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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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Analysis of Notch-induced VEGF, ID1 and GIMAP5 expression. (A) Supernatants from GFP-alone, N1ΔE, or N3ΔE-transduced Jurkat or CEM cells were used for VEGF ELISAs to analyse secreted VEGF protein levels in response to ectopic Notch. Supernatants of CEM cells transduced with DN-MAML were also analysed to show the contribution of endogenous Notch transcriptional activity on VEGF expression. * represents p < 0.05 versus GFP-alone-transduced cells (B) Jurkat cells were used for a GSI washout assay and ID1 and GIMAP5 gene expression analysed over 4 hrs following washout using cDNA from untreated cells as the calibrator sample. (C) Western blot analysis of ID1 and GIMAP5 expression using protein extracts from Jurkat cells treated with a dose range of GSI IX.
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Figure 8: Analysis of Notch-induced VEGF, ID1 and GIMAP5 expression. (A) Supernatants from GFP-alone, N1ΔE, or N3ΔE-transduced Jurkat or CEM cells were used for VEGF ELISAs to analyse secreted VEGF protein levels in response to ectopic Notch. Supernatants of CEM cells transduced with DN-MAML were also analysed to show the contribution of endogenous Notch transcriptional activity on VEGF expression. * represents p < 0.05 versus GFP-alone-transduced cells (B) Jurkat cells were used for a GSI washout assay and ID1 and GIMAP5 gene expression analysed over 4 hrs following washout using cDNA from untreated cells as the calibrator sample. (C) Western blot analysis of ID1 and GIMAP5 expression using protein extracts from Jurkat cells treated with a dose range of GSI IX.

Mentions: At the mRNA level, VEGF is expressed at low levels in GFP-alone transfected Jurkat cells and is only upregulated by ectopic Notch1 (not Notch3). To confirm this finding at the protein level, we performed ELISAs on supernatants of cells transduced with GFP-alone, N1ΔE and N3ΔE retroviruses. As can be seen in figure 8.A, virtually no basal expression of VEGF protein is detected in supernatants from GFP-alone or N3ΔE-transduced Jurkat cells, whereas N1ΔE-transduced cells produce detectable levels of VEGF. The lack of detectable basal levels of secreted VEGF protein is contrary to the gene expression data shown in figures 5 &6, where GSI treatment and expression of DN-MAML decreased VEGF mRNA levels in Jurkat cells. This lack of correlation between VEGF mRNA and secreted VEGF protein levels could be due to a number of factors including post-transcriptional regulation of VEGF expression or regulation of VEGF protein secretion in the cell supernatants. This finding suggests that although ectopic Notch1 may promote VEGF protein expression, Notch does not necessarily contribute to basal VEGF protein expression in T-ALL cells.


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)

Analysis of Notch-induced VEGF, ID1 and GIMAP5 expression. (A) Supernatants from GFP-alone, N1ΔE, or N3ΔE-transduced Jurkat or CEM cells were used for VEGF ELISAs to analyse secreted VEGF protein levels in response to ectopic Notch. Supernatants of CEM cells transduced with DN-MAML were also analysed to show the contribution of endogenous Notch transcriptional activity on VEGF expression. * represents p < 0.05 versus GFP-alone-transduced cells (B) Jurkat cells were used for a GSI washout assay and ID1 and GIMAP5 gene expression analysed over 4 hrs following washout using cDNA from untreated cells as the calibrator sample. (C) Western blot analysis of ID1 and GIMAP5 expression using protein extracts from Jurkat cells treated with a dose range of GSI IX.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698846&req=5

Figure 8: Analysis of Notch-induced VEGF, ID1 and GIMAP5 expression. (A) Supernatants from GFP-alone, N1ΔE, or N3ΔE-transduced Jurkat or CEM cells were used for VEGF ELISAs to analyse secreted VEGF protein levels in response to ectopic Notch. Supernatants of CEM cells transduced with DN-MAML were also analysed to show the contribution of endogenous Notch transcriptional activity on VEGF expression. * represents p < 0.05 versus GFP-alone-transduced cells (B) Jurkat cells were used for a GSI washout assay and ID1 and GIMAP5 gene expression analysed over 4 hrs following washout using cDNA from untreated cells as the calibrator sample. (C) Western blot analysis of ID1 and GIMAP5 expression using protein extracts from Jurkat cells treated with a dose range of GSI IX.
Mentions: At the mRNA level, VEGF is expressed at low levels in GFP-alone transfected Jurkat cells and is only upregulated by ectopic Notch1 (not Notch3). To confirm this finding at the protein level, we performed ELISAs on supernatants of cells transduced with GFP-alone, N1ΔE and N3ΔE retroviruses. As can be seen in figure 8.A, virtually no basal expression of VEGF protein is detected in supernatants from GFP-alone or N3ΔE-transduced Jurkat cells, whereas N1ΔE-transduced cells produce detectable levels of VEGF. The lack of detectable basal levels of secreted VEGF protein is contrary to the gene expression data shown in figures 5 &6, where GSI treatment and expression of DN-MAML decreased VEGF mRNA levels in Jurkat cells. This lack of correlation between VEGF mRNA and secreted VEGF protein levels could be due to a number of factors including post-transcriptional regulation of VEGF expression or regulation of VEGF protein secretion in the cell supernatants. This finding suggests that although ectopic Notch1 may promote VEGF protein expression, Notch does not necessarily contribute to basal VEGF protein expression in T-ALL cells.

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