Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression data analysis from RPMI 8226 cells irraidated to C-ions.

ABSTRACT: Multiple Myeloma (MM) is an hematological malignancy. MM cells are resistant to X-ray irradiations. We irradiated RPMI 8226 cancer cells with C-ions, which are more energetic than X-ray irradiations. We found that MM cells, RPMI 8226, are also resistant to C-ion irradiations. We used microarrays to investigate the mechanisms of MM cell resistance to C-ion irradiations. MM cells, RPMI 8226, were subjected to 0 or 6 Gy doses of C-ion irradiations. RNA were extratred from each batch of cells one day after irradiations and subjected to microarray analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Brigitte Sola

PROVIDER: E-GEOD-76471 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although new therapies have doubled the survival of multiple myeloma (MM) patients, this remains an incurable disease. It has been postulated that the so-called MM Cancer Stem Cells (MM-CSC) would be responsible for tumor initiation and relapse but their unequivocal identification remains unclear. Here, we investigated in a panel of MM cell lines the presence of CD20+ cells harboring a MM-CSC phenotype. Among the multiple cell lines investigated, only a small population of CD20dim+ cells (0.3%) in the RPMI-8226 cell line was found. CD20dim+ RPMI-8226 cells expressed the plasma cell markers CD38 and CD138 and were CD19-CD27-. Additionally, CD20dim+ RPMI-8226 cells did not exhibit stem-cell markers as shown by gene expression profiling and the aldehyde dehydrogenase (ALDH) assay. Moreover, we demonstrated that CD20dim+ RPMI-8226 cells are not essential for CB17-SCID mice engraftment and show lower self-renewal potential than the CD20- RPMI-8226 cells. These results do not support CD20+ expression for the identification of MM-CSC. For detailed description of Material and Methods, see supplementary Methods. The human MM cell lines RPMI-8226, U266, MM1S, MM1R, NCI-H929, RPMI-LR5, U266-LR7 and U266-Dox4 were studied. Cells were cultured as previously described [Ocio, 2009, 3781]. MM cell lines were immunophenotyped using a 7-color immunofluorescence technique [Paiva, 2011, 697], with the following combination of monoclonal antibodies (Pacific Blue (PB)/ anemonia majano cyan (AmCyan)/ fluorescein isothiocyanate (FITC)/ peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5)/ PE-cyanin 7 (PE-Cy7)/ allophycocyanin (APC)/ alexafluor 700 (AF700)): CD19/CD45/CD20/CD138/CD27/CD56/CD38. Data were stored for a minimum of 3x105 events. CD20dim+ and CD20- RPMI-8226 cells were sorted after incubation with CD20-APC/7AAD and acquisition on a FACSAria cytometer (Becton Dickison Biosciences). Sorting was performed only for viable cells (7AAD-) and debris were excluded by scatter properties. The CD20- and CD20dim+ RPMI-8266 sorted cells had a final purity of 98% and 88%, respectively. CD20dim+ and CD20- RPMI-8226 cells were extensively characterized. Morphological characterization was done with May-Grünwald-Giemsa staining. Characterization of VDJH and IGKappa rearrangements was performed as described elsewhere [BIOMED-2, BMH4-CT98-3936]. The expression of aldehyde dehydrogenase (ALDH) was assessed using the Aldefluor Kit (StemCell Technologies) following manufacturer’s instructions with further staining with a CD20-APC antibody. For gene expression profile studies, RNA was isolated, labeled and hybridized to Human Gene 1.0 ST array (Affymetrix) according to Affymetrix protocols [Gutiérrez, 2005, 402]. The arrays were analyzed using the DNA-Chip Analyzer software (DChip). Fold change ≥2 was considered significant.

Project description:Our previous studies have shown that inhibition of the IGF-1R pathway by the IGF-1RTK inhibitor picropodophyllin (PPP) can be achieved and also constitutes a favorable therapeutic window in multiple myeloma (MM). As no complete remission using in vivo models of MM could be obtained, a combinatorial drug screen (HTS) was performed to select the most performant combination with PPP. The HDAC inhibitor LBH589 was shown to act in synergy with PPP on survival of MM cells. The contribution from both drugs and the combination were further monitored for apoptosis, cell cycle distribution, and the impact on downstream gene and protein expression in human and mouse MM models in vitro. In the RPMI 8226 human MM cell line, simultaneous treatment with both compounds for 48h caused a 5-fold increase of apoptotic and late apoptotic/necrotic cells as compared to controls, while treatment with either compound alone only induced a 3-fold increase. After 24h cleavage of apoptotic proteins caspase -9, -8 and -3 could be found in RPMI 8226 cells treated with both drugs individually, but in the combination we observed an additive effect on the cleavage of the active forms of caspase 8 as compared to single drug treatments. The combination of LBH589 and PPP could be monitored as an accumulation of cells in the G2/M phase, and subsequent down-regulation of cell cycle regulated proteins. The effect of both compounds on the expression of cyclin B1, -E and -D2 was additive, as demonstrated by western blot. These data were also confirmed in the mouse 5T33MM cells in vitro. Gene expression analysis and validations of the RPMI 8226 cells reveal that the drug combination has better effects than the single drug alone. Combined treatment in vivo resulted in a significant prolonged survival of 5T33MM inoculated mice when compared to the control group and to treatment with the drugs alone. In conclusion, the results indicate an improved MM treatment opportunity in using a combination of PPP and LBH589. We used whole genome Microarray to decipher the changes occuring after treatment of the single and combination of the drugs. And see if the combination has better effects.

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Expression data analysis from RPMI 8226 cells irraidated to C-ions.

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