Proteases and their inhibitors as prognostic factors for high-grade serous ovarian cancer.

Ovarian carcinoma is one of the most lethal malignancies, but only very few prognostic biomarkers are known. The degradome, comprising proteases, protease non-proteolytic homologues and inhibitors, have been involved in the prognosis of many cancer types, including ovarian carcinoma. The prognostic significance of the whole degradome family has not been specifically studied in high-grade serous ovarian cancer. A targeted DNA microarray known as the CLIP-CHIP microarray was used to identify potential prognostic factors in ten high-grade serous ovarian cancer women who had early recurrence (<1.6 years) or late/no recurrence after first line surgery and chemotherapy. In women with early recurrence, we identified seven upregulated genes (TMPRSS4, MASP1/3, SPC18, PSMB1, IGFBP2, CFI - encoding Complement Factor I - and MMP9) and one down-regulated gene (ADAM-10). Using immunohistochemistry, we evaluated the prognostic effect of these 8 candidate genes in an independent cohort of 112 high-grade serous ovarian cancer women. Outcomes were progression, defined according to CA-125 criteria, and death. Multivariate Cox proportional hazard regression models were done to estimate the associations between each protein and each outcome. High ADAM-10 expression (intensity of 2-3) was associated with a lower risk of progression (adjusted hazard ratio (HR): 0.51; 95% confidence interval (CI): 0.29-0.87). High complement factor I expression (intensity 2-3) was associated with a higher risk of progression (adjusted HR: 2.30, 95% CI: 1.17-4.53) and death (adjusted HR: 3.42; 95% CI: 1.72-6.79). Overall, we identified the prognostic value of two proteases, ADAM-10 and complement factor I, for high-grade serous ovarian cancer which could have clinical significance.

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence.

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (

Non-small cell lung cancer cells produce a functional set of complement factor I and its soluble cofactors.

The complement system is important for protection from invading pathogens, removal of waste products and guidance of the immune response. Furthermore, complement can be also targeted to cancer cells. However, membrane-bound inhibitors over-expressed by certain types of tumor cells restrict the cytotoxic activity of complement. Herein we report that non-small cell lung cancer (NSCLC) cells produce soluble complement inhibitors factor I (FI) and C4b-binding protein (C4BP). FI is a serine protease capable of degrading the activated complement components C3b and C4b, whilst C4BP acts as its cofactor. Furthermore, NSCLC cells express membrane-bound regulators and shed membrane cofactor protein (MCP), which shares cofactor function with C4BP. Secretion of FI from NSCLC cells was higher than previously reported for any non-hepatic source and FI produced by these cells could efficiently support cleavage of C3b and C4b. In vitro functional assays revealed that additional FI significantly decreased C3 deposition and complement-dependent lysis, particularly when cofactors were added. Our results demonstrate that soluble inhibitors produced by NSCLC cells may provide further protection from complement beyond the level ensured by membrane-bound inhibitors and, as such, contribute to the aggressive phenotype of these lung cancer cells.

Human keratinocytes produce the complement inhibitor factor I: Synthesis is regulated by interferon-gamma.

Extrahepatic complement synthesis is believed to play an important role in host defense and inflammation at tissue and organ level. In the epidermis the most abundant cell type, keratinocytes have been shown to produce C3, factor B and factor H. In the present study, we investigated the synthesis of factor I by human keratinocytes. We also studied whether proinflammatory cytokines IL-1alpha, IL-6, TGF-beta1, TNF-alpha and IFN-gamma regulate factor I synthesis in keratinocytes. Human keratinocytes constitutively expressed factor I mRNA and produced factor I protein. Amongst the above-mentioned cytokines, only IFN-gamma regulated the synthesis of factor I, and this effect occurred predominantly at pre-translational level. Factor I produced by keratinocytes was functionally active in cleaving C3b. In conclusion, we demonstrate that keratinocytes are capable of synthesizing factor I, and that this synthesis is regulated by IFN-gamma.

Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome.

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy with manifestations of hemolytic anemia, thrombocytopenia, and renal impairment. Genetic studies have shown that mutations in complement regulatory proteins predispose to non-Shiga toxin-associated HUS (non-Stx-HUS). We undertook genetic analysis on membrane cofactor protein (MCP), complement factor H (CFH), and factor I (IF) in 156 patients with non-Stx-HUS. Fourteen, 11, and 5 new mutational events were found in MCP, CFH, and IF, respectively. Mutation frequencies were 12.8%, 30.1%, and 4.5% for MCP, CFH, and IF, respectively. MCP mutations resulted in either reduced protein expression or impaired C3b binding capability. MCP-mutated patients had a better prognosis than CFH-mutated and nonmutated patients. In MCP-mutated patients, plasma treatment did not impact the outcome significantly: remission was achieved in around 90% of both plasma-treated and plasma-untreated acute episodes. Kidney transplantation outcome was favorable in patients with MCP mutations, whereas the outcome was poor in patients with CFH and IF mutations due to disease recurrence. This study documents that the presentation, the response to therapy, and the outcome of the disease are influenced by the genotype. Hopefully this will translate into improved management and therapy of patients and will provide the way to design tailored treatments.

Complement factor I is upregulated in rat hepatocytes by interleukin-6 but not by interferon-gamma, interleukin-1beta, or tumor necrosis factor-alpha.

Complement factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b and thus prevents the assembly of the C3 and C5 convertases. We have investigated the proinflammatory cytokines IL-6, IL-1beta, TNF-alpha and IFN-gamma for their potential role in the regulation of FI expression. Of the investigated cytokines, only IL-6 increased the FI-specific RT-PCR signal in isolated hepatocytes, in the two rat hepatoma-derived cell lines FAO and H4IIE or in HUVECs. Quantitative competitive RT-PCR showed an IL-6 induced upregulation of FI-specific mRNA by about ten-fold. These data are in accord with Northern blot analyses in which the FI-mRNA was upregulated by IL-6 between five- and seven-fold. IL-6, but not IL-1beta, TNF-alpha or IFN-gamma also increased FI-protein levels in cell culture supernatants by about five-fold as determined by a semiquantitative immunoblot using a novel monoclonal antibody specific for rat FI.

Expression and regulation of complement factors H and I in rat and human cells: some critical notes.

The complement factors I (FI) and H (FH) are complement regulatory proteins. FI, a highly glycosylated serine protease of 88 kDa cleaves the alpha-chains of both complement components C3b and C4b, thereby inactivating them. Complement FH, a glycoprotein of 150 kDa which is composed of 20 short consensus repeats synergizes with FI by increasing the affinity of FI for C3b in the C3b/FH complex by about 15-fold as compared to free C3b. Furthermore, FH prevents factor B from binding to C3b and promotes the dissociation of the C3bBb complex. Both, FI and FH are mainly synthesized in the liver. According to the quantification of specific mRNA of both factors, various amounts are produced by different liver cell types, i.e. hepatocytes (HC) and Kupffer cells (KC). Investigations of cultured primary HC and KC from rat liver showed that FI is exclusively synthesized and secreted by HC whereas FH is synthesized by both HC and KC. Using quantitative-competitive PCR for the quantification of FH-specific mRNA, its constitutive rate of synthesis was found to be nearly ten times higher in KC than in HC. An extrahepatic source of both proteins are human umbilical vein endothelial cells (HUVEC) in which the synthesis of FI is upregulated by IL-6 which is in accord with the upregulation observed in rat HC and two rat hepatoma cell lines (FAO and H4IIE). Three other proinflammatory cytokines, IL-1beta, IFN-gamma and TNF-alpha, were alone or in combination, without any effect on the regulation of FI. This demonstrates that the regulation of FI is similar in HUVEC and HC. These results are in contrast to a previously described IFN-gamma-mediated upregulation of FI in HUVEC and suggest, in accordance with other investigations on extrahepatic sources of FI (e.g. myoblasts), that IFN-gamma has probably no prominent role in the regulation of FI. Instead, IL-6 appears to be the main upregulating cytokine of FI mRNA and of FI protein synthesis in HC as well as in rat and human hepatoma cells and in HUVEC. Of note are experiments by others and us who could not identify FI-specific mRNA in peripheral blood-derived monocytes, granulocytes, or B- and T-cells of man or rat and in rat peritoneal macrophages. FI-specific mRNA could also not be detected in B- or T-cell lymphoma cells, whereas FH-specific mRNA was easily detectable in both human and rat monocytes, and in rat peritoneal macrophages. These data support the notion that FI in contrast to FH is not expressed by cells of the monocyte-macrophage lineage or by other leukocytes of peripheral blood, at least in the absence of additional stimulants.

Transcriptional and post-transcriptional regulation of complement factor I (CFI) gene expression in Hep G2 cells by interleukin-6.

We have investigated the effects of IL-1 and IL-6 on human complement factor I (CFI) production by Hep G2 cells. IL-6 treatment caused a dose- and time-dependent increase in CFI secretion while IL-1 did not demonstrate such effects. The increase in CFI synthesis correlated with increase in CFI mRNA levels. The half-life of CFI mRNA in untreated cells was approx. 23 h and this was increased to 31 h (26% increase) following induction with IL-6. The IL-6 induced increase in CFI gene expression was inhibited by actinomycin D indicating regulatory effects at the level of transcription. Nuclear run-on experiments showed that IL-6 increased the rate of CFI gene transcription 4.2-fold. Transient transfection analysis of chloramphenicol acetyltransferase reporter gene constructs containing truncated segments of the 5'-flanking region of CFI gene showed that the cis-acting sequence(s) controlling the IL-6 inducible transcription resides in an 83 bp region located between -738 bp and -655 bp relative to the transcription start site. Our results indicate that the upregulation of CFI gene expression by IL-6 involves a coordinate effort at the level of transcription and mRNA stability, with the enhanced rate of transcription being the principal mechanism.

Human complement factor I: its expression by insect cells and its biochemical and structural characterisation.

Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.

Expression of the complement alternative pathway by human myoblasts in vitro: biosynthesis of C3, factor B, factor H and factor I.

In this study, we demonstrate expression in vitro of complement alternative pathway components C3, factor B, factor H and factor I by normal human myoblasts and human rhabdomyosarcoma cell lines CRL1558 and HTB153. Proteins in culture supernatants were detected by Western (protein) blot analysis and biosynthetic labeling followed by immunoprecipitation experiments, and quantified by ELISA. Newly secreted proteins were structurally and functionally similar to their serum counterparts. An additional polypeptide of 43 kDa with factor H immunoreactivity was detected, which could correspond to the N-terminal truncated form found in plasma. Protein expression was correlated with mRNA expression by reverse transcriptase-polymerase chain reaction analysis. The major proteins of complement alternative pathway C3, factor B and factor H were produced constitutively by skeletal muscle cells at a rate of 50 to 150 ng/10(6) cells/ml and factor I was expressed 20 ng/10(6) cells/ml. These syntheses in vitro were regulated by inflammatory cytokines. Interferon-gamma significantly upregulated C3, factor B and factor H expression, but had no effect on factor I production. Interleukin-1 beta strongly enhanced C3 and factor B production and had a weak enhancing or no effect on factor I and factor H secretion. Human myoblast cell lines constitute an interesting model to analyze complement biosynthesis by human skeletal muscle cells. Local complement expression by skeletal muscle in vivo may be implicated in some muscular inflammatory or pathological processes.

Processing of human factor I in COS-1 cells co-transfected with factor I and paired basic amino acid cleaving enzyme (PACE) cDNA.

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed form of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.

Characterization of Xenopus laevis complement factor I structure--conservation of modular structure except for an unusual insert not present in human factor I.

Factor I (C3b/C4b inactivator) is a regulatory protein of the classical and alternative complement pathways. In this paper, we report the sequence of Xenopus factor I cDNA and the deduced protein structure. The basic structure of human preprofactor I, NH2-heavy chain-cleavage peptide-light chain-COOH, is conserved in the frog. However, the frog heavy chain contains a highly charged segment of 29 amino acids, encoded by a poly dA-rich mRNA insert, which is not found in human factor I. The modular structure of the frog heavy chain was analyzed, and found to differ vis-à-vis previously published analyses of human factor I. We also evaluate the timing of factor I transcription during frog embryogenesis.

Complement factors H and I synthesized by B cell lines function to generate a growth factor activity from C3.

B lymphocytes and transformed B lymphoblastoid cell lines express CR2 (CD21, C3d/EBV-receptor) that is specific for C3 fragments generated by cleavage of C3b or spontaneously hydrolyzed native C3 (C3i) by the serum enzyme factor I and its cofactor, factor H. It had been shown previously that the Raji B cell line could be cultivated in serum-free medium supplemented with only transferrin and either OKB7 anti-CR2 mAb, C3d, or C3d-derived peptides containing the CR2 binding site. Because these agents appeared to function through ligation of CR2, it was unclear how native C3 could also serve as a growth factor, because C3 does not bind to CR2. It appeared possible that Raji cells might be able to use endogenous factors H and I to generate a CR2 ligand from C3, because previous studies had shown that Raji cells synthesized factor H and probably also synthesized factor I. PCR analysis was used to demonstrate factor I mRNA in Raji cells. Secretion of Raji cell factor I protein was confirmed by a sensitive mAb ELISA. Several B cell lines were examined for C3-dependent growth. Raji cells required both C3 (or OKB7) and transferrin for growth, whereas Wil-2 cells grew with transferrin alone and C3 enhanced the growth-promoting activity of transferrin. Two other B cell lines (Daudi and U698M), the T cell line 8402, and the U937 monocytoid cell line could not be sustained with transferrin plus C3. The C3-dependent growth of Raji cells was inhibited almost completely by either OX-23 anti-factor H or 052.11.3 anti-factor I mAb that also blocked the activity of serum-derived factor H or I, respectively. By contrast, there was no inhibition of growth by either OX-24 anti-factor H or OX-21 anti-factor I mAb that did not block factors H and I activity. After the spontaneous hydrolysis of native C3 to C3i, it is hypothesized that Raji cells convert C3i to iC3i with endogenous factors H and I, and then this iC3i serves as a growth factor by binding to membrane CR2.