JOURNAL OF PATHOLOGY, VOL. 179: 3 9 4 2 (1996) IN SITU ANALYSIS OF TISSUE DYNAMICS AND p53 EXPRESSION IN HUMAN GASTRIC MUCOSA AKIRA IMATANI*t, HIRONOBU SASANO*, NORITAKA YABUKI*t, KATSUAKI KATOt , SHUICHI OHARAt, SHIGERU ASAKIT, TAKAYOSHI AND HIROSHI NAGURA* TOYOTAT Departments of Pathology* and Medicine?, Tohoku University School of Medicine, Sendai, Japan SUMMARY In situ tissue dynamics were studied in 12 cases of human gastric mucosa, including normal gastric body mucosa and gastric glands with intestinal metaplasia, obtained from gastrectomy specimens of adenocarcinoma. Cell proliferation was determined by Ki67 immunoreactivity. DNA fragmentation was studied in sits by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In addition, p53 expression was examined by both immunohistochemistry and mRNA in situ hybridization. In the oxyntic gastric glands, Ki67 immunoreactivity was observed exclusively in the proliferative zone and TUNEL-positive cells were present predominantly in the surface foveolar epithelium. In the gastric glands with complete intestinal metaplasia, Ki67-positive cells were present in the lower portion of the glands and TUNEL-positive cells in the superficial epithelium. In the gastric glands with incomplete intestinal metaplasia, TUNEL-positive cells were detected in the lower gastric glands adjacent to cells immunoreactivefor Ki67; the proportion of these gastric glands with TUNEL-positive cells (40 out of 108 glands) was significantly higher than for oxyntic glands (94 out of 620 glands) or for glands with complete metaplasia (31 out of 254 glands). Relatively strong p53 immunoreactivity and mRNA hybridization were also observed in the proliferative and apoptotic areas of gastric glands with incomplete intestinal metaplasia. These results indicate that incomplete intestinal metaplasia is associated with increased cell turnover and p53 overexpression, possibly in response to various noxious or DNA-damaging stimuli. KEY woms-stomach; cell proliferation; Ki67; apoptosis; p53 INTRODUCTION A multifocal gastritis associated with diminution in the number of glands and intestinal metaplasia is the most common type worldwide and is endemic in some parts of the world, including Japan.' In addition, an increased incidence of this type of chronic gastritis correlates with the geographical distribution of populations at increased risk for gastric carcinoma. 1,2 Although controversy exist^,^ several groups of investigators have proposed that this type of chronic atrophic gastritis, especially involving the presence of intestinal metaplasia, is a precursor of the intestinal type of adenocarcinoma of the stomach.'.* Little is known, however, about the biological features of this type of gastritis, and especially about the in situ tissue dynamics of the lesion. This study, therefore, initially examined cell proliferation by immunoreactivity for Ki67, one of the cell cycle-related antigens, and DNA fragmentation, which is associated with apoptosis or programmed cell death, by the 3'-OH nick end labelling technique, or TdTmediated dUTP-biotin nick end labelling (TUNEL) m e t h ~ dThe . ~ aim of the study was to elucidate the tissue topography of these markers in human gastric mucosa, including gastric mucosa with complete and/or incomplete intestinal metaplasia. p53 protein and mRNA expression were then examined in order to study the possible involvement of either mutated or overexpressed p53 in these lesions. Addressee for correspondence: Hironobu Sasano, MD, Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryoumachi, Aobaku, Sendai, Japan 980. CCC 0022-341 7/96/050039-04 01996 by John Wiley & Sons, Ltd. MATERIALS AND METHODS Tissue source and preparation Gastric mucosa was obtained from the gastric body in 12 patients (eight men and four women, mean age 61 years) undergoing total or partial gastrectomy for intestinal-type adenocarcinoma at Tohoku University Hospital and Sendai City Hospital, Sendai, Japan. Tissue strips 3-5mm in width were made from the non-pathological gastric body along the line of the incision. They were fixed in 8 per cent paraformaldehyde (pH 7-4) for 18 h at 4°C and paraffin-embedded following fixation. Sections 3pm thick were then cut and placed on glass slides. Immunohistochemistry Immunohistochemistry was performed by a modified strept avidin biotin method using a Histofine kit (Nichirei, Tokyo, Japan), as previously described by the author^.^ The primary antibodies employed were monoclonal antibodies MIB-1 for Ki67 (Immunotech SA, Marseille, France) and DO-7 for p53 (Novocastra Ltd., Newcastle upon Tyne, U.K.). The negative control of immunostaining was 0.01 M PBS and normal mouse IgG instead of primary antibodies; immunoreactivity was not observed in these tissue sections. TUNEL (3'-0H nick end labelling) DNA fragmentation associated with apoptosis was detected in situ by nick end labelling (transfer of biotinylated nucleotide to the 3'-OH end) according to the Received 3 April 1995 Accepted 4 December 1995 40 A. IMATANI ET AL. Fig. l-(A) Ki67 immunohistochemistry in the gastric mucosa without atrophy or inflammation. Nuclear immunoreactivity is present in the proliferative zone. x 40. (B) TUNEL or 3'-OH nick end labelling in the same field as (B). Reactive cells can be observed predominantly in the superficial foveolar epithelium (arrow-heads). x 40 Fig. 2--(A) Ki67 immunohistochemistry in the gastric mucosa with incomplete intestinal metaplasia. x 40. (B) TUNEL or 3'-OH nick end labelling in the same case as (A). TUNEL-reactive cells (arrow-heads) are not present in the areas occupied by Ki67-immunoreactive cells. x 40 method of Gavrieli et ~ 1with . ~some m~difications.~ For the positive control, tissue sections were treated with DNase I (Stratagene Co., La Jolla, CA, U.S.A.) in 0.7pglml potassium cacodylate buffer (pH 7.2) for 10 min prior to treatment with TdT. Tissue sections incubated with TdT buffer without DNase I were used as the negative control. We defined TUNEL-positive glands as those which were longitudinally sectioned from surface to base and which contained at least one TUNELpositive cell. We then obtained the TUNEL index, by examining the percentage of glands containing TUNELpositive cells among gastric glands, including 620 oxyntic glands, 108 glands with incomplete intestinal metaplasia, and 254 glands with complete intestinal metaplasia. An overall comparison was performed with a one-way analysis of variance (ANOVA) by the Kruskal-Wallis rank-sum test, and the Bonferroni method was applied to the significance of simultaneous multiple comparisons." RESULTS Histopathology We classified gastric glands from the gastric body into oxyntic glands and glands with intestinal metaplasia by histological examination. We further subclassified gastric oxyntic glands with intestinal metaplasia into incomplete and complete metaplasia, by the presence or absence of a brush border and Paneth cells.3 fnzmlcnohisrochemistry Both Ki67 and p53 immunoreactivity were observed in the nuclei of the cells in normal gastric mucosa and chronic atrophic gastritis with complete or incomplete intestinal metaplasia (Figs lA, 2A, 3, and 4A). In situ hybridization TUNEL DNA fragmentation in situ detected by the TUNEL method was confined to the nucleus (Figs I B and 2B). Pretreatment with DNase I caused intense staining of all nuclei in the preparation. Tissue sections incubated in the absence of TdT did not demonstrate nuclear staining. The in situ hybridization procedures for p53 mRNA used in this study, including the oligonucleotide probes employed, have been previously described by Sasano et a1.7 In situ hybridization p53 hybridization signals were observed as black silver dots on autoradiography (Fig. 4B). Sections 41 TISSUE TOPOGRAPHY IN HUMAN STOMACH Table I-The mucosa percentage of TUNEL glands in human gastric Oxyntic glands Incomplete metaplasia Complete metaplasia Fig. 3-Ki67 immunohistochemistry in the gastric rnucosa with complete intestinal metaplasia. Nuclear immunoreactivity is observed in the lower part of the glands. x 40 hybridized with a sense oligonucleotide did not yield accumulation of hybridization signal. Oxyntic glands Ki67 immunoreactivity was observed in all the oxyntic glands and was confined to the neck or the proliferative zone (Fig. 1A). TUNEL-positive cells were observed at the tip of the foveolar glands, among the superficial epithelial cells and exfoliated cells in the lumen, and very sporadically at the base of the glands, in 94 of 620 oxyntic glands observed (Fig. 1B). Very weak nuclear p53 immunoreactivity and accumulation of p53 mRNA hybridization signal were sporadically observed in the proliferative zone of oxyntic glands. Gastric glands with incomplete intestinal metapfasia Ki67 immunoreactivity was observed in almost all of the cells in the areas below the goblet cells and above the base of the gland (Fig. 2A). Ki67-immunoreactive cells in the glands with incomplete intestinal metaplasia appeared generally to be more numerous than those in oxyntic glands and glands with complete metaplasia. TUNEL-reactive cells were observed among the superficial cells and more frequently in the cells at the base of the glands (Fig. 2B). TUNEL-reactive cells included cells with apoptotic bodies. TUNEL-reactive glands No. of glands examined TUNELreactive glands Percentage 620 108 254 94 40 31 15.16 37.03* 12.2 (“4 *There are statistically significant differences between oxyntic glands and incomplete metaplasia (P<0.01) and between complete and incomplete metaplasia (P<0.05) by the Bonferroni multiple comparison test. comprised 40 out of 108 glands with incomplete intestinal metaplasia (Table I). The proportion of TUNEL reactive glands in incomplete intestinal metaplasia was significantly higher than for oxyntic glands and glands with complete intestinal metaplasia (Table I). Relatively strong p53 nuclear immunoreactivity was observed in 28 out of 96 glands with incomplete intestinal metaplasia (Fig. 4A). Some of the p53immunoreactive cells demonstrated mild to moderate degrees of dysplasia, but none was severely dysplastic. The gastric glands containing p53-positive cells were associated generally with marked inflammatory cell infiltration, including lymphocytes and neutrophils. Accumulation of p53 mRNA was also observed in these glandular cells (Fig. 4B). Serial tissue sections revealed that cells immunoreactive for p53 protein product were distributed between Ki67-immunoreactive and TUNELreactive cells, but cells with p53 mRNA accumulation were observed generally among the latter cell type. The cells with accumulation of p53 mRNA included both p53-positive and p53-negative cells. Gastric glands with complete intestinal metaplasia Ki67 immunoreactivity was confined to the lower part of the glands (Fig. 3). Ki67 immunoreactivity was observed in all the glands which had a complete longitudinal profile on histological section. TUNEL-reactive cells were observed predominantly in the superficial Fig. &(A) Imrnunohistochemistry of p53 in the gastric glands with incomplete intestinal metaplasia. x 45. (B) In situ hybridization of p53 mRNA in the gastric glands with incomplete intestinal metaplasia in the same field as (A). Accumulation of hybridization signal is demonstrated (arrows). Signals are observed in both p53-immunopositive and -negative cells 42 A. IMATANI ET AL. layer and among exfoliated cells in the lumen. TUNELreactive cells were observed in 31 out of 254 glands. Weak pS3 nuclear immunoreactivity and mRNA hybridization were observed sporadically in the lower part of the glands where Ki67-positive cells were located. DISCUSSION There has been controversy as to whether intestinal metaplasia, especially incomplete intestinal metaplasia, represents a precancerous lesion for gastric carcin ~ m a . ~On, ~ the other hand, intestinal metaplasia appears to represent a reaction to a more advanced degree of chronic gastritis and possibly an adaptation to long-standing damage to gastric mucosa.8 In our study, patterns of the intramucosal distribution of proliferating and apoptotic cells in gastric glands with complete intestinal metaplasia were not markedly different from those in the non-metaplastic oxyntic glands; Ki67positive cells were located beneath the area of goblet cells, corresponding to the proliferative zone, and TUNEL-reactive cells were present predominantly in the superficial epithelium. However, in oxyntic glands with incomplete metaplasia, cells with DNA fragmentation were present adjacent to the proliferating areas. In addition, the proportion of glands with cells positive for nick end labelling was significantly higher among glands with incomplete intestinal metaplasia than in complete intestinal metaplasia and oxyntic glands. The proliferative zones in the oxyntic glands with incomplete intestinal metaplasia were also much larger than those in non-metaplastic oxyntic glands and glands with complete intestinal metaplasia. These results all indicate that the oxyntic glands with incomplete intestinal metaplasia are associated with rapid cell turnover, with high proliferative and apoptotic activities. Abnormalities of p53 are considered to represent the most common molecular change in human cancer. Abnormalities have been reported not only in cancer, but also in precancerous gastric lesions, including gastric dysplasia.’,1° In our present study, relatively strong pS3 immunoreactivity and mRNA hybridization signals were observed only in oxyntic glands with incomplete intestinal metaplasia, especially in the areas of active cell proliferation and apoptosis. Wild-type p53 protein is involved in normal cellular proliferation and transcription of its mRNA increases by 10- to 20-fold late in the G1 phase.” In addition, wild-type p53 has also been considered to play a critical role in the process of apoptosis,I2 its gene product being required to induce apoptosis following exposure to various DNAdamaging agents; l 3 such agents, in turn, have been demonstrated to induce nuclear accumulation of wild-type p53 protein. l4 Apparent abnormalities in the development and differentiation of the stomach have not been reported in studies of pS3 null mice.IS The low background level of apoptosis in the intestine of these mice appeared to be p53-independent, but pS3 was required for the induction of apoptosis following DNA damage. l 6 Increased wild-type p53 expression may, therefore, also represent a cellular response to DNA damage. Marked inflammatory cell infiltration of the gastric mucosa has been considered to be one of the sources of nitric oxide-related mutagens or DNA-damaging agents in gastric carcinogenesis. Infiltration of inflammatory cells, including lymphocytes and neutrophils, was observed around the glands which contained p53reactive cells. p53 expression observed in human oxyntic glands with incomplete intestinal metaplasia is therefore considered to reflect adaptation of the gastric mucosa to various noxious stimuli. Further studies, including analysis of the DNA damage and of pS3 mutations in these cells, will be necessary to clarify the relationship between noxious stimuli to gastric mucosa, p53 expression, and increased cell turnover. It remains a possibility that the overexpressed p53 in gastric glands with incomplete intestinal metaplasia examined in this study could represent a mutant form of the protein. Even if this were so, various DNA-damaging agents which result in increased cell turnover, possibly associated with elevated wild-type p53, can still be considered to play an important role in the development of genetic instability, including p53 mutations. REFERENCES I . Hirota T, Ming S-C. Early gastric carcinoma. In: Ming S-C, Goldman H, eds. Pathology ofthe Gastrointestinal Tract. Philadelphia: W. B. Saunders. 1992; 570-583. 2. Jaskiewicz K, Louwrens HD. Chronic atrophic gastritis in a population at risk for gastric carcinoma. Anticancer Re.y 1991; 11: 835-840. 3. Kato Y, Kitagawa T, Yanagisawa A, er al. Site-dependent development of complete and incomplete intestinal metaplasia types in the human stomach. Jpn J Cancer Res 1992; 8 3 178--183. 4. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in sifuvia specific labelling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493-501. 5. Sasano H, lmatani A, Shizawa S, Suzuki T, Nagura H. Cell proliferation and apoptosis in normal pathologic human adrenal. Mod Puthol 1995; 8: 11-17, 6. Wallenstein S, Zucker CI, Ferris JL. Some statistical methods useful in circulation research. Circ Res 1980: 47: 1-26. I. Sasano H, Goukon Y, Nishihira T, Nagura H. In sifu hybridization of p53 tumor suppressor gcne in human esophageal carcinoma. A m J Putkol 1992; 141: 545-550. 8. Tsutsumi Y, Nagura H, Watanabe K. Immune aspects of intestinal metaplasia of the stomach: an Immunohistochemical study. Virchoivs Arch A [Pathol AnafJ 1984; 403: 345-359. 9. Sasano H, Date F, Imitani A, Asaki S, Nagura H. Double immunostaining for c-erb-B2 and p53 in human stomach cancer cells. Hum Puthol 1993; 2 4 584-589. 10. Shiao Y-H, Rugge M, Correa P, Lehmann HP, Scheer WD. p53 alterations in gastric precancerous lesions. Am J Pathol 1994: 144: 511-517. 11. Reich NC, Levine AJ. Growth regulation of a cellular tumor antigen, p53. in nontransformed cells. Nature 1984: 308 199 201. 12. Shaw P, Bovey R, Tardy S, Sahli R, Sardat B, Costa J. Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Nuti Acud Sci USA 1992; 8 9 44954499. 13. Caelles C, Helmberg A, Karin M. p53 dependent apoptosis in the absence of transcriptional activation of p53 target genes. Nutuw 1994 3 7 0 220-223. 14. Fritsche M, Haessler C, Brandner G. Induction of nuclear accumulation of the tumor suppressor protein p53 by DNA damaging agents. Oncogene 1993; 8: 307-318. 15. Donehower LA, Harvey M, Slagle BL, et ul. Mice deficient for p53 are developmentally normal but susceptible to spontaneous turnours. Nufure 1992; 3 5 6 215-221. 16. Clarke AR, Gledhill S, Hooper ML, Bird CC, Wyllie AH. p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation. Oncogene 1994; 9 1767~1773. 17. Correa P. Human gastric carcinogenesis: a multistep and multifactorial process. First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cuncer Res 1992: 52: 6735-6740.