ABSTRACT: Biological and molecular characterization of CMA03/06, a newly established interleukin-6 independent variant of the CMA03 human myeloma cell line.
Project description:Biological and molecular characterization of CMA03/06, a newly established interleukin-6 independent variant of the CMA03 human myeloma cell line.
Project description:We performed a biological and molecular characterization of the novel human multiple myeloma cell line CMA03/06, an IL-6-independent variant of CMA03 cell line previously established in our Institution. We showed that the CMA03/06 cells grows in the absence of IL-6 with a doubling time comparable to CMA03; the addition of IL-6 to the culture medium or co-culture with multipotent mesenchymal stromal cells does not induce an increase in proliferation rate. Interestingly, we provided evidence that IL-6 independence of CMA03/06 cells is not a consequence of the development of an autocrine IL-6 loop, even though the cells maintain the IL-6 signaling pathway responsiveness as demonstrated by STAT1 and STAT3 induction by IL-6. A slight constitutive activation of STAT3 has been observed in CMA03/06 cell line; however STAT3 silencing in CMA03/06 cells did not affect their viability and proliferation suggesting that this pathway is not the only responsible for the IL-6 independency of CMA03/06 cell line. The new cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA03 cells, whereas an increased induction of apoptosis was observed in CMA03/06 cell line after Bortezomib treatment which may suggest the involvement NF-kB pathway in the IL-6 independent growth and survival of CMA03/06 cells. Finally, global gene expression profiling analysis allowed the identification of a list of 308 modulated genes in CMA03/06 versus CMA03 cells, many of which particularly relevant for MM biology. Overall, the novel CMA03/06 cell line may 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.
Project description:We performed a biological and molecular characterization of the novel human multiple myeloma cell line CMA03/06, an IL-6-independent variant of CMA03 cell line previously established in our Institution. We showed that the CMA03/06 cells grows in the absence of IL-6 with a doubling time comparable to CMA03; the addition of IL-6 to the culture medium or co-culture with multipotent mesenchymal stromal cells does not induce an increase in proliferation rate. Interestingly, we provided evidence that IL-6 independence of CMA03/06 cells is not a consequence of the development of an autocrine IL-6 loop, even though the cells maintain the IL-6 signaling pathway responsiveness as demonstrated by STAT1 and STAT3 induction by IL-6. A slight constitutive activation of STAT3 has been observed in CMA03/06 cell line; however STAT3 silencing in CMA03/06 cells did not affect their viability and proliferation suggesting that this pathway is not the only responsible for the IL-6 independency of CMA03/06 cell line. The new cell line showed a lower susceptibility to camptothecin-induced apoptosis compared to CMA03 cells, whereas an increased induction of apoptosis was observed in CMA03/06 cell line after Bortezomib treatment which may suggest the involvement NF-kB pathway in the IL-6 independent growth and survival of CMA03/06 cells. Finally, global gene expression profiling analysis allowed the identification of a list of 308 modulated genes in CMA03/06 versus CMA03 cells, many of which particularly relevant for MM biology. Overall, the novel CMA03/06 cell line may 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. This series of microarray experiments contains the gene expression profiles of 3 independent replicas of CMA03 grown in in normal culture conditions and in absence of IL-6, respectively, and 3 independent replicas of CMA03/06 in normal culture conditions. Cell pellets were resuspended in TRIzol Reagent (Life Technologies, Inc., Rockville, MD, USA), total RNA was then purified using the RNeasy® total RNA Isolation Kit (Qiagen, Valencia, CA).5.5 micrograms of single-stranded DNA target obtained from 100 ng of purified total RNA was fragmented and then labeled using the WT Terminal Labeling Kit according to the standard Affymetrix protocol (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual).The fragmented labeled single-stranded DNA target was hybridized for 16 hr 30 minutes at 45°C on GeneChip® Gene 1.0 ST array according to the standard Affymetrix protocol. The arrays were washed and stained in the Affymetrix Fluidics Station 450.
Project description:Bone marrow monocytes are primarily committed to osteoclast formation. It is, however, unknown whether potential primary alterations are specifically present in bone marrow monocytes from patients with multiple myeloma, smoldering myeloma or monoclonal gammopathy of undetermined significance. We analyzed the immunophenotypic and transcriptional profiles of bone marrow CD14+ monocytes in a cohort of patients with different types of monoclonal gammopathies to identify alterations involved in myeloma-enhanced osteoclastogenesis. The number of bone marrow CD14+CD16+ cells was higher in patients with active myeloma than in those with smoldering myeloma or monoclonal gammopathy of undetermined significance. Interestingly, sorted bone marrow CD14+CD16+ cells from myeloma patients were more pro-osteoclastogenic than CD14+CD16-cells in cultures ex vivo Moreover, transcriptional analysis demonstrated that bone marrow CD14+ cells from patients with multiple myeloma (but neither monoclonal gammopathy of undetermined significance nor smoldering myeloma) significantly upregulated genes involved in osteoclast formation, including IL21RIL21R mRNA over-expression by bone marrow CD14+ cells was independent of the presence of interleukin-21. Consistently, interleukin-21 production by T cells as well as levels of interleukin-21 in the bone marrow were not significantly different among monoclonal gammopathies. Thereafter, we showed that IL21R over-expression in CD14+ cells increased osteoclast formation. Consistently, interleukin-21 receptor signaling inhibition by Janex 1 suppressed osteoclast differentiation from bone marrow CD14+ cells of myeloma patients. Our results indicate that bone marrow monocytes from multiple myeloma patients show distinct features compared to those from patients with indolent monoclonal gammopathies, supporting the role of IL21R over-expression by bone marrow CD14+ cells in enhanced osteoclast formation.
Project description:The sialic glycoprotein, Mucin1, is known to be involved in the pathogenesis of various types of cancers. In a fraction of patients with multiple myeloma, their myeloma cells have high Mucin1 expression. We established a myeloma cell line designated EMM1 from a myeloma patient whose myeloma cells have high Mucin1 expression. Then we performed knockdown of Mucin1 to elucidate the role of the high Mucin1 expression.
Project description:Purpose: Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome. Experimental Design: CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for one to six months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA-seq and ATAC-seq. Chromatin accessible regions were used to identify regulatory RNA expression in over 700 samples from newly diagnosed patients in the MMRF CoMMpass trial (NCT01454297). Results: Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. Over 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with IGH translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma. Conclusions: These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.
Project description:Purpose: Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome. Experimental Design: CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for one to six months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA-seq and ATAC-seq. Chromatin accessible regions were used to identify regulatory RNA expression in over 700 samples from newly diagnosed patients in the MMRF CoMMpass trial (NCT01454297). Results: Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. Over 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with IGH translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma. Conclusions: These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.
Project description:ENCODE ChIP/chip study using human lymphoblastoid cell line GM06990; human cervix carcinoma cell line HeLaS3; human fetal lung fibroblast cell line HFL1; human T cell line MOLT4; chimpanzee lymphoblastoid cell line PTR8; and anti Histone H3K4me1 (Abcam; ab8895); H3K4me2 (Abcam; ab7766); H3K4me3 (Abcam; ab8580); H3ac (Upstate; 06-599) and H4ac (Upstate; 06-866) antibodies. The experiment was conducted in three biological replicates (1;2;3) with up to two technical duplicates (a;b).