Università degli Studi di Milano, 2011-01-17

The growth and survival of multiple myeloma (MM) cells in the bone marrow microenvironment is regulated by functional complex interactions between the tumor cells and the surrounding bone marrow stromal cells mediated by adhesion molecules and the production of several cytokines of which interleukin-6 (IL-6) has been identified as the most important. Major advances in the investigation of MM biology were made possible by the availability of human myeloma cell lines (HMCLs). The IL-6-dependent CMA-03 cell line was established in our laboratory from a peritoneal effusion of a refractory relapsed MM patient. By gradually decreasing the IL-6 added to the culture, an IL-6-independent variant, CMA-03/06, could be obtained. The aim of this study was to perform a biological and molecular characterization of this novel cell line and to provide insights into the signaling pathways and target genes involved in the growth and survival of CMA-03/06. The growth, immunophenotypic, cytogenetic and fluorescence in situ hybridization (FISH) characterization of CMA-03/06 cell line was performed by means of standard procedures. IL-6 production into the culture media was determined using a high sensitivity IL-6 specific ELISA. Genome-wide profiling data were generated by means of Affymetrix GeneChip Human Mapping 250K NspI arrays; copy number (CN) alterations were calculated using the DNAcopy Bioconductor package, based on circular binary segmentation method. Global gene expression profiling (GEP) was performed by means of the GeneChip Human Gene 1.0 ST Arrays (Affymetrix); the supervised analyses were done using the rank Prod method and a 2 fold change cut off. The immunophenotypic analysis of the two cell lines showed a significant difference in the expression of three antigens: CD45 was considerably reduced in CMA-03/06 cells, whereas they were found positive for both chains of IL-6 receptor, CD126 and CD130, almost undetectable in CMA-03. Conventional cytogenetic and FISH analyses did not reveal differences between the 2 HMCLs. Unlike CMA-03, the addition of IL-6 to the culture medium of CMA-03/06 cells or co-culture with multipotent mesenchymal stromal cells did not induce an increase in CMA-03/06 proliferation. IL-6 was not detected in the supernatants from either CMA-03 or CMA-03/06 cell lines within 48 h, suggesting that the IL-6 independence of CMA03/06 cells is not a result of the development of an autocrine IL-6 loop. Nevertheless, IL-6 induced the activation of STAT3 and STAT1 in both cell lines, even if a slight constitutive STAT3 phosphorylation was found in CMA-03/06. The genome-wide analysis allowed the identification of about 100 altered chromosomal regions common to both HMCLs, mostly DNA gains. Comparison of CMA-03/06 and CMA-03 cells evidenced a different CN in only 15 small chromosomal regions, 8 of which did not contain any transcript, whereas few genes were located on the other ones. GEP analysis of CMA-03/06 compared with CMA-03 identified 140 upregulated and 168 downregulated genes, many of which particularly relevant for MM biology, mainly involved in cellular signaling, cell cycle, cell adhesion, cell development, regulation of transcription, immunologic, inflammatory or defense activity, cytokine receptor activity, apoptosis. None of the genes differentially expressed in CMA-03/06 compared with CMA-03 except 2 were positioned on the chromosomal regions showing a different CN. Finally, CMA-03/06 cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA-03 cells. The novel CMA03/06 cell line may thus represent a suitable model for studies investigating molecular mechanisms involved in clonal evolution towards IL-6 and/or stroma-independent growth and survival of myeloma cells.

diritti: info:eu-repo/semantics/closedAccess
tutor: Antonino Neri ; coordinatore: Paolo Corradini
Settore MED/15 - - Malattie del Sangue

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD16000224