Objectives:The presence of CD44 isoforms was evaluated in ascitic fluid and serum samples of patients with gynecologic malignancies. Previously, the shedding of tumor-associated cell surface antigens has been demonstrated in the blood and malignant effusions of gynecologic cancer patients. Thus, the shedding of CD44 was also studied in ascitic fluids and sera of these patients, to address variant isoform expression as a biomarker of gynecologic cancer.
Methods:The expression of CD44 isoforms by ovarian tumor cells was examined by flow cytometry using variant-specific monoclonal antibodies. The release of these isoforms into the peripheral circulation and ascites was assayed by Western immunoblot analysis.
Results:Flow cytometric analysis of ovarian tumor cell lines revealed a strong expression of CD44 with significant levels of v4/5 and v6 isoforms. The presence of circulating CD44 isoforms was detectable in the sera of six of eight cancer patients, as well as in 12 of 16 ascitic fluids. Of the CD44-positive specimens, all six positive sera expressed detectable levels of variant CD44. The CD44v6 was present in all of the positive sera samples tested. In the ascites, the “shed” CD44 appeared to be associated predominantly with shed particles (vesicles) of plasma membranes (membrane fragments). Of ten CD44-positive ascites samples, all expressed significant levels of variant CD44.
Conclusions:In addition to mediating metastasis, the differential expression and shedding of CD44 isoforms into the circulation may represent important deteminants in the escape of tumors from immune surveillance, and their detection may be a diagnostic or prognostic marker.
1. Honn KV, Tang DG. Adhesion molecules and tumor cell interaction with endothelium and subendothelial matrix. Cancer Metastasis Rev1992;11:353-375.
2.
2. Hamilton MS, Ball J, Bromidge E, Franklin IM. Surface antigen expression of human neoplastic plasma cells includes molecules associated with lymphocyte recirculation and adhesion. Brit J Haematol1991;78:60-65.
3.
3. Shimizu Y, Van Seventer GA, Siraganian R, Wahl L, Shaw S. Dual role of the CD44 molecule in T cell adhesion and activation. J Immunol1989;143:2457-2463.
4.
4. Zhou DFH, Ding JF, Picker LJ, Bargatze RF, Butcher EC, Goeddel DV. Molecular cloning and expression of Pgp-I. The mouse homology of the human H-CAM (Hermes) lymbocyte homing receptor. J Immunol1989;143:3390-3395.
5.
5. Denning SM, Le PT, Singer KH, Haynes BF. Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation. J Immunol1990;144:7-15.
6.
6. Miyake K, Medina KL, Hayashi S, Ono S, Hamaoka T, Kincade PW. Monoclonal antibodies to Pgp-I/CD44 block lymphohemapoiesis in long-term bone marrow cultures. J Exp Med1990;171:477-488.
7.
7. Arch R, Wirth K, Hofmann M, et al. Participation in normal immune responses of a metastasis-inducing splice variant of CD44. Science1992;257:682-685.
8.
8. Brown TA, Bouchard T, St John T, Wayner E, Carter WG. Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons. J Cell Biol1991;113:207-221.
9.
9. Stamenkovic I, Aruffo A, Amiot M, Seed B. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronare bearing cells. EMBO J1991;10:343-348.
10.
10. Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, Bell Jl. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 altematively spliced exons. Proc Natl Acad Sci U S A1992;89:12160-12164.
11.
11. Peach RJ, Hollenbaugh D, Stamenkovic I, Aruffo A. Identification of hyaluronic acid binding sites in the extracellular domain of CD44. J Cell Biol1993;122:257-264.
12.
12. Thomas L, Byers HR, Vink J, Stamenkovic I, CD44H regulates tumor cell migration on hyaluronate-coated substrate. J Cell Biol1992;118:971-977.
13.
13. Pandolfi F, Trentin L, Boyle LA, et al. Expression of cell adhesion molecules in human melanoma cell lines and their role in cytotoxicity mediated by tumor-infiltrating lymphocytes. Cancer1992;69:1165-1173.
14.
14. Taylor DD, Homesley HD, Doellgast GJ. Identification of antigenic components recognized by “membrane bound” antibodies from ovarian cancer patients. Am J Reprod Immunol1984;6:179-184.
15.
15. Taylor DD, Black PH. Inhibition of macrophage Ia antigen expression by shed plasma membrane vesicles from metastatic murine melanoma lines. J Natl Cancer Inst1985;74:859-867.
16.
16. Taylor DD, Levy EM, Black PH. Shed membrane vesicles: a mechanism for tumor-induced immunosuppression. In: Immunity to Cancer. Mitchell MS, Reif AE, eds. New York: Academic Press, 1985:369-373.
17.
17. Owens K, Gercel-Taylor C, Taylor DD, Day TG, Doering DL. Isolation and characterization of an ovarian carcinoma cell line as a model for drug resistance. Proc Am Assoc Cancer Res1993;34:26-26.
18.
18. Taylor DD, Chou IN, Black PH. Isolation of plasma membrane fragments from cultured murine melanoma cells. Biochem Biophys Res Comm1983;113:470-476.
19.
19. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem1951;193:265-275.
20.
20. Laemmli UK. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4, Nature1970;227:680-685.
21.
21. Birch M, Mitchell S, Hart IR. Isolation of human melanoma cell variants expressing high and low levels of CD44. Cancer Res1991;51:6660-6667.
22.
22. Sy MS, Guo YJ, Stamenkovic I. Distinct effects of two CD44 isoforms on tumor growth in vivo. J Exp Med1991;174:859-866.
23.
23. HOfmann M, Rudy W, Zoller M, et al. CD44 splice variants confer metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines. Cancer Res1991;51:5292-5297.
