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Hausherr-Bohn, Amparo (2009): Cellular and Molecular Characterisation of Novel Lipopeptides with Anit-Myeloma Activities. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The pleiotropic cytokine interleukin-6 (IL-6) is one of the major growth factors for multiple myeloma cells. It was previously shown that IL-6 induces the activation of Src family kinases Hck, Lyn and Fyn and that Hck is associated with the IL-6-receptor beta chain (gp130) via an acidic domain in gp130. In the first part of this thesis the acidic domain is narrowed down from 41 to 18 amino acids by a peptide-based functional screening assay. A derivative of the acidic domain, an 18mer lipopeptide, peptide 18AD, is characterised on the cellular and molecular level. IL-6-dependent growth of human and murine myeloma cells is inhibited with an IC50 of 25-30 µM by the addition of peptide 18AD to the growth medium. These cells remain unaffected by treatment with a control peptide with scrambled sequence (18sc). Furthermore, growth of IL-6-independent myeloma cells is not inhibited by peptide 18AD. In IL-6-dependent cells, peptide 18AD causes the same degree of apoptosis induction as IL-6 deprivation. On the molecular level it is shown by peptide competition assays that the association of Hck and gp130 is inhibited by peptide 18AD in a concentration dependent way. Peptide 18AD blocked the IL-6-induced activity of the Src family kinases Hck, Lyn and Fyn. Results from different factor-dependent cell lines demonstrate a common mechanism of the molecular peptide effects on Src family kinase activity, but the involved pathways downstream of the kinases appear to differ among species and cell lines tested. Treatment of human IL-6-dependent myeloma cells with peptide 18AD reduced the activating tyrosine phosphorylation of the signal transducer and activator of transcription 3 (STAT3), while STAT3 activation remains unaffected in murine cells. The Co-immunoprecipitations from different overexpressed receptor-deletionmutants and Hck confirms that the association is mainly due to the interaction between the kinase and a 9 amino acids spanning region within the acidic domain which carries the highest accumulation of acidic residues. Apparently, a sequence of 8-9 amino acids within gp130 with the prevalence of acidic side chains resembles a potential pseudosubstrate domain of tyrosine kinases and is responsible for localisation and activation of Src family kinases in response to receptor stimulation. The identification of sequence motives similar to the acidic domain of gp130 in other cytokine receptors, receptor tyrosine kinases and adapter proteins by an in silicio motive scan might suggest a general role of such motives. This could be the efficient recruitment of cytoplasmic kinases to signalling complexes at the time of ligand stimulation. Here, the importance of the acidic domain-kinase interaction for the IL-6-signaling pathway is shown by the growth-inhibiting effect of peptide 18AD on myeloma cells. In the second part, a novel sequence and celltype-specific anti-myeloma agent, peptide 1A, is characterised. It was initially designed as the negative control for a "reverse alanine scan" to define the role of the acidic residues within the sequence of peptide 18AD. Unexpectedly it turned out to be at least a 25-fold more potent growth inhibitor of myeloma cells than peptide 18AD. Similar molecular bases of the observed effects of peptide 18AD and peptide 1A are very unlikely, because in contrast to peptide 18AD, peptide 1A efficiently kills IL-6-independent myeloma cells as well. Moreover, an excess of IL-6 fails to rescue the cells from peptide 1A-induced cell death. Peptide 1A shows, as tested so far, no effects on cells derived from non-tumor tissue or tumor cells of various origins other than multiple myeloma. Peptide 1A specifically kills myeloma cells by the induction of apoptosis and severely disturbs cell cycle progression. Apoptotic cell death induction by peptide 1A is shown by peptide 1A-induced caspase-3 activation and the cleavage of PARP, a substrate of effector caspases. Peptide-induced cell death can partly be inhibited by co-treatment with the pan-caspase inhibitor ZVAD-fmk. Major survival pathways like the STAT3, PI3K/Akt and the MAPK pathway are inhibited in peptide 1A treated cells. If a biotinylated version of the peptide is added to the growth medium, it is incorporated into human myeloma cells and localised in defined regions of the cytoplasm of myeloma cells. Here some of the molecular mechanisms of peptide 1A-induced cell death are elucidated. However, the direct molecular target(s) are still unknown. Despite the potential difference in the molecular mechanisms, both peptides 18AD and 1A are expected to prove useful for developing derivatives with possible applications for the treatment of multiple myeloma.