Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

Abstract

Background: During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L) expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period.

Methods: RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat) and three mouse (CMT93, CT26, B16F10) cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody.

Results: Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent.

Conclusion: These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.

Figure 1

Characterisation of FasL expression in tumor cell lines of human origin. A: FasL mRNA expression by each cell line was analyzed at the indicated times by RT-PCR. β-actin RT-PCR was performed to monitor RT-PCR amplification efficiency, with all samples yielding equivalent levels. B: FasL protein expression was monitored by immunoblot analysis using the Ab-1 anti-FasL antibody (Oncogene). Results are representative of three independent experiments.

Figure 2

Analysis of FasL expression in murine tumor cell lines. A. FasL expression by murine tumor cells was analyzed by RT-PCR at the indicated times. B. FasL protein expression was monitored by Western blotting using the Ab-1 anti-FasL antibody. C. After 48 hrs culture, FasL expression was detected by immunofluorescence on paraformaldehyde-fixed cell monolayers. Representative cell lines are shown – (a) EL-4 (murine T-cell line – positive control), (b) CMT93 and (c) B16F10. Results are representative of three independent experiments.

Figure 3

Confirmation of the specificity of FasL protein detection. A. FasL protein was detected by Western blotting using both the Ab-1 and N-20 anti-FasL antibodies. Results from three representative cell lines are shown. B. FasL protein production was also detected by Western blotting using the Ab-1 antibody following PHA activation of Jurkat cells and treatment of SW620 and SW480 cells with MMP inhibitors. Results are representative of three independent experiments.

Figure 4

CT26 cell morphology during the course of FasL analysis. Cell lines were seeded at ~2 × 10⁵ cells/ml in 6 well plates to achieve 20% confluency 18 hours later. This point was defined as 0 hr. After 72 hrs, cells had reached ~80–90% confluency.