Carina Seidel

Serglycin Constitutively Secreted by Myeloma Plasma Cells Is a Potent Inhibitor of Bone Mineralization in Vitro

Although the biological significance of proteoglycans (PGs) has previously been highlighted in multiple myeloma (MM), little is known about serglycin, which is a hematopoietic cell granule PG. In this study, we describe the expression and highly constitutive secretion of serglycin in several MM cell lines. Serglycin messenger RNA was detected in six MM cell lines. PGs were purified from conditioned medium of four MM cell lines, and serglycin substituted with 4-sulfated chondroitin sulfate was identified as the predominant PG. Flow cytometry and confocal microscopy showed that serglycin was also present intracellularly and on the cell surface, and attachment to the cell surface was at least in part dependent on intact glycosaminoglycan side chains. Immunohistochemical staining of bone marrow biopsies showed the presence of serglycin both in benign and malignant plasma cells. Immunoblotting in bone marrow aspirates from a limited number of patients with newly diagnosed MM revealed highly increased levels of serglycin in 30% of the cases. Serglycin isolated from myeloma plasma cells was found to influence the bone mineralization process through inhibition of the crystal growth rate of hydroxyapatite. This rate reduction was attributed to adsorption and further blocking of the active growth sites on the crystal surface. The apparent order of the crystallization reaction was found to be n = 2, suggesting a surface diffusion-controlled spiral growth mechanism. Our findings suggest that serglycin release is a constitutive process, which may be of fundamental biological importance in the study of MM.

Prognostic evaluation in multiple myeloma: an analysis of the impact of new prognostic factors.

We have analysed the prognostic information for survival of presenting features in an unselected series of 394 myeloma patients. 15 variables with significant prognostic information were identified, among these were some not previously or only recently reported: serum levels of hepatocyte growth factor (HGF), interleukin‐6 (IL‐6), C‐terminal cross‐linked telopeptide of collagen I (ICTP) and soluble interleukin‐6 receptor (sIL‐6R). In a multivariate Cox analysis six variables were significantly and independently associated with poor survival: high age, low W.H.O.‐performance status (PS), high serum levels of calcium, β‐2‐microglobulin (β‐2M), IL‐6 and sIL‐6R. A risk score formed to predict survival for each percentile of the patient population allowed an efficient separation of prognostic groups. The discriminating power of the model compared favourably with three other previously published staging systems applied to the study population. Exclusion of IL‐6 and sIL‐6R from the model only marginally decreased the efficacy of the separation. The predictive value of some variables (sIL‐6R, β‐2M and W.H.O.‐PS) decreased significantly over time. We conclude that formation of a risk score based on independent variables is an efficient way to separate prognostic groups, that the contribution of new and not easily available parameters should be thoroughly evaluated before inclusion in prognostic models for clinical use and that the predictive value of parameters may decrease over time.

Hepatocyte growth factor in myeloma patients treated with high-dose chemotherapy.

Summary. Hepatocyte growth factor (HGF) is a cytokine produced by myeloma cells. We examined serum HGF levels in a population of young myeloma patients (median age 52 years) treated with high‐dose chemotherapy. Sera from 128 myeloma patients at diagnosis and serial samples from 16 patients were analysed. Compared with 62 healthy controls, HGF was elevated at diagnosis in 25% of patients (median 0·48 and 1·08 ng/ml respectively; P < 0·0001). The 95 patients who completed therapy were analysed for the impact of HGF on survival. Median survival was not reached after 77 months in the patient group with normal HGF values (< 1·7 ng/ml, n = 69). In the group with elevated HGF (≥ 1·7 ng/ml, n = 26), median survival was 63 months (P = 0·08). In 16 patients, serum was drawn at diagnosis and at the time of expected disease remission (6 weeks to 3 months after chemotherapy). HGF values declined after treatment in 14 of these patients, from a median of 0·9 ng/ml (0·49–1·65) to 0·42 ng/ml (0·32–0·73) (P = 0·005). Our results show that in young myeloma patients HGF is elevated, and that patients with higher levels had a trend towards poorer prognosis. Treatment with high‐dose chemotherapy reduced HGF in the serum of the majority of patients.

The role of hepatocyte growth factor and its receptor c-Met in multiple myeloma and other blood malignancies.

The cytokine hepatocyte growth factor (HGF) and its receptor c-Met are a ligand-receptor pair with important functions in a communicative interplay between HGF-producing, mesenchymal cells and c-Met-expressing target cells. HGF is cytoprotective and causes regeneration of parenchyma after tissue damage in several organs. The receptor c-Met was first characterized as an oncogene product being responsible for the transformation of an osteosarcoma cell line. HGF or c-Met is overexpressed in several human cancers, including various carcinomas. Some cells of hematopoietic origin also seem to be capable of c-Met expression, but the precise role of HGF in normal hematopoiesis is yet to be determined. In blood malignancies like acute myelogenous leukemia and, notably, multiple myeloma, HGF is overproduced and has implications for the prognosis of the patients. Biological significance of HGF overexpression in multiple myeloma is discussed and is likely to include effects on bone turnover and angiogenesis.

Expression of urokinase plasminogen activator and the urokinase plasminogen activator receptor in myeloma cells.

Binding of urokinase (uPA) to its receptor (uPAR; CD87) focuses proteolytic activity on the cell surface and this system is of importance in malignant matrix degradation and tumour invasion. By immunocytochemistry and flow cytometry, we found that primary myeloma cells and myeloma cell lines expressed uPA and uPAR. Soluble uPA was present in cell line supernatants and lysates in low concentrations. In cell lines, uPA and uPAR were located both on the cell surface and intracellularly, but the expression of both proteins was low. Higher levels of uPAR was detected on the cell surface of primary myeloma cells. When primary myeloma cells were gated by CD45 expression, stronger expression was found on immature CD45+ cells than on mature CD45−/dim cells. Finally, both myeloma cell lines and primary cells were able to cleave a uPA‐specific substrate showing that the uPA system is functionally active. We conclude that myeloma cells are able to produce uPA and uPAR. This opens up a possible role of the uPA system in myeloma cell invasion and in the proteolytic digestion of bone matrix.

Apoptosis, proliferation and NF‐κB activation induced by agonistic Fas antibodies in the human myeloma cell line OH‐2: amplification of Fas‐mediated apoptosis by tumor necrosis factor

Abstract: Tumor necrosis factor (TNF) is known to be a growth factor for several myeloma cell lines. However, in the presence of the agonistic Fas antibody CHI 1, TNF enhanced the level of apoptosis in cultures of the human myeloma cell line OH‐2. This pro‐apoptotic effect of TNF was explained at least in part by a TNF‐mediated enhancement of Fas expression. TNF induces proliferation of OH‐2 by activating nuclear transcription factor kappa‐B (NF‐κB). The proliferative effect of TNF on OH‐2 cells was abrogated by CH11, but this was not caused by an inhibition of the translocation of NF‐κB. On the contrary, CH11 could by itself activate NF‐κB in OH‐2 cells, and in the presence of an inhibitor of caspase‐1 induce proliferation of the cells. The relationship between stimulation of TNF receptors and Fas and the level of NF‐κB activation was also examined in three other myeloma cell lines. RPMI‐8226 cells showed NF‐κB activation by TNF, but contrary to OH‐2, not by CHI 1. Unstimulated U‐266 and JJN‐3 cells had high levels of activated NF‐κB. This shows that NFκ‐B is either constitutively activated or inducible in myeloma cells. Modulation of Fas expression and inhibition of NF‐κB activation can potentially be of therapeutic importance in multiple myeloma.

Hepatocyte growth factor in serum after injection of unfractionated and low molecular weight heparin in healthy individuals

Soluble heparin displaces the cytokine hepatocyte growth factor (HGF) from heparan sulphate proteoglycans on the cell surface and in the extracellular matrix into the circulation. We examined serum HGF elevation after heparin injections, and whether there is a difference between unfractionated heparin (UH) and low molecular weight heparin (LMWH) in their ability to increase serum HGF. 20 healthy individuals were randomized to a single injection of intravenous or subcutaneous UH or LMWH. There was a significant increase in HGF from pretreatment values. This HGF was bioactive. When these preparations were compared on the basis of their serum concentrations (anti‐factor Xa activity or molar concentrations), the increase in HGF was greater in individuals receiving UH than LMWH. When UH or LMWH were administered over a 5 d period, the increase in HGF, as well as the difference between treatments to induce HGF, remained stable throughout the treatment. In five patients treated with continuous intravenous heparin infusion HGF was increased throughout the treatment period of 5–7 d. In summary, the rise in bioactive HGF after heparin treatment was stable during continued treatment. UH was more potent in inducing HGF increase than LMWH, both after a single injection and after several days of treatment.

Elevated serum concentrations of hepatocyte growth factor in acute myelocytic leukaemia

Abstract:  Serum concentrations of hepatocyte growth factor (HGF) were measured in 60 patients suffering from acute myelocytic leukaemia (AML). At the time of diagnosis elevated HGF concentrations (>1.25 ng/ml) were found in 28% of the patients. HGF levels correlated with the presence of disseminated intravascular coagulation (DIC), levels of lysozyme, creatinine, peripheral blood blast counts and lactic dehydrogenase. In the group of patients with high HGF (>1.25 ng/ml) we found a tendency towards an increased early mortality; 41 % of them died within 15 d from diagnosis, as opposed to 5% of the patients with normal HGF (log rank test p = 0.07). DIC‐related bleeding or thrombosis contributed to this early mortality. In responders, HGF levels normalized after treatment. HGF levels are low in neutropenia and neutropenic infections.

A sandwich ELISA for the estimation of human syndecan-2 and syndecan-4 in biological samples

Syndecans are integral membrane proteoglycans containing both chondroitin and heparan sulphate side chains. Syndecans are shed from the cell surface, and may be detected in cellular supernatant or body fluids. In this study, we wished to design ELISA methods for syndecan-2 and syndecan-4, with the future aim of analyzing syndecans in large number of patient materials. Briefly, sandwich ELISAs for human syndecan-2 and syndecan-4 were designed employing monoclonal mouse antibodies as capture and commercially available polyclonal rabbit anti-syndecan as secondary antibodies. Although no quantified standard was available, relative estimation of syndecan-2 and syndecan-4 levels was possible in a small pilot material. The paper presents the ELISA method for these proteoglycans. We suggest that this may be a useful tool in the analysis of patient materials and in the detection of syndecans during proteoglycan purification.