CA125 is an ovarian cancer antigen that is the basis for a widely used serum assay for the monitoring of patients with ovarian cancer; however, detailed information on its biochemical and molecular nature is lacking. We now report the isolation of a long, but partial, cDNA that corresponds to the CA125 antigen. A rabbit polyclonal antibody produced to purified CA125 antigen was used to screen a ZAP cDNA library from OVCAR-3 cells in Escherichia coli. The longest insert from the 54 positive isolated clones had a 5797-base pair sequence containing a stop codon and a poly(A) sequence but no clear 5 initiation sequence. The deduced amino acid sequence has many of the attributes of a mucin molecule and was designated CA125/MUC16 (gene MUC16). These features include a high serine, threonine, and proline content in an N-terminal region of nine partially conserved tandem repeats (156 amino acids each) and a C-terminal region non-tandem repeat sequence containing a possible transmembrane region and a potential tyrosine phosphorylation site. Northern blotting showed that the level of MUC16 mRNA correlated with the expression of CA125 in a panel of cell lines. The molecular cloning of the CA125 antigen will lead to a better understanding of its role in ovarian cancer.

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation, we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [ 3 H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, and HB-2) derived from cells cultured from human milk, with three breast cancer cell lines (MCF-7, BT-20, and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1, and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern, with NeuAc␣2-3Gal␤1-3GalNAc-ol, Gal␤1-3GalNAc-ol, and GalNAc-ol predominating; in contrast, MUC-1 from MTSV1-7 had more complex structures, including a number of disialo, core 2 species, i.e. NeuAc␣2-3Gal␤1-4GlcNAc␤1-6[NeuAc␣2-3Gal␤1-3]GalNAc-ol and NeuAc␣2-3Gal␤1-4GlcNAc␤1-6[NeuAc␣2-3Gal␤1-4GlcNAc␤1-3Gal␤1-3]GalNAc-ol. Double-labeling experiments with [ 3 H]GlcN and 14 C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler, and fewer, carbohydrate chains than MUC-1 from normal breast epithelial cells, and that these differences, combined or separately, explain the differential tumor specificity of some MUC-1 antibodies and T cells.

Serum assays based on the CA125 antigen are widely used in the monitoring of patients with ovarian cancer; however very little is known about the molecular nature of the CA125 antigen. We recently cloned a partial cDNA (designated MUC16) that codes for a new mucin that is a strong candidate for being the CA125 antigen. This assignment has now been confirmed by transfecting a partial MUC16 cDNA into 2 CA125-negative cell lines and demonstrating the synthesis of CA125 by 3 different assays. Of the 3 antibodies (OC125, M11 and VK-8) tested on the transfected cells, only the first 2 were strongly positive, indicating the differential expression of the CA125 epitopes in these cells. CA125 is a tumor antigen that forms the basis for a serum assay that is widely used in the monitoring of ovarian and other gynecologic cancers. Subsequent to the development of the first mouse monoclonal antibody (OC125) that detected this antigen in 1981 1 a large number of other anti-CA125 antibodies have been developed. 2 These antibodies have been divided into 3 families (OC125-like, M11-like and Ov197-like) that recognize 3 domains of nonoverlapping epitopes. 2 Most clinical assays use a double determinant assay with 2 antibodies recognizing different epitopes; however a single determinant assay can also be used. 3 CA125 levels are routinely used in gynecologic practice to monitor the clinical status of patients with ovarian cancer. For example, rising CA125 levels during or after treatment often indicate a poor therapeutic response and an unfavorable prognosis. 4 -6 On the other hand, CA125 is of limited use in the initial diagnosis of ovarian cancer because of its elevation in some benign conditions, 4 -6 although possible ways around this limitation are being examined. 7,8 Information on the molecular nature of the CA125 antigen could lead to improved assay methods as well as to an understanding of its biological role in normal and malignant tissues.Most biochemical studies have suggested that the CA125 antigen is a high molecular weight glycoprotein, probably a mucin, although conflicting views have been presented. 9,10 Progress in understanding the molecular nature of CA125 has been hampered by difficulties encountered in attempting to clone this antigen. We have, however, recently isolated a partial cDNA (MUC16) from ovarian cancer cells that is a strong candidate for being the gene coding for the peptide moiety of CA125. 11 The identity of MUC16 as CA125 was based on (i) the isolation of peptides from purified CA125 that were contained in the deduced amino acid sequence of MUC16 and (ii) a precise correlation between MUC16 mRNA expression, determined by Northern blotting and CA125 expression, determined by serological analysis, in a panel of cancer cell lines. We now confirm this identity by demonstrating that CA125-negative cells transfected with MUC16 cDNA synthesize CA125 antigen. We also show the differential expression of CA125 epitopes in the transfected cells. MATERIAL AND METHODS MaterialsAnti-CA125 monoclonal antibod...

To provide further information on the biochemical nature of the cellular and secreted forms of the mucin-like CA 125 ovarian cancer antigen. Pulse-chase experiments were performed in the NIH:OVCAR-3 ovarian cancer cell line with [³⁵S]Met/Cys radiolabeling. After pulsing the cells with radioisotope for 30 min and analyzing cell lysates by immunoprecipitation with anti-CA 125 antibodies, a doublet species (form B, approximately 400 kD) and a ladder of slower-moving components were detected by SDS-PAGE and autoradiography. After a 4-hour chase period, a much larger species (form A) became evident. With further culture, the B form disappeared and the A form accumulated, suggesting a ‘precursor-product’ relationship between the two forms. The putative precursor species did not appear in the culture supernatant, but secretion of the mature species (form A) began after about 1 h of synthesis. Lectin-binding experiments demonstrated that the B form is a glycoprotein and not an early apomucin precursor. In contrast to other reports, no smaller species of the mature form of CA 125 were detected in this study. Trypsin digestion severely degraded the antigen but discrete smaller fragments were not formed. CA 125 antigen is synthesized through a glycosylated 400-kD precursor species. The mature form of the antigen appears in the cell after about 1 h of synthesis and in the culture medium after 1–4 h.

A new murine monoclonal antibody (MAb VK-8), detecting the CA 125 ovarian cancer antigen, was used to purify this antigen from OVCAR-3 ovarian cancer cells by affinity chromatography. The biochemical properties of the purified antigen are characteristic of a mucin-type glycoprotein: (1) the molecule is highly glycosylated (77% w/w), mainly with galactose, N-acetylglucosamine, and N-acetylgalactosamine, (2) the protein moiety is rich in serine, threonine and proline, (3) many of the serine and threonine residues are glycosylated, (4) the glycan chains are almost entirely O-linked, with core 2 [Galb1 = 3(GlcNAcb1 = 6)GalNAc] structures predominating and (5) these chains carry fucosylated Type 2 (Le y and Le x , and H type 2) blood group structures. The antigen exhibited a very high m.w. (G10 3 kDa) in aqueous buffer as well as in urea, but was degraded by proteolytic enzymes to smaller fragments that no longer reacted with the antibody. Although this result, and other immunochemical data, indicate that OC125, the original MAb to CA125, and VK-8 antibodies detect epitopes on the protein portion of the molecule, the involvement of carbohydrate cannot be ruled out. Further insight into the structure and function of the CA125 antigen will come from cloning the gene coding for the peptide backbone, and from more detailed carbohydrate structural analysis. Int.