Notch protection against apoptosis in T-ALL cells mediated by GIMAP5☆
Introduction
Dysregulation of the Notch signalling pathway has been identified in patients with T cell acute lymphoblastic leukaemia (T-ALL). The human homologue of Drosophila Notch was first identified as a chromosomal translocation in a rare subset of patients with T-ALL [1]. Mice transgenic for the Notch1 intracellular domain (ICD) [2] develop aggressive T cell lymphomas, revealing the potential of aberrant Notch signalling to induce T cell neoplasia. More recently, a study by Bellavia et al. showed over-expression of Notch3 and the pre-T cell receptor alpha to be a characteristic of all T-ALL samples [3]. Weng et al. have identified mutations in the heterodimerisation domain of Notch1 which result in ligand-independent activation, and in the PEST domain, which result in prolonged activity of the cleaved form of Notch1 [4]. These gain-of-function mutations in Notch1 were found to be present in half of the T-ALL patients studied, and in many of the cell lines derived from T-ALL patients.
The functional consequences of aberrant Notch1 signalling in the context of T-ALL include the induction of proliferation [4], [5] and the inhibition of apoptosis [6], [7]. Notch has been shown to promote proliferation by the upregulation of c-myc [8], [9] and cyclin D1 [10], while mechanisms of Notch-protection against apoptosis include downregulation of the glucocorticoid receptor [11], and activation of the PI-3 K/AKT pathway [6], [7], [12]. The latter mechanism has been shown to lead to mTOR activation and p53 inhibition in breast cancer cell lines, while inhibition of this pathway promoted apoptosis in human and mouse T-ALL cell lines [6].
To date the studies of Notch signalling have focussed on Notch1 and the comparative effects of Notch1 and Notch3 have not yet been studied. Both Notch1 and Notch3 are expressed during normal thymocyte development where Notch signalling is thought to act synergistically with signalling through the pre-TCR alpha chain to mediate positive selection [13]. Also, Deftos et al. have demonstrated Notch-associated resistance to glucocorticoid-mediated apoptosis in a mouse CD4/CD8 double-positive (DP) thymocyte cell line [14] and in primary DP thymocytes [14].
In this study, we investigated the role Notch in T-ALL cell apoptosis and found that inhibiting endogenous Notch signalling using gamma secretase inhibitors (GSIs) promoted apoptosis. We also found that ectopic expression of either Notch1 or Notch3 protected T-ALL cells from apoptosis. We showed that expression of GIMAP5 (a GTPase associated with intracellular membranes) is upregulated by Notch signalling and that knockdown of this gene using siRNA attenuates Notch-associated protection against apoptosis. These data provide evidence of a novel mechanism mediating Notch protection against apoptosis in T-ALL.
Section snippets
Plasmid constructs, cell lines and retroviral transductions
“∆E” constructs of Notch were generated which lack extracellular ligand binding domains and are activated constitutively by gamma secretase at the plasma membrane. N1∆E (base pairs 5143-7671) and N3∆E (base pairs 4942-7045) cDNAs were cloned into the bicistronic retroviral vector, pMX-eGFP (a kind gift from T. Kitamura, Tokyo, Japan). Similarly, Notch1 ICD, which is constitutively active and gamma-secretase-independent, was cloned into pMX as described previously [15]. Dominant-negative
GSI washout assay
Jurkat cells were incubated with 10 uM GSI IX for 48 hrs then cells were washed twice with growth medium and seeded in growth medium plus 10 uM GSI IX (mock washout) or 0.1% DMSO (GSI washout) in the presence or absence of 20 uM cycloheximide to inhibit protein synthesis. After 4 hrs, RNA was isolated and cDNA used for gene expression analysis.
Notch signalling protects T-ALL cells from dexamethasone-induced apoptosis
Inhibition of the Notch signalling pathway using GSIs has been shown to inhibit proliferation of some T-ALL cell lines [4], [5], [17]). Given previous studies showing a protective role of Notch in T cell apoptosis [6], [7], we investigated whether GSI-mediated inhibition of endogenous Notch signalling could increase the susceptibility of T-ALL cells to dexamethasone-mediated apoptosis. Glucocorticoids are an important therapeutic tool in lymphoid malignancies and resistance of malignant cells
Discussion
In this study, we have confirmed the results of previous studies showing that inhibition of Notch signalling promotes dexamethasone-induced apoptosis and we have extended these findings by showing that both Notch1 and Notch3 are capable of protecting T-ALL cells from dexamethasone-induced apoptosis. Notch1 and Notch3 are the predominant forms of endogenous Notch expressed in T-ALL cell lines [21] and the relative role of these two forms of Notch in protecting cells against dexamethasone-induced
Acknowledgments
The authors would like to thank Andy Hayes and the University of Manchester Microarray Core Facility for help with Affymetrix microarray experiments. We would also like to acknowledge the help and advice of Professor Andrew Sharrocks, Songbi Chen, Apolinar Maya-Mendoza, Federico Dajas-Bailador, Fiona Warrander and Dorota Feret. We thank the Leukaemia Research Fund for their support.
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Dedication: This manuscript is dedicated to the memory of Anne-Marie Buckle.