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Secretion of soluble complement inhibitors factor H and factor H-like protein (FHL-1) by ovarian tumour cells.

Junnikkala S, Hakulinen J, Jarva H, Manuelian T, Bjørge L, Bützow R, Zipfel PF, Meri S - Br. J. Cancer (2002)

Bottom Line: We observed that the soluble complement regulators factor H and factor H-like protein were abundantly present in ascites samples as well as in primary tumours of patients with ovarian cancer.In ascites samples the mean level of factor H-like protein (130+/-55 microg ml(-1)) was 5.5-fold higher than in normal human serum (24+/-3 microg ml(-1)).Subsequently, the PA-1 and SW626 cell lines were found to secrete a soluble form of the membrane cofactor protein (CD46).

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Immunology, Haartman Institute, University Central Hospital, FIN-0014 Helsinki, Finland.

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Cofactor activity of the ovarian cell growth supernatants for factor I-mediated cleavage of 125I-labelled C3b. Cell supernatants (concentrated 50-fold) were first preincubated with or without polyclonal anti-factor H (50 μg ml−1) or the GB24 anti-MCP (25 μg ml−1) antibody (37°C, 30 min) and then for 90 min at 37°C with 125I-labelled C3b and factor I (25 μg ml−1). In the positive controls 125I-C3b and factor I were incubated with factor H (25 μg ml−1). The mixtures were analysed by SDS–PAGE and autoradiography under reducing conditions. The protein compositions in the mixtures are marked beneath the lanes. The α′-chain of 125I-C3b becomes cleaved into fragments with apparent Mrs of 68, 46 and 43 kDa. Because of the relatively high concentration of factor H in the positive control (second lane from the left) the second factor I-mediated cleavage had fully converted the 46 kDa fragment into the 43 kDa α′-chain fragment. The cofactor activity of the SW626 and PA-1 supernatants could be inhibited with the anti-MCP mAb whereas the activities of the SK-OV-3 and Caov-3 supernatants were inhibited with the polyclonal anti-factor H antibody. The results indicate that all the ovarian cell supernatants have cofactor activity for C3b cleavage. In SK-OV-3 and Caov-3 cells this is due to functionally active H/FHL-1, whereas in the SW626 and PA-1 cell supernatants it is due to soluble MCP.
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fig5: Cofactor activity of the ovarian cell growth supernatants for factor I-mediated cleavage of 125I-labelled C3b. Cell supernatants (concentrated 50-fold) were first preincubated with or without polyclonal anti-factor H (50 μg ml−1) or the GB24 anti-MCP (25 μg ml−1) antibody (37°C, 30 min) and then for 90 min at 37°C with 125I-labelled C3b and factor I (25 μg ml−1). In the positive controls 125I-C3b and factor I were incubated with factor H (25 μg ml−1). The mixtures were analysed by SDS–PAGE and autoradiography under reducing conditions. The protein compositions in the mixtures are marked beneath the lanes. The α′-chain of 125I-C3b becomes cleaved into fragments with apparent Mrs of 68, 46 and 43 kDa. Because of the relatively high concentration of factor H in the positive control (second lane from the left) the second factor I-mediated cleavage had fully converted the 46 kDa fragment into the 43 kDa α′-chain fragment. The cofactor activity of the SW626 and PA-1 supernatants could be inhibited with the anti-MCP mAb whereas the activities of the SK-OV-3 and Caov-3 supernatants were inhibited with the polyclonal anti-factor H antibody. The results indicate that all the ovarian cell supernatants have cofactor activity for C3b cleavage. In SK-OV-3 and Caov-3 cells this is due to functionally active H/FHL-1, whereas in the SW626 and PA-1 cell supernatants it is due to soluble MCP.

Mentions: Since SK-OV-3 and Caov-3 cells were found to produce and secrete both factor H and FHL-1 into their growth supernatants we wanted to study whether they were capable of promoting the degradation of C3b to iC3b, i.e. whether the secreted proteins acted as cofactors for factor I. Cofactor activity analysis demonstrated that the supernatants of all the cell lines, including those of PA-1 and SW626, promoted factor I-mediated cleavage of 125I-labelled C3b (Figure 5Figure 5


Secretion of soluble complement inhibitors factor H and factor H-like protein (FHL-1) by ovarian tumour cells.

Junnikkala S, Hakulinen J, Jarva H, Manuelian T, Bjørge L, Bützow R, Zipfel PF, Meri S - Br. J. Cancer (2002)

Cofactor activity of the ovarian cell growth supernatants for factor I-mediated cleavage of 125I-labelled C3b. Cell supernatants (concentrated 50-fold) were first preincubated with or without polyclonal anti-factor H (50 μg ml−1) or the GB24 anti-MCP (25 μg ml−1) antibody (37°C, 30 min) and then for 90 min at 37°C with 125I-labelled C3b and factor I (25 μg ml−1). In the positive controls 125I-C3b and factor I were incubated with factor H (25 μg ml−1). The mixtures were analysed by SDS–PAGE and autoradiography under reducing conditions. The protein compositions in the mixtures are marked beneath the lanes. The α′-chain of 125I-C3b becomes cleaved into fragments with apparent Mrs of 68, 46 and 43 kDa. Because of the relatively high concentration of factor H in the positive control (second lane from the left) the second factor I-mediated cleavage had fully converted the 46 kDa fragment into the 43 kDa α′-chain fragment. The cofactor activity of the SW626 and PA-1 supernatants could be inhibited with the anti-MCP mAb whereas the activities of the SK-OV-3 and Caov-3 supernatants were inhibited with the polyclonal anti-factor H antibody. The results indicate that all the ovarian cell supernatants have cofactor activity for C3b cleavage. In SK-OV-3 and Caov-3 cells this is due to functionally active H/FHL-1, whereas in the SW626 and PA-1 cell supernatants it is due to soluble MCP.
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Related In: Results  -  Collection

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fig5: Cofactor activity of the ovarian cell growth supernatants for factor I-mediated cleavage of 125I-labelled C3b. Cell supernatants (concentrated 50-fold) were first preincubated with or without polyclonal anti-factor H (50 μg ml−1) or the GB24 anti-MCP (25 μg ml−1) antibody (37°C, 30 min) and then for 90 min at 37°C with 125I-labelled C3b and factor I (25 μg ml−1). In the positive controls 125I-C3b and factor I were incubated with factor H (25 μg ml−1). The mixtures were analysed by SDS–PAGE and autoradiography under reducing conditions. The protein compositions in the mixtures are marked beneath the lanes. The α′-chain of 125I-C3b becomes cleaved into fragments with apparent Mrs of 68, 46 and 43 kDa. Because of the relatively high concentration of factor H in the positive control (second lane from the left) the second factor I-mediated cleavage had fully converted the 46 kDa fragment into the 43 kDa α′-chain fragment. The cofactor activity of the SW626 and PA-1 supernatants could be inhibited with the anti-MCP mAb whereas the activities of the SK-OV-3 and Caov-3 supernatants were inhibited with the polyclonal anti-factor H antibody. The results indicate that all the ovarian cell supernatants have cofactor activity for C3b cleavage. In SK-OV-3 and Caov-3 cells this is due to functionally active H/FHL-1, whereas in the SW626 and PA-1 cell supernatants it is due to soluble MCP.
Mentions: Since SK-OV-3 and Caov-3 cells were found to produce and secrete both factor H and FHL-1 into their growth supernatants we wanted to study whether they were capable of promoting the degradation of C3b to iC3b, i.e. whether the secreted proteins acted as cofactors for factor I. Cofactor activity analysis demonstrated that the supernatants of all the cell lines, including those of PA-1 and SW626, promoted factor I-mediated cleavage of 125I-labelled C3b (Figure 5Figure 5

Bottom Line: We observed that the soluble complement regulators factor H and factor H-like protein were abundantly present in ascites samples as well as in primary tumours of patients with ovarian cancer.In ascites samples the mean level of factor H-like protein (130+/-55 microg ml(-1)) was 5.5-fold higher than in normal human serum (24+/-3 microg ml(-1)).Subsequently, the PA-1 and SW626 cell lines were found to secrete a soluble form of the membrane cofactor protein (CD46).

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology and Immunology, Haartman Institute, University Central Hospital, FIN-0014 Helsinki, Finland.

Show MeSH
Related in: MedlinePlus