Research ArticleNotch signaling induces SKP2 expression and promotes reduction of p27Kip1 in T-cell acute lymphoblastic leukemia cell lines
Introduction
T-cell acute lymphoblastic leukemia (T-ALL) is a malignant disease, originating in thymocytes. T-ALL constitutes a substantial fraction of ALL tumors, both in children and in adults [1]. Current treatment is based on combination chemotherapy, but long-term survival rates are reduced, particularly in older patients, emphasizing the need for improved therapy. The molecular mechanisms underpinning T-ALL are likely to be complex, as a number of genes, including c-MYC, HOX 11, TAL1 and LMO, are involved in chromosomal translocations with the T-cell receptor locus (for review see [1]). Mutations in the Notch signaling pathway have recently emerged as an important genetic component in T-ALL. The involvement of Notch was first observed in rare t(7,9)(q34;q34.3) translocations, which brings an activated form of the NOTCH 1 receptor gene (TAN 1) under control elements from the T-cell receptor gene [2]. More recently, it was shown that more than 50% of all T-ALL patients carry NOTCH1 gain-of-function mutations that, similar to TAN 1, generate an activated form of Notch [3].
The Notch signaling pathway is important for cell fate decisions in many different cell types, including the T-cell lineage [4]. Notch signaling is initiated when the transmembrane Notch receptor interacts with DSL ligands on juxtaposed cells. This leads to two proteolytic processing events in the receptor, first at the extracellular side and subsequently in the plasma membrane. The latter cleavage is executed by the gamma-secretase complex and leads to liberation of the Notch intracellular domain (Notch ICD), which translocates to the nucleus. In the nucleus, Notch ICD binds to the DNA-binding protein CSL, and converts CSL from a repressor to a transcriptional activator [5].
T-ALL mutations lead to aberrantly high Notch signaling and affect two different regions of the NOTCH 1 receptor [3]. One class of mutations falls in the heterodimerization domain (HD), making the receptor more prone to proteolytic processing and thus less dependent on ligand-activation. The other class of mutations occurs in the PEST domain of NOTCH 1, generating truncated versions of the NOTCH 1 ICD, which increases the stability of the normally short-lived intracellular domain [6]. Further mutational analysis of the PEST domain has revealed four serine residues that serve as a negative regulatory sequence, and deletion of these residues makes the Notch ICD more active [7]. The link between deregulated Notch signaling and T-ALL receives further support from mouse models of T-ALL, where Notch 1 mutations occur as secondary hits in mice engineered for deregulation of c-Myc, TAL1, Ikaros and Pbx1 expression [8], [9], [10], [11], [12].
The downstream response of Notch signaling is only partially understood. There is a wealth of data supporting the notion that Hes and Hey genes, which encode bHLH negative transcriptional regulators [13], [14], are immediate downstream genes, but there are situations where Notch signaling exert biological effects without affecting Hes and Hey expression [15]. It is therefore likely that other immediate downstream genes exist that are directly activated by Notch ICD via CSL in specific cellular contexts, and several such genes have recently been identified, e.g. Nrarp, GFAP, GATA2, c-Myc and p21cip1 (see [16] for review).
In T-ALL, our understanding of which downstream genes are activated, and how this affects cell cycle regulation and leads to cellular transformation, is more limited. The finding that HES 1 activation is not always required for Notch-induced growth promotion [17], may suggest that other downstream responses could be relevant for T-ALL. In keeping with this, c-MYC has recently been identified as an important new direct target gene in some T-ALL cell lines [18]. c-MYC was, however, not activated in all T-ALL cell lines [18], suggesting the existence of other immediate Notch downstream effectors.
In this report, we have addressed the downstream aspects of Notch signaling in T-ALL. We find that T-ALL tumors and cell lines have low levels of HES1 and HEY2 expression. Instead, increased Notch signaling results in elevated levels of SKP2, the F-box component in the SCFSkp2 E3 ubiquitin ligase complex [19]. The high level of SKP2 was paralleled by low levels of the CDK inhibitor (CKI) p27Kip1, a target substrate for ubiquitinylation and thus negatively regulated by the SCF–Skp2 complex [20] (for review see [19]). Blocking Notch signaling pharmacologically reversed the situation, and this “Notch off” state was associated with reduced SKP2 and increased p27Kip1 expression and subsequent G1 cell cycle arrest. In conclusion, our data suggest that a NOTCH/SKP2/p27Kip1 axis may contribute to the development of T-ALL.
Section snippets
Cell lines, tumor samples and plasmids
Malignant T-ALL and pre B-ALL cell lines were cultured as described [21]. JM-JURKAT, MOLT4, CEM, peer, RS4 and REH6 cell lines were derived from the ATCC collection. Samples of cryopreserved lymphoblasts were collected from children with the diagnosis of T-ALL and B-precursor-cell phenotype, treated at the Karolinska Hospital, Stockholm, Sweden, and leukemic cells were purified as previously described [22]. Approval was obtained from the Institutional Review Board for these studies. Normal T-
The canonical Notch downstream genes HES1 and HEY2 are expressed at low levels in T-ALL patients and cell lines
We first analyzed whether NOTCH 1 ICD accumulated in T-ALL cell lines, as a reflection of high level Notch signaling. We observed clearly detectable levels of NOTCH 1 ICD in the three T-ALL cell lines JM-JURKAT (JM), MOLT4 and CEM, but not in the B-ALL cell line RS4 (Fig. 1A). The observed bands in the JM and CEM cells were approximately 100 kDa, which corresponds to the expected length of the wild type NOTCH 1 ICD. This is in line with that these cell lines carry missense mutations in the HD
Discussion
In this report we have addressed how the deregulated Notch signaling leads to altered cell cycle progression in T-ALL cells. Our data support the notion that expression of SKP2 and p27Kip1 is under the control of Notch signaling, and that this Notch/SKP2/p27Kip1 axis regulates G1/S cell cycle transition in T-ALL cells. This line of reasoning receives support from several experimental observations. First, the link between Notch and SKP2 is based on two findings: Notch 1 ICD binds to the SKP2
Acknowledgments
We are indebted to Susanne Bergstedt for cell culture work. The financial support from the Swedish Cancer Society, the Swedish Research Council (OS and UL), the EU project EuroStemCell, the Foundation for Strategic Research (CEDB) (UL), the Cancer Society of Stockholm and the Children Cancer Foundation (OS) is gratefully acknowledged.
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