Interleukin-1beta expression in human gastric carcinoma with Epstein-Barr virus infection

Abstract

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1beta (IL-1beta) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1beta mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1beta increased the cell growth of a GC cell line, TMK1. Thus, IL-1beta may act as an autocrine growth factor in EBVaGC.

FIG. 1.

Northern blot analysis of the GCs associated or unassociated with EBV. Total RNA extracted from the GC strains in SCID or nude mice (a) and from GC cell lines (b) was subjected to Northern blot analysis using IL-1β and glyceraldehyde 3-phosphate dehydrogenase cDNA probes. Expression of IL-1β was observed only in the KT tumor, a transplantable strain of EBVaGC; the level of expression was high. On the other hand, there was no expression in other strains or in seven GC cell lines, all of which are EBV negative. Normal gastric mucosa does not show any messaging of IL-1β.

FIG. 2.

Western blot analysis of the GCs associated or unassociated with EBV. The lysate of the GC strains in SCID mice were subjected to Western blot analysis using anti-hIL-1β antibody. There are three forms of proIL-1β protein in the KT tumor cells, including 31- and 28-kDa proteins, which are similarly observed in the cell lysate and the supernatant of LPS-stimulated human PBM. There are additional extra bands at a lower molecular weight and a mature form of IL-1β at 17 kDa in the cell lysate and the supernatant of LPS-stimulated PBM. There is no band in the EBV-negative GC strains (KN-91 and JS-1), except the bands at the higher molecular weight, which correspond to the immunoglobulin heavy chain.

FIG. 3.

IL-1β ISH of the GCs associated or unassociated with EBV. IL-1β ISH was applied to the formalin-fixed and paraffin-embedded tissue sections of the tumor strains and the surgically resected carcinomas. With an IL-1β antisense probe, a positive signal of IL-1β mRNA was observed in the cytoplasm of the KT tumor cells (a), whereas no signal was observed in KN-91 (c). There was no signal in the KT tumor cells when the ISH probe was replaced with the sense probe (b). Similarly, a positive signal was specifically observed in the cytoplasm of the surgically resected carcinoma, which is associated with EBV (d). On the other hand, there was no signal in the surgically resected carcinoma, which was EBV negative (e). The normal gastric mucosa was negative for IL-1β (f). Magnification, ×66.