Abstract
Purpose: :
To investigate the role of complement factor H (CFH) in the protection of uveal melanoma cells.
Methods: :
Three cell lines OCM-1, OMM2.3 and MEL 290 were grown as monolayers in RPMI 1640 with 3 mM L-glutamine, 10% heat inactivated fetal calf serum (FCS), 100 IU/ml penicillin and 100 µg/ml streptomycin. In some experiments the cells were grown in absence of FCS with serum free supplement HL-1. Expression of CFH was investigated using flow cytometry, immunofluorescence and RT-PCR. Levels of CFH and C3 split products were also monitored using Western blot analysis. Specific antibodies and siRNA were used to block the function and expression CFH. Complement mediated cytotoxicity was measured by detecting the release of lactose dehodrogenase (LDH) by melanoma cells after the treatment with normal human serum (NHS). The levels of TGF-beta were measured by RT-PCR. The cells were treated with purified human factor H protein to see the effect of exogenous CFH on uveal melanoma cells.
Results: :
Flow cytometry and RT-PCR analysis demonstrated that all three cell lines OCM-1, OMM2.3 and MEL 290, express CFH when cultured in the presence of complement. Interestingly, uveal melanoma cells grown in absence of complement also expressed factor H protein as detected by Western blot analysis. Increased levels of C3 split products could be detected in the supernatant of uveal melanoma cells grown in absence of complement. Inhibition of function and expression of CFH resulted in dramatic increase in complement mediated cytotoxicity. The immunoflurescent staining detected low level of iC3b on the surface of uveal melanoma cells. Furthermore the expression of TGF-beta by uveal melanoma cells was up regulated when CFH and C3b were added to the culture medium.
Conclusions: :
Our results suggest that CFH play an important role in not only protecting the uveal melanoma cells from complement-mediated cytotoxicity but also by up regulating the expression of TGF-beta by the melanoma cells.
Keywords: melanoma • uvea