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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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Identification of direct notch targets using a GSI washout experiment. Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p < 0.01 versus untreated cells (effect of Notch inhibition). ** prepresents p < 0.01 versus GSI-treated cells (effect of Notch signalling). *** represents p < 0.01 versus washout cells (effect of cyclohexamide treatment).
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Figure 5: Identification of direct notch targets using a GSI washout experiment. Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p < 0.01 versus untreated cells (effect of Notch inhibition). ** prepresents p < 0.01 versus GSI-treated cells (effect of Notch signalling). *** represents p < 0.01 versus washout cells (effect of cyclohexamide treatment).

Mentions: To investigate the response dynamics of the Notch target genes identified by Affymetrix microarray analysis, we used a GSI washout assay to measure gene expression in response to endogenous Notch signalling. This assay involves incubating cells with GSI to allow Notch to accumulate at the cell surface. Washing the cells then removes gamma secretase inhibition and leads to active Notch signalling [9]. As shown in figure 5.A, mRNA expression analysis of known Notch target genes confirmed the validity of this method by showing an increase in gene expression following the removal of gamma secretase inhibition. In all cases, GSI treatment led to a significant decrease in gene expression, although the inhibition of c-Myc expression was not as striking as other known Notch targets. As expected, expression of these known Notch target genes increased following GSI washout and in some cases, expression increased above that of the untreated cells. Some cells were also incubated in parallel with cyclohexamide to inhibit protein synthesis and allow us to determine if de novo protein synthesis is required for gene expression. This in turn would indicate whether these genes are direct or indirect Notch targets. As expected, cyclohexamide did not prevent the increased gene expression following GSI washout in known Notch target genes (which have mostly been characterised as direct transcriptional targets). On the contrary, there was a general increase in gene expression in the presence of cyclohexamide. One explanation for this could be that an inhibitor of gene expression (such as HES1) provides negative feedback for Notch target genes in normal circumstances. This is supported by the finding that HES1 physically interacts with CSL to inhibit Notch/CSL-mediated transcription [33]. Moreover, oscillations in HES1 expression have been found to be due to auto-inhibition of HES1 transcription [34]. In the presence of cyclohexamide, a reduction in the protein level of an inhibitor such as HES1 may allow Notch to increase gene expression levels without any negative feedback mechanism.


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)

Identification of direct notch targets using a GSI washout experiment. Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p < 0.01 versus untreated cells (effect of Notch inhibition). ** prepresents p < 0.01 versus GSI-treated cells (effect of Notch signalling). *** represents p < 0.01 versus washout cells (effect of cyclohexamide treatment).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Identification of direct notch targets using a GSI washout experiment. Jurkat cells were treated with 10 uM GSI (or DMSO (untreated)) for 48 hours to accumulate cell surface Notch before washing to permit Notch signalling. After washing, cells were treated with 20 uM cyclohexamide (CHX) or ethanol (vehicle control) to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA made for real-time PCR analysis of known Notch target genes (A) and novel Notch target genes (B). Expression values were calculated using cDNA from untreated cells as the calibrator sample. * represents p < 0.01 versus untreated cells (effect of Notch inhibition). ** prepresents p < 0.01 versus GSI-treated cells (effect of Notch signalling). *** represents p < 0.01 versus washout cells (effect of cyclohexamide treatment).
Mentions: To investigate the response dynamics of the Notch target genes identified by Affymetrix microarray analysis, we used a GSI washout assay to measure gene expression in response to endogenous Notch signalling. This assay involves incubating cells with GSI to allow Notch to accumulate at the cell surface. Washing the cells then removes gamma secretase inhibition and leads to active Notch signalling [9]. As shown in figure 5.A, mRNA expression analysis of known Notch target genes confirmed the validity of this method by showing an increase in gene expression following the removal of gamma secretase inhibition. In all cases, GSI treatment led to a significant decrease in gene expression, although the inhibition of c-Myc expression was not as striking as other known Notch targets. As expected, expression of these known Notch target genes increased following GSI washout and in some cases, expression increased above that of the untreated cells. Some cells were also incubated in parallel with cyclohexamide to inhibit protein synthesis and allow us to determine if de novo protein synthesis is required for gene expression. This in turn would indicate whether these genes are direct or indirect Notch targets. As expected, cyclohexamide did not prevent the increased gene expression following GSI washout in known Notch target genes (which have mostly been characterised as direct transcriptional targets). On the contrary, there was a general increase in gene expression in the presence of cyclohexamide. One explanation for this could be that an inhibitor of gene expression (such as HES1) provides negative feedback for Notch target genes in normal circumstances. This is supported by the finding that HES1 physically interacts with CSL to inhibit Notch/CSL-mediated transcription [33]. Moreover, oscillations in HES1 expression have been found to be due to auto-inhibition of HES1 transcription [34]. In the presence of cyclohexamide, a reduction in the protein level of an inhibitor such as HES1 may allow Notch to increase gene expression levels without any negative feedback mechanism.

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