Cancer Therapy Vol 2, 463-468, 2004
Genetic variation in the P2X7
apoptosis purinoreceptor correlated with anti – nuclear and cytoskeleton autoantibodies induction in
nasopharyngeal carcinoma
Majida Jalbout1,
Noureddine Bouaouina1,2, and Lotfi Chouchane1*
1Laboratoire
dImmuno-Oncologie Molculaire, Facult de Mdecine de Monastir, Universit du
Centre, Tunisia,
2Department of Cancrologie Radiothrapie CHU Farhat
Hached, Sousse, Tunisia
__________________________________________________________________________________
*Correspondence: Pr. Lotfi Chouchane, Laboratoire dImmuno-Oncologie Molculaire, Facult
de Mdecine de Monastir, 5019 Monastir, Tunisie; Tel: 216-3-462 200; Fax:
216-3-460 737; E-mail: lotfi.chouchane@planet.tn
Key words: Apoptosis, Nasopharyngeal carcinoma,
Autoantibodies, Purinoreceptor P2X7.
Abbreviations:
antinuclear antibodies, (ANA); bovine serum albumin, (BSA); Epstein-Barr virus,
(EBV); nasopharyngeal carcinoma, (NPC); nuclear antigens, (NA); odds ratio,
(OR); single nucleotide polymorphism, (SNP); Statistical Package of Social
Sciences, (SPSS); undifferentiated carcinoma of nasopharyngeal type, (UCNT)
This work was supported by le
Ministre de la Recherche Scientifique et de Technologie, by le Ministre de
lEnseignement Suprieur, by le Ministre de la Sant Publique de la Rpublique
Tunisienne.
Summary
Apoptosis is
initiated by the stimulation of numerous cellular receptors. The P2X7 receptor
is expressed on immune cells and induces their ATP-mediated apoptosis. The
A1513C polymorphism in P2X7 gene causes a loss of function of the
receptor, impairing the death of cells which express these mutated receptors.
Recently, we showed high frequency of autoantibodies to cytoskeleton and
nuclear proteins in sera of patients with Epstein-Barr virus (EBV)-related
nasopharyngeal carcinoma. We investigated the potential association between P2X7
genetic variation and anti – nuclear and cytoskeleton autoantibodies
induction in patients with nasopharyngeal carcinoma.We developed an RFLP-PCR to
analyze the A1513C polymorphism of the P2X7 gene, reflecting a
deficit in apoptosis, in 88 patients with nasopharyngeal carcinoma.A highly
significant association was found between lack of autoantibodies to
cytoskeleton and nuclear proteins and the wild P2X7-A/A homozygote
genotype (p = 0.0018). The heterozygote genotype A1513C (A/C) was significantly
more frequent in patients presenting autoantibodies in their sera (p = 0.014).
We suggest that patients with nasopharyngeal carcinoma carrying the homozygote
genotype A/A of the P2X7 gene might be less susceptible to produce
autoantibodies against the cytoskeleton and nuclear antigens. This genotype reflecting
a fully functional receptor might inhibit or eliminate autoreactive B
lymphocytes by an appropriate process of apoptosis. Conversely, the
heterozygote P2X7 -A/C genotype might be associated to the immune
response deregulation observed in nasopharyngeal carcinoma due to the potential
deficiency in purging autoreactive B cells.
Recent investigations estimated that over half of the
major medical diseases can be attributed directly and indirectly to defective
regulation of programmed cell death mechanisms (Reed et al, 2001). Defects in
the regulation of apoptosis make important contributions to the pathogenesis or
severity of many diseases, including cancer and autoimmunity.
Under pathological conditions such as stress or cell
damages, large amount of ATP and other nucleotides can be rapidly released from
different cellular sources. Particularly in the immune system, extracellular
ATP triggers a variety of biological responses including suicide of immune
cells (Rassendren et al, 1997; Humphrey et al, 2000; Kusner et al, 2000).
Apoptosis regulates B cell maturation and differentiation, as well as the
development of memory B cells (Di Virgilio et al, 1995; Buell et al, 1996;
Nihei et al, 2000).
This effect of ATP is mediated through the activation
of specific surface molecules called P2 purinoreceptors (Ralevic et al, 1998;
Di Virgilio et al, 2001). The latest cloned member of the P2X family, the P2X7
receptor (previously termed P2Z), is unique in being constituvely highly
expressed in immune cells, particularly B and T lymphocytes (Di Virgilio et al,
1995; Rassendren et al, 1997; Khakh et al, 1999). Depending on cell background,
activation of the P2X7 receptors triggers diverse physiologic
activities including cytokine secretion and induction of cell death (Chused et
al, 1996; Surprenant et al, 1996).
Ferrari et al, (1999) have shown that activation of
the P2X7 receptor, following the exposure to ATP, rapidly induced
apoptotic DNA fragmentation, accompanied by the proteolytic processing of
multiple caspases and caspase substrates (Ferrari et al, 1999). The genomic
structure of P2X7 consists of 13 exons with exon 12 and exon 13
encoding for the carboxyl terminal tail of this molecule. There is strong
evidence that this long carboxyl terminus is necessary for the permeability
properties of the P2X7 channel (Surprenant et al, 1996; Wiley et al,
1998). Recently, a single nucleotide polymorphism (SNP) has been identified in
the exon 13 of the P2X7 gene. This polymorphism (A1513C) encodes for
a glutamic acid to alanine substitution at amino acid 496 (Glu496Ala).
Homozygosity for the rare P2X7 allele (C/C) produces non-functional
P2X7 protein while the heterozygous state gives cells with half the
function of cells with germline P2X7 protein (Gu et al, 2001).
The Epstein-Barr virus (EBV) - associated
nasopharyngeal carcinoma (NPC) has a striking geographic and ethnic
distribution in different populations of the world. While it is rare in western
countries, its incidence is high in certain regions such as Southeast Asia. In
North Africa, the NPC has an intermediate incidence. In Asia, NPC mainly
affects patients in the 4th or 5th decade of their life, whereas in North
Africa an additional peak of incidence is found confined to young population.
The association of NPC with widely distributed factors implies that exceptional
circumstances combining some interactions between Epstein- Barr virus infection
and a particular environment, lead to the development of a carcinoma of the
nasopharynx, favored by a genetic disposition. Among NPCs, the histological
type showing the most consistent worldwide association with EBV is the
undifferentiated carcinoma of nasopharyngeal type (UCNT) (Fedder et al, 1985;
Choi et al, 1993; Jeannel et al, 1999).
EBV infects B lymphocytes with very high efficiency,
both in vivo and in vitro. The virus attaches itself to B cells via interaction of
the EBV gp350/220 envelope glycoprotein with the CD21 molecule (receptor CR2 of
complement) on B cell and generates the activation, proliferation and
immortalization of these cells. The polyclonal activation of B lymphocytes is
associated with the secretion of antibodies (Henderson et al, 1993).
In a recent study, we showed that frequencies of
autoantibodies produced against nuclear and cytoskeleton proteins were markedly
higher in patients sera with nasopharyngeal carcinoma compared to those of
control subjects (Jalbout et al, 2002).
Given the importance of apoptosis in immune response
regulation, aberrant apoptosis could conceivably associate with immune system
deregulation. In this study, we hypothesize that the genetic variation in the
P2X7, reflecting a loss of function of its product, might be among
factors leading to the autoantibody induction in patients with NPC.
In line with this
hypothesis, we investigated the potential association of the P2X7
gene polymorphism in NPC patients with or without autoreactivity sera to
cytoskeleton and nuclear proteins.
A. Patients
Patients with UCNT were
recruited from the Department of Radiation Oncology and Medical Oncology of
Sousse Hospital, between 1991 and 2003. The patients with UCNT (67 males and 21
females) had a mean age of 40 21.3 years. The clinical stages ranged from II
to IV (TNM classification, 1987). The diagnosis of cancer was confirmed by
histopathology analyses. The histology was undifferentiated carcinoma (type
III, WHO classification) in all cases (Shanmugaratnam et al, 1978).
Written informed consent was
obtained from all subjects and, in the case of children, from the parents.
B. ELISA for autoantibodies
Sera autoreactivity to the
cytoskeleton and nuclear proteins was determined on serum samples obtained
prior to cancer therapy. Each serum collected was tested individually. As
negative control for the reaction, 82 sera from healthy individuals were pooled
and used in each test.
The autoantibodies detection
was carried out as previously described (Jalbout et al, 2002).
C. Immunofluorescence analysis
Identification of antinuclear antibodies (ANA) by
indirect immunofluorescence was carried out using cryostat sections prepared
from liver tissue of young rats. These liver sections were deposed on glass and
incubated overnight at -80C. Briefly, serum was diluted
at 1/100 with PBS pH7.4 and deposited on liver sections. After 30 min of
incubation and 3 washes with PBS, antibodies were revealed by anti-human Igs
antibodies (IgG, IgA, IgM) labeled with fluorescein.
D. Genomic DNA extraction
Genomic DNA was extracted
from peripheral blood leukocytes as previously described (Olerup et al, 1992).
Briefly, 10 ml of blood was mixed with Triton lysis buffer (1% Triton X, 5 mM
MgCl2, H2O, 0.32 M sucrose, 10mM Tris-HCl, PH 7.5). Leukocytes were
spun down and washed with H2O. The pellet was incubated with proteinase K at 56C and subsequently salted out
at 4C using a saturated NaCl solution. Precipitated
proteins were removed by centrifugation. The DNA in supernatant fluid was
precipitated with ethanol. The DNA pellet was dissolved in 500 ml TE (Tris-EDTA).
E. P2X7 gene polymorphism analysis
A restriction map of the P2X7
gene was assessed using the infobiogen web site (http://www.infobiogen.org) in order to
determine a couple of primers enclosing the 1513 SNP. This nucleotide variation
lies in the recognition motif of AluI
[AG½CT]. The substitution of the adenine to a
cytosine causes a loss of the restriction site of AluI.
A 91-pb DNA fragment
containing 1513 SNP was amplified by PCR using the following primers: forward,
5-GCTGCCTCCCATCTCAACTCC-3 and reverse, 5-CTCTGAGGTGGTGATGCAGGCC-3.
Primers cited above were used
to amplify DNA fragments of 91 pb enclosing the 1513 SNP by PCR. Amplification
reactions were done in a total volume of 30ml containing 100ng of genomic
DNA, 1.5 mM MgCl2 , 200mM dNTPs, 50 pmol of each
primer, and 0.5 unit of Taq (Amersham) in 1X Taq buffer.
Cycling conditions were 94C for 5 min, 30 cycles of 94C for 30 s, 60C for 60s, and 72C for 60s, and a final 10 min
extension at 72C. DNA Fragment (91 pb) were revealed on a 3%
agarose gel.
Genotyping was done using
restriction fragment-length polymorphism analysis. PCR products were digested
at 37C overnight with 4 unites of the restriction enzyme AluI (New England Biolabs) in a 20ml reaction mix containing 1 X
restriction enzyme buffer 2 and bovine serum albumin (BSA). Juxtaposed
heterozygote digested products of 46 and 45 pb were resolved on a 3% agarose
gel.
F. Statistical analysis
Genotypes frequencies were
estimated by gene counting. A Chi-Square Test (c2) was used to compare
observed number of each P2X7 genotype with those expected for a
population in Hardy- Weinberg equilibrium.
The chi-square test (or Fishers exact test when n <5) was used to
determine whether significant differences (p value) in the autoantibodies
induction to cytoskeleton and nuclear antigens were observed between carriers
of different P2X7
genotypes.
Statistical analyses were
performed using the Statistical Package of Social Sciences for Windows (SPSS
version 10.0). Differences between groups were tested for significance by the
two-tailed test. A p value less than 0.05 was considered significant.
The significance of the
association was estimated by the odds ratio (OR).
A. P2X7 polymorphism
distribution and sera autoreactivity in patients with nasopharyngeal carcinoma
We set up an RFLP-PCR to analyze the A1513C
polymorphism in the P2X7 gene. The A to C nucleotide transition at
the position 1513 causes a loss of AluI
site. We used this restriction site to genotype 88 patients for the P2X7.
After defining the serological profile of patients for autoreactivity against
cytoskeleton and nuclear proteins, we stratified them into two groups according
to their sera autoreactivity. A patient is considered as having autoreactive
serum when he presents autoantibodies produced against at least one of the
tested antigens. The cut-off value for reactivity was defined as twice the
value of the average autoreactivity in our pool of negative controls.
Table 1 shows genotype frequencies for the P2X7 gene
in the two groups of patients with nasopharyngeal carcinoma. These
distributions were in Hardy-Weinberg equilibrium for both groups.
The frequency of the wild homozygote genotype P2X7
-A/A was significantly higher in patients with non-autoreactive sera
(35/48, 72.9%) than in the group of patients having autoantibodies to nuclear
and cytoskeleton antigens (16/40, 40%) (p = 0.0018). The heterozygote P2X7
–A/C genotype was significantly more frequent in patients
presenting autoantibodies in their sera (52.5%) than those having no
autoantibodies (27.1%) (p = 0.014).
The homozygote C/C genotype was carried only by patients with autoreactive antibodies (3 versus 0 in patients with no autoreactivity).
B. P2X7 genetic variation and
autoreactivity to nuclear antigens
As previously shown (Jalbout et al, 2002), the most
frequent sera autoreactivity, found in patients sera compared to controls, was
towards nuclear antigens (NA) (22% in patients versus 1.2% in controls; p = 0.00003).
A significant association was found between the presence of ANA and the P2X7 A/C heterozygote genotype (p = 0.030). The frequency of homozygote A/A genotype, corresponding to a complete function of the P2X7 receptor, was significantly higher in patients without ANA (p=0.016)
Table 1. P2X7 polymorphism distributions in patients
with nasopharyngeal carcinoma with or without anti- nuclear and cytoskeleton
autoantibodies
|
Genotype |
Patients With autoantibodies n= 40 |
Patients Without autoantibodies n= 48 |
OR |
Confidence
Interval |
p |
||||
|
P2X7 |
n |
f |
n |
f |
|
|
|
|
|
|
A/A |
16 |
0.40 |
35 |
0.29 |
0.25 |
[0.09 - |
0.66] |
0.0018 |
|
|
A/C |
21 |
0.525 |
13 |
0.271 |
2.98 |
[1.12 - |
8.01] |
|
|
|
C/C |
3 |
0.075 |
0 |
0 |
|
|
|
NS |
|
The chi-square and the Fisher
tests were used to determine whether significant differences (p value) were
observed when patients having anti- nuclear and cytoskeleton autoantibodies
group was compared with that of those without. f, frequency.
Regardless of patient origin, nasopharyngeal carcinoma
is consistently associated with Epstein-Barr virus. The EBV genome is contained
in malignant epithelial cells (Jeannel et al, 1999; Murray et al, 2001). This
virus has a tropism for B lymphocytes and a propensity to oncogenicity.
Infection of B lymphocytes with EBV is associated with their polyclonal
activation and in vitro
immortalization (Mannick et al, 1991; Hildesheim et al, 1993).
Recently, we detected in sera of patients with NPC
autoantibodies induced against the cytoskeleton and nuclear proteins (Jalbout
et al, 2002). These antigens are involved in cell division and proliferation.
In this study, we hypothesize that the deregulation in apoptosis of autreactive
B cells might be among factors leading to the autoantibodies induction in
patients with NPC.
The P2X7 receptor is attracting increasing
interest due to its restricted cells distribution such as macrophages,
thymocytes and B cells, and its induction of these cells death (Chen et al,
1999; Ferrari et al, 2000; Gu et al, 2000). The C-terminal cytoplasmic tail of
P2X7 receptor, which is 120 amino acids longer than in other P2X
receptors, is an important modulator of the receptor functions.
In this study, frequencies of the non-functional
single nucleotide polymorphism of the C-terminal tail of P2X7 were
assessed within patients with NPC stratified according to their sera
autoreactivity to nuclear and cytoskeleton antigens.
The frequency of the wild genotype (Glu496) was
associated to the absence of significant level of autoantibodies in patients
with nasopharyngeal carcinoma.
The Glu496Ala genotype frequency is significantly
higher in patients presenting anti- nuclear and cytoskeleton proteins
antibodies compared to patients with no autoantibodies. Reflecting a deficiency
in purging the autoreactive B cells, this heterozygote genotype might be a
susceptibility marker for autoantibodies production in patients with NPC. This
observation was highlighted by the distribution of the mutant homozygote
genotype 496Ala which was found only in patients having anti- nuclear and
cytoskeleton autoantibodies.
The negative effect of this mutation on P2X7 function
was evident in all leukocytes which express surface P2X7, namely B
and T lymphocytes in patients with chronic lymphocytic leukemia (Gu et al, 2000). The transition of glutamic acid to
an alanine found in P2X7 in these patients confers resistance to
ATP-induced apoptosis of B-lymphocytes (Gu et al, 2000). Moreover, Wilson and
colleagues showed that the C-terminal domain of this receptor is implicated in
the binding of epithelial membrane proteins, which regulates cell death (Wilson
et al, 2002).
Several lines of evidence support the idea that
malignant cells are more prone to apoptosis in NPC than other head and neck
cancers. It has been reported that NPC tumor-infiltrating lymphocytes, which
often produce the anti-apoptotic CD40 ligand, may increase the survival of
malignant cells, thereby enhancing tumor growth in patients. In fact, the
interaction of the CD40 ligand with the CD40 on B cells allows these cells to
escape to CD95-mediated apoptosis (Sbih-Lammali et al, 1999). Other proteins
might be involved in the escapement of B cells to apoptosis via different
alternatives including the P2X7 pathway.
In conclusion, this study
showed a significant association between genetic variation in the P2X7 receptor
and anti- nuclear and cytoskeleton autoantibodies induction. Our data suggest
that apoptosis dysfunction might be among mechanisms responsible for the immune
deregulation observed in NPC.
We gratefully acknowledge the technical assistance of
S. Gabbouj. We thank the staff of the Departments of Radiation Oncology and
medical oncology of CHU F. Hached, Sousse, for providing samples and clinical
information.
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