Heparin and heparin derivatives can regulate tumor growth and inhibit ... (381) 494-7349; fax, +49 (381) 494-7482; e-mai
CD44, bFGF and Hyaluronan in Human Pancreatic Cancer Cell Lines JÖRG RINGEL,a,c JOACHIM RYCHLY,a BARBARA NEBE,a CHRISTIAN SCHMIDT,a PETRA MÜLLER,a JENS RINGEL,b JÖRG EMMRICH,a STEFAN LIEBE,a AND MATTHIAS LÖHRa aDivision
of Gastroenterology, Department of Internal Medicine, University of Rostock, D-18057 Rostock, Germany bDepartment of Biochemistry, Friedrich Schiller University Jena, D-07749 Jena, Germany
INTRODUCTION Altered expression of cell surface molecules and the overexpression of growth factors and their receptors may be involved in malignant progression of human exocrine pancreatic cancer. CD44 is a transmembrane glycoprotein postulated to play a functional role in tumor cell metastasis. A “standard” form of CD44 (CD44st) binds hyaluronan (HA) and is reported to be overexpressed in pancreatic tumors.2 Alternative splicing gives rise to numerous CD44 isoforms. Variants containing the exon v6 appear to be of major importance for metastatic behavior in a rat pancreas carcinoma model.1 The published data about the expression of CD44st and CD44 isoforms in pancreatic cancer are contradictive.2–4 The regulation of CD44 expression is nearly unknown. Recently, a study showed a possible role of basic fibroblast growth factor (bFGF) in endothelial cells. It has been reported that bFGF and its high-affinity receptor (FGF-R) is abundantly present in pancreatic cancer.5 bFGF is one of the heparin-binding growth factors (FGF, epidermal growth factor (EGF)). The interaction with heparin protects bFGF from proteolytic degradation. Glycosaminoglycan heparin influences the binding of bFGF to the high-affinity receptor, too. Heparin and heparin derivatives can regulate tumor growth and inhibit metastatic behavior. The mechanisms of these regulation processes are poorly understood. Heparin is found exclusively in mast cells. Interestingly, mast cells accumulate by surrounding various neoplastic tissues. We asked whether bFGF alone, bFGF in combination with heparin, heparin alone or the CD44-ligand HA—a member of the glycosaminoglycans—modulates the expression of CD44. Therefore, we used the pancreatic adenocarcinoma cell lines PancTu and Panc-1 (both grade II–III). Furthermore, we investigated the effects of bFGF/heparin on proliferation behavior.
cAddress for correspondence: Dr. Jörg Ringel, Division of Gastroenterology, Dept. of Internal Medicine, University of Rostock, E. Heydemann Str. 6, D-18057 Rostock, Germany. Phone, +49 (381) 494-7349; fax, +49 (381) 494-7482; e-mail,
[email protected]
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MATERIALS AND METHODS The influence of bFGF/heparin on proliferation was measured using the 3-(4,5dimethylthiazol-eyl)-2,5-diphenyltetrazolium bromide (MTT) test (Cell Proliferation Kit I, Boehringer). Briefly, both cell lines were cultured in 96-well microtiter plates in medium (without fetal calf serum (FCS)) containing bFGF alone (1, 2, 5, 10, or 100 ng/ml), bFGF and a constant concentration of unfractionated heparin (Sigma, 1 µg/ml) or heparin (0.5; 1, 5, 10, 25; or 100 µg/ml) alone. After 1, 2 or 3 days’ incubation, cell proliferation was measured using an enzyme-linked immunosorbent assay (ELISA)-reader. For flow cytometric analysis we used the following monoclonal antibodies (mAbs): SFF-2 (CD44st), anti-CD44v3 (R&D), VFF-8 (CD44v5), VFF-18 (CD44v6), VFF-9 (CD44v7), VFF-17 (CD44v7/8), VFF-14 (CD44v10) (all from Serva), anti-FGF receptor antibody (Chemicon), IgG control antibodies and fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Sigma). For stimulation experiments, 106 cells were incubated with bFGF (Boehringer; 1, 5, 10, 100 ng/ml), bFGF and a constant concentration of unfractionated heparin (Sigma) (1 µg/ml) or heparin (0.5; 1, 5, 10, 25; 100µg/ml). After 1, 2 or 3 days the cells were detached. Furthermore, PancTu cells were incubated with hyaluronan (Sigma) (100 µg/ml) for 1 or 2 days. The CD44 expression and CD44st, CD44v3 and v6 expression after stimulation was analysed by fluorescence-activated cell sorter (FACS) flow cytometer.
RESULTS The FACS analyses revealed the expression of CD44st,v3, v4, v5, v6, v7, v7/8 and v10 on the cell lines Panc-1 and PancTu. Both cell lines also express the flg gene product (FGF high-affinity receptor—FGF-R, data not shown). The incubation with bFGF/heparin for 3 days decreased the expression of CD44st, whereas CD44v6 and CD44v3 were unaffected. In contrast, we found that higher concentrations of unfractionated heparin alone upregulated the CD44st expression. The reasons for this interesting effect are unknown. Furthermore, the incubation with hyaluronan as a CD44-ligand had no influence on CD44st and v6 expression. Various concentrations of bFGF alone and bFGF in combination with 1 µg heparin had no effect on the proliferation of adherent growing Panc-1 and PancTu cells. The MTT proliferation tests also showed that heparin alone had no influence. The blocking of FGF-binding with antibodies reduced the proliferation of PancTu (40%) and Panc1 (30%).
DISCUSSION We have found that bFGF, heparin and the combination of both have no effect on cell proliferation. However, the blocking of FGF binding with antibodies reduced the proliferation of both pancreatic tumor cell lines. This suggests autocrine loop effects. We found an expression of CD44st,v3, v4, v5, v6, v7, v7/8 and v10 on the protein level in our human pancreatic cancer cell lines. In correlation with the important cell
FIGURE 1. FACS analysis of CD44st expression of PancTu cells after stimulation with 5 ng bFGF/ml and (b) 1 µg unfractionated heparin (n = 3) and (c) 25 µg unfractionated heparin alone (n = 3) for 72 hours; (a) control; black figure, CD44st expression of unstimulated cells; white figure, stimulated cells.
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FIGURE 2. MTT test: incubation with mAb (VBS1, Chemicon) blocking the binding of bFGF on FGF receptor or equial amounts of control mAb for 48 hours (n = 6).
surface molecule intercellular adhesion molecule-1 (ICAM-1), the expression of CD44st and CD44 variants is weaker on pancreatic cancer cells. But the FACS analysis of CD44 revealed similar results as in other cancer cell lines. The expression of CD44st and CD44v isoforms in abnormal amounts and compositions may play an important role in malignant transformation and cell metastasis. Because CD44 variants expressing v3 have been shown to carry binding sites for heparin binding factors like bFGF, the CD44 expression may play a role in the immobilization and regulation of growth factors in pancreatic cancer. In former studies we could show that interferon-γ (IFN-γ) can modulate the CD44st and CD44v6 expression in pancreatic adenocarcinoma cell lines. In addition, expression of CD44st and variants was unaffected by TGF-β1 using exogenous TGF-β1 incubation and TGF-β1 transfected Panc1 cells. The CD44 ligand hyaluronan does not influence the expression of CD44st and v6. The combination of bFGF and heparin downregulated the CD44st expression. bFGF and heparin had no significant effect on CD44v6 or CD44v3 expression in our cell lines, suggesting an increased gap between CD44st and CD44v6 expression. The reasons for the slightly upregulation of CD44st expression after heparin incubation are unknown. It is possible that added heparin may trigger the biological effects of other heparin-binding factors. Recently, we could show that unfractionated heparin alone can activate mitogen-activated protein (MAP) kinases in a time- and dose-dependent manner in pancreatic adenocarcinoma cell line PaCa44.6 Whether bFGF/heparin may affect the metastatic behavior of pancreatic carcinomas by the mechanism of up- or downregulation of CD44/CD44 variants has to be investigated further. The mechanism of bFGF/heparin modulation effects has to be proved in further analyses. REFERENCES 1. G ÜNTHERT , U. et al. 1991. A new variant glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65: 13–24.
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2. T AKADA , M. et al. 1994. The significance of CD44 in human pancreatic cancer: I. High expression of CD44 in human pancreatic adenocarcionoma. Pancreas 9: 748–752. 3. C ASTELLÀ , E.M. et al. 1996. Differential expression of CD44v6 in adenoarcinoma of the pancreas: an imunohistochemical study. Virchows Arch. 429(4–5): 191–195. 4. G ANSAUGE , F., et al. 1995. Differential expression of CD44 splice variants in human pancreatic adenocorcinoma and in normal pancreas. Cancer Res. 55: 5499–5503. 5. L EUNG , H.Y. et al. 1994. Expression and functional activity of fibroblast growth factors and their receptors in human pancreatic cancer. Int. J. Cancer 59: 667–675. 6. R INGEL , J. et al. 1998. The effects of unfractionated heparin on the human pancreatic cancer cell line PaCa44. J. Mol. Med. 76: A64.
