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https://www.abbs.info ISSN 0582-9879 |
Cyclin
D1 Polymorphism and the Susceptibility to NPC Using DHPLC
DENG
Lin, ZHAO Xiao-Rong, PAN Kai Feng1, WANG Yi1, DENG Xi-Yun, LÜ You-Yong1, CAO Ya*
（
Laboratory of Molecular Biology,
Cancer Research Institute,
Xiang-ya School
of
Medicine, Central South
University, Changsha
410078, China;
1Beijing
Institute for Cancer Research,
Peking University School of Oncology, Beijing 100034, China ）
Abstract Cyclin D1 is a key cell cycle regulator
and a candidate proto-oncogene,
whose deregulation has been implicated in pathogenesis of several types
of cancers, including NPC. A common A/G polymorphism (A870G) in
exon 4 of the cyclin D1 gene, CCND1, is associated with the presence of 2
distinct mRNA transcripts for this G1/S regulatory protein, and CCND1 genotype has been
related to some phenotypes of several tumors. To investigate the influence of cyclin D1 genotypes on the
genetic susceptibility in humans from Southern China to sporadic nasopharyngeal
carcinoma, cyclin D1 genotyping
was performed by denaturing high performance liquid chromatography (DHPLC) and
DNA sequencing analysis of the PCR products from 84 NPC cases and 91 normal
controls. Gene frequency
distribution was tested by Hardy-Weinberg equilibrium and comparison of cyclin
D1 gene frequencies between the patient and control groups was performed by c2 test.
Results showed that in NPC patients, the AA genotype of CCND1 was significantly lower
(20.24%) than in normal controls (38.46%), and the GG and AG genotypes (GG + AG) were significantly
higher in NPC group than in the control group (c2=6.946, Pcorrected=0.016, OR=2.463, 95% CI=1.249―4.859). These suggest that the A/G polymorphism of CCND1 was
associated with the susceptibility to NPC, and the GG and AG genotypes in NPC patients were
significantly higher than those in normal controls.
Key
words cyclin D1; polymorphism; nasopharyngeal carcinoma; DHPLC; genotype
Nasopharyngeal
carcinoma (NPC) is one of the most common malignant tumors in Southern
China. Genetic factors, Epstein-Barr virus (EBV) infection as
well as environmental factors have been reported to be associated with the
etiology of NPC[1, 2]. However, the molecular mechanism for NPC tumorigenesis remains
unclear.
A
molecule that possibly participates in NPC tumorigenesis is cyclin D1, which is involved in both the normal
regulation of the cell cycle and the neoplasm. Cyclin D1 is a key cell cycle regulator essential for G1
progression and a candidate proto-oncogene, whose deregulation has been implicated in pathogenesis of
several types of cancers[3],
including NPC[4, 5]. Our previous work demonstrated that
cyclin D1 protein was overexpressed in NPC and was regulated by another
oncoprotein, the latent membrane
protein 1 encoded by Epstein-Barr virus,
which is often present in NPC and functions as a constitutively activated
growth factor receptor[6―9].
Recently, it was reported that the cyclin D1
gene, CCND1, has a G to A polymorphism at codon 242
in exon 4, that increases
alternative splicing. However, both the G and A alleles can produce
altered transcripts due to the altered splicing. Both the normal and the altered transcripts encode a protein
that contains amino acids thought to be responsible for the cyclin D1
function, but the protein encoded
by the alternate transcript may have a longer half-life[10]. The AA and AG genotypes are associated
with early age of HNPCC (hereditary nonpolyposis colorectal cancer) onset[11]
and the AA genotype with poorer clinical prognosis in patients with NSCLC[10]
(non-small cell lung cancer). On
the other hand, the GG genotype of
cyclin D1 gene is associated with poor differentiation in histopathology and
reduced latent periods in patients with squamous cell carcinoma of the head and
neck, including oral cavity cancer
and laryngeal, oropharyngeal and
hypopharyngeal carcinomas[12].
These suggest that personal differences in the levels of various CCND1
transcripts may influence tumor behavior.
The aim of this study was to investigate whether CCND1 genotype
affect genetic susceptibility in sporadic NPC patients in Southern China. For this, cyclin D1 genotyping was performed by DHPLC (denaturing high
performance liquid chromatography) and was confirmed by DNA sequencing.
1 Materials and Methods
1.1
Subjects
This
investigation was conducted on 84 newly diagnosed NPC patients hospitalized to
Tumor Hospital of Hunan province,
Xiang-ya Hospital and Xiang-ya Second Hospital, Hunan, China. 82 cases
of NPC were histopathologically diagnosed as poorly differentiated squamous
cell carcinomas and the other 2 cases were diagnosed as non differentiated
carcinoma and large round cell carcinoma,
respectively. The patients
were 62 males and 22 females, aged
from 20 to 77 with an average of 47.25 years. 91 healthy volunteers were the students from Xiang-ya School
Medicine, Central South
University, Hunan, China. The normal controls were 61 males and 30 females, aged from 18 to 22 with an average of
19.73 years.
1.2
Cyclin D1 genotyping
Genomic
DNA was extracted from peripheral blood mononuclear cells by the routine high
salt method[13]. PCR
combined with DHPLC analysis was used to genotype the G/A polymorphism in exon
4 of cyclin D1 gene.
We
used previously published primers to amplify a 216 bp fragment containing exon
4 of the cyclin D1 gene. The
forward primer was 5′-TCCCTG-CTCACAGCCTCCT-3′; the reverse primer was 5′-CTGCCTGGGACATCACCCTC-3′[14]. PCR frag-ments were amplified from 120
ng of genomic DNA in a 25 ml
reaction mixture containing 0.2 mmol/L each of dATP, dTTP, dCTP and
dGTP (SABC, China), 0.5 pmol/L each primer, and 1.75 unit of Pfu polymerase
(Sangon Company, China). PCR was performed with a touch-down
protocol: initial denaturation at
94 ℃ for 3 min; followed by 10 cycles of 30 s at 94 ℃
for denaturation, 45 s at 67.5 ℃
(decreasing 0.5 ℃
by every cycle) for annealing, 50
s at 72 ℃
for elongation and 20 cycles of 30 s at 94 ℃
for denaturation, 45 s at 63 ℃
for annealing, 50 s at 72 ℃
for elongation and another 10 cycles of 30 s at 94 ℃
for denaturation, 45 s at 60.5 ℃
for annealing, 50 s at 72 ℃
for elongation; with a final
extension of 8 min at 72 ℃.
DHPLC
is a novel high-throughput technique for screening single nucleotide
polymorphisms and inherited mutations.
Polymorphism/mutation scanning by DHPLC involves subjecting PCR products
to ion-pair reverse-phase liquid chromatography in a column containing
alkylated non-porous particles.
Under conditions of partial heat denaturation in a linear acetonitrile
gradient, heteroduplexes in PCR
sample that have internal sequence variations display reduced column retention
time relative to their homoduplex counterparts, resulting in the difference in elution profiles of such
samples from those having homozygous sequence. The major advantages of this method include its high
sensitivity, efficiency and the
automated operation[15].
DHPLC analysis was carried out on the Transgenomic WAVE®
System (Transgenomic Incorporation,
USA) identical to that described by Gross et al[15]. Three ml
of each PCR product was denatured for 1 min at 94 ℃
and then gradually reannealed by decreasing sample temperature from 94 ℃
to 45 ℃
over a period of 30 min to form homo- and/or hetero- duplexes. PCR products were eluted with a linear
acetonitrile gradient at a flow rate of 0.9 ml/min. The column mobile phase consisted of a mixture of different
ratios of buffer A (containing 0.1 mol/L triethylamine acetate and 0.1 mmol/L
EDTA, pH 7.0) and buffer B
(containing 0.1 mol/L triethylamine and 25% acetonitrile, pH 7.0). Different combination of buffer A (ranging from 49%―40%)
and buffer B (ranging from 51%―60%)
would result in a mixture with varying acetonitrile concentrations. The values of separation times and
mobile phase temperature were 4.5 min and 60 ℃, respectively. From the first DHPLC,
we found the heterozygous genotype (AG) of cyclin D1 gene based on the
appearance of the chromatogram. To
determine the genotype of homozygotes,
each homozygous DNA was mixed with a known homozygous reference sample
(genotype “AA”, known from sequence analysis) at almost
equal molarity, then was denatured
and was gradually reannealed and the second DHPLC was carried out as
before. If the genotype of one
homozygote is the same as the reference sample, the chromatogram should appear as a single peak. However, when the mixture contains two different genotypes, the appearance of chromatogram should
be the same as that of heterozygotes.
So we could discriminate the two types of homozygotes.
After
that, DNA sequencing analysis was
used to further confirm the genotype determined by DHPLC. The PCR fragments were amplified in a
100 ml
reaction mixture with the forward and the reverse primers. The DNA sequences of PCR products were
determined by using reverse primer on an Applied Biosystems model 377
sequencer.
1.3
Statistical analysis
Data
were analyzed using the SPSS statistical software. Distribution of gene frequencies of cyclin D1 was tested by
Hardy-Weinberg equilibrium.
Differences of cyclin D1 allele frequencies between patients and normal
controls were compared by c2
test.
2 Results
2.1
Distribution of cyclin D1 gene frequencies
A
216 bp PCR fragment was generated and three different genotypes of the cyclin
D1 gene were distinguished by DHPLC and DNA sequencing analysis (Fig.1). The distribution of cyclin D1 gene in
NPC patients and normal controls was shown in Table 1. Each observed value was
very close to the expected value,
indicating that the distribution of cyclin D1 gene frequencies agreed
with Hardy-Weinberg equilibrium (P >0.05 with 1 degree of freedom).
Fig.1 Representative screening for the cyclin
D1 genotypes by DHPLC and DNA sequencing analysis
(A) At the first DHPLC, we discriminated the heterozygous
genotype (AG) of cyclin D1 gene from the homozygous genotypes according to the
appearance of the chromatogram;
(B) To determine the genotype of homozygotes, each homozygous DNA was mixed with a known homozygous
reference sample (genotype “AA”)
and the second DHPLC was performed,
then the two types of homozygotes (“AA”
genotype and “GG”
genotype) were discriminated through the appearance of chromatograms; (C) Results of nucleotide sequence
analysis by using reverse primer that allowed us to confirm the genotypes
determined by DHPLC. Arrows
indicate the locations of the nucleotide at which the polymorphism occurs.
2.2 Association of cyclin D1 genotype with
susceptibility to NPC
c2
analysis showed that the AA genotype of cyclin
D1 gene was significantly lower in NPC patients (20.24%, 17/84) than that in normal controls
(38.46%, 35/91), and the GG and AG genotypes (GG+AG)
were significantly higher in NPC group than that in the control group (c2=6.946, Pcorrected=0.016, OR=2.463, 95%CI=1.249―4.859, Table 2). These suggest that the subjects with the AG and GG genotypes
were more susceptible to NPC during their lives than those with the AA
genotype.
3
Discussion
Cyclin
D1 is one of the major cyclins involved in transition from G1 to S
phase, associating with CDK 4/6 in
mid to late G1 phase. Both gene
activation (due to amplification or chromosomal rearrangement) and/or protein
overexpression of cyclin D1 have been described in a variety of tumor types, including NPC[4, 5]. Our previous work demonstrated that EBV-LMP1 could regulate
cell growth by activation of cyclin D1 expression via NF-kB
pathway in NPC[6, 7]. In the present study, we showed that an intragenic A/G polymorphism of the
cyclin D1 gene was associated with susceptibility to NPC; the GG and AG genotypes in NPC patients were significantly higher
than those in normal controls.
This novel finding implies that cyclin D1 may take part in the
carcinogenesis of NPC.
The
mechanism by which the cyclin D1 genotype influences tumor susceptibility in
NPC is unclear. Cyclin D1 mRNA is
alternately spliced to produce two transcripts, a and b.
Transcript a is identical to the reported cyclin D1 cDNA. However, transcript b fails to be spliced at the exon 4/intron 4
boundary, it does not contain exon
5, and terminates downstream of
exon 4. Splicing of cyclin D1 mRNA seems to be modulated by the A/G
polymorphism. The AA homozygotes
express more transcript b than GG homozygotes and AG heterozygotes. This results in an altered protein that
lacks the PEST-rich region (destructin box) encoded by exon 5, which is responsible for rapid turnover
of the G1 cyclins. If
that increases the half-life of the altered protein, it should increase the steady-state levels of the cyclin D1 protein
in individuals with the AA genotype,
allowing cells to pass through the G1-S phase chckpoint more
easily and promoting the transformation of cells. However, the
mechanism underlying the individuals with GG and AG genotypes susceptible to NPC
remains ambiguous.
It
has been reported that the GG genotype of cyclin D1 is associated with poorly
differentiated histology and reduced disease-free interval in patients with
squamous cell carcinoma of the head and neck[12]. Since most of the NPC patients in
Southern China have poorly differentiated squamous cell carcinoma, and almost all of the patients’ samples
we used were poorly differentiated or non differentiated, it was difficult to determine whether
the G allele of the cyclin D1 gene was associated with the differentiation
status of NPC. It is possible that
the G allele of cyclin D1 gene is one of the molecular mechanisms underlying
the development of NPC in Southern China prone to poor differentiation. On the other hand, homozygosity for the “G”
allele was related to better prognosis in patients with NSCLC[10]
and older age of onset of HNPCC[11]. These conflicting results may be due to tissue-specific
transcription regulation.
Functional studies in the future may help to elucidate the paradoxical
experimental findings and influence of cyclin D1 genotype on tumor behavior in
different cell types.
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Received: June 27,
2001Accepted: August
24, 2001
This work was supported by the Special
Funds for Major State Basic Research (973) of China (No.G1998051201), the
National Natural Science Foundatioin of China (No.30100005) and the National Natural Science Fundation
of China for Distinguished Young Scholars (No. 39525022)
*Corresponding author: Tel, 86-731-4805448;
Fax, 86-731-4470589; e-mail, [email protected]