Multiple Myeloma: A Study of the Factors That Control Death and Cell Survival in Neoplastic Plasma Cells

Livingston, Margo Mhairi (1996) Multiple Myeloma: A Study of the Factors That Control Death and Cell Survival in Neoplastic Plasma Cells. PhD thesis, University of Glasgow.

Abstract

Since malignant plasma cells in myeloma patients home to the bone marrow, a study was undertaken firstly to define the phenotypic profile of a panel of myeloma cell lines and then to investigate their ability to adhere to various extracellular matrix molecules. The extracellular matrix molecules studied were- collagen, fibronectin and FN-RGD, a synthetic peptide consisting of multiple arginine-glycine-aspartic acid (RGD) repeats which mimics the part of the fibronectin molecule that binds to the integrin VLA-5, expressed on some myeloma cells. Adhesion blockade was attempted using monoclonal antibodies directed against various myeloma surface antigens in order to ascertain the importance or otherwise of these molecules in the binding of myeloma cells to members of the extracellular matrix. It was found that, although the panel of eight myeloma cell lines tested all expressed VLA-4 and showed a varying expression of VLA-5, their ability to adhere to fibronectin differed. None of the cell lines tested adhered to FN-RGD or collagen. Adhesion blockade using anti- VLA-4 and anti-VLA-5 antibodies was only successful in half of the lines tested with a combination of the two antibodies resulting in improved blockade compared with either antibody alone. It has been demonstrated that CD40 crosslinking on the surface of B cells and myeloma cells rescues them from apoptosis and induces IL-6 secretion. This is usually, but not always, accompanied by up-regulation of the hcI-2 gene. Since the Fas antigen has homology with CD40 and has been reported to act in opposition to bcl-2, it was decided to study these genes for mRNA and protein expression in myeloma cells in an effort to elucidate any potential link between expression of these antigens and the onset of multiple myeloma. A more complete knowledge of the interactions between IL-6, CD40 and its ligand, and the expression of hcl-2 or Fas and its ligand is necessary in order to determine possible improvements in treatment strategies for this disease. The panel of eight myeloma cell lines used in this study was examined for CD40 and Fas antigen expression by flow cytometric analysis. All the myeloma cell lines tested positive for CD40 and Fas albeit at different intensities. It has been shown that resting tonsil B cells, expressing either low or absent levels of Fas, are induced to express Fas after ligation of CD40 using the CD40Lig-L culture system. Anti-Fas monoclonal antibody was shown to inhibit the later phases of CD40-induced B cell growth due to apoptosis. Fas ligation was shown to inhibit proliferation and immunoglobulin secretion of CD40 activated B cells in response to recombinant cytokines. This implies that engagement of CD40 antigen on B cells induces Fas expression and sensitises them to Fas-mediated apoptosis. It was decided, therefore, to investigate whether myeloma plasma cells could be stimulated to proliferate in the same manner and whether they, too, would be sensitive to Fas-mediated apoptosis. It was noted from the literature that the myeloma cell lines resistant to apoptosis were mostly dependent on IL-6 and it was therefore proposed to investigate this further by broadening the study by looking at several myeloma cell lines and comparing IL-6 dependent and independent passages of the same myeloma cell line (ANBL-6) for their susceptibility to Fas-mediated apoptosis. The fact that fresh myeloma samples were also resistant to activation induced cell death (AICD) is interesting since these are likely to be dependent on IL-6 for their growth also. It has previously been reported that Fas antigen is not expressed on normal plasma cells. Two culture systems originally established in an effort to generate factor-dependent B cell lines were adapted for the study of myeloma cell activation of proliferation via CD40 (and the cytokines IL-4 and IL-6) and induction of apoptosis via Fas activation. Although myeloma cell lines are obviously no longer dependent on stromal layers for their propogation it was decided that the use of the CD40 and CD40Lig-L culture systems may potentiate the response to CD40 activation. Cross- linkage of this antigen on the surface of myeloma cells using mouse fibroblasts transfected with either the human immunoglobulin Fc receptor (FcyRII/CDw32) or with the human CD40 ligand constitute the CD40 and CD40Lig-L culture systems respectively. Interleukin-4 was used to test for proliferation as a comparison to interleukin-6 in these studies since it is a potent B cell stimulation factor. It was initially attempted to establish a fast, reliable, cost-effective and informative method of assessing apoptosis and then to develop an effective assay system for Fas and CD40 activation in myeloma cell lines. Finally, a comprehensive study was undertaken to examine, in these cell lines, the role of activation of CD40 and/or Fas with or without co-stimulatory cytokines (IL-6 and IL-4).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Ian Franklin
• Keywords: Medicine, Oncology
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-75850
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 17:43
• Last Modified: 19 Nov 2019 17:43
• URI: https://theses.gla.ac.uk/id/eprint/75850