No abstract

Gastrokine 1 (GKN1) is involved in the replenishment of the surface lumen epithelial cell layer, in maintaining the mucosal integrity, and could play a role in cell proliferation and differentiation. In fact, after injury of the gastric mucosa, restoration may occur very rapidly in the presence of GKN1. In contrast, if the protein is downregulated, the repair process may be hampered; however, application of GKN1 to gastrointestinal cells promoted epithelial restoration. Because GKN1 possesses some mitogenic effects on intestinal epithelial cells (IEC-6) whereas this protein was also capable of inhibiting proliferation in gastric cancer cells (MKN28), we decided to study its involvement in apoptosis to understand the role of GKN1 in the modulation of inflammatory damage or tumorigenesis in gastric mucosa. We found by cytofluorimetry, Western blot and RT-PCR that the overexpression of GKN1 in gastric cancer cell lines (AGS and MKN28) stimulated the expression of Fas receptor. Moreover, compared to control cells, a significant increase of apoptosis, evaluated by TUNEL, was observed when GKN1 transfected cells were treated with a monoclonal antibody (IgM) anti-Fas. The activation of Fas expression was also observed by the overexpression of GKN1 in other cancer cell lines. Moreover, in GKN1-overexpressing gastric cancer cells exposed to FasL, the activation of caspase-3 was also observed by Western blot and fluorescence assays. Our data represent the first report for GKN1 as modulator of apoptotic signals and suggest that GKN1 might play an important role for tissue repair during the early stages of neoplastic transformation.

We have analyzed the expression of Gastrokine 1 (GKN1), one of the most abundant protein of gastric mucosa, in Helicobacter pylori-related preneoplastic and neoplastic gastric lesions.The GKN1 expression was downregulated in 36 H. pyloripositive patients with respect to 29 H. pylori-negative subjects as evaluated by Western blot, reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry, showing a progressive decrease from chronic gastritis to atrophy and intestinal metaplasia. Interestingly, in gastric cancer (GC) patients, GKN1 was undetectable in tissue tumoral areas but was instead expressed in the corresponding tissue non-tumoral areas. In conclusion our data suggests that GKN1 expression is related to the inflammatory damage of gastric mucosa and could be the related to the gastric cellular phenotype.

The gastrokine 1 (GKN1) protein is important for maintaining the physiological function of the gastric mucosa. GKN1 is down-regulated in gastric tumor tissues and derived cell lines and its over-expression in gastric cancer cells induces apoptosis, suggesting a possible role for the protein as a tumor suppressor. However, the mechanism by which GKN1 is inactivated in gastric cancer remains unknown. Here, we investigated the causes of GKN1 silencing to determine if epigenetic mechanisms such as histonic modification could contribute to its down-regulation. To this end, chromatin immunoprecipitation assays for the trimethylation of histone 3 at lysine 9 (H3K9triMe) and its specific histone-lysine N-methyltransferase (SUV39H1) were performed on biopsies of normal and cancerous human gastric tissues. GKN1 down-regulation in gastric cancer tissues was shown to be associated with high levels of H3K9triMe and with the recruitment of SUV39H1 to the GKN1 promoter, suggesting the presence of an epigenetic transcriptional complex that negatively regulates GKN1 expression in gastric tumors. The inhibition of histone deacetylases with trichostatin A was also shown to increase GKN1 mRNA levels. Collectively, our results indicate that complex epigenetic machinery regulates GKN1 expression at the transcriptional level, and likely at the translational level.

A full-length cDNA aldolase A clone was isolated from a human fibroblast cDNA library and completely sequenced. Excluding the poly(A) tail, the clone covers 1095 base pairs (bp) bf the coding region, plus 199 bp downstream for the termination codon and 146 bp upstream for the initiation codon, within a total of 1440 bp.Primer extension experiments performed with human cultured fibroblast mRNA indicate an elongated product of a further 40 bp. These results evaluated together with those obtained in a concurrent study concerning aldolase A mRNA isolated from human liver are direct evidence of aldolase A mRNA multiplicity in man. The data also suggest the existence in mammals of three different classes of aldolase A mRNA, which would account for tissue specificity and resurgence of foetal expression in tumors.Three tissue-specific aldolase isoenzymes (A, B and C) are found in higher organisms [l]. At least three distinct genes code for these isoenzymes. The aldolase isoenzyme system (see [l, 21 for reviews) lends itself to the study of the tissuespecific and developmental regulatory mechanisms of gene expression and can provide information on some aspects of molecular evolution. Furthermore, diseases related to alterations at the gene level, such as hereditary fructose intolerance [3], may be studied via the analysis of such alterations.Within the framework of a study of the aldolase system, we have completely sequenced a full-length cDNA for human liver aldolase B [4] and a partial cDNA for mouse brain aldolase C [5]. Other groups have elucidated the mRNA structure and expression of aldolase B in rat [6] and in humans [7 -91, and have described the aldolase B gene organization and structure in chicken [lo] and in rat [ll].The muscle-type aldolase A has been studied at the mRNA level in rabbit [12], and more recently in rat [13] and in man [14, 151. The data obtained in rat support a multiplicity of mRNA for aldolase A and its tissue-specific patterns. Until now only one human mRNA sequence has been reported [14] and that was obtained from a liver cDNA library.In this paper we report the nucleotide sequence of a new human aldolase A mRNA, obtained from a fibroblast cDNA library. The sequence analysis of the 5'-non-coding region and its comparison with other known sequences demonstrate the multiplicity of aldolase A mRNA in man.Correspondence to F. Salvatore, Istituto di Scienze Biochimiche, I1 Facolta di Medicina e Chirurgia, Universita degli Studi di Napoli, Via Sergio Pansini, $1-80131 Napoli, ItalyAbbreviations. bp, base pairs in DNA; SSC, standard saline citrate, i.e. 0.15 M NaC1, 0.015 M sodium citrate, pH 7.0; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.Enzyme. Fructose-1,6-bisphosphate aldolase (EC 4.1.2.13). MATERIALS AND METHODS Mu teriulsChemicals were from Sigma, Merck or Serva. All the radionuclides, M13 primers and restriction enzymes were supplied by Amersham International plc (UK), AMV reverse transcriptase was from Boehringer Mannheim, deoxynucleotides were from P-L Biochemia Inc. (Milwau...