Notch1 regulates chemotaxis and proliferation by controlling the CC-chemokine receptors 5 and 9 in T cell acute lymphoblastic leukaemia.

Tumour cells often express deregulated profiles of chemokine receptors that regulate cancer cell migration and proliferation. Notch1 pathway activation is seen in T cell acute lymphoblastic leukaemia (T-ALL) due to the high frequency of Notch1 mutations affecting approximately 60% of patients, causing ligand-independent signalling and/or prolonging Notch1 half-life. We have investigated the possible regulative role of Notch1 on the expression and function of chemokine receptors CCR5, CCR9 and CXCR4 that play a role in determining blast malignant properties and localization of extramedullary infiltrations in leukaemia. We inhibited the pathway through γ-Secretase inhibitor and Notch1 RNA interference and analysed the effect on the expression and function of chemokine receptors. Our results indicate that γ-Secretase inhibitor negatively regulates the transcription level of the CC chemokine receptors 5 and 9 in T-ALL cell lines and patients' primary leukaemia cells, leaving CXCR4 expression unaltered. The Notch pathway also controls CCR5- and CCR9-mediated biological effects, ie chemotaxis and proliferation. Furthermore, engaging CCR9 through CCL25 administration rescues proliferation inhibition associated with abrogation of Notch activity. Finally, through RNA interference we demonstrated that the oncogenic isoform in T-ALL, Notch1, plays a role in controlling CCR5 and CCR9 expression and functions. These findings suggest that Notch1, acting in concert with chemokine receptors pathways, may provide leukaemia cells with proliferative advantage and specific chemotactic abilities, therefore influencing tumour cell progression and localization.

The non-proteinogenic amino acids L-methionine sulfoximine and DL-phosphinothricin activate mTOR.

L-Methionine sulfoximine (MSO) and DL-Phosphinothricin (PPT), two non-proteinogenic amino acids known as inhibitors of Glutamine Synthetase, cause a dose-dependent increase in the phosphorylation of the mTOR substrate S6 kinase 1. The effect is particularly evident in glutamine-depleted cells, where mTOR activity is very low, but is detectable for PPT also in the presence of glutamine. The stimulation of mTOR activity by either MSO or PPT is strongly synergized by essential amino acids. Thus, the non-proteinogenic amino acids MSO and PPT are mTOR activators.

Reciprocal regulation of Notch and PI3K/Akt signalling in T-ALL cells in vitro.

Notch signalling plays an important role in hematopoiesis and in the pathogenesis of T-ALL. Notch is known to interact with Ras and PTEN/PI3K (phosphoinositide-3 kinase)/Akt pathways. We investigated the interaction of Notch with these pathways and the possible reciprocal regulation of these signalling systems in T-ALL cells in vitro. Our analyses indicate that the PI3K/Akt pathway is constitutively active in the four T-ALL cell lines tested. Akt phosphorylation was not altered by the sequestration of growth factors, that is, Akt activation seems to be less dependent on but not completely independent of growth factors, possibly being not subject to negative feedback regulation. PTEN expression was not detected in 3/4 cell lines tested, suggesting the loss of PTEN-mediated Akt activation. Inhibition of the PI3K/Akt pathway arrests growth and enhances apoptosis, but with no modulation of expression of Bax-alpha and Bcl-2 proteins. We analysed the relationship between Notch-1 and the PI3K/Akt signalling and show that inhibition of the Akt pathway changes Notch expression; Notch-1 protein decreased in all the cell lines upon treatment with the inhibitor. Our studies strongly suggest that Notch signalling interacts with PI3K/Akt signalling and further that this occurs in the absence of PTEN expression. The consequences of this to the signalling outcome are yet unclear, but we have uncovered a significant inverse relationship between Notch and PI3K/Akt pathway, which leads us to postulate the operation of a reciprocal regulatory loop between Notch and Ras-PI3K/Akt in the pathogenesis of T-ALL.

Down-regulation of Notch1 expression is involved in HL-60 cell growth inhibition induced by 4-hydroxynonenal, a product of lipid peroxidation.

The role of the Notch1 pathway has been well assessed in leukemia. Notch1 mutations are the most common ones in T acute lymphoblastic leukaemia patients which carry either oncogenic Notch1 forms or ineffective ubiquitin ligase implicated in Notch1 turnover. Abnormalities in the Notch1-Jagged1 system have been reported also in acute myelogenous leukaemia (AML) patients where Jagged1 is frequently over-expressed. Moreover, activating Notch1 mutations, as well, can occur in human AML and in leukemia cases with lineage infidelity. As a result, Notch1 signalling inhibition is an attractive goal in leukaemia therapy. Blockage/delay in cell differentiation and/or increase of proliferation are the main results of Notch1 signalling activation in several leukemic cell lines. Moreover, specific genes involved in cell growth control have been identified as Notch1 transcriptional targets, i.e. Cyclin D1 and c-Myc. 4-Hydroxynonenal (HNE), an aldehyde produced during lipid peroxidation, is involved in several pathological and physiological conditions, including inflammation; atherosclerosis; and neurodegenerative and chronic liver diseases. Moreover HNE has an antiproliferative/ differentiative effect in several cell lines, by affecting the expression of key genes, such as oncogenes (e.g. c-Myc, c-Myb), cyclins and telomerase. This prompted us to study the effect of HNE on Notch1 expression and its related signalling in HL-60 cells, a leukemic cell line widely used for differentiation studies. RT-PCR as well as Western blot assay showed Notch1down-regulation in HNE-treated HL-60 cells. The expression of Hes1, a Notch1 target gene, was concomitantly down-regulated by HNE treatment, reflecting Notch1 signalling inhibition. DAPT, an inhibitor of Notch activity, when added contemporary to HNE, further increased cell growth inhibition, without affecting apoptosis. Moreover, DAPT treatment reversed the HNE-induced differentiation. Overall these results suggest that Notch1 is a target for HNE and its down regulation is a key event in HNE-mediated inhibition of cell proliferation in the HL-60 cell line. By contrast our data do not support a role for Notch1 in HNE- induced differentiation or apoptosis.

Resveratrol-induced apoptosis in human T-cell acute lymphoblastic leukaemia MOLT-4 cells.

Resveratrol (RES) is a natural occurring phytoalexin that has been shown to have chemopreventive activity. Resveratrol acts both by suppressing cell proliferation and inducing apoptosis in a variety of cancer cell lines. In this study, we show that RES induces apoptosis in MOLT-4 acute lymphoblastic leukaemia cells by modulating three different pathways that regulate cells survival and cell death. We show for the first time that RES inhibits the survival signalling pathways Notch and their down stream effector and modulates the operation of interacting signalling systems. It induces an increase in the levels of the pro-apoptotic proteins p53, its effector p21waf and Bax. We also show that RES inhibits the PI3K/Akt pathway and activates Gsk-3beta. The data presented here demonstrate unequivocally that RES induces apoptosis by inhibiting the Notch pathway and markedly influencing the operation of the interacting apoptosis pathways mediated by p53 and PI3K/Akt. These data support findings from other laboratories that have suggested the use of RES as a chemopreventive agent. Here, we have identified potential signalling pathways influenced by RES and this could lead to the identification of the targets of RES-induced apoptosis and growth control.

A wide role for NOTCH1 signaling in acute leukemia.

NOTCH1 is involved in the pathogenesis of T-acute lymphoblastic leukemia (T-ALL) carrying the very rare translocation t(7;9)(q34;q34.3). We analyzed the expression of genes belonging to NOTCH pathway, in acute leukemia primary samples and lymphoblastoid cell lines. NOTCH1 pathway activation represents a common feature of T-ALL when compared to acute myelogenous leukemia (AML) and B-cell precursor acute lymphoblastic leukemia. The contemporary expression of NOTCH1 and its ligands on cell surface contributes to high levels of pathway activity. AML primary samples show high levels of JAGGED1 expression despite the low NOTCH1 pathway activation, consistent with an autonomous JAGGED1 signaling in myeloid leukemogenesis.

Notch signal transduction is not regulated by SEL1L in leukaemia and lymphoma cells in culture.

The transduction of Notch signal plays an intricate role in cell differentiation and pathogenesis of haematological malignancies as well as in certain congenital conditions. The functionality of Notch signalling was tested using HES1 gene activation. SEL1 gene product has been postulated to be a negative regulator of Notch signalling. We investigated the relationship between Notch signalling and the expression of SEL1L gene in a number of leukaemia and lymphoma cells in culture. The cell lines could be separated into two groups. Group 1 contained lymphoma cell lines in which Notch signalling was intact; of these 4 cell lines were SEL1L+/HES1- and 3 SEL1L-/HES1-. Notch signalling was not subverted by EBNA2 expression in these lymphoma cells. In Group 2 cell lines Notch signalling was constitutively active but 6 out of 7 cell lines expressed SEL1L at high levels. In summary, a majority of cell lines of both groups express SEL1L and no inverse relationship is evident between SEL1L expression and the status of Notch signalling. The present investigation therefore suggests that SEL1L may not exert a negative regulatory influence on Notch signalling. No genomic alterations affecting SEL1L were detected either in the lymphoma or T-ALL cell lines tested. Taken together the present findings do not support the postulated negative regulatory role for SEL1L in Notch signalling.

Notch-ing from T-cell to B-cell lymphoid malignancies.

Notch receptors are transmembrane proteins critically determining cell fate and maintenance of progenitor cells in many developmental systems. Notch signaling is involved in stem cell self-renewal and regulates the main functions of cell life at different levels of development: cell proliferation, differentiation and apoptosis. By virtue of its involvement in the regulation of cell physiology, it is not surprising that a deregulation of the Notch pathway leads to the development of different tumors. In this review, we critically discuss the latest findings concerning Notch roles in hematologic oncology, with a special focus on T-cell acute lymphoblastic leukemia and B-cell malignancies. We also describe the molecular mediators of Notch-driven oncogenic effects and the current pharmacological approaches targeting Notch signaling.

Hyperthermia inhibits cell proliferation and induces apoptosis: relative signaling status of P53, S100A4, and Notch in heat sensitive and resistant cell lines.

The effects of hyperthermia on the expression of p53, the apoptosis-associated genes Bax and Bcl-2, Notch and S100A4 have been studied in the HepG2 cell line and the HUT cell line derived from HepG2, adapted for growth in hyperthermic conditions. Hyperthermia inhibits cell proliferation and induces apoptosis. HepG2 and HUT cells differed in respect of anchorage to growth surface, degree of proliferation and apoptosis and expression of p53, Bax, Bcl-2, Notch, and S100A4 genes. The induction of apoptosis and the inhibition of cell proliferation occurred independently of p53, and independently also of involvement of the apoptosis family genes Bax and Bcl-2. We demonstrate novel and marked differences between transient heat shock and heat adaptation in respect of pathways of signaling and generation of phenotypic effects in vitro. Different signaling patterns have been identified here. Pathways of signaling by S100A4, by its interaction with and sequestration of p53, and by Notch also seem differentially operational in the induction of apoptosis, and both appear to be activated as alternative pathways in the context of hyperthermia signaling independently of p53.

Allele frequency of two intragenic microsatellite loci of SEL1L gene in Northern Italian population.

Two cytosine-adenine (CA) repeats CAR/CAL and RepIN20 occur in the human SEL1L gene, which is regarded as a candidate gene for insulin-dependent diabetes mellitus (IDDM) and Grave's disease. We have characterized these repeats to determine if they might serve as effective microsatellite markers for linkage analysis to clarify whether SEL1L gene plays a role in the pathogenesis of these autoimmune diseases. The allele frequencies and average heterozygosity of the microsatellite repeats were analysed in 94 DNA samples from peripheral blood mononuclear (PBMC) cells from adults of Northern Italy. The average heterozygosity was 0.68 for CAR/CAL polymorphism and 0.85 for RepIN20. The size of PCR fragments of CAR/CAL ranged from 207-225 bp and the most frequent allele was 207 bp (40.4%). The size of the fragments of RepIN20 ranged from 237-255 bp and the most frequent allele was 249 bp (30.8%). In the light of the highly polymorphic nature of both microsatellites and their intragenic location in SEL1L gene, we suggest that they could provide a means for linkage analysis to clarify the potential role of SEL1L in conferring susceptibility to IDDM or Grave's disease.

Differential regulation of Notch signal transduction in leukaemia and lymphoma cells in culture.

The transduction of Notch signal plays an intricate role in cell differentiation and pathogenesis of haematological malignancies as well as in certain congenital conditions. We found no genomic changes in either gene in 34 leukaemic samples and 25 leukaemia and lymphoma cell lines. The functionality of Notch signalling was tested using HES1 gene activation. We show that Notch signalling is differentially regulated in T-acute lymphoblastic leukaemia (ALL) and B-lymphoma cells. The Notch pathway is intact in a majority of B-lymphoma cell lines, but EBNA2, which mimics notch function, can occasionally activate the pathway. In contrast, the Notch pathway is constitutively active in T-ALL. This is the first demonstration of a distinction between B-lymphomas and T-cell leukaemias in the functioning of the Notch-signalling pathway. This might be related to their pathogenesis.