Establishment and phenotypic characterization of human U937 cells with inducible P210 BCR/ABL expression reveals upregulation of CEACAM1 (CD66a)

Håkansson, Petra; Lassen, Carin; Olofsson, Tor; Baldetorp, Bo; Karlsson, Anna; Gullberg, Urban; Fioretos, Thoas

doi:10.1038/sj.leu.2403255

Cited by 18 publications

(16 citation statements)

References 57 publications

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“…8,31 Proliferative advantage of the emerged aberrant phenotype seems to strongly depend on the BCR-ABL oncoprotein as the number of aberrant cells continuously decreases after p210BCR-ABL turn off. This may explain the need for culture ficollization and the delayed doubling time of induced cells when compared to uninduced control cells observed by us and Hakansson et al 23 Similarly, a reduction in the number of aberrant cells was achieved when p210BCR-ABL oncoprotein expression was inhibited using abldirected siRNA. Thus, our results suggest that the constitutive BCR-ABL tyrosine kinase activity is sufficient for either inducing centrosomal aberrations or granting proliferative advantage of a distinct aneuploid phenotype with aberrant centrosomes in U937 cells in vitro.…”

Section: Discussionmentioning

confidence: 63%

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Expression of the p210BCR-ABL oncoprotein drives centrosomal hypertrophy and clonal evolution in human U937 cells

et al. 2007

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Centrosomes play fundamental roles in mitotic spindle organization, chromosome segregation and maintenance of genetic stability. Recently, we have shown that centrosome aberrations occur early in chronic myeloid leukemia (CML) and are induced by imatinib in normal fibroblasts in vitro. To investigate the influence of BCR-ABL on centrosomes, we performed longterm in vitro experiments employing the conditionally p210BCR-ABL-expressing (tetracycline-inducible promoter) human monocytic cell line U937p210BCR-ABL/c6 as a model of CML chronic phase. Centrosome hypertrophy was detectable after 4 weeks of transgene expression onset, increasing up to a rate of 25.7% aberrant cells within 13 weeks of propagation. This concurred with clonal expansion of aneuploid cells displaying a hyperdiploid phenotype with 57 chromosomes. Partial reversibility of centrosome aberrations (26-8%) was achieved under prolonged propagation (14 weeks) after abortion of induction and bcr-abl silencing using small interfering RNA. Therapeutic doses of imatinib did not revert the aberrant phenotype, but counteracted the observed reverting effect of bcr-abl gene expression switch off. Suggesting a mechanistic model that features distinct abl-related tyrosine kinase activity levels as essential determinants of centrosomal integrity, this is the first report mechanistically linking p210BCR-ABL oncoprotein activity to centrosomal hypertrophy.

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Section: Discussionmentioning

confidence: 63%

“…23 The control cell line K562 and untransfected U937 monocytic cells (both American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in the same medium omitting hygromycin B and geneticin. For induction of BCR-ABL protein expression, 1 mg/ml doxocycline (Gibco) was added to the culture medium.…”

Section: Cell Lines and Cell Culture Conditionsmentioning

confidence: 99%

Expression of the p210BCR-ABL oncoprotein drives centrosomal hypertrophy and clonal evolution in human U937 cells

et al. 2007

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“…The U937/Tet-On/A38 clone, carrying the pTET-On regulator plasmid conferring resistance to geneticin was used as control cells. 9 Primary cells from chronic myeloid leukemia patients were collected after obtaining informed consent to the use for basic in vitro research and under the approval of the Ethics Committee of Azienda Ospedaliero/Universitaria Careggi (AOUC; University Hospital) at the Division of Haematology of that institute. Mononuclear cells were obtained by centrifugation on a FicollHypaque gradient (Cedarlane Laboratories, Ontario, Canada) and cultured in the presence of Flt-3 ligand (50 ng/mL) and TPo (20 ng/mL) in LC1 and of SCF (50 ng/mL), G-CSF (100 ng/mL), and IL-6 (20 ng/mL) in LC2 (all from PeproTech, Rocky Hill, NJ, USA).…”

Section: Cells and Culture Conditionsmentioning

confidence: 99%

Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate

Giuntoli¹,

Tanturli²,

Gesualdo³

et al. 2010

Haematologica

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BackgroundIncubation of chronic myeloid leukemia cells in hypoxia inhibits growth and selects BCR/Ablindependent cells with stem cell properties which are refractory to imatinib-mesylate. This study aimed to characterize the relationship of this refractoriness with glucose availability in the environment. Design and MethodsK562 or primary chronic myeloid leukemia cells were cultured at 0.1% O2, different cell densities and glucose concentrations. The stem and progenitor cell potential of these cultures at different times of incubation in relation to BCR/Ablprotein expression and sensitivity to imatinibmesylate was explored by transferring cells to growth-permissive secondary cultures in normoxia, according to the Culture-Repopulating Ability assay methodology. ResultsHypoxia-resistant cells maintained BCR/Ablprotein expression until glucose was no longer available in primary hypoxic cultures, where glucose availability appeared to regulate cell number and the balance between the enrichment of cells with kinetic properties typical of stem or progenitor cells. Cells surviving merely hypoxic conditions were, upon transfer to secondary cultures, immediately available for numerical expansion due to the maintained BCR/Ablprotein expression, and were consequently sensitive to imatinib-mesylate. Instead, BCR/Ablprotein-negative cells selected in primary cultures under oxygen/glucose shortage underwent a delayed numerical expansion in secondary cultures, which was completely refractory to imatinib-mesylate. Cells with the latter properties were also found in primary chronic myeloid leukemia explants. ConclusionsGlucose shortage in hypoxia was shown to represent the condition selecting BCR/Ablprotein-negative cells refractory to imatinib-mesylate from either chronic myeloid leukemia lines or patients. These cells, exhibiting stem cell properties in vitro, are metabolically suited to home to stem cell niches in vivo and so may represent the chronic myeloid leukemia cell subset responsible for minimal residual disease.Key words: imatinib-mesylate, chronic myeloid leukemia, glucose, hypoxia.Citation: Giuntoli S, Tanturli M, Di Gesualdo F, Barbetti V, Rovida E, and Dello Sbarba P. Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate. Haematologica 2011;96(2):204-212. doi:10.3324/haematol.2010 Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate

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“…Protein lysates were prepared and western blotting was performed as previously described, 30 with the following exception: from transduced CD34 þ cells lysate was prepared from 200 000 sorted GFP þ cells. The following primary antibodies were used: anti-phospho-STAT5A/B (Y694/Y699) (Upstate Biotechnology, Lake Placid, NY, USA), anti-STAT5B (Upstate Biotechnology), anti-WT1 (180; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-P-p70 S6K (T389) (Cell signalling Technology) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (6C5; Ambion, Austin, USA).…”

Section: Western Blot Analysismentioning

confidence: 99%

Deregulation of the Wilms' tumour gene 1 protein (WT1) by BCR/ABL1 mediates resistance to imatinib in human leukaemia cells

et al. 2007

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The Wilms' tumour gene 1 (WT1) protein is highly expressed in most leukaemias. Co-expression of WT1 and the fusion protein AML1-ETO in mice rapidly induces acute myeloid leukaemia (AML). Mechanisms behind expression of WT1, as well as consequences thereof, are still unclear. Here, we report that the fusion protein BCR/ABL1 increases expression of WT1 mRNA and protein via the phosphatidylinositol-3 kinase (PI3K)-Akt pathway. Inhibition of BCR/ABL1 or PI3K activity strongly suppressed transcription from WT1 promoter/enhancer reporters. Forced expression of BCR/ABL1 in normal human progenitor CD34 þ cells increased WT1 mRNA and protein, further supporting the notion of BCR/ABL1-driven expression of WT1 in human haematopoietic cells. Forced expression of WT1 in K562 cells provided protection against cytotoxic effects of the ABL1 tyrosine kinase inhibitor imatinib, as judged by effects on viability measured by trypan blue exclusion, metabolic activity, annexin V and DAPI (4 0 , 6-diamidino-2-phenylindole) staining. None of the isoforms provided any detectable protection against apoptosis induced by arsenic trioxide and only very weak protection against etoposide, indicating that WT1 interferes with specific apoptotic signalling pathways. Our data demonstrate that WT1 expression is induced by oncogenic signalling from BCR/ABL1 and that WT1 contributes to resistance against apoptosis induced by imatinib.

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Establishment and phenotypic characterization of human U937 cells with inducible P210 BCR/ABL expression reveals upregulation of CEACAM1 (CD66a)

Cited by 18 publications

References 57 publications

Expression of the p210BCR-ABL oncoprotein drives centrosomal hypertrophy and clonal evolution in human U937 cells

Expression of the p210BCR-ABL oncoprotein drives centrosomal hypertrophy and clonal evolution in human U937 cells

Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate

Deregulation of the Wilms' tumour gene 1 protein (WT1) by BCR/ABL1 mediates resistance to imatinib in human leukaemia cells

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