Fig 1.
Expression of Notch receptor in CD34+ cells isolated from BM of normal subjects and CML patients.
(a) Conventional PCR products are shown for four bone marrow (NBM1-4) samples from normal subjects on the left panel and for four CML samples on the right panel. For each sample, the expression of Notch1, Notch2, and Notch4 within the CD34+, Thy-, Thy+ subpopulation is shown. The housekeeping gene GAPDH was used as a control to assess the quality of cDNA in each sample. The lower left panel shows human genomic DNA (HGDNA) as a positive control for each set of oligonucleotides. Bar graph shows real-time PCR analysis of Notch1 (b) and Notch2 (c) expression on CD34+ subsets from NBM and CML patients. Gene expression was normalised to the GAPDH. Notch1 showed significant upregulation in the CD34+ Thy+ cell subset (p<0.05). Notch2 showed significant (p<0.05) upregulation in all the CD34+ CML primary subset cells compared with NBM.
Fig 2.
Expression of Notch receptors and its target genes in CD34+ cells isolated from BM of normal subjects and CML patients.
(a) Conventional PCR products are shown for four NBM (left panel) and four CML samples (right panel). GAPDH was used to assess the quality of cDNA. The lower left panel shows human genomic DNA (HGDNA) as a positive control for each set of oligonucleotides. (b) Real-time PCR analysis of Hes1 expression on CD34+ cell subsets from NBM and CML patients. Results showed significant (* = p<0.05, ** = p≤0.01) upregulation of Hes1 in all the CD34+ CML primary subset cells compared with NBM. (c) Summary of Notch1 expression profile in different cell lineages in CML and NBM. FACS analysis of Notch1 in different myeloid, lymphoid, and more primitive lineages in CML was done by co-staining mononuclear cells with both extracellular Notch1 (ECN1-EA1) antibody and a lineage-specific cell surface marker. Results shown here are representative of the total CD34+ cells in each sample. The mean of expression refers to the percentage of each cell population in the left column that was positive for EA1. The means of expression were measured from four different CML samples (n = 4).
Fig 3.
Hes1 gene expression profile in CD34+ cells isolated from CML patients.
The expression profiles of the Notch target gene Hes1 was investigated by real-time PCR. Data is shown from CD34+ cells isolated from six CML patients (pt) in chronic phase and CD34+ control cells from three NBM samples. Relative gene expression was calculated using the DDCt method. ** = p<0.01; significant expression of Hes1 in each CML sample was compared with Hes1 expression in the NBM samples.
Fig 4.
Evaluation of BCR-ABL activity in primary CD34+ CML cells.
(a) Inhibition of BCR-ABL activity by IM in CD34+ primary cells isolated from CML patients. CD34+ primary cells were cultured in the presence of 10 μM IM for 72h and then stained for P-crkl expression. (b) Primary CD34+ cells from two CML patients show resistance to IM, assessed by BCR-ABL activity. (c) Hes1 gene expression in IM-sensitive CD34+ primary CML cells (*p<0.01). (d) Hes1 gene expression in CD34+ cells isolated from IM resistant CML patients.
Fig 5.
Assessment of cross-talk between Notch and BCR-ABL activity in CD34+ primary CML cells.
(a) Hes1 gene expression in CD34+ cells isolated from GSI-responder CML patients. CD34+ cells were isolated from CML patients and cultured in the presence of 10 μM GSI for 72h. Live CD34+ cells were then sorted, and the gene expression of the Notch target gene Hes1 was investigated by real-time PCR (*p<0.01). (b) Hes1 gene expression in CD34+ cells isolated from GSI-nonresponder CML patients. (c) Overexpression of P-crkl in CD34+ CML cells treated with GSI. CD34+ cells from five CML patients in chronic phase were cultured in the presence of 10 μM GSI. The change in BCR-ABL activity was assessed by the FACS-based P-crkl assay. (d) P-crkl expression was measured by mean fluorescence intensity (MFI) units in each condition. MFI of P-crkl in GSI-treated CD34+ cells were compared to a no-drug control in each sample, and the percentage of increase in P-crkl was calculated. Data shown here represent the mean of six CML samples (**p<0.01).
Fig 6.
Expression of Notch1 in the K562 cell line.
(a) Expression of Notch1 in the K562 cell line model at transcriptional level. cDNA was prepared from K562 cells. The CEM cell line was used as a positive control for active Notch signalling. Transcript levels were measured by RT-PCR. RT-PCR products were resolved by agarose gel electrophoresis and visualised by Vistra Green (n = 3). (b) Analysis of Notch1 expression in K562 cells at protein level. Cells were stained with EA1 antibody to detect the extracellular domain of Notch1 (ECN1) and bTAN 20 antibody to detect intracellular domain of Notch1 (ICN1) using FACS. Appropriate isotype controls were used in each staining (n = 4). (c) Inhibition of Notch signalling by γ-seretase inhibitor (GSI) in K562 cells. The cDNA was prepared from cells treated with vehicle control (DMSO) and 10 μM GSI for 24h. Real-time PCR of the Notch target gene Hes1 is shown (n = 5). ** = p<0.01. (d) The effect of Notch inhibition on BCR-ABL activity. K562 cells were cultured for 24h in the presence of GSI (10μM) and BCR-ABL activity was assessed by FACS based P-crkl assay (n = 4).
Fig 7.
Cross-talk between Notch and BCR-ABL in the K562 and ALL-SIL cell line model.
(a) Assessment of IM efficacy in K562 cells using P-crkl assay. K562 cells were cultured in increasing concentrations of IM (10, 5, 1, 0.5, and 0.1 μM) for 48h. P-crkl expression in cells treated with IM is shown. Data shown is from one experiment representative of three separate experiments (n = 4). (b) Dose-dependent effect of IM on the expression of P-crkl in K562 cells. P-crkl expression of IM treated and untreated K562 cells represented as mean fluorescence intensity (MFI) uing FACS as described in (a) (n = 4). (c) Concentration-dependent effect of IM on P-crkl protein. K562 and Jurkat cells cultured in increasing concentrations of IM (10, 5, 1, 0.5, and 0.1 μM) for 48h. P-crkl protein levels were measured by western blotting. (d) Expression of Hes1 in K562 cells, 48h posttreatment. Notch target gene Hes1 was assessed after 48h treatment with 10 μM IM (**p<0.01). (e) Hes1 expression in K562 cells post valproic acid (VPA) treatment. K562 cells were treated with 4mM VPA for 72h and Hes1 expression was measured by real-time PCR. Gene expression was normalised to the GAPDH (n = 3). Statistical significance was calculated using student t-test. (** = p <0.01). (f) Effect of VPA on BCR-ABL activity in K562 cells. K562 cells were treated with 4mM VPA for 72h and the activity of BCR-ABL was assessed by FACS analysis of P-crkl expression. (n = 3).
Fig 8.
Cross-talk between Notch and BCR-ABL in the ALL-SIL cell line models.
(a) Assessment of expression of P-crkl levels in the ALL-SIL cell line. (i) Cells were stained with P-crkl primary antibody and PE secondary antibody (red) and isotype control (blue). (ii) Expression of P-crkl in ALL-SIL cells after incubating the cells for 48h with 10 μM IM shown in green and isotype control in blue. (iii) P-crkl expression of IM-treated cells is shown in green as compared with untreated cells in red (n = 3). (b) Expression of Hes1 at 48h posttreatment. Notch target gene Hes1 mRNA was evaluated in ALL-SIL cells at 48h posttreatment with 10 μM IM using qRT-PCR (n = 4). ** = p<0.01, statistical significance difference between treated and untreated group.
Fig 9.
Analysis of microarray datasets from CML CD34+ patients and NBM donors.
(a) Shows PCA plots of three gene expression datasets from the GSE library. Datasets comprise gene expression profiles of CD34+ haematopoietic stem cells from normal donors (n = 14), donors in chronic phase of CML (n = 15), donors treated with seven days of IM following a diagnosis of chronic phase CML (n = 6), and donors treated with IM for a median duration of 28 months (range 11–39 months) (n = 8). To identify the role of Notch signalling in chronic phase CML and following treatment with IM, gene set enrichment analysis was performed by comparing 14 bone marrow samples from normal subjects with 6 paired samples obtained from chronic phase CML patients before IM and after 7 days of IM treatment. (b) Shows regulation of genes involved in Notch signalling pre- and post-IM and its correlation with cell cycle pathways and DNA replication pathways.
Fig 10.
Blue-pink O'gram of altered Notch signalling is CML patient samples.
(a) Shows Notch signalling pre-IM in CD34+ haematopoietic stem cells (HSCs) during chronic phase CML and (b) shows the effect of IM on Notch signalling in CD34+ HSCs during chronic phase CML.
Fig 11.
(a) Proposed model for Notch and BCR-ABL cross-talk in CML. Both BCR-ABL and Notch activate the PI3K/AKT-mTOR pathway that may trigger the survival and proliferation signals to CML cells. Blocking BCR-ABL kinase activity may not be sufficient to induce apoptosis as this may switch to survival signals to the PI3K pathway activated by Notch in CML cells. In this model, IM may be upregulating Notch by modulating Wnt component GSK3β and/or Dishevelled. IM-induced activation of GSK3β or inhibition of Dishevelled stabilises ICN in the cytoplasm, which in turn activates the PI3K/AKT-mTOR signalling by upregulation of Hes1 which abolish the inhibitory effect of PTEN on PI3K pathway. (b) Cooperative model of activated Notch and BCR-ABL signalling in chronic phase CML. Both Notch and BCR-ABL are activated in chronic phase CML and may activate survival signalling pathways to inhibit apoptosis in CD34+ CML cells. In vitro inhibition of Notch by GSI-induced BCR-ABL activity to keep the same level of survival signals is required for CML cell. Treatment with IM leads to activation of Notch signalling to maintain the same level of survival signals needed by CML cells to inhibit apoptosis. The net effect is maintenance of balanced levels of survival signals that protect CD34+ CML cells from apoptosis in the chronic phase of CML (GSI: γ-secretase inhibitor, IM: imatinib mesylate).