Expression of hepatocyte growth factor, transforming growth factor alpha, apoptosis related proteins Bax and Bcl-2, and gastrin in human gastric cancer.

BACKGROUND: Gastric cancer is one of the most frequent neoplasms and a leading cause of the death world-wide. In recent years, epidemiological and animal studies demonstrated a link between gastric cancer and chronic infection with H. pylori. The exact mechanism responsible for the development of gastric cancer in H. pylori-infected patients still remains unclear. There is evidence that the up-regulation of certain growth factors could play an important role in the promotion of the gastric carcinogenesis. AIMS: The present study was designed to determine the gene expression of major known growth factors such as transforming growth factor alpha (TGFalpha), hepatocyte growth factor (HGF) and gastrin in the gastric cancer tissue, the surrounding mucosa and, for comparison, in the normal gastric mucosa. Furthermore, the luminal and plasma levels of gastrin in patients with gastric cancer were determined. In addition, the gene and protein expressions of apoptosis-related proteins such as Bax and Bcl-2 were investigated by reverse transcription-polymerase chain reaction and Western blot. Twenty-five gastric cancer patients and 40 age- and gender-matched control subjects hospitalized with non-ulcer dyspepsia were included into this study. RESULTS: An overall H. pylori-seropositivity among gastric cancer patients was about 72% and was significantly higher than in the controls (56%). The prevalence of CagA-positive strains was also significantly higher among gastric cancer patients than in controls (56% vs. 32%). The gene expression of HGF and TGFalpha was detected more frequently in gastric cancer tissue samples than in normal gastric mucosa (52% vs. 12% for HGF and 48% vs. 24% for TGFalpha). The extent of protein expression in Western blotting analysis for HGF and TGFalpha correlated with the mRNA expression of these factors. Gene expression of gastrin was detected in the antrum of all tested patients and in the majority (84%) of gastric cancer patients. The median plasma and luminal concentrations of gastrin in gastric cancer patients were significantly higher than in controls. The gene expression of bcl-2 was detected in all (100%) and that of proapoptotic bax only in 56% of gastric cancer samples. In comparison to the surrounding non-tumorous tisssue, the gene expression of bax was significantly down-regulated and the gene expression of bcl-2 was up-regulated in gastric cancer tissue. At the protein level, Bax was not detectable and Bcl-2 was seen in 80% of gastric cancer samples. CONCLUSIONS: It is concluded that the patients infected with H. pylori, especially with CagA-positive strains, are at a higher risk of developing a gastric cancer. An increased production and release of gastrin, as well as an over-expression of growth factors such as HGF and TGFalpha, might contribute to the gastric carcinogenesis. In addition, a dysregulation of the Bax/Bcl-2 system with significant up-regulation of Bcl-2 is observed in gastric cancer.

Role of leptin in ulcer healing.

Leptin was shown to exhibit similar to cholecystokinin (CCK) cytoprotective activity against acute gastric lesions, but its role in ulcer healing has not been examined. The aims of this study were: (1) to compare the effects of exogenous leptin to those of CCK on the course of healing of chronic gastric ulcers; (2) to study the gene and protein expression of leptin at the ulcer margin during ulcer healing; and (3) to assess the effects of leptin administration on the mucosal gene expression of main growth factor such as transforming growth factor alpha (TGFalpha). Gastric ulcers were produced in rats by the acetic acid method. Rats with ulcers were divided in following treatment groups: (1) vehicle; (2) leptin (10 microg/kg i.p.); (3) CCK (10 microg/kg s.c.); and (4) leptin or CCK with or without tyrphostin A46 (200 microg/kg i.p.), an inhibitor of epidermal growth factor (EGF)-receptor tyrosine kinase or NG-nitro-L-arginine (20 mg/kg i.g.), a blocker of nitric oxide synthase. Animals were euthanized 9 days after ulcer induction. The area of gastric ulcers and the gastric blood flow at the ulcer area were determined. In addition, mucosal biopsy samples were taken from the ulcer area for histological evaluation as well as for the determination of mRNA and protein expression for leptin and constitutive nitric oxide synthase (cNOS) and inducibile nitric oxide synthase (iNOS) by reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. In addition, the gene expression for TGFalpha was analyzed by RT-PCR. Both leptin and CCK reduced significantly the ulcer area as compared to vehicle-treated group by approximately 50%. The treatment with tyrphostin or N(G)-nitro-L-arginine reversed in part the acceleration of ulcer healing by leptin and CCK. The expression of leptin mRNA and protein was significantly increased at the ulcer edge. The leptin-induced acceleration of ulcer healing was associated with increased expression of transcripts for TGFalpha as well as increased mRNA and protein expression for cNOS and iNOS at the ulcer margin. We conclude that leptin accelerates ulcer healing by mechanisms involving the up-regulation of TGFalpha and increased production of nitric oxide due to up-regulation of cNOS and iNOS in the ulcer area.

Exon 14-skipping of the adenomatous polyposis coli gene in purified epithelial cells of colonic mucosa and tumors.

Adenomatous polyposis coli (APC) exon 14-skipped transcripts encode putative APC proteins of low molecular weight. To prove that exon 14-skipped mRNA variants do not simply represent tissue culture artifacts, expression of these APC transcript variants was demonstrated in native colorectal epithelium. Fresh surgical specimens of human colon were processed and epithelial cells were affinity-purified with the dynabead-immobilized monoclonal antibody Ber-EP4. Epithelium derived cDNA was PCR-amplified in the range of linear accumulation. RT-PCR products with and without APC exon 14 were evaluated by densitometry. Analyses of normal mucosa (n = 8) and matching mucosa--tumor samples (n = 4) revealed a consistent 4 to 1 ratio of APC exon 14-positive to exon 14-negative mRNA levels. We conclude, a) that APC exon 14-skipped transcripts are physiologically expressed in native human colon mucosa, and b) that ratios of exon 14-negative to exon 14-positive isoforms were not altered when colorectal tumor cells were compared with matching normal mucosa (p = 0.80).

Enhanced expression of leptin following acute gastric injury in rat.

Leptin, a product of ob-gene plays an important role in the regulation of food intake. Recently, leptin expression has been detected in gastric epithelium, but the physiologic role of gastric leptin remains unknown. The purpose of this study was: 1) to determine the effect of gastric injury by ethanol and aspirin on the expression of leptin in gastric mucosa and 2) to investigate whether exogenous leptin affects the integrity of gastric mucosa exposed to noxious agents such as ethanol or aspirin. In Wistar rats the acute gastric lesions were induced by intragastric application of 1.5 ml of 75% ethanol or acidified aspirin (100 mg/kg in 0.2 N HCl). Rats were divided into two groups and pretreated either with leptin (1-10 microg/kg i.p.) or vehicle (saline). Rats were anesthetized 1 h after i.g. induction of acute gastric lesions and the gastric blood flow (GBF) was measured by H2 gas clearance method. Then the rats were sacrificed, the stomach was excised and the mean lesions area was assessed by planimetry. In addition, mRNA and protein expression for leptin was analyzed in the gastric mucosa by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Both ethanol and acidified aspirin induced acute gastric lesions and led to significant reduction in GBF. In the intact gastric mucosa, the mRNA and protein expression for leptin was small but detectable. The exposure of gastric mucosa to noxious agents such as ethanol and aspirin was associated with markedly increased expression for gastric leptin at mRNA and protein level. Application of 75% ethanol or acidified aspirin caused wide-spread mucosal lesions. The pretreatment with exogenous leptin reduced dose-dependently these ethanol or aspirin-induced gastric lesions. The protective effects of exogenous leptin were accompanied by a significant attenuation of the fall of GBF. We conclude that: 1) Exogenous leptin exerts potent gastroprotective and hyperemic actions on gastric mucosa, and 2) Acute injury of gastric mucosa is associated with increased expression of leptin suggesting a possible role of this peptide in mediating of repair process in injured gastric mucosa.

Novel exon connections of the brain-specific (BS) exon of the adenomatous polyposis coli gene.

Expression of the adenomatous polyposis coli (APC) gene derived exon BS (e.g., brain-specific exon) has been analyzed by RT-PCR. Four novel APC mRNA isoforms derived from alternative splicing of exons 1A, BS and 1 were identified, which were ubiquitously expressed. One novel cDNA was characterized by cloning and DNA sequence analysis, which combined the exon 1A (identical with exon 0.3) 3' end with nucleotide position +101 of intron 1A and continued throughout exon BS. A second cDNA isoform was isolated, which joined the 3' end of exon 1A with nucleotide position +118 of exon BS. Both novel isoforms were found to be expressed together with a third novel APC exon connection, which was specified by exon BS/2 joining. This interesting exon junction resulted in novel deduced amino terminal open reading frames, which are completely in-frame with sequences located further downstream. Systematic exon connection analyses revealed that APC transcripts with exon BS/2 junctions were predominantly detected with a fixed exon composition. RT-PCR analyses did not identify facultative skipping of exons 9, 10A and 14 in this type of mRNA, in contrast to exon 1-containing APC transcripts analyzed from the same cDNA pool under identical conditions. Hence, exon 1 skipping of exon BS-positive mRNA molecules might preferentially encode unique APC polypeptide chains, which are characterized by an alternative amino terminus and extended heptad repeat structures due to combined incorporation of exons 9 and 10A.

Constitutive APC exon 14 skipping in early-onset familial adenomatous polyposis reveals a dramatic quantitative distortion of APC gene-specific isoforms.

Adenomatous polyposis coli (APC) gene transcripts skipping exon 14 in combination with the alternatively spliced exons 9 and 10A contribute to the heterogeneity of physiological APC mRNA isoforms. Here we report on a novel genotype-phenotype correlation in familial adenomatous polyposis (FAP) with early onset of disease and malignancy due to an APC exon 14 splice defect. Compared to controls, two affected individuals of a FAP kindred presented with a significantly distorted APC mRNA isoform pattern in B lymphocytes. As a result of an A-->G transition in the canonical AG-splice acceptor dinucleotide of exon 14, expression levels of all APC mRNA isoforms without exon 14 were dramatically increased and those with exon 14 were simultaneously decreased. Skipping of exon 14 is a physiological event also seen in nonmalignant cells, which results in a frameshift to produce low-molecular-weight APC proteins. Western blot analysis of the patients' lymphoblastoid B cells revealed the identification of intracellularly stable APC protein isoforms with an Mr of 55-67 kDa and, thus, the first demonstration of APC proteins encoded by exon 14-skipped transcripts. We postulate that the quantitatively imbalanced expression of these physiological APC light chains represents a novel pathogenetic mechanism associated with predisposition to FAP.

Immunochemical identification of novel high-molecular-weight protein isoforms of the adenomatous polyposis coli (APC) gene.

Mapping analyses of monoclonal antibodies (MAbs) directed against the amino-terminus of the adenomatous polyposis coli (APC) gene product revealed that epitopes recognized by the MAbs FE9, CF11 and AC4 constitute different peptide sequences encoded by the APC exons 1, 2 and 3, respectively. The protein pattern detected with these specificity-defined immunoreagents, however, differed depending on the particular antibody used on Western blots of cellular urea extracts. APC exon 15-positive "classic" p300apc polypeptide chains were identified by the MAb FE9, MAb CF11 and the C-terminus-specific MAb IE1, but only weak signals were obtained with the AC4 MAb, which defines an exon 3-encoded epitope. In contrast with this immunoreactivity, 2 novel high m.w. products of approx. 150/160 and 200 kDa were exclusively recognized by the AC4 MAb, which was shown to bind to the APC exon 3-encoded peptide sequence SRESTGYL. A molecular form of some 400 kDa was identified to represent a disulfide-bound oligomer of the p150/160apc molecules. The novel APC-related molecules did not contain exon 1- and exon 15-encoded epitopes, as confirmed with the help of the FE9 and IE1 MAbs, respectively. This observation was corroborated by the fact that these novel proteins were not truncated in a collection of familial adenomatous polyposis patients with stop mutations in exon 15. We conclude, that APC MAb AC4-reactive p150/160 and p200 polypeptide chains represent novel genuine products of the APC gene devoid of exon 1- and exon 15-encoded protein interaction domains.

Multiple APC messenger RNA isoforms encoding exon 15 short open reading frames are expressed in the context of a novel exon 10A-derived sequence.

Intragenic splice mechanisms affecting the coding exons 8 to 15 of the human adenomatous polyposis coli (APC) gene have been analyzed. Three mechanisms within this gene area were found to contribute to mRNA heterogeneity: (i) facultative expression of exon 9-encoded sequences; (ii) in-frame insertion of a 54-nucleotide sequence encoded by a novel exon located 1.6 kb down-stream from exon 10, provisionally designated APC exon 10A; (iii) skipping of exon 14, resulting in a novel exon 13/15 connection. Interestingly the latter event provided the mRNA with a novel open reading frame, which was terminated after 19 codons of exon 15-derived sequences. Combinatorial joining of these segments yielded 7 different transcripts in addition to an mRNA species resulting from an exon 10/15 connection, as determined by cloning and sequence analysis. RT-PCR expression analyses were carried out to demonstrate that this complexity of splice variants is indeed synthesized in cell lines derived from various tissues. Furthermore, in accordance with our findings at the transcript level, we provide Western blot analyses demonstrating that moderate steady-state levels of genuine APC-specific low m.w. polypeptide chains exist. These APC "light chains", however, are not identical with polypeptide chains, which have been reported to accompany apoptosis and necrosis, since the molecules described here are definitively co-expressed with p300apc at the transcript and protein levels.

A novel coding exon of the human adenomatous polyposis coli gene.

We report the genomic cloning and sequence analysis of a novel and (so far) smallest coding exon of the human adenomatous polyposis coli (APC) gene. This additional exon of 54 nucleotides in length, designated APC exon 10A, is located 1.6 kb downstream from exon 10. It resides on a 0.5-kb genomic EcoRI restriction fragment. APC exon 10A is alternatively spliced and inserted in-frame into mature transcripts; it gives an APC protein with an additional 18 amino acids. This highly conserved exon is expressed in a tissue-independent fashion. APC exon 10A flanking sequences are presented so that this novel exon can be included in future mutation screening procedures.