The materials used in this study were biopsy specimens taken from 9 patients suffering from advanced gastric cancer (Table 1). The specimens were carefully taken from the nondiseased portions of the pyloric mucosa, and 3 biopsy specimens were obtained from each individual. ³H-thymidine autoradiography was performed using the in vitro method described by Hattori and Fujita⁸⁾. Briefly, the specimens were cut into 1 mm pieces and washed in sterile saline solution. They were then immersed in MEM (modified Eagle’s medium) supplemented with 10% fetal calf serum containing 20 μCi of ³H-thymidine (methyl-³H-thymidine, Amersham, England, specific activity, 25 μCi/mmol) per 1 ml and incubated for 60 min at 37°C. After fixation with 10% formalin, they were dehydrated in a graded alcohol series and embedded in paraffin. The embedded tissues were carefully oriented and sectioned serially and longitudinally to the long axis of the glandular tubule. Sectons of about 2–3 μm in thickness were mounted on glass slides treated with tissue adhesives, dipped in SAKURA NR-M₂ nuclear emulsion, and developed in FD-111 after 6 weeks’ exposure. They were stained with hematoxylin and eosin.

The in vitro labeling of the tissues with ³H-thymidine did not provide constant results. Frequently, only the margins of the specimens were labeled with ³H-thymidine. Sometimes, however, we were successful in labeling a whole layer of the specimens in which a zone of the labeled epithelial cells was found. In this study, we used these specimens in order to interpret cell proliferation kinetics in the pyloric and intestinalized mucosa.

The materials used in the BrdUrd immunohistochemical study were resected stomachs from 4 patients suffering from advanced gastric cancer (Table 1). Informed consent for administration of BrdUrd was obtained from each patient. The method used in this study is described by Hoshino et al⁹⁾. Briefly, the patients were given a 60-min i.v. infusion of BrdUrd, 200 mg/m², at the time of surgery but before gastric resection. Non-cancerous and non-ulcerated regions of the antral mucosa were cut into rectangular pieces and embedded in paraffin. They were cut serially and mounted on glass slides treated with tissue adhesives. Tissue sections were deparaffinized for 15 min in xylene, rinsed 3 times (5 min each time) in 0.01 M phosphated buffered solution (PBS), hydrated for 30 min in 1N HCl, and incubated for 20 min at 37°C in PBS containing 0.05% proteinase type VII (Sigma, U.S.A). The sections were then stained by avidin biotin peroxidase method using Vectastain ABC kit: they were incubated for 16 hr at room temperature with 1 : 60 dilution of purified anti-BrdUrd monoclonal antibodies (Bection Dickinson, U.S.A) in bovine serum albumin, for 30 min at room temperature with biotinilated antimouse IgG (horse); and for 30 min at room temperature with ABC. The tissue sections were rinsed with PBS at each stage. Finally, the slides were reacted with 0.03% 3,3′-diaminobenzidine (DAB) and PBS containing 1% H₂O₂. Light counter stains were performed by hematoxylin.

Black-silver grains in the nuclei (Fig. 1) and dark brown colored nuclei (Fig. 2) were considered as labeled cells in autoradiography and immunohistochemistry, respectively, and a zone of labeld cells was considered as a proliferative zone of the mucosa^8,11). The site of the labeled cells was indicated by the number of counted nuclei from the surface of the gland. The labeling index (LI) was defined as the percentage of the labeled cells counted in a total number of over 1,000 nuclei in the generative cell zone between the level of the uppermost labeled cells and that of the lowermost cells in each specimen⁸⁾.

Autoradiographs of the pyloric mucosa not involved with metaplastic change showed the labeled cells to be confined to the region of the isthums between the base of the pits and the upper part of the pyloric gland (the middle one-third of the mucosa) (Fig. 4). The cell order, the number of counted nuclei from the surface of the gland of the labeled cells, was distributed between 19 and 72 (Table 2). The LI varied from 15.3 to 26.6%, and the total LI was 19.4% (Table 3).

In the autoradiographs of the intestinalized mucosa, a zone of the labeled epithelial cells was found in the lower third of the mucosa (Fig. 5). The cell order of the labeled cells of metaplastic glands was distributed between 42 and 116 (Table 2). The LI varied from 20.6 to 29.4%, and the total LI was 25.2% (Table 3).

The pattern of distribution of labeled cells revealed by in vivo BrdUrd immunohistochemistry (Fig. 6a, 6b, and 7a, 7b) was very similar to that revealed by in vitro ³H-thymidine autoradiography.

The cell order of the labeled cells in the pyloric mucosa varied from 13 to 84 and in the intestinalized mucosa between 50 and 135 (Table 2). The LI varied from 12.5 to 22.2% in the pyloric mucosa and between 21.8 and 27.2% in the intestinalized mucosa. The total LI of the pyloric mucosa was 18.0% and that of the intestinalized mucosa 24.2% (Table 3).