Study on p53 gene mutations in Mongolian gerbils infected with Helicobacter pylori

A1404 AGA ABSTRACTS

GASTROENTEROLOGY Vol. 118, No.4

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P21, BAX AND CASPASE·3 PARTICIPATE IN THE PROPAGAT· ING MECHANISM OF APOPTOSIS IN THE HUMAN GASTRIC EPITHELIAL CELLS. Kenji Noguchi, Katsuaki Kato, Ging Yang, Tatsuhiko Suzuki, Yuji Kubota, Naohiro Dairaku, Keisuke Oikawa, Yutaka Konno, Hitoshi Sekine, Shuichi Ohara, Takayoshi Toyota, Tooru Shimosegawa, Tohoku Univ Sch of Medicine, Sendai, Japan. Background and Aims: Apoptosis can be defined as the cell death programmed inherently. Bcl-2 family proteins such as Bcl-2, Bax and Bak act as preventive or promotive factors for apoptosis. Caspases are located in the common pathway to apoptosis, and act as the executors of apoptosis. Apoptosis is also closely related to the cell cycle, so that p21, a cyclin dependent kinase inhibitor, is also implicated in apoptotic process. In the human gastric mucosa, it is known that apoptotic cell death is upregulated relating to Helicobacter pylori (HP) infection, and the disintegration of apoptotic system, which excludes damaged cells may lead to carcinogenesis. However, it is unclear how apoptotic signals propagate in the human gastric mucosa.Therefore, we studied the apoptotic pathway in the human gastric mucosa. Materials and Methods: Gastric mucosal specimens were obtained from 10 HP-negative dyspepsia patients, 10 HP-positive chronic gastritis patients before and after HP eradication. Immunohistochemistry of Ki-67, p21, Caspase-3, Bax and Bak was performed and apoptotic cells were detected by the TUNEL method. Results: Ki-67 immunoreactivity was identified in the nuclei of proliferative cells located in the mucosal neck region, and the number of Ki-67 positive cells increased in HP positive gastric mucosa. Immunoreactivity of p21 was detected in the nuclei of the cells located in the area of more luminal than the cell proliferation area toward the mucosal surface. Immunoreactivities of caspase-3 and Bax increased in the cytoplasm and nuclei of the cells that were located predominantly in the upper part of surface mucosa. Bak did not show any remarkable changes. TUNEL positive cells were predominantly found at the tip of mucosa. The cells positive for p21 were also sporadically observed in the gastric gland, but they did not coincided with the caspase-3- or Bax-positive cells. The expression of all these molecules was increased in the HP positive gastric mucosa and was decreased after HP eradication. Conclusions: The present data suggested that apoptotic signal may propagate in the gastric surface epithelium through the upregulation of p21, Bax and caspase-3 in both the normal and inflamed gastric surface mucosa, which would be set in just after cell proliferation. On the other hand, apoptosis in the gastric glandular cells might be regulated by different mechanisms from the surface epithelium.

ACCUMULATION OF P53 GENE MUTATIONS IN HELICOBACTER PYLORI·ASSOCIATED GASTRITIS OF 8 YEARS INFEC· TION IN JAPANESE MONKEY. Touta Oda, Akira Nisizono, Masaaki Kodama, Reiji Kodama, Shyouji Honda, Kazunari Murakami, Toshio Fujioka, Masaru Nasu, Oita Med Univ, Oita, Japan. Background & Aims: Helicobacter pylori is considered as a carcinogenic agent producing gastric cancer by WHOIIARC in 1994. However, there has not been reported direct evidence of carcinogenicity except for using Mongolian gerbils model. We have already demonstrated that p53 protein overexpressed and various gene mutation observed in Hpylori-infected atrophic gastric mucosa in Japanese monkey. In this study we analyzed gene mutation of a monkey in time couse and aimed to investigate the relationship between long term infection of Hipylori and change of p53 gene in gastritis. Materials & Methods: The original p53 sequences were determined by the amplification of exon(Ex) 5 to 8 using DNA of gastric tissues obtained from 6 uninfected monkeys of different ages. Mutations in p53 Ex 5 to 8 were analyzed by the direct sequencing using gastric specimens of lessor curvature in stomach obtained from H pylori infected the Japanese monkey. Compared gene mutations 3 years after inoculation with 8 years of the same monkey. Results: No apparent mutations in p53 Ex 5 to 8 were observed among 6 uninfected monkeys. As the duration of H pylori infection, the number of nucleotide (amino-acid) substitutions in p53 gene accumulated as the gastric atrophy score in experimentally H pylori infected Japanese monkey. Between 3 years and 8 years after inoculation, the same mutations observed 5 cases (codon 129, codon 225, codon 286, codon 293, codon 295). These mutations 4 of 5 were G:C to A:T transitions. Mutations in p53 gene were identified in I I of 2 I Hipyloripositive patients in a biopsy sample of human gastric mucosa. 3 of 5 mutations in Japanese monkey(codon 286, 293, and 295) were agreed with 6 of 1I in human. Conclusions: There might be a relation between the occurrence of p53 mutation and gastric atrophy depended on the duration of infection. P53 mutations do not have the same tendency to mutational characteristics found in time couse. This suggests that the p53 gene is unstable and that reversible gene alteration may occure during tumor progression. Stable mutations in time couse were G:C to A:T transitions. This type of mutation is reported to be linked to exposure to nitric oxide, one of the well-established chemical carcinorgens of gastric carcinoma. There might be one of these stable mutations accumulated in duration of infection, may be one of oncogenic factor of gastric neoplasia. Follow up of p53 mutation may be useful for studying the possibility between H pylori infection and gastric carcinogenesis.

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ROLE OF INTESTINAL METAPLASIA AND EPITHELIAL DYSPLASIA IN THE PATHOGENESIS OF GASTRIC CARCINOMA. Ana Mmf Nogueira, Monica Mda Cabral, Paulo Go Salles, Liubiana A. Araujo, Luiz Gm Rodrigues, Marco Ag Rodrigues, Celso A. Oliveira, Dulciene Mm Queiroz, Gifone A. Rocha, Andreia M. Oliveira, Fed Univ of Minas Gerais, Belo Horizonte, Brazil. Intestinal metaplasia (1M) and epithelial dysplasia (ED) are sequential events in the process of gastric carcinogenesis, specially in intestinal-type carcinomas. Epithelial dysplasia has been considered the most important precancerous lesion in the stomach, however it appears to be rare in several series studied so far. We performed a topographical and histological study of 1M and ED in stomachs resected for carcinoma and peptic disease. One hundred and twenty-nine surgical specimens were studied: 100 carcinomas (43 early and 57 advanced tumors), 11 duodenal ulcers (DU), and 16 gastric ulcers (GU). Strips of gastric mucosa (3xO.3cm) taken along the lesser and greater curvatures and gastric walls, and 3-10 samples from the tumor were collected (at each 2-3 em in 71 carcinomas and in the peptic ulcers). Endoscopic biopsies (n =7) from 53 patients with chronic gastritis (34 H pylori + )were also analyzed. The samples were processed routinely; histological sections were stained by HE, PAS-Alcian blue, high iron diamine and carbolfucsin. In the cases from endoscopy H pylori was detected by culture and urease. Sixty-seven carcinomas were of intestinaltype, 19 were diffuse, and 14 were mixed. 1M was observed in 95.5% of intestinal carcinomas and in 78.9% of diffuse ones, being significantly more intense in the former (p=0.004). 1M type III was detected in 52.5% of the intestinal-type (in 32.5% around the tumors) and in 35.3% of the diffuse-type (around the tumor in 17%)(p>0.5). The presence of 1M type III correlated significantly with the intensity of the metaplastic process (p
THE FIBROBLAST PROLIFERATION, RELATIONSHIP OF HEPATOCYTE GROWTH FACTOR (HGF) AND INTERFERON·A2A. Nihat Okcu, Abdulhalim Baki, Mehmet Sari, Mehmet Arslan, Sait Kapicioglu, Karadeniz Tech Univ, Trabzon, Turkey. In this study, the effect of interferon alfa 2a and hepatocyte growth factor (HGF) on the proliferation of circumsition skin fibroblast was investigated in vitro. These agents were used separetely and in combination. Study was done on seven groups. Group I (Control): fibroblast+ media, group 2 (IFNSOO) fibroblast+media+IFN (SOOU/ml), group 3 (IFN250):fibroblast+ media+IFN (250U/ml), group 4 (HGF20):fibroblast+ media+ HGF (20ng/m!), group 5 (HGFIO):fibroblast+media+HGF (l Ong/ml), group 6 (lFN500+HGF20):fibroblast+media+IFN (500U/m1)+HGF (20ng/ml), group 7 (IFN250+ HGFIO):fibroblast+media+ IFN (250D! ml)+ HGF(l Ong/ml). All groups were incubated at 3rc for 72 hours. The effect of IFN and HGF on fibroblast development was observed by counting cell populations, analyzing cell cycles, and investigating morphological features. Cell number was decreased in group 3 (p<0,OO5) compared to group 1, and increased in groups 4,5,6 and 7.There was no significant change in group 2. Increased cell counts was seen in group 6 (p 0,00239) compared to. group 2. Similarly, increase in cell counts was seen in group 7 (p 0,00137) compared to group 3. No significant difference was seen between the control group and other groups for cell concentrations at G,phase and proliferation index (PI) by flowcytometrical analysis In this study we found that IFN plus HGF couldn t suppress the fibroblast proliferation.HGF suppressing agent in combination with IFN may be used in treatment of diseases with fibrosis.

6380 STUDY ON P53 GENE MUTATIONS IN MONGOLIAN GERBILS INFECTED WITH HELICOBACTER PYLORI. Tadayoshi Okimoto, Jiro Kagawa, Shoji Honda, Toshio Fujioka, Masaru Nasu, Oita Med Univ, Oita, Japan. Background and Aims: Mongolian gerbil is an only model for Helicobacter pylori-induced atrophic gastritis, intestinal metaplasia and gastric cancer. It is considered that p53 gene mutations playa critical role in the development of gastric carcinome. To investigate the mechanism of gastric tumorigenesis induced by H pylori infection, we detected the normal p53 DNA sequence(ex 5, 6, 7 and 8) of Mongolian gerbils and studied on p53 gene mutations of animals infected with Hipylori. Materials and methods: To detect normal p53 DNA sequence, cDNAs from spleen, liver, and stomach of a five-week-old male Mongolian gerbil was used. P53 gene of Mongolian gerbil amplified using nested PCR method with the primers for human

AGAA1405

April 2000

p53 gene . 19 animals inoculated Hipylori and 10 uninoculated control anim als, they were alive up to the end of the experiment, were sacrificed at 18 month s after H pylori inoculation . The stomach was quickly removed and used for microscopical examinations and p53 DNA sequencing. Results: All control animals showed no abnormal findings histologically and no mutat ions. I of 19 animals inoculated with H. pylori was not infected . 18, 18 and 2 of 18 animals infected with H pylori presented atrophic gastriti s, intestinal metaplasia and ga stric cancer, respectively. However, no mutations of p53 gene were found. Conclusions : P53 gene of Mongolian gerbil shows 83.6% homology to human p53 gene (ex5 , 6, 7 and 8). In this study, no mutations of p53 gene were found. Howe ver, the DNA s used for sequencing were extracted from the lesion that didn't have intestinal metapl asia nor cancer. So mutations of p53 gene may be detected onl y in such lesions.

DBTC animals(41.3:t5.4%) compared with control groups (I8.5 :t2.3%; p
6383 6381 EXPESSION OF CYCLOOXYGENASE·2 IN HUMAN GASTRIC CARCINOMAS. Ryo Oono, Takeshi Fujita, Keigo Yo shinaga , Hideaki Iseki, Kumi Hasegawa, Hidefumi Tsunozaki, Wataru Ichikawa, Yohko Takagi, Masa yuki Enomoto, Kenichi Sugihara, Tok yo Med and Dental Univ, Tokyo, Japan . Epidem iological studies show that non steroidal anti-inflammatory drugs could reduce the incidence of gastric carcinoma. Their chemopreventive effects are postulated to be based on the inhibition of cyclooxygenase (COX)-2. In the present study we inve stigated the expression of COX-2 in hum an gastric carcinoma tissues using RT-PCR method and immunohistochemistry . Materials and Methods: A total of 34 surgical specimens of gastric carcinoma were obtained at surgery and examined for COX-2 mRNA expression. PCRs were performed con comitantly for both COX-I and COX-2 in the same tube on each sample. To estimate COX -2 expression levels, COX -2 index was designated by the band densit y ratio of COX -21COX-I, because COX-I mRNA is expre ssed constitutively. The relationship between COX -2 index and clinicopathological findings was anal yzed . Immunohistochemical staining with anti-COX-2 antibod y was performed on the same samples of gastri c carcinoma. All results are sho wn as means :t SE . Result s: The COX-2 mRNA expressio n was detected in all gastric carcinomas. The COX-2 index of carci nomas was significantly higher than that of normal muco sae (carcinoma; 4.0 :t 0.8, normal mucosa 2.8 :t 0.9; p < 0.01). The COX -2 indices of pT! , pT2, pT3 and pT4 carcinoma were 0.7 :t 0.3 , 4.2 :t 1.5, 4.1 :t 1.0 and 8.8 :t 5.5, respectively ; The COX-2 indice s were significantly higher in the tumors with deeper invasion (p < 0.05). The COX-2 index was not affected by histopatholog ical type, location and size of tumor, lymph node and distant metastasis and lymphatic and vascular perm eation . In immunohistochemistry, the staining intensities of COX-2 prote in were correlated with the COX -2 mRNA levels. The COX -2 prote in was stained diffusely in the cytoplasm of cancer cells and it was observed weakl y in the noncancerous tissues. Conclusions: Both levels of the COX-2 mRNA and protein were elev ated in gastri c carcinoma tissue. The COX-2 mRNA level wa s closely correlated with the depth of tumor invasi on. These results suggest that COX -2 is associated with the growth and expan sion of gastric carcinoma. 6382 FURTHER EVIDENCE FOR THE DUCTAL ORIGIN OF DMBAINDUCED PANCREATIC NEOPLASMS IN RATS. Fumiaki Ozawa, Kaspar Z'graggen, Luca Mazzucchelli, Lierka Zipperle, Helmut Fries s, Markus W. Buechler, Dept of Visceral and Transplantation Surg, Inselspital , Bern , Switzerland; Institute of Pathology, Univ of Bern , Bern , Sw itzerland; Visceral and Transplantation Surgery; Inselspital, Bern , Switzerland. Implantation of dimethylbenzanthracene (DM BA) into the rat pancreas induce s different stages of ductal neopla sia and invasive adenocarcinoma. We investigated whether the destructi on of pancreatic parenchyma and its replacemen t by pancreatic fibrosis or the stimulation of acinar cell s by a cholecystokinin analogue changed the prevalence of DMBA-induced neoplasia in the rat pancreas . Furthermore, we studied whether indefinite for dysplasia termed lesions were neopl astic or reactive in nature . Methods: DMBA crystals (5mg) were implanted into the head of the rat pancreas(group A; 16 animal s). Pancreatic fibrosis was induced by intravenous dibut ylin dichloride (8 mglkg bw ) 60da ys before DMBA implantation (group B; 12 animal s). Caerulein(5 meg/kg body wt ) was i.p. injected twice daily for 15 day s after DMBA implantation (group C; 19 animals). After 30 days 5 -bromo-2' -deoxyuridine(BrdU) was i.p. injected 6 hours before animals were killed . Sham operated control animals were included in the study( 13 animals). Image-pro plus software was used to quantitate pancreatic fibros is. Histologies were evaluated in a blinded fashion . BrdU labeling index (LI) was correlated with histol ogy in all stages of neoplasia. Result s: Histologic analysis demonstrated that the prevalence of all stages of neoplasia was 80% in group A, 67% in group B, 47% in group C (p> 0.05) and 0 in control groups . Pancreatic fibrosis was increased in

COST-EFFECTIVENESS OF GENETIC DIAGNOSIS FOR HEREDITARY HEMOCHROMATOSIS SCREENING. Alberto Pardo, Spani sh Group for the Stud y of Hereditary Hemochromatosis, Uni v Hosp of Canary Islands, La Laguna., Spain .

It has been suggested that genetic test (C282Y mutation) could substitute conventional biochemical tests and simplify the screening strategy for Hereditary Hemochromatosis. However, this hypothesis has not been confinned. OBJECTIVE: To evaluate the role of genetic diagnosis (C28 2Y homozygosity) for HH screening in the Spanish general population. METHODS: Four screening strategies were evaluated by decision tree analysis (Markov) in a 30 years old hypothetical cohort: I) no screening; 2) Transferrin saturation (TS) : screen ing by transferrin saturation (TS) plus liver biopsy (LB) when TS > 45 % and serum ferritin (SF) levels above normal values; 3) C282Y mutation: detection of C282Y (PCR) and LB when SF > 1000 ng/ml ; and 4) TS + C282Y : initial screening by TS followed by C282Y detection when TS > 45 %. In this strategy LB was performed when SF > 1000 ng/m!. Estimates of prevalence, asymptomatic interval s, transitional probabilities between clini cal stages , and sensitivity/ specificity of screening tests were obtained from the publi shed literature. Interventional and medi cal care costs were obtained from 10 National Health Spani sh Hospit als. RESULTS : TS +C282Y screening strategy increased days of life gained and decrea sed interventional and medical care costs as compared to TS or C282Y strategies. CONCLUSION: In the Spanish population initial tran sferrin saturation analysis followed by genetic test (C282Y mutati on) when appropriate, seems to be the most cos t-effective strategy for Hereditary Hemochromatosis screening. Cost-effectiveness data fordifferent HH screeningestrategies (Costs= Euros)

Screening Strategies

ute days gained per person screened

Costs saved per person screened

Screening costs per year oflivegained

T5 C282Y TS+C282Y

12,78 16,14 59,68

·12.98 16,14 59,68

1244,72 2585,64 689,68

6384 STUDY ON THE EXPRESSION OF INSULIN-LIKE GROWTH FACTOR II (IGF-II) IN HEPATOCELLULAR CARCINOMA CELLS AND DEVELOPING RAT EMBRYO. Mooin Park, Sunggeun Shin, Sunghoon Chang, Seonja Park, Kyuwon Kim, layoung Koo, Kosin Med Ctr, Pusan , MD , South Korea; Dept of Molecular Biology , Pusan National Univ, Pusan, South Korea . The insulin-like growth factor II (IGF-II) gene, encoding a fetal sornatomedin, expresses a fam ily of transcripts in embryonic/fetal tissue, and also in the adult choroid plexu s and the leptomeninges. IGF-II is also highl y expre ssed during hepat ocellular carci nogenesis. In this stud y, we show that IGF-II mRNA and protein levels are detected in HepG2 human hepatoma cells by Northern and Western analysi s. We have also localized IGF-II gene products in sections of rat embryos of 7-17 days post coitum (d.p.c) by performing immunohi stochemical staining. IGF-II was mainly expressed in proximal endoderm and ectoplacental cone from 7 to 9 d.p.c.. From 10 d.p.c . the expression was lacalized at heart primodium as well as proximal endoderm. After the II d.p .c., IGF-II was expressed in the liver (including liver primodia) and heart . While following 12 d.p.c. and 14 d.p.c., the expres sion was also detected in the brain , muscle and bone (including myotome and sclerotome), and head mesenchyme, respecti vely. At 17 d.p .c. intense staining was detected in heart , liver and choroid plexu s, where as in normal adult liver the expression of IGF-II protein was not detected. These results sugges t that IGF-II may act as an onco fetal protein during hepatocellular carcinogenesis and embryogenesis.