1Laboratoire d'Immuno-Oncologie Moléculaire, Faculté de Médecine de Monastir, Université du Centre, Tunisia,

__________________________________________________________________________________

*Correspondence: Pr. Lotfi Chouchane, Laboratoire d'Immuno-Oncologie Moléculaire, Faculté de Médecine 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 Ministère de la Recherche Scientifique et de Technologie, by le Ministère de l'Enseignement Supérieur, by le Ministère de la Santé Publique de la République Tunisienne.

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).

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.

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.

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 -80° C. 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.

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 56° C and subsequently salted out at 4° C 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 m l TE (Tris-EDTA).

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 30m l containing 100ng of genomic DNA, 1.5 mM MgCl₂ , 200m M dNTPs, 50 pmol of each primer, and 0.5 unit of Taq (Amersham) in 1X Taq buffer.

Cycling conditions were 94° C for 5 min, 30 cycles of 94° C for 30 s, 60° C for 60s, and 72° C for 60s, and a final 10 min extension at 72° C. 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 37° C overnight with 4 unites of the restriction enzyme AluI (New England Biolabs) in a 20m l 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.

Genotypes frequencies were estimated by gene counting. A Chi-Square Test (c ²) 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 Fisher's 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).

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).

As previously shown (Jalbout et al, 2002), the most frequent sera autoreactivity, found in patient's 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

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.