Ali Gargouri

PIK3CA amplification is predictive of poor prognosis in Tunisian patients with nasopharyngeal carcinoma

PI3Ks (phosphatidylinositol 3‐kinases) are lipid kinases that regulate signalling pathways involved in cell proliferation, motility, and adhesion. Somatic mutations and amplification of the PIK3CA gene have been reported in various types of human cancers. However, little is known about the frequency and prognosis role of PIK3CA activation in nasopharyngeal carcinoma (NPC). This study was conducted with the aim to screen for PIK3CA mutations in the two hot spot regions (exons 9 and 20) and to investigate for the PIK3CA gene amplification combined with the expression analysis of the phosphorylated Akt (pAkt). We showed that among 88 specimens, none had mutation in the helical domain (exon 9) and only one (1.13%) had mutation in the kinase domain (exon 20). On the other hand, PIK3CA gene amplification was found in 21.6% of cases and was strongly associated with distant metastasis (Pu2003=u20030.002), lymph node involvement (Pu2003=u20030.032), and advanced tumor stage (Pu2003<u20030.001). Moreover, patients with PIK3CA copy number gain have a significant reduced overall survival time (P log ranku2003=u20030.02). We concluded that PIK3CA gene amplification is frequent in NPC and occurs in the advanced stage of NPC. Moreover, our finding emphasizes the association of PIK3CA gene amplification with worse prognosis in nasopharyngeal carcinoma. (Cancer Sci 2009); 00: 000–000)

Epigenetic Alteration of the Wnt Inhibitory Factor-1 Promoter Is Common and Occurs in Advanced Stage of Tunisian Nasopharyngeal Carcinoma

ABSTRACT Activation of the wingless-type (Wnt) signaling pathway is common in cancers. The Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that acts by binding to Wnt ligands. We examined by methylation-specific PCR (MSP), whether WIF-1 is inactivated in 68 nasopharyngeal carcinomas (NPC), and 10 normal mucosa. We showed that the WIF-1 promoter was methylated in 89.7% of tumors, whereas all normal mucosa were unmethylated. The WIF-1 methylation was associated with the tumor, node, and metastasis (TNM) (p = .003) and the age (p = .014). The Wnt-5a mRNA was higher in tumors and correlated with TNM (p = .012). The methylation of WIF-1 contributes to the activation of the Wnt pathway in NPC.

Molecular cloning, structural analysis and modelling of the AcAFP antifungal peptide from Aspergillus clavatus.

An abundantly secreted thermostable peptide (designed AcAFP) with a molecular mass of 5777 Da was isolated and purified in a previous work from a local strain of A. clavatus (VR1). Based on the N-terminal amino acid (aa) sequence of the AcAFP peptide, an oligonucleotide probe was derived and allowed the amplification of the encoding cDNA by RT-PCR. This cDNA fragment encodes a pre-pro-protein of 94 aa which appears to be processed to a mature product of 51 aa cys-rich protein. The deduced aa sequence of the pre-pro-sequence reveals high similarity with ascomycetes antifungal peptide. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of an open reading frame of 282 bp interrupted by two small introns of 89 and 56 bp with conserved splice site. The three-dimensional (3D) structure modeling of AcAFP exhibits a compact structure consisting of five anti-parallel beta barrel stabilized by four internal disulfide bridges. The folding pattern revealed also a cationic site and spatially adjacent hydrophobic stretch. The antifungal mechanism was investigated by transmission and confocal microscopy. AcAFP cause cell wall altering in a dose-dependent manner against the phytopathogenic fungus Fusarium oxysporum.

Haplotype analysis of p53 polymorphisms: Arg72Pro, Ins16bp and G13964C in Tunisian patients with familial or sporadic breast cancer

BACKGROUNDnThe p53 polymorphisms have been extensively studied as putative breast cancer susceptibility variants. The present study was undertaken to investigate the association of p53 Arg72Pro, Ins16bp and G13964C polymorphisms and their haplotypes with breast cancer risk in Tunisian women.nnnMETHODSnGenotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 159 patients and 132 controls.nnnRESULTSnThe G13964C intronic variant was significantly associated with familial breast cancer risk (p=0.0018) while the genotypic distribution was similar for p53 Arg72Pro and Ins16bp in patients and controls. Moreover, the (NoIns-C), (Arg-C) and (NoIns-Arg-C) haplotypes were significantly associated with familial breast cancer risk (p=0.0021, p=0.0096 and p=0.0084, respectively) while there was a trend of association between the (Ins-Arg) and (Ins-Arg-G) haplotypes and the risk of sporadic breast cancer. Only the G/C genotype as well as the (NoIns-C) haplotype remained significant after correction for multiple testing.nnnCONCLUSIONnOur data revealed an association between the G/C genotype and the (NoIns-C) haplotype and the risk of familial breast cancer in Tunisian women. However, these observations need to be confirmed due to the limited statistical power of our study and the small number of cases.

Aberrant methylation of hMLH1 and p16INK4a in Tunisian patients with sporadic colorectal adenocarcinoma

The methylation of CpG islands in the promoters is associated with loss of protein via repression of gene transcription. Several studies have demonstrated that tumour suppressor and DNA repair genes are often aberrantly hypermethylated in colorectal cancer. The present study was conducted to examine whether the methylation profile of p16INK4a and hMLH1 (human mutL homologue 1) promoters was associated with clinical features and patients survival in CRC (colorectal carcinoma). Aberrant methylation of p16INK4a and hMLH1 promoters was found in 47.2 and 53.4% of tumours respectively. For adjacent non-tumoral mucosa, p16INK4a was fully unmethylated in 30% of the cases, whereas hMLH1 was predominantly unmethylated (76%). Methylation of p16INK4a correlated with gender and tumour size (P=0.005 and 0.035 respectively), whereas those of hMLH1 significantly correlated with overall survival (P log rank=0.007). Concomitant methylation of p16INK4a and hMLH1 was associated with TNM (tumour, lymph node and metastases) stage and tumour size (P=0.024 and 0.021 respectively). Our data show that loss of hMLH1 expression through aberrant methylation could be used as a marker of poor prognosis in CRC.

Expression of HBsAg and preS2-S protein in different yeast based system: a comparative analysis.

We have expressed both S and preS2-S genes coding for the hepatitis B small (S) and medium (M) proteins, respectively, in different yeast based expression systems and compared the production level of the recombinant proteins. In Saccharomyces cerevisiae, viral genes were expressed under the inducible Gal10/cyc1 and the constitutive PGK promoters using 2mu replicating vectors. We showed that the yield of S protein was higher than M protein under both inducible (14.27 vs 10.9 mg/l) and constitutive (9.18 vs 6.39 mg/l) conditions, respectively. In the methylotrophic yeast Pichia pastoris, the viral genes were expressed in GS115 (Mut(+): Methanol Utilizing) and KM71 (Mut(S): Methanol Utilizing Slow) under the control of the alcohol oxidase promoter (AOX1). In Mut(S) background, both S and preS2-S genes were expressed at higher levels than in Mut(+). In attempt to increase the yield of recombinant viral proteins in S. cerevisiae, we have co-expressed both inducible and constitutive vectors harboring the S or preS2-S genes leading to recombinant strains called UTS (containing pDP/S+pYePIT/S) and UTP (containing pDP/preS2-S+pYePIT/preS2-S). We showed that the recombinant S and preS2-S proteins were successfully detected and the production level reached 18.31 mg/l for the S and 13.22 mg/l for the M proteins. Our comparative study provides evidence that in small scale, S. cerevisiae is more suitable for HBsAg and preS2-S proteins production than P. pastoris under inducible rather than constitutive condition.

A spontaneous direct repeat deletion in the pGEX fusion vector decreases the expression level of recombinant proteins in Escherichia coli

pGEX vectors are widely used for GST-fusion protein expression in Escherichia coli under the control of a strong IPTG inducible tac promoter. While using pGEX-4T-2 vector in heterologous protein expression we noticed that the GST or GST-fusion protein were expressed at a very low levels. Interestingly, we found a spontaneous deletion of 701 bp DNA fragment harbouring the tac promoter in both, native and recombinant pGEX-4T-2 vectors. This deletion took place between two direct repeats of 43 bases and led to the loss of a 701 bp DNA fragment. This explained the decrease in GST or GST-fused protein level since the tac promoter, that directs transcription was deleted. The lacZ promoter, located upstream of the deleted fragment, replaced tac promoter but was less efficient. The deleted DNA also specifies part of the lacZ gene coding for the N-terminal end of the beta-galactosidase (the alpha-peptide), which is slightly functional. Consequently, bacterial cells transformed with the original pGEX are of a faint blue colour while those bearing the deleted ones are white, when plated on X-Gal containing medium. The deletion, did not affect neither the sequence nor the molecular weight of GST and fusion protein since it took place just before the GST start codon. It occurred in E. coli TOP10 cells which are deficient in RecA protein, suggesting that the deletion did not require the RecA recombination system.

C-type lectin protein isoforms of Macrovipera lebetina: cDNA cloning and genetic diversity

Snake venom contains a complex protein mixture belonging to a few well-characterized protein families: disintegrins, phospholipase A2, serine protease, l-amino acid oxidase, Zn-dependent metalloproteinase, natriuretic peptides, myotoxins, cysteine-rich secretory protein (CRISP) toxins, Kunitz-type protease inhibitors and C-type lectin-like. Despite their pharmacological importance, little is known about the exact composition of each protein family. We report here the cloning of 25 complete ORFs from Macrovipera lebetina transmediterranea venom gland that encodes several isoforms and novel C-type lectins (CTLs). 16 alpha and nine beta CTL chains were identified. Based on their sequence alignment, we categorized the 16 CTL alpha subunits into five groups and the nine CTL beta subunits into four groups to deduce the phylogenetic tree of M. lebetina transmediterranea CTLs. Sequence analysis revealed that they share a high degree of similarity with each other and with other snake venom CTLs. The M. lebetina transmediterranea CTL sequences described here contain a C-lectin carbohydrate recognition domain-like fold (C-lectin CRD-like) characterized by several conserved amino acid residues in their structure, especially the cysteine. Finally, based on the comparison of some Macrovipera CTL, we propose that some new CTL gene versions should have occurred through domains shuffling from former genes.

Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli

The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with eight right-handed parallel β-helix architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 °C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole.

Methylation status and overexpression of COX-2 in Tunisian patients with ductal invasive breast carcinoma

Inflammation and hormonal signalling induce the cyclooxygenase-2 (COX-2) expression in solid tumours including breast cancer, which in turn affects cell proliferation, apoptosis and metastasis. The aim of this study was to investigate the expression of COX-2 and its association with clinical parameters, patient’s survival, hormones receptors (oestrogen, progesterone), ERBB2 and TP53 expression in 83 cases of infiltrating ductal breast carcinomas. Moreover, the methylation status at the CpG islands of the COX-2 gene promoter was also explored in 70 specimens. We showed that tumours exhibiting moderate to intense COX-2 immunostaining were significantly more frequent in patients over 45xa0years old (pu2009=u20090.027). Moreover, a high level of COX-2 expression correlated with a shorter survival time (p log-ranku2009=u20090.04) and was an independent prognostic factor (pu2009=u20090.022; HR 6.4; 95% CIu2009=u20091.3–31.4). On the other hand, hypermethylation of the COX-2 gene promoter was observed in 27% of cases and strongly associated with smaller tumours (<5xa0cm, pu2009=u20090.011). Furthermore, patients with methylated COX-2 pattern have a better 4-year disease-free survival (pu2009=u20090.022) as well as a prolonged overall survival (p log-rank testu2009=u20090.034). In conclusion, we showed that high COX-2 expression was associated with reduced survival and was an independent prognostic factor. However, hypermethylation of the COX-2 promoter correlated with a better overall survival in Tunisian patients with breast carcinoma.