The leukocyte antigen CD43 is expressed in different cell lines of nonhematopoietic origin.

CD43 is an abundant transmembrane sialoglycoprotein in leukocyte-type cell lines, but it has also been suggested to be present in colon adenomas and colon carcinomas. We have now shown that CD43 is expressed in a variety of cell lines of different origins (CaSKI, A549, 293, MTSV1-7, MCF7, HT-1080, Jurkat, K562, COLO 205, HT-29, Caco-2, DLD-1 and SW480). The level of expression of CD43 mRNA was analyzed by reverse transcriptase-polymerase chain reaction and that of the protein by immunoprecipitation and Western blot, flow cytometry and confocal microscopy using two monoclonal anti-CD43 antibodies (L10 and 4D2). As all cell lines expressed CD43, it is suggested that CD43 has a more fundamental function than previously believed and thus cannot be considered only as a specific leukocyte marker.

Detection of CD43 (leukosialin) in colon adenoma and adenocarcinoma by novel monoclonal antibodies against its intracellular domain.

CD43 is a leukocyte-associated sialoglycoprotein which is also expressed in human colon adenoma and carcinoma. To obtain monoclonal antibodies (MAbs) that would react with CD43 in a glycosylation-independent way, antibodies were raised against a peptide corresponding to a portion of the CD43 cytoplasmic domain. Hybridomas were screened on paraffin sections from CD43-positive colon tumours. The reactivity of the antibodies with CD43 was verified by Western blot analysis of lysate of CHO cells transfected with human CD43 cDNA and by immunoprecipitation of lysates from CD43+ cell lines. Epitope mapping of antibodies was done using overlapping heptameric peptides. A detailed characterisation of one of the novel antibodies (CD43-3A1) is presented. This antibody reacts with the CD43 protein regardless of its glycosylation in Western blot analysis, immunoprecipitation and immuno-histochemistry of paraffin sections. Immuno-histochemical analysis of paraffin sections from colon adenoma and carcinoma tissues as well as colon cancer cell lines revealed that CD43 was predominantly localised intracellularly, in contrast to leukocyte-type cells. The MAb reacted more efficiently with paraffin-embedded colon adenoma and carcinoma cells than previously characterised CD43-specific antibodies. This should facilitate the evaluation of a potential role of CD43 during cancer development.

Deglycosylation by gaseous hydrogen fluoride of mucus glycoproteins immobilized on nylon membranes and in microtiter wells.

Strongly reacting antibodies specific for defined mucin gene products are often directed against the mucin protein backbone of the heavily glycosylated serine/threonine rich regions. A prerequisite for the use of such antibodies is often the complete removal of the oligosaccharides from the protein. This paper describes an efficient one-step deglycosylation method using gaseous hydrogen fluoride on nylon blotting membranes and microtiter wells.

Reactivity of antibodies with highly glycosylated MUC1 mucins from colon carcinoma cells and bile.

The 55 antibodies submitted to the ISOBM TD-4 Workshop were analysed for their reactivity with core proteins of the heavily glycosylated MUC1 mucins from the colon carcinoma cell line COLO205 and bile. Both these mucins (designated as H-CanAg and SBG2) are highly glycosylated having 15 and 50 sugar residues per oligosaccharide, respectively. Only a few of the antibodies (129, 139, 153 and 162) reacted with both SBG2 and H-CanAg, and not with a control mucin (L-CanAg) having a similar glycosylation as H-CanAg. These antibodies were tested for their ability to catch soluble mucins, and the antibody 162 was found to be good also in this type of assay. The antibodies selected here should be useful for the detection of high glycosylated forms of the MUC1 mucin in tissues and serum.

Colon adenoma and cancer cells aberrantly express the leukocyte-associated sialoglycoprotein CD43.

CD43 (leukosialin) has hitherto been considered as an exclusive leukocyte marker, but now we report the expression of CD43 in the epithelial cells of all studied colorectal adenomas (21/21) and in about 50% (18/34) of adenocarcinomas as analyzed both at the mRNA and protein levels. Direct evidence showing the causal role of CD43 in colon tumorigenesis is lacking, but its involvement in leukocyte activation and impairment of apoptotic response suggests a role for CD43 in colon cancer development.

A MUC1 mucin secreted from a colon carcinoma cell line inhibits target cell lysis by natural killer cells.

The effect of two secreted mucin-type glycoproteins on natural killer (NK) cell cytotoxicity against K562 target cells has been studied. These mucins carry the carcinoma-associated sialyl-Lewis a carbohydrate epitopes and were purified from the human colon adenocarcinoma cell line COLO 205 secretions, where they lack their cytoplasmic parts. The larger one has an apoprotein encoded by the MUC1 gene, and the smaller one has CD43 (leukosialin) as the core protein. The purified MUC1 mucin could inhibit the target cell lysis by NK cells in a dose-response-dependent way, whereas other mucin domains of similar size showed no inhibition. The second mucin, CD43, inhibited lysis by NK cells, although less than the larger one. The MUC1 mucin bound to the enriched natural killer cell preparations in a partial Ca2+-dependent way as well. This mucin also bound to the target cells. The K562 cells, normally expressing high amount of CD43, showed an increased resistance to lysis by NK cells when transfected with MUC1 cDNA compared with nontransfected cells. One can speculate that mucins secreted or expressed in the plasma membrane of cancer cells could interfere with NK cell-mediated lysis.

Distinct sub-populations of carcinoma-associated MUC1 mucins as detected by the monoclonal antibody 9H8 and antibodies against the sialyl-Lewis a and sialyl-Lewis x epitopes in the circulation of breast-cancer patients.

The cancer-associated epitope defined by the monoclonal antibody (MAb) 9H8 was shown to be closely related to the T antigen (Thomsen-Friedenreich antigen) by its sensitivity to 0-glycanase treatment of a mucin glycopeptide known to express this epitope. The reactivity with this glycopeptide increased upon neuraminiclase treatment, and among several MAbs tested for ability to block binding of the 9H8 antibody, the one specific for the T antigen was the most efficient. Out of 41 serum samples from breast-cancer patients, 11 showed elevated levels of the 9H8 epitope, and several sera also showed elevated levels of the cancer-associated carbohydrate epitopes sialyl-Lewis a and sialyl-Lewis x. By the use of antibodies specific for the MUC1 apoprotein (Ma552 and HMFG-2) it could be shown that these epitopes were attached to the MUC1 apoprotein in at least 4 of the cases. By combining antibodies specific to 9H8, sialyl-Lewis a and sialyl-Lewis x in catcher and tracer positions in several types of immunofluorometric assays, it was shown that the 9H8 epitope was rarely co-expressed with sialyl-Lewis a or sialyl-Lewis x epitopes an the same molecule, though all were expressed on MUC1 mucins. In fact, they can be considered as mutually exclusive epitopes, suggesting that these sera contained different populations of MUC1 mucins distinguishable by different sets of oligosaccharides. The existence of mutually exclusive carbohydrate epitopes on different MUC1 mucins in one and the same patient should be taken into account when designing immunoassays exploiting MUC1-reactive antibodies.

No immunoglobulins in cataractous lenses.

All 47 human senile cataractous lenses studied by a direct immunohistochemical method for the presence of immunoglobulins yielded negative results. Consequently, cataract cannot be an autoimmune disease as has been suggested by some authors.

Ovarian carcinomas express a sperm acrosomal antigen, defined by monoclonal antibody 9H8.

A new monoclonal antibody (MAb 9H8, IgM class) reactive with human ovarian carcinoma has been raised after immunizing C57BL/6 mice with bovine sperm. Immunohistological studies indicated that 20/21 serous ovarian adenocarcinomas expressed 9H8-defined antigen but it was absent in benign ovarian tumors (0/11). 1/11 of breast carcinomas and 5/5 of rectal carcinomas expressed this antigen, although to a considerably lesser degree. Tumors of lung, skin, brain and mesothelium were negative. The antigen was also expressed in embryonic skin, in renal collecting tubule cells and in saliva. In bovine, human and mouse sperm the antigen is confined to the acrosomal region. The molecular weight of this antigen was determined by Western blot analysis and gel filtration. In SDS-PAGE the antigen ran as a broad band barely entering the 7% gel, indicating an apparent molecular weight > 300 kDa. In the absence of detergents and reducing agents this glycoprotein forms larger complexes (> 1,500 kDa) as determined by gel filtration on Sephacryl S300. The epitope contains carbohydrate structures recognized by lectin PNA (peanut agglutinin).