Production and characterization of monoclonal and polyclonal antibodies to human alpha 2-HS: development of a two-site ELISA test.

A convenient and sensitive indirect sandwich ELISA test was developed for measuring both 63 kDa human alpha 2-HS secreted by human hepatoma cell lines and the 59 kDa alpha 2-HS species present in serum/plasma. Monoclonal and rabbit antibodies to plasma alpha 2-HS were produced and selected by immunoprecipitation techniques using iodinated alpha 2-HS or 35S-labeled alpha 2-HS. Various monoclonal antibodies recognizing both forms of the protein were coated onto microtiter plates and after binding of alpha 2-HS, biotinylated monoclonal antibodies with compatible binding or biotinylated immunopurified F(ab')2 fragments from the rabbit antiserum were added and subsequently revealed with avidin-biotin peroxidase complex. Formats using a rabbit detector antibody were the most sensitive and one was selected for the whole study. The test developed was capable of detecting plasma alpha 2-HS devoid of connecting peptide and HepG2 hepatoma cell line derived alpha 2-HS at the ng/ml level. The test has been used to measure levels of alpha 2-HS in both serum and supernatants from HepG2 cell lines.

Characterization of a cDNA clone coding for human testis membrane cofactor protein (MCP, CD46).

Membrane cofactor protein (MCP) is a complement regulatory protein that acts as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. We have previously reported the characterization of a functional MCP molecule on the acrosomal membrane. This protein migrated as a single band with a molecular weight of 40,000 Da, which is 10,000-20,000 Da smaller than the known MCP molecules, and is devoid of N- and O-linked sugars. We have proposed that the difference in molecular weight resulted from the lack of sugars. To investigate if this is due to the absence of glycosylation sites, we have characterized a cDNA clone from a human testis cDNA library. This cDNA corresponds to a peculiar MCP form previously described, which is characterized by the presence of the serine/threonine/proline-rich exon C (STPC) and the cytoplasmic tail known as CYT2, and we conclude that the absence of mature oligosaccharide of the sperm MCP cannot be totally attributed to a defect of N- and O-glycosylation sequences but rather reflects an alteration of the mechanisms of glycosylation in spermatozoa. The presence of functional MCP on the acrosomal membrane, as well as the other complement regulatory protein, decay-accelerating factor, strongly suggests that these proteins may act concomitantly to protect the acrosome-reacted spermatozoa from the attack of the complement present in the female genital tract.

Insulin and interleukin-1 differentially regulate pp63, an acute phase phosphoprotein in hepatoma cell line.

We have reported previously that the phosphoprotein pp63, an acute phase protein, which has been recently identified as the rat fetuin, was capable of blocking the mitogenic effect of insulin on the rat Fao hepatoma cell line, without affecting metabolic effects of the hormone. Only the phosphorylated form of the protein has been shown to exhibit both anti-tyrosine kinase and growth inhibitory properties. In this study, we used the FTO-2B rat hepatoma cell line to analyze the mechanisms involved in the control of synthesis and/or phosphorylation of pp63. For this purpose, we investigated the action of effectors known to modulate hepatic functions, such as cytokines (interleukin (IL)-1 beta and IL-6), which regulate the production of acute phase proteins, and insulin, which elicits profound effects on hepatocyte metabolism. Here, we demonstrate that IL-1 beta diminished markedly the pp63 production by affecting its mRNA transcription and that the cytokine was able to modify the N-glycosylation process of the protein. In contrast, insulin did not affect the biosynthesis of pp63 but dramatically decreased its extent of phosphorylation.

Metalloproteinases in multiple myeloma: production of matrix metalloproteinase-9 (MMP-9), activation of proMMP-2, and induction of MMP-1 by myeloma cells.

Multiple myeloma is a very devastating cancer with a high capacity to destroy bone matrix. Matrix metalloproteinases (MMPs) play a critical role in bone remodeling and tumor invasion. In this study, we have investigated the involvement of interstitial collagenase (MMP-1) and gelatinases (MMP-2 and MMP-9) in the biology of multiple myeloma. We show (1) that myeloma cells express MMP-9 and (2) that this expression is not subjected to regulation either by interleukin-6 (IL-6), the major myeloma cell growth factor, or by other cytokines involved in the multiple myeloma cytokine network. In the tumoral environment, we show that bone marrow stromal cells express MMP-1 and MMP-2. Whereas MMP-1 is positively regulated by IL-1beta, tumor necrosis factor-alpha, and Oncostatin M, MMP-2 is not modulated by any of these cytokines. To evaluate whether myeloma cells can modify the bone marrow stromal environment, we have examined these MMP activities in coculture. Interestingly, we have observed an upregulation of MMP-1 and a partial conversion of the proMMP-2 into its activated form. We conclude that the increase of MMP activity produced or induced by myeloma cells in these cocultures could favor bone resorption and tumor invasion. Inhibition of such activities could represent a new therapeutical approach in multiple myeloma.