British Journal of Cancer (2002) 86, 756 – 760
ª 2002 Cancer Research UK All rights reserved 0007 – 0920/02 $25.00
www.bjcancer.com
Analysis of CHK2 in vulval neoplasia
A Reddy1, M Yuille2, A Sullivan1, C Repellin1, A Bell3, JA Tidy4, DJ Evans5, PJ Farrell1, B Gusterson3, M Gasco6
and T Crook*,1
1
Ludwig Institute for Cancer Research, Imperial College Faculty of Medicine, St Mary’s Campus, Norfolk Place, London W2 1PG, UK; 2MRC HGMP-RC,
Hinxton, Cambridge CB10 1SB, UK; 3University Department of Pathology, Glasgow University, Western Infirmary, Glasgow, UK; 4Department of
Gynaecological Oncology, University of Sheffield, Northern General Hospital, Sheffield S5 7AU, UK; 5Department of Histopathology, St Mary’s Hospital
Medical School, Norfolk Place, London W2, UK; 6UO Oncologia Medica, Azienda Ospedaliera S. Croce e Carle, Via Coppino 26, 12100, Cuneo, Italy
Molecular and Cellular Pathology
Structure and expression of the Rad53 homologue CHK2 were studied in vulval neoplasia. We identified the previously
described silent polymorphism at codon 84 (A4G at nucleotide 252) in the germ-line of six out of 72, and somatic mutations
in two out of 40 cases of vulval squamous cell carcinomas and none of 32 cases of vulval intraepithelial neoplasia. One
mutation introduced a premature stop codon in the kinase domain of CHK2, whereas the second resulted in an amino acid
substitution in the kinase domain. The two squamous cell carcinomas with mutations in CHK2 also expressed mutant p53. A
CpG island was identified close to the putative CHK2 transcriptional start site, but methylation-specific PCR did not detect
methylation in any of 40 vulval squamous cell carcinomas, irrespective of human papillomavirus or p53 status. Consistent with
this observation, no cancer exhibited loss of CHK2 expression at mRNA or protein level. Taken together, these observations
reveal that genetic but not epigenetic changes in CHK2 occur in a small proportion of vulval squamous cell carcinomas.
British Journal of Cancer (2002) 86, 756 – 760. DOI: 10.1038/sj/bjc/6600131 www.bjcancer.com
ª 2002 Cancer Research UK
Keywords: vulval cancer; p53; CHK2
Although vulval squamous cell carcinoma (SCC) is less common
than cervical cancer, it is nevertheless of interest since a proportion of cases contain sequences from high-risk human
papillomavirus (HPV) types (principally HPV 16), whereas a
further, substantial subset of cancers arise via HPV-independent
pathways. Pathobiological differences exist between HPV positive
and HPV negative cancers (Crum, 1992), but allelotype analysis
suggests that there are no significant differences in sites of loss
of heterozygosity (LOH) (Pinto et al, 1999). A number of
studies have however revealed that mutation in p53 is more
common in HPV negative cancers (Lee et al, 1994; Brooks et
al, 2000). Mechanistically, it is hypothesised that mutation in
p53 functionally compensates for the absence of HPV 16E6,
since this protein mediates inactivation of p53 via promotion
of ubiquitin-dependent proteolysis. Despite the more common
mutation of p53 in HPV negative cases, a substantial number
of vulval SCC occur which lack both mutation and HPV.
The mechanism, if any, by which p53 function is compromised
in such cases is not known.
The CHK2 kinase, the human homologue of yeast RAD53, is
located on chromosome 22q which is a common site for LOH in
vulval cancer (Worsham et al, 1991; Pinto et al, 1999). CHK2 functions downstream of ATM (ataxia telangiectasia-mutated protein)
in response to DNA damage to phosphorylate p53 and BRCA1
and thereby regulate the tumour suppressor functions of these
proteins (Chehab et al, 2000; Lee et al, 2000; Matsuoka et al,
2000). Furthermore, CHK2-deficient mice fail to maintain G2
arrest following irradiation (Hirao et al, 2000). Taken together
*Correspondence: T Crook; E-mail: t.crook@ic.ac.uk
Received 5 September 2001; revised 3 December 2001; accepted 4
December 2001
these data suggest that CHK2 functions at both G1 and G2 cell
cycle checkpoints. The protein contains functionally important fork
head-associated and kinase domains.
Somatic mutations in CHK2 have been reported in both solid
tumours (Haruki et al, 2000) and in myelodysplastic syndrome
(Hofmann et al, 2001). Furthermore, analysis of individuals with
the Li – Fraumeni cancer predisposition syndrome revealed that a
subset of individuals with the syndrome, but lacking mutations
in p53, harbour heterozygous germ-line mutations in CHK2 (Bell
et al, 1999). These observations support the assertion that p53
and CHK2 function in a common pathway of tumour suppression and raise the interesting possibility that abrogation of
CHK2 function via mutation or loss of expression might functionally compensate for mutations in cancers with wild-type
p53. To investigate this hypothesis, we have performed analysis
of the structure and expression of CHK2 in a series of vulval
cancers and pre-malignant lesions characterised for both HPV
and p53 status.
MATERIALS AND METHODS
Tissues
Fresh-frozen tumour tissue was available from 40 cases of vulval
SCC, each with matched normal vulval epithelium. The diagnosis
in each case was determined by histopathological analysis. Genomic
DNA was isolated from frozen tissues (normal and tumour) by
proteinase K digestion and RNA by RNAzol B. Paraffin sections
of vulval intraepithelial neoplasia (VIN) were obtained from the
pathology archives of St Mary’s Hospital (London). The diagnosis
and presence of adequate neoplastic tissue was verified for each
case and DNA isolated from suitable samples by extended digestion
in proteinase K.
CHK2 status in vulval cancer
A Reddy et al
757
Gene analysis
Each genomic DNA was initially checked by amplification of
globin using the PCO3/PCO4 primer pair (Greer et al, 1991).
HPV DNA was sought using the HPV consensus primer pair
CPI/CPIIG which detects a broad range of HPV types and allows
detection of HPV in DNA from paraffin sections (Smits et al,
1992). Positive cases were typed by direct sequencing of amplified products. The p53 status of each cancer was determined
by SCCP and DNA sequencing as described previously (Brooks
et al, 2000). Mutations in CHK2 were sought in genomic
DNA by SSCP and, in cases where RNA was available, by
RT – PCR SSCP. In total, 36 SCC were examined by RT – PCR
SSCP. Primers for SSCP from genomic DNA were designed from
AL117330 and AL121825 (Table 1). Primers for RT – PCR SSCP
were as described (Haruki et al, 2000). cDNA was synthesized in
50 ml reaction from 3 mg of total RNA using the Stratagene
ProStar system. Two ml of this solution was used for each
PCR. In all cases, PCR products were resolved on 6% native
polyacrylamide gels with and without 10% glycerol. When SSCP
amplimers exhibited aberrant mobility, the fragment was reamplified with Pfx DNA polymerase and multiple plasmid clones
in the pCRBlunt vector were sequenced.
lated 5’-TACAACAACCCATAAAACCCCAAACAAA-3’ and 5’TAGATTTTGATGTGTTTTTTGTTTGGGTTT-3’, giving a product
of 161 bp. Methylated 5’-GACGACCCATAAAACCCCGAACGAA3’ and 5’-TTTCGACGTGTTTTTCGTTCGGGTTC-3’, giving a
product of 154 bp. Following PCR, reactions were resolved on
2% agarose gels and visualised under UV. Each PCR included
control methylated and unmethylated DNA samples.
Immunocytochemistry
Five-mm sections were cut from archival paraffin blocks, stained
with haematoxylin and eosin, and the diagnosis in each case verified by two pathologists. For immunocytochemical analysis, the
sections were subjected to antigen retrieval by microwaving in
citrate buffer, then incubated with anti-human CHK2 antibody
(Santa Cruz, sc-8812). The specificity of this antibody was
confirmed by Western blotting prior to immunocytochemical
studies. Sections were reviewed by two pathologists to score expression. A negative control section, lacking the primary antibody, was
included in each immunocytochemical analysis.
RESULTS
A CpG island is located in AL117330 between 26038 – 26731. Using
a combination of 5’RACE and searching of the human genome
database (http://genome.ucsc.edu/goldenPath/hgTracks.html), we
established that this is close to the 5’ end of the CHK2 mRNA.
Methylation in the CHK2 promoter was analysed using MSP
(Herman et al, 1996). Primers were designed from the sequence
of the CpG island using a commercially available software
programme (Intergen). Primer sequences are as follows: Unmethy-
Table 1 Primers for SSCP of CHK2 from genomic DNA
CHK2
Primer sequence (5’43’)
Exon 1a
ACAACAAAGGGTCTTACCAAGATT
AGGGCATATCCAGCTCCTCTAC
AGTGAGAGGACTGGCTGGAGT
GGCCCATCATTTACTTTTTAATTTT
TGACCAAATTACCAGCTCTCCTA
TACATGAAATTCAACAGCCCTCT
TCCTCCTATGAGAGAGTGGAAAA
TCCCACTATAAATCTCTGCTATTCAA
AATCAGAAATGAGAAACCACCAA
AGTGATCGCCTCTTGTGAATAAA
AGGTGATCAGCCTTTTATTGGTA
ACCCAGGAGTGGTAGGTCTCATA
ACTGAAAGGCTTTATACTCTTCTCATA
CCTTGAGTCAACTGAGTTTAACTGT
AAAGCATTTGAATGGAAACAGAA
CTCTGGGCAGATGTTCTAAGCTC
ACTAAAAGAAAGGCAGCTGTCAA
GTTTTCCCCCAGGAATGAAC
TTTCTGAACAAGAATCTACAGGAAT
TTAAGTATCTACTGCATGAATCTGAGG
ATCACCTCCTACCAGTCTGTGC
GCAAGTTCAACATTATTCCCTTTT
GCACATACACATTTTAGCATACCA
TGAGAATGCCACTTGATTTCTTT
CATGTCTCTCAGGCAGCAG
TTTATCCTTTTCACTGTGATTTGC
AGCTCCTTAAGCCCAGACTACAT
GGAGTTTATTATCCTTCAGACACAGC
CATCAGTGACTGTGAAAAAGCAA
TTTTGAACATTTCTCCATTTTCC
Exon 1b
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9
Exon 10
Exon 11
Exon 12
Exon 13
Exon 14
ª 2002 Cancer Research UK
Annealing (8C)
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We performed analysis of the sequence of CHK2 in vulval
neoplasia. Primers for analysis of CHK2 sequence by SSCP from
genomic DNA were designed by reference to the intron – exon
structure of the gene (Accession No. AL117330, and
AL121825). Primer pairs and annealing temperatures are shown
in Table 1. The previously described silent polymorphism at
nucleotide 252 in codon 84 (GAA4GAG) (Bell et al, 1999;
Haruki et al, 2000; Hofmann et al, 2001) was observed in four
out of 40 individuals with vulval SCC and two of 32 cases of
VIN. Analysis of matched normal epithelium from each individual with SCC revealed that each was heterozygous for this
polymorphism in the germline. Mutations in CHK2 were
detected in two vulval SCC by RT – PCR SSCP and subsequent
sequencing of cloned RT – PCR products (Figure 1 and Table 2).
Both mutations occurred in the kinase domain of CHK2. One
introduced a premature stop at codon 377 (gAg4TAg), whereas
the second was a substitution (gAA4AAA; Glu4Lys) at codon
394 (Table 2). During these studies a number of highly
conserved 3’ fragments of CHK2 were identified which map
to several autosomes and the X chromosome (Sodha et al,
2000). These fragments are homologous to sequences in exons
10 – 14 of CHK2. We therefore performed additional analyses
to confirm that the sequence changes we had detected represent
bona fide CHK2 mutations rather than rare polymorphisms
within the homologous fragments. We used forward primers
located upstream of exon 9 which are unique to CHK2 together
with reverse primers from exons 10 and 11 to amplify CHK2specific genomic sequences and sequenced these. These
confirmed the presence of the mutations. We also performed
sequence analysis of matched normal tissue for each cancer with
mutation. The mutations were not detected in the normal
tissue, confirming their authenticity as somatic CHK2 mutations.
We were interested to determine whether there was loss of
heterozygosity (LOH) in the cases with CHK2 mutations. In
an attempt to address this, we performed direct sequencing of
the RT – PCR products in the two cases with mutations. In
one case (codon 377 gAg4TAg) the mutated allele was clearly
the predominant one present, consistent with loss of the wildtype allele (Figure 1B). In the remaining case, there was no
evidence of LOH, both alleles being equally represented (Figure
1D). It should be noted, however, that the presence of normal
tissue may mask LOH. As such, we cannot be certain that this
is a heterozygous mutation.
British Journal of Cancer (2002) 86(5), 756 – 760
Molecular and Cellular Pathology
Somatic mutations in CHK2 in vulval neoplasia
Analysis of CpG methylation in CHK2
CHK2 status in vulval cancer
A Reddy et al
758
CHK2 mRNA and the CHK2 promoter. These studies identified a
CpG island 5’ of, and including the non-coding first exon of the
CHK2 mRNA. We designed primers for MSP to determine whether
aberrant hypermethylation occurred in the CpG island in vulval
cancer. The precise positions of the primers are given in Materials
and Methods. We did not detect methylation in the CHK2 promoter
in any of the 40 vulval SCC analysed, despite detection of methylation
in the control DNA samples (Figure 2).
CHK2 mutations co-exist with p53 mutations in vulval SCC
The first reported examples of human – tumour associated CHK2
mutations were in Li – Fraumeni patients in which p53 was wildtype (Bell et al, 1999). We therefore checked the p53 sequence in
the vulval SCC with CHK2 mutations. Both cancers with mutations
in CHK2 were mutant for p53 (Table 2).
The CHK2 promoter is not hypermethylated in vulval
neoplasia
CHK2 expression is not down regulated in vulval neoplasia
We were interested to determine whether CHK2 might be inactivated
by alternative mechanisms in this series of cancers. Using a combination of data base searching and 5’ RACE, we mapped the 5’ end of the
Despite the absence of methylation in the CHK2 promoter, it was
clearly important to examine expression of the gene in cancers. We
therefore performed both RT – PCR and immunocytochemical
Molecular and Cellular Pathology
A
C
B
D
Figure 1 Somatic mutation in CHK2 in vulval cancer. (A) Sequence analysis of plasmid clone containing the arrowed mutation at codon 377 of CHK2
GAG4TAG (Glu4Ter); (B) Direct sequencing of cDNA from this tumour reveals loss of heterozygosity; (C) Sequence of plasmid clone containing the
arrowed mutation at codon 394 of CHK2 GAA4AAA (Glu4Lys); (D) Direct sequencing of cDNA from this tumour reveals no evidence for loss of
heterozygosity.
Table 2
1
Chk2 mutations co-exist with p53 mutations in vulval cancer
Tumour
p53
CHK2
HPV
SCC (metastasis)
SCC
282 CGG4TGG (Arg4Trp)
152 CCG4CAG (Pro4Gln)
377 GAG4TAG (Glu4Ter)
394 GAA4AAA (Glu4Lys)
-ve
-ve
M
U
M
U
M
U
M
U
M
U
M
U
M
U
M
U
C1
C2 C3 C4
Figure 2 Absence of CpG methylation in the CHK2 promoter in vulval cancer. MSP was performed on bisulphite-treated genomic DNA isolated from
vulval SCC, using the primers for MSP as described in Materials and Methods. Lane 1 is 100 bp ladder. For each carcinoma M=methylated, U =unmethylated. C1= unmethylated control DNA with U primers; C2=unmethylated control DNA with M primers; C3=methylated control DNA with U primers;
C4=methylated control DNA with M primers.
British Journal of Cancer (2002) 86(5), 756 – 760
ª 2002 Cancer Research UK
CHK2 status in vulval cancer
A Reddy et al
Discussion
Abrogation of p53 function is a critical event in tumorigenesis. p53
is directly phosphorylated by CHK2 in response to DNA damage.
NI
T1 N2 T2 N3 T3 N4 T4 N5 T5 N6 T6
CHK2
GAPDH
Figure 3 RT – PCR analysis of CHK2 expression in vulval neoplasia. RT –
PCR was performed on RNA isolated from matched pairs of normal vulval
epithelium (N) and tumour (T). CHK2 and the control RNA (GAPDH) are
indicated.
A
Furthermore, germ-line mutations in CHK2 occur in individuals
with Li – Fraumeni syndrome which express wild-type p53. Taken
together, these observations suggested that loss of function in
CHK2 might represent an alternative mechanism by which the
p53 pathway can be inactivated or attenuated in cancers lacking
other recognised means of abrogation of p53 function. The
mechanism by which p53 function is compromised in vulval cancer
is of considerable interest since only a subset of cancers have HPV
or p53 mutation (Crum, 1992; Lee et al, 1994; Brooks et al, 2000).
We show in this report that somatic mutation of CHK2 occurs
in a small proportion of vulval cancers. Both of the mutations we
detected are in the kinase domain of CHK2, and, therefore highly
likely to compromise the function of the protein. One of the
mutants introduced a premature termination codon whereas the
other mutation resulted in an amino acid substitution. Interestingly, both cancers with CHK2 mutations also contained mutant
p53. The simultaneous presence of mutations in CHK2 and in
p53 has been reported previously in colon cancer (Bell et al,
1999) and small cell lung cancer (Haruki et al, 2000) but not in
a case of myelodysplastic syndrome with CHK2 mutation in which
p53 was wild-type (Hofmann et al, 2001). Taken together with the
present analysis of vulval cancer, these data imply that CHK2 and
p53 mutations are not mutually exclusive events in cancer. Our
results also suggest that mutation of CHK2 is not a common
mechanism by which vulval cancers negative for HPV and lacking
p53 mutations abrogate the tumour suppressor function of p53.
To investigate alternative potential mechanisms by which CHK2
might be altered in vulval neoplasia, we analysed the CHK2
promoter and 5’ end of the CHK2 transcript. The CpG island
located close to the transcription start site of CHK2 represents a
B
C
Figure 4 Immunocytochemical analysis of expression of CHK2 in vulval neoplasia. The sections shown are (A) control with no primary antibody; (B)
normal vulval epithelium; (C) VIN II. Sections were prepared and stained as described in Materials and Methods.
ª 2002 Cancer Research UK
British Journal of Cancer (2002) 86(5), 756 – 760
Molecular and Cellular Pathology
759
analyses to compare expression in normal and neoplastic vulval
epithelium. Expression levels of CHK2 assessed by RT – PCR were
variable between normal and tumour pairs (Figure 3). However, we
did not identify a single case in which CHK2 expression was lost.
To further confirm that loss of expression does not occur commonly
in vulval cancer, we performed immunocytochemical analysis of a
series of vulval neoplastic lesions. These samples comprised six cases
of vulval SCC, four cases of VIN I, six cases of VIN II and 22 cases of
VIN III. There was expression of CHK2 protein throughout normal
vulval epithelium (Figure 4B) but no loss of expression in any of
the VIN or vulval SCC analysed (Figure 4C).
CHK2 status in vulval cancer
A Reddy et al
760
potential site for aberrant methylation and transcriptional silencing, but we found no evidence for CpG hypermethylation in
any of the vulval SCC in our series. Consistent with this, there
was abundant expression of CHK2 at both mRNA and protein
levels in normal, pre-malignant and malignant vulval epithelium
and no case was observed in which expression was lost.
In conclusion, our results reveal that CHK2 is a target for
somatic mutation in a small proportion of cases of vulval cancer
and further confirm its status as a bona fide human tumour
suppressor gene. They do not, however, support a model in which
mutation or loss of expression of CHK2 might functionally substitute for inactivation of p53 via mutation or expression of HPV E6.
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