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Biological and molecular characterization of CMA03/06, a newly established interleukin-6 independent variant of the CMA03 human myeloma cell line.

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE39685 | GEO | 2014/11/21

SECONDARY ACCESSION(S): PRJNA171398

REPOSITORIES: GEO

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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:Introduction Disruption of the epithelial barrier can induce eosinophilic esophagitis (EoE), a TH2-mediated food- and aeroallergen associated chronic inflammatory disease 1, 2. The expression of IL-20 subfamily cytokines, IL-19, IL-20 and IL-24, is increased in TH2-mediated diseases, yet their function in EoE is unknown. Methods Combining RNA-sequencing (RNA-seq) and proteome analysis, we have examined the effects of the IL-20 subfamily on esophageal epithelial cells using patient-derived organoids and an experimental EoE mouse model. Results When stimulated with IL-20 subfamily cytokines patient-derived esophageal organoids showed decreased expression of Filaggrin (FLG) and Filaggrin 2 (FLG2) responsible for epithelial cornification. In agreement, EoE patients with active inflammation showed elevated IL-20 subfamily cytokines and decreased FLG and FLG2 expression. Topical corticosteroid treatment in EoE patients restored filaggrin expression, while reducing IL-20 subfamily cytokines. Abolishment of IL-20 subfamily signalling in Il20R2-/- animals attenuated experimental EoE in an OVA-induced mouse model. In the esophageal keratinocyte cell line, KYSE-180, we identified IL-20 subfamily-induced STAT3 and MAPK (ERK1/2) pathway activation. However, Stat3fl/flKrt5cre mice with an epithelium specific deletion of Stat3 developed exacerbated experimental EoE with a pronounced decrease of Flg2. In line, combined stimulation of patient-derived esophageal organoids with IL-20 subfamily cytokines and pharmacological inhibition of STAT3 resulted in aggravated decrease of FLG and FLG2. In addition, pharmacological STAT3 inhibition resulted in reduced transepithelial electrical resistance (TEER) and increased macromolecular flux in patient-derived air liquid interface (ALI) cultures. Whereas, inhibition of the MAPK (ERK1/2) pathway hampered IL-20 subfamily induced TEER reduction and paracellular flux increment. Finally, MAPK (ERK1/2) inhibitor treatment reduced infiltrating CD45+ immune cells in the esophagus and alleviated experimental EoE in mice. Conclusion We discovered a novel regulatory function of the IL-20 subfamily for epithelial barrier integrity. Aberrant IL-20 subfamily signaling disturbs the epithelial barrier function, while abrogation of IL-20 subfamily and ERK1/2 signaling restores epithelial integrity and attenuates experimental EoE. Therefore, we propose that targeting the IL-20 subfamily pathway could present a novel therapeutic strategy for EoE treatment.

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Biological and molecular characterization of CMA03/06, a newly established interleukin-6 independent variant of the CMA03 human myeloma cell line.

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