Immunohistochemical identification of type I procollagen in tumour cells of scirrhous adenocarcinoma of the stomach

Abstract

Summary Human gastric carcinomas were tested for their immunohistochemical reactivity with anti-type I procollagen antiserum. In all specimens of scirrhous carcinomas, staining of the tumour cells was strongly positive, while in medullary carcinomas staining of the tumour cells was generally poor. These results suggest that the tumour cells in scirrhous carcinomas produce collagen in their stroma.Scirrhous carcinoma of the stomach is characterized by extensive fibrosis with sparse tumour cell infiltration in desmoplastic stroma, and clinically by the worst prognosis of any type of gastric cancer. The fibrous stroma is composed mainly of type I and type III collagens (Kohda et al., 1984; Nagi et al., 1985). Both of these are found in most normal connective tissues, but type I is more prevalent. Generally, mesenchymal cells are responsible for the synthesis and deposition of collagen in connective tissues. In an attempt to determine which cell types are responsible for the synthesis of collagen in tissue of scirrhous carcinoma, the immunohistochemical localization of procollagen type I, a precursor form of collagen type I, was investigated. Materials and methodsPurification of type I procollagen Normal human lung fibroblasts (IMR90) (Uitto et al., 1976) in the 20-30th generation with 3rd-5th passage, were grown at 37°C in culture flasks, (T 75, T 150; Corning) or roller bottles (area 490 cm2, Corning), in Dulbecco's modified Eagle medium (DMEM, Grand Island Biological), supplemented with 10% foetal calf serum and 50pgml-1 Geramycin (Schering). When the cells reached the early confluence they were washed 3 times with PBS and cultured another 24 h in serum-free DMEM containing ascorbate (75 pg ml -1) and L(2,3)3H-proline (20-40 Ci mmol -1, New England Nuclear) 4 pCi ml -1. The serum-free medium (-. 1 1) was then collected and cooled on ice, and to it were added various protease inhibitors (Uitto et al., 1976) to give the following final concentrations: 25 mm disodium ethylene diaminetetraacetate (EDTA, Baker); 10 mm N-ethylmaleimide (NEM, Sigma); 1.0 mm phenylmethylsulfonyl-fluoride (PMSF, Sigma) and 1 mm p-aminobenzamide-HCI (Sigma). Proteins in the medium were precipitated by adding 176mgml-1 ammonium sulphate (Baker) with stirring overnight at 4°C. The precipitate was centrifuged at 30,000g for 30min to remove supernatant. The pellet was then dissolved in 20 ml of ice-cold of 0.1 M Tris HCI buffer containing 0.4 M NaCl, pH7.5, and was centrifuged at 30,000g for 30min. The supernatant was dialyzed against 0.1 M Tris HCI buffer, pH7.8, containing 2M urea, 2.5mm EDTA (starting buffer A) at 4°C for overnight, prior to the application to a DEAE Sephacel column (1.6 x 1Ocm) which had been equilibrated with the starting buffer A.Chromatography was carried out with a linear salt gradient prepared with 150ml of the starting buffer A and equal amounts of the same buffer containing 0.2M NaCl. Small portions (200 ul) of each fraction (2.5 ml) from the column were mixed with Hydrofluor (National Diagnostics) and counted in a...

Overexpression of TGF-β by infiltrated granulocytes correlates with the expression of collagen mRNA in pancreatic cancer

“…The origin of type I and type III collagens mRNA has been assumed to be cancer stroma (Gress et al, 1995), a finding supported by our studies in pancreatic cancer cell lines. Indeed, cancer cells are capable of synthesis and production of extracellular matrix proteins (Niitsu et al, 1988;Yoshida et al, 1989;Lohr et al, 1994), although the expression of collagen mRNAs in the pancreatic cancer cell lines was somewhat lower in comparison to that of fibroblast cell lines ( Figure 3A). Furthermore, the expression of collagens from pancreatic cancer cells does not increase in vivo, that is, subcutaneous xenotransplantation in immunodeficient mice (unpublished data).…”

Breast tumour cell-induced down-regulation of type I collagen mRNA in fibroblasts

“…There has been considerable controversy as to whether the tumour cells themselves (Al-Adanani et al, 1975;Niitsu et al, 1988), or the fibroblasts in the vicinity of the tumour cells, are stimulated to produce type I collagen (Barsky et al, 1982;Ohtani et al, 1992;Hewitt et al, 1993). Early studies claimed that the excess production of type I collagen is not due to increased collagen synthesis by the stromal fibroblasts, but that it is produced by the breast cancer cells themselves, which suggested that this response was an inappropriate rather than a deliberate host response (Al-Adanani et al, 1975).…”

Transforming growth factor β 1 secreted from scirrhous gastric cancer cells is associated with excess collagen deposition in the tissue

“…The mechanisms of interstitial proliferation in scirrhous gastric cancer still remain obscure in many respects. In a previous immunohistological study we showed that scirrhous gastric cancer cells themselves produce collagen in large amounts (Niitsu et al, 1988a). That finding does not necessarily preclude the possibility that, while producing collagen themselves, scirrhous gastric cancer cells release some humoral factors that stimulate fibroblasts in the interstitial space to synthesise collagen, and thus promote fibrosis.…”