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Identification of novel Notch target genes in T cell leukaemia.

Chadwick N, Zeef L, Portillo V, Fennessy C, Warrander F, Hoyle S, Buckle AM - Mol. Cancer (2009)

Bottom Line: As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach.These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1).The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Life Sciences, Manchester Interdisciplinary Biocenter, University of Manchester, Manchester, UK. n.chadwick@manchester.ac.uk

ABSTRACT

Background: Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat.

Results: RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1DeltaE or N3DeltaE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1).

Conclusion: The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.

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CD28 expression is downregulated by inhibition of Notch signalling. (A) T-ALL cell lines were treated with 10 uM GSI IX (or DMSO) for 24 hrs and cell surface CD28 expression analysed by flow cytometry. * represents p < 0.05 verus DMSO-treated cells. (B) GSI washout experiment as described in figure 2 and CD28 mRNA expression analysis in Jurkat cells. * represents p < 0.05 verus cells at the zero timepoint. ** represents p < 0.05 verus cyclohexamide- cells (C) Real-time PCR expression analysis of CD28 using cDNA from GFP- (untransduced) or GFP+ (transduced) Jurkat cells transduced with GFP-alone or N1ΔE retrovirus. * represents p < 0.05 verus GFP-alone transduced cells. (D) Cell surface CD28 expression using the cells described above. (E) CD28 expression in primary CD3+ cells transduced with GFP-alone, N1ΔE, or N1ΔE+GSIs.
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Figure 3: CD28 expression is downregulated by inhibition of Notch signalling. (A) T-ALL cell lines were treated with 10 uM GSI IX (or DMSO) for 24 hrs and cell surface CD28 expression analysed by flow cytometry. * represents p < 0.05 verus DMSO-treated cells. (B) GSI washout experiment as described in figure 2 and CD28 mRNA expression analysis in Jurkat cells. * represents p < 0.05 verus cells at the zero timepoint. ** represents p < 0.05 verus cyclohexamide- cells (C) Real-time PCR expression analysis of CD28 using cDNA from GFP- (untransduced) or GFP+ (transduced) Jurkat cells transduced with GFP-alone or N1ΔE retrovirus. * represents p < 0.05 verus GFP-alone transduced cells. (D) Cell surface CD28 expression using the cells described above. (E) CD28 expression in primary CD3+ cells transduced with GFP-alone, N1ΔE, or N1ΔE+GSIs.

Mentions: CD28 was of interest to us because of its well characterised role in T cell activation [25] and its ability to positively or negatively regulate thymocyte apoptosis ((([26-30]. CD28 was found to be upregulated by both Notch1 and Notch3 in Jurkat cells based on Affymetrix data (figure 1) and we validated this finding by real-time PCR using transduced Jurkat, CEM and Molt4 cells as described above (figure 2.A). We investigated Notch-induced CD28 upregulation at the protein level by flow cytometry. Analysis of GFP-alone- or Notch-transduced Jurkat cells showed a clear upregulation of CD28 expression at the cell surface while untranfected GFP negative cells in the same culture did not show Notch-induced CD28 upregulation (figure 2.B). This effect was seen more clearly in CEM cells where very little basal CD28 expression was seen. The majority of Notch1-transduced cells were CD28 positive, while untransduced cells in the same culture remained negative. Treatment of all T-ALL cell lines with GSIs resulted in a downregulation of cell surface CD28 expression (figure 3.A), showing that endogenous Notch signalling contributes to CD28 expression. This was confirmed using a GSI-washout experiment (figure 3.B) which showed that Notch-induced CD28 upregulation is not affected by cyclohexamide and so does not require de novo protein synthesis. Finally, DN-MAML downregulated CD28 mRNA and cell surface expression (figure 3.C&3D), confirming the contribution of endogenous Notch to basal CD28 expression and also showing that the transcriptional activity of Notch is necessary for this effect. Together, the upregulation of CD28 in the absence of de novo protein synthesis and the requirement of the transcriptional activity of Notch shows that CD28 is a direct transcriptional target of Notch. This finding is in agreement with a recent study by Margolin et al. which used ChIP-on-chip to identify direct transcriptional targets of Notch1 [31] and found that there was a high degree of significance in the affinity of Notch1 for the CD28 promoter (see Additional file 4).


Identification of novel Notch target genes in T cell leukaemia.

Chadwick N, Zeef L, Portillo V, Fennessy C, Warrander F, Hoyle S, Buckle AM - Mol. Cancer (2009)

CD28 expression is downregulated by inhibition of Notch signalling. (A) T-ALL cell lines were treated with 10 uM GSI IX (or DMSO) for 24 hrs and cell surface CD28 expression analysed by flow cytometry. * represents p < 0.05 verus DMSO-treated cells. (B) GSI washout experiment as described in figure 2 and CD28 mRNA expression analysis in Jurkat cells. * represents p < 0.05 verus cells at the zero timepoint. ** represents p < 0.05 verus cyclohexamide- cells (C) Real-time PCR expression analysis of CD28 using cDNA from GFP- (untransduced) or GFP+ (transduced) Jurkat cells transduced with GFP-alone or N1ΔE retrovirus. * represents p < 0.05 verus GFP-alone transduced cells. (D) Cell surface CD28 expression using the cells described above. (E) CD28 expression in primary CD3+ cells transduced with GFP-alone, N1ΔE, or N1ΔE+GSIs.
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Figure 3: CD28 expression is downregulated by inhibition of Notch signalling. (A) T-ALL cell lines were treated with 10 uM GSI IX (or DMSO) for 24 hrs and cell surface CD28 expression analysed by flow cytometry. * represents p < 0.05 verus DMSO-treated cells. (B) GSI washout experiment as described in figure 2 and CD28 mRNA expression analysis in Jurkat cells. * represents p < 0.05 verus cells at the zero timepoint. ** represents p < 0.05 verus cyclohexamide- cells (C) Real-time PCR expression analysis of CD28 using cDNA from GFP- (untransduced) or GFP+ (transduced) Jurkat cells transduced with GFP-alone or N1ΔE retrovirus. * represents p < 0.05 verus GFP-alone transduced cells. (D) Cell surface CD28 expression using the cells described above. (E) CD28 expression in primary CD3+ cells transduced with GFP-alone, N1ΔE, or N1ΔE+GSIs.
Mentions: CD28 was of interest to us because of its well characterised role in T cell activation [25] and its ability to positively or negatively regulate thymocyte apoptosis ((([26-30]. CD28 was found to be upregulated by both Notch1 and Notch3 in Jurkat cells based on Affymetrix data (figure 1) and we validated this finding by real-time PCR using transduced Jurkat, CEM and Molt4 cells as described above (figure 2.A). We investigated Notch-induced CD28 upregulation at the protein level by flow cytometry. Analysis of GFP-alone- or Notch-transduced Jurkat cells showed a clear upregulation of CD28 expression at the cell surface while untranfected GFP negative cells in the same culture did not show Notch-induced CD28 upregulation (figure 2.B). This effect was seen more clearly in CEM cells where very little basal CD28 expression was seen. The majority of Notch1-transduced cells were CD28 positive, while untransduced cells in the same culture remained negative. Treatment of all T-ALL cell lines with GSIs resulted in a downregulation of cell surface CD28 expression (figure 3.A), showing that endogenous Notch signalling contributes to CD28 expression. This was confirmed using a GSI-washout experiment (figure 3.B) which showed that Notch-induced CD28 upregulation is not affected by cyclohexamide and so does not require de novo protein synthesis. Finally, DN-MAML downregulated CD28 mRNA and cell surface expression (figure 3.C&3D), confirming the contribution of endogenous Notch to basal CD28 expression and also showing that the transcriptional activity of Notch is necessary for this effect. Together, the upregulation of CD28 in the absence of de novo protein synthesis and the requirement of the transcriptional activity of Notch shows that CD28 is a direct transcriptional target of Notch. This finding is in agreement with a recent study by Margolin et al. which used ChIP-on-chip to identify direct transcriptional targets of Notch1 [31] and found that there was a high degree of significance in the affinity of Notch1 for the CD28 promoter (see Additional file 4).

Bottom Line: As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach.These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1).The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculty of Life Sciences, Manchester Interdisciplinary Biocenter, University of Manchester, Manchester, UK. n.chadwick@manchester.ac.uk

ABSTRACT

Background: Dysregulated Notch signalling is believed to play an important role in the development and maintenance of T cell leukaemia. At a cellular level, Notch signalling promotes proliferation and inhibits apoptosis of T cell acute lymphoblastic leukaemia (T-ALL) cells. In this study we aimed to identify novel transcriptional targets of Notch signalling in the T-ALL cell line, Jurkat.

Results: RNA was prepared from Jurkat cells retrovirally transduced with an empty vector (GFP-alone) or vectors containing constitutively active forms of Notch (N1DeltaE or N3DeltaE), and used for Affymetrix microarray analysis. A subset of genes found to be regulated by Notch was chosen for real-time PCR validation and in some cases, validation at the protein level, using several Notch-transduced T-ALL and non-T-ALL leukaemic cell lines. As expected, several known transcriptional target of Notch, such as HES1 and Deltex, were found to be overexpressed in Notch-transduced cells, however, many novel transcriptional targets of Notch signalling were identified using this approach. These included the T cell costimulatory molecule CD28, the anti-apoptotic protein GIMAP5, and inhibitor of DNA binding 1 (1D1).

Conclusion: The identification of such downstream Notch target genes provides insights into the mechanisms of Notch function in T cell leukaemia, and may help identify novel therapeutic targets in this disease.

Show MeSH
Related in: MedlinePlus