Molecular cloning of the CA125 ovarian cancer antigen: identification as a new mucin, MUC16.

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 lambdaZAP 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.

Synthesis and secretion of the ovarian cancer antigen CA 125 by the human cancer cell line NIH:OVCAR-3.

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 [(35)S]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.

MUC-6 mucin is a major component of "blood group substance" from human ovarian cyst fluid.

Ovarian cyst fluid has been a valuable source of the mucins (traditionally termed "blood group substances") that were used for the elucidation of the structures of the ABO Lewis blood group determinants, but the identity of the mucin peptide core(s) carrying these carbohydrate specificities is not known. An ovarian cyst fluid mucin was purified, deglycosylated with HF and digested with trypsin or chymotrypsin to yield a number of peptides. Amino acid sequencing of these peptides yielded five different sequences which showed complete or partial homology to the MUC-6 apomucin deduced from DNA sequencing. As no other sequences were identified, it is concluded that MUC-6 is the major mucin core structure of ovarian cyst fluid mucin.

Initial immunochemical characterization of MX35 ovarian cancer antigen.

Monoclonal antibody (mAb) MX35 reacts with approximately 90% of ovarian epithelial cancers and has been studied in localization and biodistribution trials in ovarian cancer patients. This study shows that mAb MX35 recognizes a cell surface antigen of about 95,000 D on OVCAR-3 ovarian cancer cells. The antigen could be immunoprecipitated from lysates of cells metabolically labeled with [3H]glucosamine and it bound to concanavalin A and wheat germ agglutinin lectins, showing that it is a glycoprotein. MX35 antigen can also be detected in detergent lysates of OVCAR-3 cells by Western blotting. Using this technique the MX35 epitope(s) was shown to be heat stable but susceptible to reduction by 2-mercaptoethanol. Protease digestion of the antigen resulted in smaller fragments (42-52 kDa) that still reacted with antibody. We conclude that MX35 antigen is a 95 kDa glycoprotein, stabilized by disulfide bonds, with a large protease-resistant region that carries the MX35 epitopes.

Isolation and characterization of ovarian cancer antigen CA 125 using a new monoclonal antibody (VK-8): identification as a mucin-type molecule.

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 [Galbeta1 --> 3(GlcNAcbeta1 --> 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. (> 10(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.

Comparison of O-linked carbohydrate chains in MUC-1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines. Demonstration of simpler and fewer glycan chains in tumor cells.

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 [3H]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 NeuAcalpha2-3Galbeta1-3GalNAc-ol, Galbeta1-3GalNAc-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. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-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.

Serological and immunochemical analysis of Lewis y (Ley) blood group antigen expression in epithelial ovarian cancer.

The expression of Ley blood group antigen in epithelial ovarian cancer tissues and cell lines has been studied using a Ley-specific monoclonal antibody (MAb 3S193). In ovarian cancer specimens, Ley was expressed in 75% of the 140 tumor specimens examined, with strong or moderate expression being observed in 56% of the samples. Seven of the 11 ovarian cancer cell lines studied were Ley-positive. Using immunochemical approaches, Ley epitopes were found to be expressed on 4 types of carrier molecules: CA125 ovarian cancer antigen, MUC-1 mucins, lower m.w. glycoproteins and glycolipids. In cell lines, Ley was more commonly expressed on MUC-1 mucin than on CA125, whereas in tumor specimens Ley was commonly found on both CA125 and MUC-1. The biochemical nature of the smaller Ley glycoproteins was not determined, but it was shown that they were not CEA and LAMP-1, known Ley carriers in some other tumor types. Glycolipids carrying Ley epitopes were detected in both ovarian cancer cell lines and tumor specimens. The presence of Ley epitopes on a number of different molecular carriers, including 2 major ovarian cancer antigens (CA125 and MUC-1), explains the high incidence of Ley in ovarian cancer. The high expression of Ley in ovarian cancer and the availability of specific murine and humanized MAbs make Ley an attractive candidate target for clinical studies.

Some monoclonal antibody reagents (C219 and JSB-1) to P-glycoprotein contain antibodies to blood group A carbohydrate determinants: a problem of quality control for immunohistochemical analysis.

Monoclonal antibodies (MAb) C219 and JSB-1 have been used extensively in the analysis of P-glycoprotein expression in normal and malignant tissues. This study demonstrates that some commercial lots of these MAb, even those supplied as purified immunoglobulins, contain contaminating anti-A blood group antibodies. In both sources of reagent, the antibody was specific for a particular A structure, known as repetitive or Type 3 A. These observations may account for earlier studies showing polymorphic variation in P-glycoprotein expression in epithelial tissues and an apparent correlation with the A blood type of the donor. Such reactivity can be eliminated by absorption of anti-P-glycoprotein reagents with A erythrocytes. These data re-emphasize the importance of evaluating MAb samples for unsuspected contaminating antibodies.

Increased yields of IgG2a- and IgG3-secreting hybridomas after fusion of B cells from mice with autoimmune diseases.

Hybridoma technology has made the production of antigen-specific monoclonal antibodies feasible and almost routine, but the production of certain biologically desirable antibody isotypes has remained difficult. Three strains of autoimmune mice (MRL/l, NZB, and BXSB) were compared to a normal strain (BALB/c), in fusions with a BALB/c myeloma (NS-1) in order to study the rescue of relevant isotypes with the desired antigenic specificities. Mice from these four strains were immunized with colon carcinoma cells, and the hybridoma supernatants from thirty fusions were analyzed for (1) reactivity with cell surface determinants on the immunizing cell line; and (2) Ig class and subclass isotypes. We found that compared to BALB/c mice, MRL/l mice produced greater numbers, and NZB and BXSB mice comparable numbers, of cell surface-reactive hybridoma clones per fusion. MRL/l mice produced the largest number and highest percentage of cell-surface reactive IgG2a (22.4%) and IgG3 (10.6%) producing clones, followed by NZB mice which produced predominantly IgG2a clones (12.3%). BXSB mice, which have latent autoimmune disease, showed no significant difference from normal BALB/c controls (IgG2a:0.7% and IgG3:1.9% vs. IgG2a:4.8% and IgG3:4.8%). The increase in IgG2a and IgG3 clones derived from MRL/l mice was age-dependent, correlating with the age at which abnormal proliferation of T cell and splenic enlargement occurs (2-4 months). We conclude that MRL/l mice are useful for generating monoclonal antibodies of the IgG2a or IgG3 isotype, provided fusions are performed at the time of maximal lymphoproliferation.

Analysis of the fine specificities of 11 mouse monoclonal antibodies reactive with type 2 blood group determinants.

The specificity of 11 mouse monoclonal antibodies reacting selectively with type 2 blood group structures was analyzed in detail by studying their reactivities with a panel of standard glycolipids, glycolipids from erythrocytes and blood group glycoproteins. The antibodies reacted with monofucosyl type 2 H, difucosyl type 2 structures (Le gamma) or both; none of the antibodies reacted with type 1 (H, Lea, or Leb) structures. Only a small proportion of the antibodies were completely specific for either type 2H or Le gamma structures. None of the antibodies had identical patterns of reactivity and their specificities were individually distinct. Seven antibodies preferentially agglutinated O and A2 erythrocytes. Anti-Le gamma-specific antibodies, except mAb101, did not agglutinate erythrocytes or react with glycolipids from erythrocytes, indicating the absence of Le gamma structures in erythrocyte glycolipids. The ability of some antibodies to react with A erythrocytes was shown to be due to cross-reactivity of the antibodies with type 3 (repetitive) A structures. The study demonstrates that monoclonal anti-carbohydrate antibodies tend to react with a range of related, and even distantly related, structure in a pattern characteristic of each antibody and that very few antibodies have extremely restricted specificities.

Expression of Lewisa, Lewisb, X, and Y blood group antigens in human colonic tumors and normal tissue and in human tumor-derived cell lines.

Serological and immunopathological analysis of the expression of Lea, Leb, X, and Y blood group antigens on cell lines and tissues was performed using a panel of mouse monoclonal antibodies. The distribution of the antigens was determined on 155 malignant tumor cell lines of various types and 10 short term cultures of normal fibroblasts and kidney cells. Among colon cancers, all four blood group antigens were expressed on the majority of cell lines. On lung, breast, bladder, and ovarian cancer cell lines, X and Y antigens were the main specificities found, whereas few of the renal and hematopoietic tumor cell lines demonstrated any of the four blood group antigens. No blood group antigens could be detected on astrocytoma or melanoma cell lines. The expression of the antigens was also analyzed on frozen sections of colon carcinoma and adjacent normal colon tissue from 42 patients using the immunoperoxidase method. Lea and X were detected throughout the normal colon and on most colonic tumors. In poorly differentiated colon cancer and in metastatic cancer, decrease of Lea antigen was observed. Leb and Y expression was observed in only 20-45% of normal tissue samples but in almost all colonic carcinoma tissues. A selected number of tumor and normal specimens from patients whose secretor status was known were examined in more detail. Both the staining of the tissues and the reactivity of blood group glycolipids from the same specimens were determined. These studies confirmed the above findings and demonstrated the unexpected ability of tumors of nonsecretors to express Leb and/or Y antigens. In such individuals, in whom the expression of Leb and Y antigens in normal tissues is absent or minimal, these antigens provide possible targets for immunodiagnosis and therapy.

Analysis of the expression of H, Lewis, X, Y and precursor blood group determinants in saliva and red cells using a panel of mouse monoclonal antibodies.

Salivary glycoproteins from 33 normal individuals were analyzed with a panel of mouse monoclonal antibodies to H-1, H-2, Lea, Leb, X, Y and precursor blood group determinants. Samples from 19/33 individuals co-expressed Leb and Y-determinants (secretors) and 6/33 co-expressed Lea and X-determinants (non-secretors). Erythrocytes of these individuals were typed Le (a-b+) and Le (a + b-), respectively. In seven other salivas, only one specificity, either Lea, Leb, X or Y, was expressed and in one sample none of these determinants could be detected. Only one saliva sample expressed H-1 specificity and none expressed H-2 or type 1 precursor determinants. The absence of H-1 and H-2 structures in secretors and the resulting expression of difucosylated Leb and Y-structures is probably a tissue-specific trait of salivary gland secretions. The strict co-expression of Leb with Y and Lea with X supports the conclusion that only one 2-O-fucosyl-galactose transferase, which can fucosylate both type 1 and type 2 chains, exists in salivary glands. The finding that a number of individuals expressed neither X- nor Y-specificities was unexpected in view of previous work showing that the 3-O-fucosyl N-acetylglucosamine transferase involved in forming this structure is a ubiquitous enzyme. The individualistic expression of blood group phenotypes in tissues should be considered when the altered expression of blood groups in malignancy and other diseases is studied.