GDF-15 signalling in multiple myeloma
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Abstract Multiple myeloma (MM), also known as bone marrow cancer, is a malignancy of hypermutated antibody-producing plasma cells in the bone marrow. An accumulation of this antibody (M-protein) can be detected in blood- and/or urine samples, and is used for diagnosis of MM. The cause of this antibody overproduction is still unknown. Multiple myeloma is a malignancy that occurs most often among older adults, with a median age of diagnosis at 66 years and the incidence increases with the age. In recent years there were about 380 cases of MM registered per year in Norway. Patients diagnosed with this disease have an overall survival of 5-7 years, with the right treatment. Multiple myeloma is currently an incurable disease, and the treatment is thus given to prolong and improve quality of life. A typical hallmark of MM is a weaker bone structure, and osteolytic lesions represent a major problem in a majority of myeloma patients. Growth and differentiation factor-15 (GDF-15) is a signaling protein that has been shown to constitute a positive effect on the myeloma cells and a negative effect on the bone tissue. Even though GDF-15 exists at a low level in almost all tissues, little is clear about its biological functions. In contrast, it is clear that elevated levels of GDF-15 is associated with MM, which could be measured in serum. The aim of this thesis was to determine the pathway of GDF-15 and try to inhibit it. Studies of GDF-15 were performed in human myeloma cell lines with well-characterized transforming growth factor-β (TGF-β) receptor expression. Primary cells and a monocytic cell line were also studied. Proliferation studies were used to determine if GDF-15 affected proliferation or apoptosis of myeloma cell lines. Experiments were also performed to determine which receptors GDF-15 used for signaling. Based on these studies and former publications ALK5, TGFBR2 and betaglycan seemed to be important. Experiments concerning the downstream signaling pathway through SMADs were also performed. SMAD2 was found to be phosphorylated by GDF-15. However at a later stage, reconsidered, due to new results. The GDF-15 signaling pathway was showed inhibited by using anti-TGFBR2 and SB431542. In addition an effort was made to see if GDF-15 could compete with TGF-β1, since GDF-15 and TGF-β ligands seemed to have a similar signaling pathway. Unfortunately, it turned out that almost all batches of the recombinant GDF-15 protein used in this thesis were more or less contaminated with TGF-β from the manufacturer. In the latest experiments much purer batches of GDF-15 were used, and the conclusion was that GDF-15 did not activate SMADs and thus did not signal through the putative TGF-β receptors. This means that it is still open how GDF-15 affects cells. The results confirm previous knowledge about TGF-β and show the importance of knowing the quality of reagents that are used in biomedical research.