Characterization of human gastric adenocarcinoma cell lines established from peritoneal ascites

Abstract

The three cell lines, designated as gastric cancer (GC)1401, GC1415 and GC1436 were derived from peritoneal effusions from patients with gastric adenocarcinoma. Cell lines were established in tissue culture and in immunodeficient, non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. These cell lines were grown as an adherent monolayer with doubling time ranging between 25 h (GC1436 cell line) and 30-34 h (GC1401 and GC1415, respectively). All cells showed morphological features of epithelial-like cells, forming sheets of polygonal cells. Chromosomal analysis showed that the modal numbers ranged from 52 (GC1401), 51-56 (GC1415) and 106 (GC1436). High heterogeneity, resulting from several structural and numerical chromosomal abnormalities were evident in all cell lines. The surface marker expression suggested a tumor origin of the cells, and indicated the intestinal phenotype of a GC (CD10⁺, MUC1). All three cell lines were tumorigenic but not metastatic, in vivo, in NOD/SCID mice. The lack of metastatic potential was suggested by the lack of aldehyde dehydrogenase 1A1 activity. In conclusion, these newly established GC cell lines widen the feasibility of the functional studies on biology of GC as well as drug testing for potential therapeutic purposes.

Keywords: ALDH; Her-2/neu; gastric adenocarcinoma.

Figure 1.

Growth curves of GC1401, GC1415 and GC1436 cell lines. Cells were cultured in duplicates and counted every 24 h. The differences in doubling time were not statistically significant.

Figure 2.

Phase-contrast photomicrographs of monolayers of (A) GC1401, (B) GC1415 and (C) GC1436 cell lines. Original magnification ×400.

Figure 3.

Expression of EMMPRIN, panCEA and MAGE-A1 in gastric cell line detected by Western blotting. 1-GC1401, 2-GC1415 and 3-GC1436. Lane 1, GC1401; lane 2, GC1415; and lane 3, GC1436.

Figure 4.

Aldehyde dehydrogenase (ALDH) protein activity and expression in gastric cancer (GC) cell lines. (A) Western blot analysis was used to compare the expression of different ALDH isozymes GC1401 (lane 1), GC1415 (lane 2) and GC1436 (lane 3) GC cell lines. GAPDH served as loading control. (B) ALDH activity in GC cell lines. Flow cytometric graphs show the fluorescence intensity of reacted ALDH substrate in the absence and presence of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. Gated regions indicated ALDH⁺ cells.

Figure 5.

Expression of mRNA for tumor markers: (A) Her-2/neu and (B) MAGE-1 in GC1401 (1), GC1415 (2), GC1436 (3) tumor cells. Negative control is out of scale. One representative experiment of 3 performed is presented.

Figure 6.

Tumor growth of GC1401, GC1415 and GC1436 cells in NOD/SCID mice. Tumor volume was calculated every 3 days and presented in time relation. One experiment of two performed is shown. The observed differences were not statistically significant. NOD/SCID, non-obese diabetic/severe combined immunodeficiency.

Figure 7.

Subcutaneous injection of GC cells into NOD/SCID mice led to primary tumor development at the site of injection. Histological analysis of section stained with hematoxylin and eosin of tumor established in NOD/SCID is presented (A) GC1401, (B) GC1415 and (C) GC1436. Magnification ×200. NOD/SCID, non-obese diabetic/severe combined immunodeficiency.