Oncofetal expression of the human intestinal mucin glycoprotein antigens in gastrointestinal epithelium defined by monoclonal antibodies

Abstract

A mucin preparation from a colonic adenocarcinoma was used to prepare monoclonal antibodies (MAbs) that reacted specifically either with normal adult small-intestine mucin antigen(s) (SIMA), or normal adult large-intestine mucin antigen(s) (LIMA). Both SIMA and LIMA show a unique oncofetal pattern of expression. Thus SIMA was expressed in early fetal stomach, large and small intestines but thereafter only in the normal small intestine. SIMA expression was detected immunohistochemically in cancers of the colorectum (82/112) and stomach (48/86). LIMA was detected in the stomach of the early fetus but thereafter only in the normal large intestine. LIMA expression was detected in 61/86 cancers of the stomach. Moreover, both SIMA and LIMA were expressed inappropriately in mucosa adjacent to tumors, indicative of the detection of possible pre-malignant epithelium. We used a sandwich ELISA and biochemical procedures to show that the SIMA and LIMA molecules were large extensively glycosylated multi-unit mucin glycoproteins that differed markedly from each other. SIMA, whether extracted from normal small-intestine or colonic cancers, had a molecular weight above 1.000 kDa, a mean buoyant density 1.33 g/ml and s value of 4.8. LIMA had a molecular weight above 10.000 kDa, a mean buoyant density 1.45 g/ml and an s value 9.5. The SIMA and LIMA epitopes were judged to be carbohydrate in nature by reason of their resistance to harsh physical chemical treatments or protease digestion, and sensitivity to periodate oxidation, neuraminidase or beta elimination. Only the SIMA epitope was sensitive to neuraminidase. In conclusion, MAbs to carbohydrate-dependent epitopes on SIMA and LIMA identify the oncofetal pattern of expression of these distinct intestinal mucin glycoproteins in colonic and gastric carcinoma. These MAbs will be useful in further studies of the significance of oncofetal mucin expression during carcinogenesis.

The adenoma-carcinoma sequence in the colorectum – early appearance of a hierarchy of small intestinal mucin antigen (SIMA) epitopes and correlation with malignant potential

“…In terms of epitope structure, previous studies have shown that the four anti-SIMA MAbs recognise distinct neuraminidase-sensitive carbohydrate epitopes, two which are distinct from sialosyl-Tn and 2 which partially cross-react (Hertzog et al, 1991a;Pilbrow et al, 1992a). Additive ELISAs have suggested that MAb 4D3 recognises a core carbohydrate structure, and the other three MAbs, more peripheral epitopes (Pilbrow et al, 1992a).…”

“…Anti-SIMA MAb 4D3 and anti-LIMA MAb 2C3 were prepared by immunisation of mice with a mucin extract from a colorectal cancer (Hertzog et al, 1991a and b), and anti-SIMA MAb 3D4, 5C5 and 6C5 by immunisation with a mucin extract of post-mortem tissue from normal adult small intestine (Pilbrow et al, 1992a in three areas where present: neoplastic adenoma tissue, base of adenoma and immediately adjacent perineoplastic mucosa. A previously described scoring method was used: a score of 0-3 was given for staining intensity and similarly, for distribution [0 (negative, 1 (<25% of crypt positive), 2 (25-75%) or 3 (75-100%)].…”

Aberrant expression of intestinal mucin antigens associated with colorectal carcinoma defined by a panel of monoclonal antibodies

“…Mucin extraction andfractionation Mucins were extracted from specimens of resected cancers or normal gastrointestinal tract using 4 M guanidine hydrochloride in phosphate buffered saline (PBS), containing the protease inhibitors: 10 mm N-ethyl maleimide, 1 mM benzamidine hydrochloride, and 25 mM EDTA (Hertzog et al, 1991). Homogenates were centrifuged at 20,000 x g for 20 min, and the supernatants subjected to three cycles of CsCl density gradient centrifugation for purification of mucin fractions.…”

“…Mucin glycoproteins have also been demonstrated to be differentiation-associated or oncofetal antigens in the gastrointestinal tract (Gold & Miller, 1978;Ma et al, 1980;Bara et al, 1980;Hertzog et al, 1991). Similar abnormalities of mucin glycoprotein expression have also been identified in other epithelial tissues, including the breast (Griffiths et al, 1987) and ovary (Bhattacharya et al, 1982).…”

“…We have described the production of two MAb to intestinal cancer mucins and the preliminary chemical characterisation of the mucin antigens (Hertzog et al, 1991). One MAb designated anti-LIMA 2C3 reacted in the normal adult specifically with mucin of the large intestine, yet showed oncofoetal reactivity with the gastric epithelium.…”