct sequence analyses of the
CDKN2 gene in patients with non-Hodgkin’s lymphoma
(NHL) or chronic lymphocytic leukemia (CLL).
HE CYCLIN-DEPENDENT kinases (CDKs) bindto
G1-cyclins and control the GUS transition of the cell
cycle by phosphorylation of the retinoblastoma protein. CDK
inhibitory proteins bind to the CDKs and inhibit the catalytic
activity of the CDIUGl-cyclin complex.’,2Several inhibitors
of CDK-cyclin activity have been identified to date in mammalian cells: p1.5,’p16:
~ 1 8 , ~ ~ and
2 1 ,~~2 -7 *. ~ CDK4”
,’”
and its closest homologue CDK6” are activated by binding
to cyclin D l , which is one of the G1-cyclins. The p16 protein
specifically inhibits CDK4-cyclin D l and CDK6-cyclin D1
kinase activity in ~ i t r o ,and
~ , ~perhaps prevents inappropriate
phosphoryration of the retinoblastoma protein.* Recently the
CDKN2 (also known as multiple tumor suppressor 1: MTSI,
p16INK4*,
CDK4 Inhibitor: CDK41) gene was found on chromosome 9 ~ 2 1 , where
’~
molecular, genetic, and cytogenetic
abnormalities have been reported in many kinds of malignancies, including malignant melanoma^,'^ gliomas,15lung cancers,I6 and 1e~kemias.l~
The in vivo importance of p16 was
suggested by the discovery that p16 protein is encoded by
this gene.” Recent studies showed frequent homozygous
deletions and point mutations of the CDKN2 gene in many
tumor cell lines.”.” These results imply that the CDKN2
gene functions as a tumor suppressor and that mutations in
the CDKN2 gene are involved in tumorigenesis in many
human cancers.
The involvement of CDK inhibitors in cancer development was first demonstrated by studies on p21, which is a
potent and universal inhibitor of G1 cyclin-dependent kinases6-’ However, it was reported recently that mutations
of the p21 encoding gene WAF1 (wild-type p53-activated
MATERIALSANDMETHODS
From the First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan.
Submitted March 27, 1995; accepted May 30, 1995.
Supported in part by Grants-in-Aid for Cancer Research (4-5)
from the Ministry of Health and Welfare of Japan.
Address reprint requests to Tomomitsu Hotta, MD, First Department of Internal Medicine, Nagoya Universiry School of Medicine,
65 Tsurumai-cyo, Showa-ku, Nagoya 466, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/9.5/8607-00I6$3.00/0
2124
Patient profiles. We analyzed a total of 52 patients, 10 with
CLL/small-cell lymphocytic lymphoma and 42 with NHL, other
than small-cell lymphocytic lymphoma of B-cell origin, who were
admitted to our hospital between July 1987 and September 1994.
Sixteen of these patients were treated in our affiliated hospitals and
seven patients were admitted at relapse. The profiles of these patients
are shown in Table l . Histologic and surface marker studies using
immunohistochemical techniques showed B-cell phenotype in all
patients.
Preparation of DNA. DNA extraction was performed bythe
standard p r ~ c e d u r eDNA
. ~ ~ samples from lymph nodes or extranodal
tumors were obtained at biopsy performed after informed consent
was obtained. We also obtained DNA samples from mononuclear
cells, which were separated on Ficoll-Paque (Pharmacia, Uppsala,
Blood, Vol 86, No 7 (October l ) , 1995: pp 2724-2731
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MUTATIONALANALYSISOF
THE CDKNZ (MTSl/p7GfNKa)GENE
with a laser image analyzer (FUJIX BAS2000; Fuji Photo Film).37
We measured phosphostimulated luminescence of bands, correNo. patients
52
sponding to the CDKN2/MTSI gene, the MTS2 gene, and the c-myb
CLL
10
gene.
Age (yrs)
54-78
As a normal control, we used DNA obtained from normal B cells
Median
63
of the spleen excised from a patient with gastric cancer, as described
Clinical stage of CLL
in detail previously.'* We also used DNA of MOLT4 and Jurkat
A
1
cells as controls, inbothofwhich
homozygous deletion of the
B
2
CDKN2 gene were reported previou~ly.~~
C
7
PCR-SSCP analysis. Analysis by PCR-SSCP was performed esNHL
42
sentially as described p r e v i o ~ s l y .We
~ ~ .analyzed
~~
exon 1 and exon
Age (yrs)
15-83
2 of the CDKN2 gene, comprising 97% of the coding sequence. For
Median
56
exon 1, weused the primers published previo~sly.'~
Because the
Clinical stage of NHL
fragment size of exon 2 seemed to be too long for PCR-SSCP
l
1
analysis, we divided it into three fragments, designated as CDKN2II
9
exon 2A, CDKN2-exon 2B, andCDKN2-exon2C.We
amplified
111
11
genomic DNAofCDKN2
gene by PCR in the presence of5%
IV
21
dimethylsulfoxide. For exon 2, we performed nested PCR to exclude
Histopathology of N H L
amplification of the MTS2 gene. Nucleotide sequences of the primers
Follicular small cleaved
are shown in Table 2. Thirty-five cycles of the PCR reaction at 94,
Follicular mixed
55,and 72°Cfor 0.5,0.5, and 1 minute, respectively, were performed
Follicular large
in a Thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). In amplifiDiffuse small cleaved
cation of exon 2, 1 p L of the first PCR products was subjected to
Diffuse mixed
the
second roundofPCR
under the same conditions as the first
Diffuse large
reaction except for the use of 30 cycles and the second-round PCR
The diagnosis of CLL was based on the criteria proposed by the
primer set. The products were diluted 100 times with98% forInternational Workshop on CLL,= and the clinical stage was decided
mamide, 20 mmoliL EDTA, 0.05% bromophenol blue, and 0.05%
according to a revised prognostic staging system also proposed by
xylene cyanol, and heated at 94°C for 4 minutes. Aliquots of 2
the International Workshop on CLL.33 The Modified WorkingFormulapL were then promptly applied to 5% polyacrylamide gels without
tion Classificationwas applied for histological classificationof NHL.=
glycerol. After electrophoresis at room temperature for 1 to 2 hours
at a constant power of 40 W with vigorous air cooling, the gels
were dried on Whatman 3MM paper (Whatman International, Maidstone, UK) and exposed to x-ray filmfor appropriate times at -70°C.
Sweden) density gradient centrifugation of peripheral blood, pleural
These conditions for electrophoresis were selected as the best for
effusions, or ascites with lymphoma cell invasion. By morphologic
the separation of products among several conditions examined emexamination and surface marker analysis, we confirmed that the
pirically in a preliminary study (data not shown).
proportion of tumor cells exceeded 70% in all the specimens examDirect DNA sequencing. Sequences of samples showing mobilined.
ity shifts by PCR-SSCP analysis were confirmed by direct sequencSouthern blor analysis. Ten micrograms ofDNAwas digested
ing as described previo~sly.~'
Briefly, a small area of the gel correwith EcoRI (Boehringer Mannheim, Mannheim, Germany), sepasponding to the position of the band was cut out, and single-stranded
rated by electrophoresis through 0.7% agarose gels, and blotted onto
DNA from the dried gel was eluted into 50 pL of distilled water.
nylon membranes (Hybond N; Amersham, Buckinghamshire, UK).
Four microliters of the eluted solution was subjected to asymmetrical
p26 cDNA was used as a probe for detection of the EcoRI-digested
amplification by PCR. The amplified reaction mixture was purified
fragments of the CDKN2 genomic DNA. The 471-bp p26 cDNA
in a micro-concentrator Centricon 30 (Amicon; W.R. Grace and CO,
probe was produced by reverse transcriptase (RT)-PCR, using cDNA
Beverly, MA), and the single-stranded DNA was annealed to a 5'from normal human peripheral blood lymphocytes as a template.
end labeled primer. Chain elongation and termination were perThe primers for RT-PCR were designed to amplify the region
formed using a Sequenase kit (Sequenase Ver. 2.0; US Biochemical
covering the entire coding sequence of the p16 cDNA reported
Corp. Cleveland, OH), and the products were run in 6% polyacrylprevio~sly.~
These sequences were as follows: 5"CGAGGCAGCATGGAGCCTT-3' and 5'-GCCTCTCTGGTTCITTCAAT-3'. amide gels containing 7 m o m urea. The products from bands with
a mobility shift wererun in parallel with those without mobility
Oligonucleotide primers in the PCR reactions were synthesized by
shift in the same gel. After electrophoresis at room temperature for
the phosphoroamide method with a 391 DNA synthesizer (Applied
appropriate times at a constant current of 30 mA, the gels were
Biosystems, Foster City, CA). This probe can identify fragments of
dried on Whatman 3MM paper and exposed to x-ray film at room
both the CDKN2IMTS2 gene and the multiple tumor suppressor 2
temperature.
(MTS2, also known as ~ 1 5 " ~gene.
~ ~ The
) p16 cDNA probe was
labeled with [a-32P]dCTPby the random primer method, and hybridRESULTS
ization was performed for 2 hours at 65°C using a Rapid-hyb buffer
(Amersham). After washing at high stringency with 0.1 X SSC and
Analysis of the CDKN2 gene by Southern blotting. We
0.1% sodium dodecyl sulfate (SDS) at 6 5 T , membranes were exinvestigated the genomic DNAcorresponding to the CDKN2
posed toKodakXARfilm
(Eastman Kodak, Rochester, NY) at
gene by Southern blotting to detect gross alterations. The
-70°C. The same membranes were then rehybridized with labeled
p16 cDNA probe hybridized with EcoRI digestion fragments
human c-myb cDNA probe36to confirm the integrity of the sample
of about 4.2- and 6.0-kb corresponding to the CDKN2MTS1
DNA and completeness of EcoRI digestion.
gene and the MTS2 gene, respectively. Figure 1 shows part
To evaluate deletions of the CDKN2 gene, we also exposed the
blots to phosphor imaging plates (Imaging plate BAS 111; Fuji Photo
of the results of Southern blot analysis. The band reflecting
Film, Minami-ashigara, Japan), whichwere subsequently studied
the CDKN2lMTSI gene disappeared clearly in patients 7
Table 1. Patient Profiler
of
2725
nt
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2726
UCHIDA ET AL
Table 2. Primers for PCR-SSCP Analysis and Direct Sequencing of the CDKN2 Gene
Amplified
(bp)
Exon 1
343
Exon 2A
210
Sequence
5'-GAAGAAAGAGGAGGGGCTG-3'
5"GCGCTACCTGAlTCCAAlTC-3'
~"CTCTACACAAGC~TCCTT-~'
5"CAAAlTCTCAGATCATCA-3'
5'-CTCTACACAAGClTCClT-3'
5'-AGCACCACCAGCGTGTCCAG-3'
5"CTCTACACAAGClTCClT-3'
5'-CAAATTCTCAGATCATCA-3'
5"CCGTGCACGACGCTGC-3'
5'-GCATCGCGCACGTCCA-3'
5"CCGTGCACGACGCTGC-3'
5"TGAGClTGGAAGCTCT-3'
5'-lTCCTGGACACGCTGGT-3'
5'-CAAAlTCTCAGATCATCA-3'
2F
1108R
For first PCR
For second PCR
83
Exon 28
For first PCR
For second PCR
Exon 2C
For first PCR
216
For second PCR
(FigIA, lane 6), 25 (Fig l A, lane 3), and 26 (Fig IA,
lane 2). We found no significant increase or decrease in
radioactivity of the band reflecting the MTS2 gene in any
specimens, and no rearranged band was shown in any of the
patients. Thus, we detected three homozygous deletions in
patients with B-NHL (3 of 42, 7.l%), and no deletions in
patients with B-CLL.
Analysis of the CDKN2 gene by the PCR-SSCP method.
To determine whether CDKN2 mutations were present in
primary B-cell lymphomas, PCR-SSCP analysis was performedusing the same specimens as those examined by
Southern blotting of all 52 CLLNHL patients. In 5 of 42
patients with B-NHL, we detected band shifts. These
changes were also confirmed under other electrophoresis
D V V
V
conditions (data not shown). Three such aberrantly migrating
bands were found by analysis of the CDKN2-exon 2A fragment (patient 3, 12, and 14) and one was found by analysis
of the CDKN2-exon 2C fragment (patient 41). We also found
aberrantly migrating bands in one patient (patient 2) by analysis of exon I fragment. But, we found no aberrantly migrating bands by analysis of theCDKN2-exon 2B fragment.
Figure 2 shows part of the results of PCR-SSCP analysis
using the primer sets for the exon 1 and the CDKN2-exon 2A
fragment. Two aberrantly migrating bands with no normally
migrating bands were detected in patient 3 (Fig 2B, lane 9).
Aberrantly migrating bands with residual normally migrating
bands were found in patient 2 (Fig 2A, lane 7). patient 12
(Fig 2B, lane 3), patient 14 (Fig 2B, lane 13), and patient
C
D
V
c-myb
C
---
23.1 kb
9.4kb
6.6 kb
4.4 kb
2.3 kb
2.0 kb
l 2 3 4 5 6 7 8 9 1 01 2 3 4 5 6 7 8
A
B
Fig 1. Southern blot analysis of the CDKNUMTSl and MTS2 genes. The CDKNWMTSl and MTS2 genes were detected as two bands of
approximately 4.2 and 6.0-kb, respectively, in EcoRI-digested DNA. The positions of molecular weight markers electrophoresed in parallel and
one of EcoRl fragments recognized by c-myb cDNA probe are also shown. D, samples from cell lines showinghomozygous deletion of the
CDKNZ gene; C, splenic B cell as a normal control. (A)Lane 1, MOLT4; lanes 2 through 9, patient samples; lane 10, splenic B cells as a normal
control. Lanes 2, 3, and 6 show homozygous deletions of theCDKNZ/MTSl gene without deletions of the MTS2 gene in patients 26,25, and
7, respectively (V).(B) Lane 1, Jurkat; lanes 2 through 7, patient samples; lane 8, splenic B cells. Lane 6 shows possible monoallelic loss of
the CDKN2/MTS1 gene in patient 3 ( W .
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MUTATIONAL ANALYSIS OF THE CDKNZ (MTS7/p76'NKa)GENE
v
2727
v
v
v
v
B
A
l 2 3 4 5 6 7 8 9 1 0
1 2 3 4 59876
CDKNZ-exon 1
10
11121314151617181920
CDKNZ-exon 2A
Fig 2. PCR-SSCP analysis of the CDKN2gene in B-NHL. Mobility shift pattern of
DNA fragments amplifiedby theprimer sets of exon 1 and
CDKfV2-exon 2A are shown. (A) Lane 7 shows an aberrantly migrating fragmentin addition t o wild-type fragments in patient 2 W ) . (B) Lane
3 shows an aberrantly migrating fragment
in addition t o wild-type fragments in patient 12 (V).Lane 9 shows aberrantly migrating fragments
with no bands corresponding t o wild-type fragments in patient 3 (V).Lane 13 shows four bands of wild-type and aberrantly migrating
fragments in patient 14 (V).Lanes 15 and 17 show the absence of bands in patients 25 and 7, respectively (V), as the result of failure of PCR
amplification due t o homozygous deletions of theCDKNZ gene.
41 (data not shown). No bands were detected by analysis of
any of these fragments in patients 7 (Fig 2B, lane 15), 25
(Fig 2B, lane 17), or 26 (data not shown). Analysis of other
samples of patient 3 containing normal tissue showed predominantly normal pattern (Fig 3A). To determine the sensitivity of our PCR-SSCP assay for mobility shift, we also
performed a mixing experiment (Fig 3B). We performed
PCR-SSCP assay using both mutated sample (patient 3).
which showed only aberrantly migrating bands and control
sample. As a result, we could detect positive aberrant bands
when 5% of mutated sample was mixed with control sample.
Nucleotide sequence analysis of the CDKN2
gene.
Nucleotide sequences of the CDKN2 gene in patients with
mobility shifts on PCR-SSCP analysis were determined by
direct DNA sequencing. Six nucleotide changes were identitied by direct sequencing in five patients with B-NHL. One
B
A
Fig 3. PCR-SSCP analysis of the CDKNZ gene in
patient 3. (A) Lymph nodesample (LN) showed only
aberrantly migrating band without normally migrating band. Mononuclear cells of bone marrow (BM)
andperipheralblood
(PB) showedpredominantly
normal pattern. Because a small number of tumor
cells were involved in BM and PB, mobility shifts
were also detected. (B) Sensitivity of PCR-SSCP
method. Detection of only5% mutated sample was
possible.
r
z
Patient 3
Blotting
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2728
UCHIDA ET AL
Table 3. Results of Analysis of the C D K W Gene in B-NHL
-
Nucleotide Sequence of Mutated Allele
No.
Southern
Codon
PCA-SSCP
2
ti+
MM
Patient
Nucleotide
3-t
GAGCC-.
Change'
. . . . CGGGGTCGG
L
GAGCCIGTCGG
(35-bp deletion)
ACCCGACCC-ACCC&4CCC
CS
Histopathology
NI*
FL
IV
FSC
IV
DL
DL
--t
3
7
14
25
26
+i-
-1+I+
-1-
-1-
MI-
-1MM
-1-1-
72
(Arg)
-
49- t
-
-
(GM
AGCGCCCGAGTGGCGGAGCT
--t AGCIAGCT
(13-bp deletion)
111
-
II*
-
1
l
DM
DL
Abbreviations: +I+, n o deletion; +I-, hemizygous deletion; -I-, homozygous deletion; M, mutated allele; W, wild-type allele; CS, clinical
stage at presentation; FL, follicular large; FSC. follicular small cleaved; DL, diffuse large; DM, diffuse mixed.
Italic and bold type with underline indicates mutatedsites.
t First codon of deletion.
Sample at relapse was used
in this study.
*
nucleotide change in exon 1 and three nucleotide changes
in exon 2 were found individually in four patients, and two
base changes in intron 1 were found in one patient. Three
of these six nucleotide changes led to amino acid changes
(Table 3). One frameshift caused by a 35-bp deletion arising
at codon 3 (nucleotides 27 to 61) was detected in patient 2,
which introduced a translational stop codon at codon 23 of
exon l (nucleotide numbering system is as described by
Serrano et a14). In exon 2, one missense mutation at codon
72 (CGA to CAA, nucleotide 233, Arg to Gln) was detected
in patient 3, and one frameshift caused by a 13-bp deletion
arising at codon 49 (nucleotides 163 to 175) was detected
in patient 14, which introduced a translational stop codon at
codon 133. Figure 4 shows the results of direct sequence
analysis of the CDKN2 gene around these mutations in these
three patients. Two base changes in intron 1 (G to A, and
G to T substitutions at 4th and 14th nucleotides 5' of the
intron l-exon 2 splice junction, respectively) were detected
in patient 12, and a silent base change at codon 127 (GGG
to GGA, nucleotide 399, Gln to Gln) was detected in patient
41. In patients showing normally migrating bands, nucleotide
sequences corresponded with those of the CDKN2 gene reported previ~usly.~
We confirmed three mutations among 42
patients with B-NHL (7.1%) and no mutations in patients
with CLL by PCR-SSCP analysis and direct sequencing.
DISCUSSION
Tumor-suppressor genes are inactivated by alterations in
the properties of the gene product (qualitative changes) or
in a decrease or complete loss of gene products (quantitative
changes). Mutations or gene rearrangements within the coding region can induce qualitative changes, whereas downregulation caused by other genes or homozygous deletion can
lead to quantitative changes. Most tumor-suppressor genes
frequently involved in human cancers, for example p53 or
RB genes, are known to be inactivated by mutation^^^'^'
whereas the CDKN2 alterations seem to frequently involve
quantitative, rather than qualitative, changes. Previous re-
ports showed the frequent occurrence of homozygous deletions of this
We performed Southern blotting to
detect mainly quantitative changes and PCR-SSCP analysis
to detect qualitative changes in this study. As a result, we
could detect both qualitative and quantitative changes of the
CDKN2 gene in B-NHL,. Six patients (14.3%) with B-NHL
showed alterations of this gene. Three of these patients
showed CDKN2 homozygous deletions (7.1%). This frequency was similar to previous reports.30344
The remaining
three of these patients showed mutations in the CDKN2 gene
(7.1%), one missense mutation and two frameshifts caused
by a small deletion.
Published studies in hematologic malignancies have
shown frequent homozygous deletions of the CDKN2 gene.
However, with regard to point mutation, to our knowledge,
only one missense mutation has been reported in a patient
with B-precursor ALL.30In the present study, we found three
mutations in B-NHL. First, we identified a missense mutation at codon 72, which is a hotspot for CDKN2 m~tation,'~.~'
in patient 3. It was suggested that this nucleotide change
was not a polymorphism because PCR-SSCP analysis used
normal tissue samples of this patient and showed normal
pattern (Fig 3A). In addition, this patient showed a possible
hemizygous deletion because of the low phosphostimulated
luminescence intensity of the bandreflecting the CDKN2
gene by laser image analysis (Fig l B , lane 6). These results
suggested that the CDKN2 gene inactivated by not only a
quantitative change, but also a qualitative change, and its
inactivation contributes to tumorigenesis of B-cell malignancies. Second, we showed other mutation in patient 14. Direct
sequencing of the DNA from the abnormally migrating band
confirmed a frameshift due to a 13-bp deletion. The first
codon of this deletion was the same codon at which a m i s sense mutation was reported in B-ALL.30 As the fragment
amplified by the CDKN2-exon 2B and exon 2C primer set
showed a normal mobility pattern on PCR-SSCP analysis,
this frameshift may cause alteration of the normal 148 amino
acid p16 protein to a 133 amino acid truncated variant. Third,
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MUTATIONAL ANALYSIS OF THE CDKNZ ( I V T S I / ~ ~ ~GENE
’~‘(~~)
2729
A
A
T “. C
G
A --
G
C
T
G
A
C
T
G
C
A
C
G
G
C
I
C
C
G
G
G
d
T
C
G
G
G
T
A
I
35 bp deletion
Patient 2
G
B
T
C
G
T
A
C
T
C
A
C
. .-.
.
.L
..
C
C
G
A
C
C
I”
C
G
C
C
C
A
A
C
C
C
Fig 4. Nucleotide sequencing of samples showing theCDKNZ gene mutations in B-NHL. Bold type
indicates the nucleotides of mutation sites. (A) The
nucleotide sequence of patient 2 shows a 35-bp deletion arising
at
codon 2 IGAGCCTCG
CGGGGTCGG to GAGCCIGTCGG, nucleotide 27 t o
61, open arrow). (B) The nucleotide sequence of patient 3 shows a point mutation atcodon 72 (CGA t o
CAA Arg t o Gln, nucleotide 233, closed a r r o q . IC)
T r e nucleotide sequence of patient 14 shows a 13bp deletion arising at
codon 49 IAGCGCCCGAGTGGCGGAGCT t o AGCIAGCT, nucleotide 163 t o 175,
open arrow).
T
C
G
A
V
b.
T
G
G
Patient 3
A
,
T C
-
G
A
c-
G
C
E\
C
:
...
although mutations in exon 1 are rare, we also found a mutation in exon 1 in patient 2. A 35-bp deletion led a frameshift,
which led stop codon at codon 23, and may alter p16 protein
to a small protein. We could not confirm hemizygous deletion in these two patients by Southern blot analysis (Fig 1 B,
lanes 5 and 7). Two alternative explanations for the findings
in these patients are possible. One is that hemizygous deletion in tumor cells ismasked by contamination with the
normal component in this lymph node sample. This possibility implies CDKN2 inactivation because one allele of tumor
G
G
T
Wild-type
C
C
C
T
C
A
G
A
G
C
Wild-type
T
I
\:
d
C
A
G
C
T
Patient 14
cells is deleted and the other allele has a small deletion. The
other possibility is that tumor cells have an abnormal allele
with a small deletion and another allele. If a small alteration
exists in the other allele, which cannot be detected by Southern blotting and PCR-SSCP analysis, the latter possibility
also implies that the CDKN2 gene is inactivated as a result
of alterations in both alleles.
As the existence of two major proliferation control pathways by p16 and p15 have been ~uggested,’.~deletions of
both of these genes, rather than loss of only one gene, may
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2730
UCHIDA ET AL
induce tumorigenesis. Previous reports that the majority of
homozygous deletions in many kinds of cell lines removed
both the CDKN2/MTSl and MTS2 gened3 confirmed this
hypothesis. However, we could show no gross alterations of
the MTS2 gene by Southern blotting. Recently, it was reported that two of three patients with B-cell type ALL showing CDKN2MTSI deletion did notshow MTS2 deletion,
similar to our results, whereas in contrast most patients with
T-cell type ALL showed homozygous deletions of both these
genes.3’ These results suggest that the CDKN2/MTSl alteration frequently does not involve the MTS2 gene in B-cell
lineage lymphoid malignancies, although we and previous
investigators did not yet analyze the MTS2 gene to detect
mutations. As we also found a small deletion in two patients
in this study, the deleted region containing the CDKN2 gene
in B-cell malignancies may be smaller than that in T-cell
malignancies. CDKN2 alterations may be more important
in the tumorigenesis of B-cell lymphomas than the MTS2
alterations.
Although a relationship between p53 mutations and disease progression was reported in B-cell lymphoma,4’ we did
notfind a definite relationship between the CDKN2 alterations and patients’ profiles (Table 3 ) . Patients 7 and 14
had lymphomas that were highly aggressive and resistant to
chemotherapy. Patients 25 and 26 had early stage lymphomas at presentation, but soon relapsed. In contrast, patient
3 has enjoyed long survival despite advanced stage at presentation. Recently, the appearance of possible allelic loss at
relapse was reported in B-ALL.22However, no significant
differences in outcome were observed between patients with
and without homozygous deletions of the CDKN2 gene in
ALL.3oItis still unclear whether the CDKN2 alterations
contribute to disease progression of hematologic malignancies.
In conclusion, we detected alterations in the CDKN2 gene
in some patients with B-NHL in this study and confirmed
the contribution of this gene to tumorigenesis of B-NHL.
However, we found no significant relationship between
CDKN2 alteration and disease progression. Recently, it has
been suggested that decreased CDKN2 expression may arise
in nasopharyngeal carcinoma cell lines without point mutat i ~ nTo
. ~clarify
~
the characteristics of the CDKN2 alterations
in hematologic malignancies, in particular lymphoid malignancies, detailed investigations of the CDKN2 gene, including RNA/protein expressions, in a large number of patients
and at different clinical stages in the same patient are necessary.
ACKNOWLEDGMENT
We thank Drs A. Ichikawa (Gifu Tajimi Prefecture Hospital) and
Y. Morishita (Showa Hospital) for allowing us to examine and for
providing information about their patients with NHL or CLL. We
also thank Dr H. Nagai (Jefferson Cancer Institute).
REFERENCES
1. Sherr CJ: Mammalian G1 cyclins. Cell 73:1059, 1993
2. Peter M, Herskowitz I: Joining the complex: Cyclin-dependent
kinase inhibitory proteins and cell cycle. Cell 79:181, 1994
3. Hannon GJ, Beach D: ~ 1 5 is” a potential
~ ~ ~ effector of TGFP-induced cell cycle arrest. Nature 371:257, 1994
4. Serrano M, Hannon GJ, Beach D: A new regulatory motif in
cell-cycle control causing specific inhibition of cyclin DICDK4. Nature 366:704, 1993
5. Guan KL, Jenkins CW, Li Y, Nichols MA, Wu X, O’Keefe
CL, Matenv AG, Xiong Y: Growth suppression by p18, a
p161NK4A/MTS1- and p141NK4B/MTS2-related CDK6 inhibitor, correlates
with wild-type pRb function. Genes Dev 8:2939, 1994
6. Harper J W , Adami GR, Wei N, Keyomarsi K, Elledge SJ: The
p21 Cdk-interacting protein Cipl is a potent inhibitor of G1 cyclindependent kinases. Cell 755305, 1993
7. El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R,
Trent JM, Lin D, Mercer E, Kinzler KW, Vogelstein B: Wafl, a
potential mediator of p53 tumor suppression. Cell 75:817, 1993
8. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach
D: p21is a universal inhibitor of cyclin kinases. Nature 366:701,
I993
9. Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts
’
JM, Tempst P, Massague J: Cloning of ~ 2 7 “ a~ cyclin-dependent
kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 7859, 1994
IO. Toyoshima H, Hunter T: p27, a novel inhibitor of G1 cyclinCdk protein kinase activity, is related to p21. Cell 78:67, 1994
I I . Matsushime H, Ewen ME, Strom DK, Kat0 JY, Hanks SK,
Roussel MF, Sherr CJ: Identification and properties of atypical catalytic subunit (p34PSK”3/cdk4)
from mammalian D type G1 cyclins.
Cell 71:323, 1992
12. Meyerson M, Harlow E: Identification of G1 hnase activity
for cdk6, a novel cyclin D partner. Mol Cell Bioi 14:2077, 1994
13. KambA, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman
K, Tavtigian SV, Stockert E, Day RS 111, Johnson BE, Skolnick
MH: A cell cycle regulator potentially involved in genesis of many
tumor types. Science 264:436, 1994
14. Fountain JW, Karayiogou M, Emstoff MS, Kirkwood JM,
Vlock DR, Titus-Emstoff L, Bouchard B, Vijayasaradhi S, Houghton
AN, Lahti J, Kidd VJ, Housman DE, Dracopoli NC: Homozygous
deletions within human chromosome band 9p21 in melanoma. Proc
Natl Acad Sci USA 89:10557, 1992
15. Olopade 01, Jenkins RB, Ransom DT, Malik K, Pomykala
H, Nobori T, Cowan JM, Rowley JD, Dim MO: Molecular analysis
of deletion of short arm of chromosome 9 in humangliomas. Cancer
Res 52:2523, 1992
16. Olopade 01, Buchhagen DL, Malik K, Sherman J, Nobori T,
Bader S, Nau MM, Gazder AF, Minna JD, Dim MO: Homozygous
loss of the interferon gene defines the critical region on 9p that is
deleted in lung cancer. Cancer Res 53:2410, 1993
17. Chilcote RR, Brown E, Rowley JD: Lymphoblastic leukemia
with lymphomatous features associated with abnormalities of the
short arm of chromosome 9. N Engl J Med 313:286, 1985
18. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson
DA: Deletions of the cyclin-dependent kinase-4 inhibitor gene in
multiple human cancers. Nature 368:753, 1994
19. Shiohara M, El-Deiry WS, Wada M, Nakamaki T, Takeuchi
S, Yang R, Chen DL, Vogelstein B, Koeffler HP: Absence of WAF1
mutations in a variety of human malignancies. Blood 84:3781, 1994
20. Mori T, Miura K, Aoki T, Nishihara T, Mori S, Nakamura
Y: Frequent somatic mutation of the MTSKDK4I (multiple tumor
suppressor/cyclin-dependentkinase 4 inhibitor) gene in esophageal
squamous cell carcinoma. Cancer Res 54:3396, 1994
21. Caldas C, Hahn SA, Costa LT, Redston MS, Schutte M,
Seymour AB, Weinstein CL, Hruban RH, Yeo CJ, Kern SE: Frequent somatic mutations and homozygous deletions of the p16
(MTSI) genein pancreatic adenocarcinoma. Nature Genet 8:27,
1994
22. Hebert J, Cayuela JM, Berkeley J, Sigaux F: Candidate tumorsuppressor genes MTSI ( ~ 1 6 ’ and
~ ~MTS2
~ ~ )( ~ 1 5 ~ display
~ ~ ” )
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
MUTATIONAL ANALYSIS OF THE CDKNZ (MTS7/p76‘NKa)GENE
frequent homozygous deletions in primary cells from T- but not
from B-cell lineage acute lymphoblastic leukemias. Blood 84:4038,
1994
23. Carins P, Mao L, Merlo A, Lee DJ, Schwab D, Eby Y, Tokino
K,van der Riet P, Blaugrund JE, Sidransky D, Kamb A, Liu Q,
Harshman K, Tavtigian S, Cordon-Cardo C, Skolnick MH: Rates of
p16 (MTSI) mutations in primary tumors with 9p loss. Science
265:415, 1994
24. Ogawa S, Hirano N, Sato N, Takahashi T, Hangaishi A, TanakaK, Kurokawa M, Tanaka T, Mitani K, Yazaki Y, Hirai H:
Homozygous loss of the cyclin-dependent kinase 4-inhibitor (p16)
gene in human leukemias. Blood 84:2431, 1994
25. Spruck CH 111, Gonzales-Zulueta M, Shibata A, Simoneau
AR, Lin MF, Gonzales F, Tsai YC, Jones PA: p16 gene in uncultured
tumors. Nature 370:183, 1994
26. Zhang SY, Klein-Szanto AJP, Sauter ER, Shafarenko M, Mitsunaga S, Nobori T, Carson DA, Ridge JA, Goodrow TL: Higher
frequency of alterations in the p16/CDKN2 gene in squamous cell
carcinoma cell lines than in primary tumors of head andneck. Cancer
Res 54:5050, 1994
27. Cheng JQ, Jhanwar SC, Klein WM, Bell DW, Lee WC, Altomare DA, Nobori T, Olopade 01, Bucker AJ, Testa JR: p16 alterations and deletion mapping of 9p21-p22 in malignant mesothelioma. Cancer Res 545547, 1994
28. Ohta M, Nagai H, Shimizu M, Rasio D, Berd D, Mastrangelo
M, Singh AD, Shields JA, Shields CL, Croce CM, Huebner K:
Rarity of somatic and germline mutations of the cyclin-dependent
kinase 4 inhibitor gene, CDK41, in melanoma. Cancer Res 545269,
1994
29. Xu L, Sgroi D, Sterner CJ, Beauchamp RL, Pinney DM, Keel
S, Ueki K, Rutter JL, Buckler AJ, Louis DN, Gusella JF, Ramesh
V: Mutational analysis of CDKN2 ( M T S I / P I ~ ’in
~ ~human
~ ) breast
carcinomas. Cancer Res 545262, 1994
30. Quesnel B, Preudhomme C, Philippe N, Vanrumbeke M, Dervite I, Lai JL, Bauters F, Wattel E, Fenaux P: p16 gene homozygous
deletions in acute lymphoblastic leukemia. Blood 85:657, 1995
31. Cayuela JM, Hebert J, Sigaux F: Homozygous MTSl
(p16INK4*)
deletion in primary tumor cells of 163 leukemic patients.
Blood 85354, 1995 (letter)
32. Binet JL, Catovsky D, Dighiero G, Gale RP, Montserrat E,
Rai KR: Chronic lymphocytic leukemia: Recommendations for diagnosis, staging, and response criteria. Ann Intern Med 110:236, 1989
33. Binet JL, Catovsky D, Chandra P, Dighiero G, Montserrat E,
Rai KR, Sawitsky A: Chronic lymphocytic leukaemia: Proposals for
a revised prognostic staging system. Br J Haematol 43:365, 1981
2731
34. Nathwani BN, Winberg CD: Non-Hodgkin’s lymphomas: An
appraisal of the “Working Formulation” of non-Hodgkin’s lymphomas for clinical usage, in Sommers SC, Rosen PP (eds): Malignant
Lymphomas: A Pathology Annual Monograph. Norwalk, CT, Appleton-Century-Crofts, 1983, p 1
35. Ohashi H, Ichikawa A, Takagi N, Hotta T, Naoe T, Ohno R,
Saito H: Remission induction of acute promyelocytic leukemia by
all-trans-retinoic acid: Molecular evidence of restoration of normal
hematopoesis after differentiation and subsequent extinction of leukemic clone. Leukemia 6:859, 1992
36. Majello B. Kenyon LC, Dalla-Favera R: Human c-myb protooncogene: Nucleotide sequence of cDNA and organization of the
genomic locus. Proc Natl Acad Sci USA 83:9636, 1986
37. Amemiya Y, and Miyahara J: Imaging plate illuminates many
fields. Nature 336239, 1988
38. Watanabe T, Hotta T, Ichikawa A, Kinishita T. Nagai H,
Uchida T, Murate T, Saito H: The MDM2 oncogene overexpression
in chronic lymphocytic leukemia and low-grade lymphoma of Bcell origin. Blood 84:3158, 1994
39. Saiki RK, Gelfand DH, Stoffel S, Scraf SJ, Higuchi R, Horn
GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification
ofDNAwith a thermostable DNA polymerase. Science 239:487,
1988
40. Orita M, Suzuki Y, Sekiya T, Hayashi K: Rapid and sensitive
detection of point mutations andDNA polymorphisms using the
polymerase chain reaction. Genomics 5:874, 1989
41. Suzuki Y, Sekiya T, Hayashi K: Alle-specific PCR: A method
for amplification and sequence determination of a single component
among a mixture of sequence variants. Anal Biochem 192:82, 1990
42. Weinberg RA: Tumor suppressor gene. Science 2S4:I 138,
1991
43. Hollstein M, Sidransky D, Vogelstein B, Hams CC: p53 mutations in human cancers. Science 253:49, 1991
44. Stranks G, Height SE, Mitchell P, Jadayel D, Yuille MAR,
De Lord C, Clutterbuck RD. Treleaven JG, Powles RL, Nacheva E,
Oscier DG, Karpas A, Lenoir GM, Smith SD, Millar JL, Catovsky
D, Dyer MJS: Deletions and rearrangement of CDKN2 in lymphoid
malignancy. Blood 85:893, 1995
45. Ichikawa A, Hotta T, Takagi N. Tsushita K, Kinoshita T,
Nagai H, Murakami Y, Hayashi K, Saito H: Mutations of p53 gene
and their relation to disease progression in B-cell lymphoma. Blood
79:2701, 1992
46. Sun Y, Hildesheim A, Lanier AEP, Cao Y, Yao KT, RaabTraub N, Yang CS: No point mutation but decreased expression of
p 16/MTS1 tumor suppressor gene in nasopharyngeal carcinomas.
Oncogene 10:785, 1995
From www.bloodjournal.org by guest on January 20, 2015. For personal use only.
1995 86: 2724-2731
Mutational analysis of the CDKN2 (MTS1/p16ink4A) gene in primary
B-cell lymphomas
T Uchida, T Watanabe, T Kinoshita, T Murate, H Saito and T Hotta
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