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Cluzeau T.,French Institute of Health and Medical Research | Cluzeau T.,University of Nice Sophia Antipolis | Cluzeau T.,Nice University Hospital Center | Dubois A.,French Institute of Health and Medical Research | And 16 more authors.
Oncotarget | Year: 2014

In the present study, we provide a comparative phenotypic and genotypic analysis of azacitidine-sensitive and resistant SKM-1 cell lines. Morphologically, SKM1-R exhibited increase in cell size that accounts for by enhanced ploidy in a majority of cells as shown by cell cycle and karyotype analysis. No specific Single Nucleotide Polymorphism (SNP) alteration was found in SKM1-R cells compared to their SKM1-S counterpart. Comparative pangenomic profiling revealed the up-regulation of a panel of genes involved in cellular movement, cell death and survival and down-regulation of genes required for cell to cell signaling and free radical scavenging in SKM1-R cells. We also searched for mutations frequently associated with myelodysplastic syndromes (MDS) and found that both cell lines harbored mutations in TET2, ASLX1 and TP53. Collectively, our data show that despite their different morphological and phenotypic features, SKM1-S and SKM1-R cells exhibited similar genotypic characteristics. Finally, pangenomic profiling identifies new potential pathways to be targeted to circumvent AZA-resistance. In conclusion, SKM1-R cells represent a valuable tool for the validation of new therapeutic intervention in MDS.

Cluzeau T.,French Institute of Health and Medical Research | Cluzeau T.,University of Nice Sophia Antipolis | Cluzeau T.,Equipe labellisee par la Ligue Nationale Contre le Cancer 2011 2013 | Cluzeau T.,Nice University Hospital Center | And 23 more authors.
Oncotarget | Year: 2012

Azacitidine is the leading compound to treat patients suffering myelodysplasticsyndrome (MDS) or AML with less than 30% of blasts, but a majority of patientsis primary refractory or rapidly relapses under treatment. These patients have adrastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 family member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients. © Cluzeau et al.

Robert G.,French Institute of Health and Medical Research | Robert G.,Equipe labellisee par la Ligue Nationale Contre le Cancer 2011 2013 | Robert G.,University of Nice Sophia Antipolis | Jullian V.,University Paul Sabatier | And 21 more authors.
Oncotarget | Year: 2012

Simalikalactone E (SkE) is a quassinoid extracted from a widely used Amazonian antimalarial remedy. Although SkE has previously been shown to have cytostatic and/or cytotoxic activities in some tumor cell lines, its mechanism of action has not yet been characterized. We show here that SkE in the high nanomolar range inhibited the growth of various leukemic and solid tumor cell lines. Importantly, SkE was highly efficient at inhibiting chronic myelogenous leukemia (CML) cells that exhibit constitutive activation of the MAPK pathway and, accordingly, it impaired the phosphorylation of ERK1/2. SkE also abrogated MEK1/2 and B-Raf phosphorylation but had no effect on Ras activity. Moreover, SkE was particularly effective against melanoma cell lines carrying the B-Raf-V600E mutation. Importantly, SkE resensitized the PLX-4032-resistant 451Lu melanoma cell line (451Lu-R) and was more efficient than U0126, a MEK inhibitor, and PLX-4032 (PLX) at inducing the apoptosis of two Hairy Cell Leukemia (HCL) patient samples carrying the B-Raf-V600E mutation. Finally, SkE was as efficient as imatinib at inhibiting tumor formation in a xenograft model of CML cells in athymic mice. In conclusion, we show that SkE, a very potent inhibitor of B-Raf-V600E, is highly effective against cancer cell lines that exhibit constitutive activation of the ERK1/2 pathway. © Robert et al.

Belhacene N.,French Institute of Health and Medical Research | Belhacene N.,University of Nice Sophia Antipolis | Belhacene N.,Equipe labellisee par la Ligue Nationale Contre le Cancer 2011 2013 | Gamas P.,French Institute of Health and Medical Research | And 25 more authors.
PLoS ONE | Year: 2012

Transgenic mice expressing the caspase-cleaved form of the tyrosine kinase Lyn (LynΔN) develop a TNFα-dependent skin disease that accurately recapitulates human psoriasis. Participation of lymphocytes in this disease was confirmed by backcrossing LynΔN mice on a Rag-1 deficient background. The present study was therefore conducted to analyze whether modification of lymphocyte homeostasis does occur and participate in the phenotype of LynΔN mice. We show here that LynΔN mice consistently exhibit thymic atrophy that correlates with both a net decrease in the CD4+/CD8+ Double Positive (DP) and an increase in Single Positive (SP) thymocyte sub-populations, but also display an increase of splenic mature B cell. Interestingly, a normal immune phenotype was rescued in a TNFR1 deficient background. Finally, none of these immune alterations was detected in newborn mice before the onset of inflammation. Therefore, we conclude that chronic inflammation can induce thymic atrophy and perturb spleen homeostasis in LynΔN mice through the increased production of TNFα, LTß and TNFR1 signaling. © 2012 Belhacéne et al.

Dufies M.,French Institute of Health and Medical Research | Dufies M.,University of Nice Sophia Antipolis | Dufies M.,Equipe labellisee par la Ligue Nationale Contre le Cancer 2011 2013 | Jacquel A.,French Institute of Health and Medical Research | And 19 more authors.
Oncotarget | Year: 2011

AXL is a receptor tyrosine kinase of the TAM family, the function of which is poorly understood. We previously identified AXL overexpression in Imatinib (IM)-resistant CML cell lines and patients. The present study was conducted to investigate the role of AXL and the mechanisms underlying AXL overexpression in Tyrosine Kinase Inhibitor (TKI)-resistant CML cells. We present evidence that high AXL expression level is a feature of TKI-resistant CML cells and knockdown of AXL sensitized TKIresistant cells to IM. In addition, expression of wild-type AXL but not a dominant negative form of AXL confers IM-sensitive CML cells the capacity to resist IM effect. AXL overexpression required PKCα and β and constitutive activation of ERK1/2. Accordingly, GF109203X a PKC inhibitor, U0126 a MEK1 inhibitor and PKCα/β knockdown restore sensitivity to IM while PKCα or PKCβ overexpression in CML cells promotes protection against IM-induced cell death. Finally, using luciferase promoter activity assays we established that AXL is regulated transcriptionally through the AP1 transcription factor. Our findings reveal an unexpected role of AXL in resistance to TKI in CML cells, identify the molecular mechanisms involved in its overexpression and support the notion that AXL is a new marker of resistance to TKI in CML. © Dufies et al.

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