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Link to original content: https://pubmed.ncbi.nlm.nih.gov/19538730
Characterization of the tumor marker muc16 (ca125) expressed by murine ovarian tumor cell lines and identification of a panel of cross-reactive monoclonal antibodies - PubMed Skip to main page content
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. 2009 Jun 18;2(1):8.
doi: 10.1186/1757-2215-2-8.

Characterization of the tumor marker muc16 (ca125) expressed by murine ovarian tumor cell lines and identification of a panel of cross-reactive monoclonal antibodies

Cara Ar Goodell  1 , Jennifer A Belisle, Jennifer Aa Gubbels, Martine Migneault, Claudine Rancourt, Joseph Connor, Muthusamy Kunnimalaiyaan, Rachel Kravitz, Ward Tucker, Michael Zwick, Manish S Patankar
Affiliations

Characterization of the tumor marker muc16 (ca125) expressed by murine ovarian tumor cell lines and identification of a panel of cross-reactive monoclonal antibodies

Cara Ar Goodell et al. J Ovarian Res. .

Abstract

Objectives: The ovarian tumor marker CA125 is expressed on human MUC16, a cell surface bound mucin that is also shed by proteolytic cleavage. Human MUC16 is overexpressed by ovarian cancer cells. MUC16 facilitates the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Additionally, MUC16 also is a potent inhibitor of natural killer cell mediated anti-tumor cytotoxic responses. Extensive studies using human as well as murine ovarian tumor cell models are required to clearly define the function of MUC16 in the progression of ovarian tumors. The major objective of this study was to determine if the murine ovarian tumor cells, MOVCAR, express Muc16 and to characterize antibodies that recognize this mucin.

Methods: RT-PCR analysis was used for detecting the Muc16 message and size exclusion column chromatography for isolating Muc16 produced by MOVCAR cells. Soluble and cell-associated murine Muc16 were analyzed, respectively, by Western blotting and flow cytometry assays using a new panel of antibodies. The presence of N-linked oligosaccharides on murine Muc16 was determined by ConA chromatography.

Results: We demonstrate that murine Muc16 is expressed by mouse ovarian cancer cells as an ~250 kDa glycoprotein that carries both O-linked and N-linked oligosaccharides. In contrast to human MUC16, the murine ortholog is primarily released from the cells and cannot be detected on the cell surface. Since the released murine Muc16 is not detected by conventional anti-CA125 assays, we have for the first time identified a panel of anti-human MUC16 antibodies that also recognizes the murine counterpart.

Conclusion: The antibodies identified in this study can be used in future purification of murine Muc16 and exhaustive study of its properties. Furthermore, the initial identification and characterization of murine Muc16 is a vital preliminary step in the development of effective murine models of human ovarian cancer. These models will aid in the further elucidation of the role that human MUC16 plays in the etiology and progression of ovarian tumors.

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Figures

Figure 1
Figure 1
RT-PCR verification of Muc16 mRNA expression in four MOVCAR cell lines. GAPDH gene was used as a housekeeping control.
Figure 2
Figure 2
Purification of Soluble Muc16 on a Sepharose CL-4B Column. Concentrated MOVCAR-2 spent media was separated as described in Methods. Fractions located under the bar stained positive for mucin with Alcian Blue and Western blotting.
Figure 3
Figure 3
Identification of soluble Muc16 and MUC16 by Western blotting. Purified MUC16 (25 μg total protein/lane) from MOVCAR-2 (lane 1) and OVCAR-3 cells (lane 2) was electrophoresed by SDS-PAGE and probed with a panel of anti-MUC16 monoclonal antibodies. Arrows indicate migration of 250 kDa molecular weight marker and identity of antibody used is shown on the right of each blot.
Figure 4
Figure 4
Specificity of 618F for human MUC16. (A) Purity of human MUC16 isolated from spent media of OVCAR-3 cells as determined by Coomassie Blue (1) and silver staining (2) of SDS-PAGE gel. Western blot analysis of the purified human MUC16 was conducted using the 618F (3) and the VK-8 (4) antibodies. (B) MUC16 expression on OVCAR-3, CAOV-3, and SKOV-3 was determined by flow cytometry using 653F, 618F, or VK-8 as the primary antibodies. Mean fluorescence intensity of the corresponding isotype controls was subtracted in each case. Data shown is mean of two independent experiments. Note that the binding of 618F, 653F and VK-8 to these three cell lines is comparable.
Figure 5
Figure 5
Binding of murine Muc16 to the lectin ConA. (A) Concentrated MOVCAR-2 spent media was separated on a ConA affinity column. Fractions eluted with ConA buffer were combined in pools 1 and 2. Fractions eluted with 100, 250, and 500 mM α-Me-Man concentrations were combined in pools 3–5, respectively. (B) Pooled fractions were electrophoresed by SDS-PAGE and probed with 653F. The arrow indicates migration of 250 kDa molecular weight marker. Murine Muc16 was detected primarily in pools 3–5.
Figure 6
Figure 6
Extra-and intracellular Muc16 expression by MOVCAR cells. (A) MOVCAR-10 cells were labeled with 618F (grey line) and analyzed for cell surface expression of Muc16 by flow cytometry. Isotype control is shown by dark line. (B) MOVCAR-10 cells were fixed and expression of intracellular Muc16 was detected by using the 618F antibody (grey line). Dark line shows isotype control. (C) Expression of cell surface and intracellular Muc16 in the four MOVCAR cell lines was determined by flow cytometry using the 618 F antibody. The mean fluorescence intensity for the binding of 618F to the cell surface and intracellular Muc16 is plotted after subtracting the mean fluorescence intensity for the matched isotype controls. Each measurement is a mean of two independent experiments. (D) Same as in (C) except 653F was used for detection of murine Muc16.
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