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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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Related in: MedlinePlus

Affymetrix microarray array analysis of Notch1-transduced Jurkat cells. Jurkat cells were transduced with GFP-alone, N1ΔE, or N3ΔE retrovirus and after 48 hrs, GFP+ cells were isolated by flow cytometry and total RNA extracted for Affymetrix array analysis. Data presented represents mean of 4 independent experiments. Graphical representation of microarray data showing the 15 genes most upregulated by Notch1. * represents p < 0.05 vesus GFP-alone-transduced cells based on p-values from n = 4 experiments. Using false discovery rate analysis, none of these changes were significant.
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Figure 1: Affymetrix microarray array analysis of Notch1-transduced Jurkat cells. Jurkat cells were transduced with GFP-alone, N1ΔE, or N3ΔE retrovirus and after 48 hrs, GFP+ cells were isolated by flow cytometry and total RNA extracted for Affymetrix array analysis. Data presented represents mean of 4 independent experiments. Graphical representation of microarray data showing the 15 genes most upregulated by Notch1. * represents p < 0.05 vesus GFP-alone-transduced cells based on p-values from n = 4 experiments. Using false discovery rate analysis, none of these changes were significant.

Mentions: GFP-alone, N1ΔE and N3ΔE retroviruses were used to infect the T-ALL Jurkat cell line with a transduction efficiency of approximately 30% and GFP+ cells were sorted by flow cytometry at 48 hrs to generate a pure (> 95%) population of transduced cells for gene expression analysis. This relatively early time-point was used to identify genes directly upregulated by Notch signalling rather those associated with secondary effects of Notch-induced differentiation. Total RNA was made from sorted cells and used for Affymetrix analysis. This procedure was performed in quadruplicate and the Affymetrix data was used to generate mean fold changes in gene expression using the GFP-alone-transduced cells as the calibrator sample. Statistical analysis using false discovery rate correction showed no genes differentially expressed. However, known targets of Notch signalling such as HES1 [23], Notch3 [24], HERP1 and HERP2 [15] were in the top 50 genes ranked by fold change. The 15 genes most upregulated by Notch1 based on analysis of microarray data are shown in figure 1. A high degree of overlap was found with genes upregulated by Notch3 (see Additional file 3). This led us to select the top 10 upregulated genes (as well as CD28; a putative Notch target gene of interest to us) for further analysis. Below we present the results of these validation studies.


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)

Affymetrix microarray array analysis of Notch1-transduced Jurkat cells. Jurkat cells were transduced with GFP-alone, N1ΔE, or N3ΔE retrovirus and after 48 hrs, GFP+ cells were isolated by flow cytometry and total RNA extracted for Affymetrix array analysis. Data presented represents mean of 4 independent experiments. Graphical representation of microarray data showing the 15 genes most upregulated by Notch1. * represents p < 0.05 vesus GFP-alone-transduced cells based on p-values from n = 4 experiments. Using false discovery rate analysis, none of these changes were significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Affymetrix microarray array analysis of Notch1-transduced Jurkat cells. Jurkat cells were transduced with GFP-alone, N1ΔE, or N3ΔE retrovirus and after 48 hrs, GFP+ cells were isolated by flow cytometry and total RNA extracted for Affymetrix array analysis. Data presented represents mean of 4 independent experiments. Graphical representation of microarray data showing the 15 genes most upregulated by Notch1. * represents p < 0.05 vesus GFP-alone-transduced cells based on p-values from n = 4 experiments. Using false discovery rate analysis, none of these changes were significant.
Mentions: GFP-alone, N1ΔE and N3ΔE retroviruses were used to infect the T-ALL Jurkat cell line with a transduction efficiency of approximately 30% and GFP+ cells were sorted by flow cytometry at 48 hrs to generate a pure (> 95%) population of transduced cells for gene expression analysis. This relatively early time-point was used to identify genes directly upregulated by Notch signalling rather those associated with secondary effects of Notch-induced differentiation. Total RNA was made from sorted cells and used for Affymetrix analysis. This procedure was performed in quadruplicate and the Affymetrix data was used to generate mean fold changes in gene expression using the GFP-alone-transduced cells as the calibrator sample. Statistical analysis using false discovery rate correction showed no genes differentially expressed. However, known targets of Notch signalling such as HES1 [23], Notch3 [24], HERP1 and HERP2 [15] were in the top 50 genes ranked by fold change. The 15 genes most upregulated by Notch1 based on analysis of microarray data are shown in figure 1. A high degree of overlap was found with genes upregulated by Notch3 (see Additional file 3). This led us to select the top 10 upregulated genes (as well as CD28; a putative Notch target gene of interest to us) for further analysis. Below we present the results of these validation studies.

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