Interleukin 1 and interleukin 1beta converting enzyme (caspase 1) expression in the human colonic epithelial barrier. Caspase 1 downregulation in colon cancer

Abstract

Background: Interleukin (IL) 1beta converting enzyme (now known as caspase 1) is able to process pro-IL-1beta into its active form and is involved in proapoptotic signalling.

Aims: To characterise IL-1 and caspase 1 expression in human colonic epithelial cells.

Methods: Intracellular IL-1 content (IL-1alpha and IL-1beta) was measured by ELISA in freshly isolated human normal colonocytes. Caspase 1 expression was determined both at the mRNA level using in situ hybridisation and reverse transcription polymerase chain reaction, and at the protein level by immunoblotting experiments using antibodies specific for the proform of caspase 1 and for its cleavage forms.

Results: Low amounts of IL-1beta were found in nearly all preparations (92%), and IL-1alpha was detected in only 45% of human colonocyte preparations. The normal colonic epithelium strongly expressed caspase 1, both at the mRNA level and at the protein level in its latent form. In contrast, caspase 1 was not expressed in colon cancer (primary colonic adenocarcinomas and cancer cell lines).

Conclusions: The demonstration that the human colonic epithelial barrier is able to express caspase 1 and its substrate IL-1beta reinforces the concept that, under certain conditions, the epithelium could trigger an inflammatory reaction. In addition, the finding that caspase 1 was downregulated in colonic adenocarcinomas supports the concept that disrupted apoptosis pathways may be involved in tumour formation and/or may confer resistance to treatment.

Figure 1

Intracellular IL-1 concentrations in isolated human colonocytes (n=49). Means are shown by horizontal bars and standard errors by vertical bars.

Figure 2

Detection of caspase 1 by in situ hybridisation in the normal human colonic mucosa. Colonocytes strongly expressed caspase 1 mRNA (A,B). Control section hybridised with a caspase 1 sense probe revealed no specific signal (C). Original magnification × 350 (A), × 700 (B,C).

Figure 3

RT-PCR analysis of caspase 1 expression in human normal isolated colonocytes. Lane 1 shows DNA weight marker ϕX174-HaeIII. Lanes 2, 4, 6 show caspase 1 amplification products from three different colonocyte preparations; lanes 3, 5, 7 show β actin amplification products from the same preparations.

Figure 4

Caspase 1 expression in human normal isolated colonocytes at the protein level. Eight different cell lysates were subjected to immunoblotting experiments. They were run on SDS-PAGE gels, blotted, and probed with either an anticaspase 1 p45 antibody (left) or with an anticaspase 1 p10 antibody (right). Numbers between the two panels indicate molecular size of standards in kDa.

Figure 5

Downregulation of caspase 1 expression in colon cancer. (A) In situ hybridisation performed on cryostat sections of a primary colon adenocarcinoma shows that tumour cells (T) do not express caspase 1 mRNA, whereas normal crypts (N) in the vicinity of the tumour are labelled with the antisense caspase 1 probe (original magnification × 350). (B) Immunoblot analysis of caspase 1 protein expression using an anti-p45 antibody in several digestive cancer cell lines: the gastric cell line HGT-1 and the colonic cell lines Colo320DM, SW480, HT-29 and its clonal derivatives Cl.19A and Cl.16E. The control lane is a lysate from an ulcerative colitis mucosa, showing strong expression of the 45 kDa proform. Numbers on the left indicate molecular size of standards in kDa.