The Notch1/c-Myc Pathway in T Cell Leukemia

The transmembrane receptor Notch1 is a member of the evolutionarily conserved family of developmental regulators originally identified in Drosophila melanogaster. Notch signaling plays essential roles in regulating cell fate in thymic, intestinal, vascular and neuronal development (1-5). Recent studies detect mutations in the Notch1 receptor in roughly half of patients with T cell acute lymphoblastic leukemia (T-ALL) (6). Although expression of an activated Notch1 allele has been shown to cause leukemia in mice, the molecular mechanisms whereby Notch1 mediates cellular transformation are unknown (7). To understand how Notch1 contributes to T cell leukemogenesis, we generated mouse leukemic cell lines where the expression of activated Notch1 was doxycycline-regulated. This cell line was used for gene expression profiling to specifically identify Notch1-regulated genes in leukemia. These studies revealed that Notch1 directly induces the expression of c-myc and that inhibition of Notch1 results in cell cycle arrest and apoptosis and decreased c-myc levels (8). These studies and those performed by Aster, Pear and colleagues in human T-ALL cell lines demonstrate that the direct Notch1-mediated activation of c-myc is required to maintain leukemic growth (8-10). Interestingly, the Notch1/c-Myc oncogenic pathway does not appear limited to T-ALL, as studies by the Efstratiadis group show that expression of intracellular Notch1 leads to mammary tumorigenesis and importantly, transformation appears at least partially c-myc dependent (11). Collectively, these studies begin to delineate how Notch1 mediates cellular transformation and raises the possibility that the Notch1/c-Myc pathway may contribute to human breast cancer and potentially other solid tumors.     

In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein

Hematopoietic stem cells (HSCs) can self-renew extensively after transplantation. The conditions supporting their in vitro expansion are still being defined. Retroviral overexpression of the human homeobox B4 (HOXB4) gene in mouse bone marrow cells enables over 40-fold expansion of HSCs in vitro. To circumvent the requirement for retroviral infection, we used recombinant human TAT-HOXB4 protein carrying the protein transduction domain of the HIV transactivating protein (TAT) as a potential growth factor for stem cells. HSCs exposed to TAT-HOXB4 for 4 d expanded by about four- to sixfold and were 8-20 times more numerous than HSCs in control cultures, indicating that HSC expansion induced by TAT-HOXB4 was comparable to that induced by the human HOXB4 retrovirus during a similar period of observation. Our results also show that TAT-HOXB4-expanded HSC populations retain their normal in vivo potential for differentiation and long-term repopulation. It is thus feasible to exploit recombinant HOXB4 protein for rapid and significant ex vivo expansion of normal HSCs.     

Discrete Notch signaling requirements in the specification of hematopoietic stem cells

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.     

The proto-oncogene c-myc acts through the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) to facilitate the activation of Cdk4/6 and early G(1) phase progression

Progression through the early G(1) phase of the cell cycle requires mitogenic stimulation, which ultimately leads to the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6). Cdk4/6 activity is promoted by D-type cyclins and opposed by Cdk inhibitor proteins. Loss of c-myc proto-oncogene function results in a defect in the activation of Cdk4/6. c-myc(-/-) cells express elevated levels of the Cdk inhibitor p27(Kip1) and reduced levels of Cdk7, the catalytic subunit of Cdk-activating kinase. We show here that in normal (c-myc(+/+)) cells, the majority of cyclin D-Cdk4/6 complexes are assembled with p27 and remain inactive during cell cycle progression; their function is presumably to sequester p27 from Cdk2 complexes. A small fraction of Cdk4/6 protein was found in lower molecular mass catalytically active complexes. Conditional overexpression of p27 in c-myc(+/+) cells caused inhibition of Cdk4/6 activity and elicited defects in G(0)-to-S phase progression very similar to those seen in c-myc(-/-) cells. Overexpression of cyclin D1 in c-myc(-/-) cells rescued the defect in Cdk4/6 activity, indicating that the limiting factor is the number of cyclin D-Cdk4/6 complexes. Cdk-activating kinase did not rescue Cdk4/6 activity. We propose that the defect in Cdk4/6 activity in c-myc(-/-) cells is caused by the elevated levels of p27, which convert the low abundance activable cyclin D-Cdk4/6 complexes into unactivable complexes containing higher stoichiometries of p27. These observations establish p27 as a physiologically relevant regulator of cyclin D-Cdk4/6 activity as well as mechanistically a target of c-Myc action and provide a model by which c-Myc influences the early-to-mid G(1) phase transition.     

Notch in the niche

Notch signaling is essential for hematopoietic stem cell (HSC) formation during embryogenesis, and hitherto it was also thought to be required for HSC maintenance. However, in this issue of Cell Stem Cell, Maillard et al. (2008) demonstrate rather conclusively that inactivation of the Notch pathway in HSCs does not interfere with their self-renewal.     

Chromosome Instability Underlies Hematopoietic Stem Cell Dysfunction and Lymphoid Neoplasia Associated with Impaired Fbw7-Mediated Cyclin E Regulation

The Fbw7 ubiquitin ligase critically regulates hematopoietic stem cell (HSC) function, though the precise contribution of individual substrate ubiquitination pathways to HSC homeostasis is unknown. In the work reported here, we used a mouse model in which we introduced two knock-in mutations (T74A and T393A [changes of T to A at positions 74 and 393]) to disrupt Fbw7-dependent regulation of cyclin E, its prototypic substrate, and to examine the consequences of cyclin E dysregulation for HSC function. Serial transplantation revealed that cyclin E^{T74A T393A} HSCs self-renewed normally; however, we identified defects in their multilineage reconstituting capacity. By inducing hematologic stress, we exposed an impaired self-renewal phenotype in cyclin E knock-in HSCs that was associated with defective cell cycle exit and the emergence of chromosome instability (CIN). Importantly, p53 deletion induced both defects in self-renewal and multilineage reconstitution in cyclin E knock-in HSCs with serial transplantation and CIN in hematopoietic stem and progenitor cells. Moreover, CIN was a feature of fatal T-cell malignancies that ultimately developed in recipients of cyclin E^{T74A T393A}; p53-null HSCs. Together, our findings demonstrate the importance of Fbw7-dependent cyclin E control to the hematopoietic system and highlight CIN as a characteristic feature of HSC dysfunction and malignancy induced by deregulated cyclin E.     

Notch signaling regulates mammary stem cell function and luminal cell-fate commitment

The recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.     

Notch1 Signaling Regulates the Proliferation and Self-Renewal of Human Dental Follicle Cells by Modulating the G1/S Phase Transition and Telomerase Activity

Multipotent human dental follicle cells (HDFCs) have been intensively studied in periodontal regeneration research, yet the role of Notch1 in HDFCs has not been fully understood. The aim of the current study is to explore the role of Notch1 signaling in HDFCs self-renewal and proliferation. HDFCs were obtained from the extracted wisdom teeth from adolescent patients. Regulation of Notch1 signaling in the HDFCs was achieved by overexpressing the exogenous intracellular domain of Notch1 (ICN1) or silencing Notch1 by shRNA. The regulatory effects of Notch1 on HDFC proliferation, cell cycle distribution and the expression of cell cycle regulators were investigated through various molecular technologies, including plasmid construction, retrovirus preparation and infection, qRT-PCR, western blot, RBP-Jk luciferase reporter and cell proliferation assay. Our data clearly show that constitutively activation of Notch1 stimulates the HDFCs proliferation while inhibition of the Notch1 suppresses their proliferation in vitro. In addition, the HDFCs proliferation is associated with the increased expression of cell cycle regulators, e.g. cyclin D1, cyclin D2, cyclin D3, cyclin E1, CDK2, CDK4, CDK6, and SKP2 and the decreased expression of p27 ^kip1. Moreover, our data show that the G1/S phase transition (indicating proliferation) and telomerase activity (indicating self-renewal) can be enhanced by overexpression of ICN1 but halted by inhibition of Notch1. Together, the current study provides evidence for the first time that Notch1 signaling regulates the proliferation and self-renewal capacity of HDFCs through modulation of the G1/S phase transition and the telomerase activity.     

Recombinant Human Adenovirus-p53 Injection Induced Apoptosis in Hepatocellular Carcinoma Cell Lines Mediated by p53-Fbxw7 Pathway,Which Controls c-Myc and Cyclin E

F-box and WD repeat domain-containing 7 (Fbxw7/hAgo/hCdc4/Fbw7) is a p53-dependent tumor suppressor and leads to ubiquitination-mediated suppression of several oncoproteins including c-Myc, cyclin E, Notch, c-Jun and others. Our previous study has indicated that low expression of Fbxw7 was negatively correlated with c-Myc, cyclin E and mutant-p53 in hepatocellular carcinoma (HCC) tissues. But the role and mechanisms of Fbxw7 in HCC are still unknown. Here, we investigated the function of Fbxw7 in HCC cell lines and the anti-tumor activity of recombinant human adenovirus-p53 injection (rAd-p53, Gendicine) administration in vitro and in vivo. Fbxw7-specific siRNA enhanced expression of c-Myc and cyclin E proteins and increased proliferation in cell culture. rAd-p53 inhibited tumor cell growth with Fbxw7 upregulation and c-Myc and cyclin E downregulation in vitro and a murine HCC model. This effect could be partially reverted using Fbxw7-specific siRNA. Here, we suggest that the activation of Fbxw7 by adenoviral delivery of p53 leads to increased proteasomal degradation of c-Myc and cyclin E enabling growth arrest and apoptosis. Addressing this pathway, we identified that rAd-p53 could be a potential therapeutic agent for HCC.     

The elements of stem cell self-renewal: a genetic perspective

Every day, the body produces billions of new blood cells. Each of these is derived from a rare cell in the bone marrow called the hematopoietic stem cell (HSC). Because most mature blood cells have a limited lifespan, the ability of HSCs to self-renew and replenish the mature cell compartment is critical to sustaining life. While great progress has been made in isolating HSCs and defining their functional and phenotypic characteristics, the molecular mechanisms that regulate their self-renewal remain a mystery. Over the last few years, alterations in HSC frequency and self-renewal capacity in transgenic and knock-out mice have led to the identification of novel mediators of HSC homeostasis in vivo. These genetically modified mice have revealed that maintenance of survival, proliferation, quiescence, and normal telomere length all contribute to the self-renewal of HSCs. They also highlight the need to test in context of the normal microenvironment the role of signaling molecules such as Notch and Wnt, which have emerged recently as important regulators of HSC self-renewal. The emerging picture these data provide of the regulation of self-renewal in HSCs has provided a better understanding of the basic biology of stem cells and holds promise for designing strategies to improve bone marrow transplantation.     

Adult mouse hematopoietic stem cells: purification and single-cell assays

Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.     

The HOXB4 homeoprotein promotes the ex vivo enrichment of functional human embryonic stem cell-derived NK cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34(+)CD45RA(+) precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.     

Studies on HOXB4 Expression During Differentiation of Human Cytomegalovirus-infected Hematopoietic Stem Cells into Lymphocyte and Erythrocyte Progenitor Cells

We investigated the role of homeobox B4 (HOXB4) mRNA/protein expression induced by human cytomegalovirus (HCMV) and/or all-trans retinoic acid (ATRA) in proliferation and committed differentiation of human cord blood hematopoietic stem cells (HSCs) into colony-forming-units of T-lymphocyte (CFU-TL) and erythroid (CFU-E) progenitors in vitro. Twelve cord blood samples were collected from the fetal placenta umbilical vein and cultured in vitro. The proliferation and differentiation of cord blood HSCs into CFU-TL and CFU-E were continuously disrupted with HCMV-AD169 and/or 6 × 10(-8) mol/l of ATRA. HOXB4 mRNA/protein expression in CFU-TL and CFU-E was detected in control, ATRA, HCMV and ATRA + HCMV groups on days 3, 7, and 12 of culture by fluorescent qRT-PCR/western blot. We found that HOXB4 mRNA/protein expression was detectable on day 3, increased on day 7 and was highest on day 12. HOXB4 mRNA/protein expression in HCMV group was downregulated compared with control group (P < 0.05). However, the levels were significantly upregulated in HCMV + ATRA group compared with HCMV group (P < 0.05). We concluded that the abnormal HOXB4 mRNA/protein expression induced by HCMV could play a role in hematopoietic damage. ATRA, at the concentration used, significantly up-regulated HOXB4 mRNA/protein expression in normal lymphocyte and erythrocyte progenitor cells as well as in HCMV-infected cells.     

NCAM as a cystogenesis marker gene of PKD2 overexpression

ADPKD (Autosomal Dominant Polycystic Kidney Disease) is characterized by the progressive expansion of multiple cystic lesions in the kidneys. ADPKD is caused by mutations in Ed-pl. consider PKD1 and PKD2. Recently a relation between c-myc and the pathogenesis of ADPKD was reported. In addition, c-Myc is a downstream effector of PKD1. To identify the gene regulated by PKD2 and c-Myc, we performed gene expression profiling in PKD2 and c-Myc overexpressing cells using a human 8K cDNA microarray. NCAM (neuronal cell adhesion molecule) levels were significantly reduced in PKD2 overexpressing systems in vitro and in vivo. These results suggest that NCAM is an important molecule in the cystogenesis induced by PKD2 overexpression.