Cytotoxic reactions of CC531s towards liver sinusoidal endothelial cells: a microscopical study
© Vekemans et al; licensee BioMed Central Ltd 2004
Published: 14 January 2004
Colorectal cancer cells can induce apoptosis in cells of various tissues . Apoptosis can be induced by a number of factors such as Fas inducing apoptosis through the Fas/FasL pathway; other factors involve the TRAIL pathway and TNF. It is known that metastasizing colon cancer cells express more FasL then primary carcinoma cells . We investigated whether a rat colon carcinoma cell line CC531s could induce apoptosis in liver sinusoidal endothelial cells (LSECs). LSECs and CC531s were co-cultured for 18 hrs and cells were visualized by SEM and TEM. Apoptosis was visualized by markers such as Hoechst and Propidium iodide. Furthermore, cells were recorded by time lapse video microscopy with and without an antagonistic antibody for FasL.
Animals, antibodies & reagents
Male Wistar rats (8–12 weeks old) were purchased at the Center of Laboratory Animals (Leuven, Belgium). Animals had free access to food and water. 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) (catalogue no. D-275), Hoechst 33342 (HO 33342) (Catalogue no. H-3570) and Propidium iodide (PI) (catalogue no. P-3566) were purchased at Molecular Probes (Leiden, The Netherlands). Anti-FasL blocking antibody, MFL-4, was obtained from BD Pharmingen (Erembodegem, Belgium, catalogue no. 555021).
Co-culture of CC531s cells and LSECs
LSECs were isolated , purified and seeded to confluence on collagen-S coated wells and RPMI- 1640 supplemented with 10% heat inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 micrograms/ml streptomycin. The cells were kept in culture for 4 hrs. Subsequently, cells were washed with prewarmed RPMI. CC531s cells were seeded on top of LSECs at a ratio of 1:10. In order to visualize apoptosis, cells were stained with Hoechst 33342 and propidium iodide . In order to discriminate cells, CC531s were stained with DiO . For TEM and SEM, cells were prepared  and viewed with F. E. I. Tecnai 10 and Philips SEM 505. For time lapse recordings, cells were recorded up to 1 hr. Cells were cultured in medium only and/or with an antagonistic antibody against FasL (20 micrograms/ml).
Results and Discussion
In conclusion: CC531s cells can induce apoptosis in LSECs and and antagonistic antibody against FasL abrogates this apoptosis.
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