Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling.

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8(+) cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.

Ligand-independent signaling functions for the B lymphocyte antigen receptor and their role in positive selection during B lymphopoiesis.

Signal transduction through the B cell antigen receptor (BCR) is determined by a balance of positive and negative regulators. This balance is shifted by aggregation that results from binding to extracellular ligand. Aggregation of the BCR is necessary for eliciting negative selection or activation by BCR-expressing B cells. However, ligand-independent signaling through intermediate and mature forms of the BCR has been postulated to regulate B cell development and peripheral homeostasis. To address the importance of ligand-independent BCR signaling functions and their regulation during B cell development, we have designed a model that allows us to isolate the basal signaling functions of immunoglobulin (Ig)alpha/Igbeta-containing BCR complexes from those that are dependent upon ligand-mediated aggregation. In vivo, we find that basal signaling is sufficient to facilitate pro-B --> pre-B cell transition and to generate immature/mature peripheral B cells. The ability to generate basal signals and to drive developmental progression were both dependent on plasma membrane association of Igalpha/Igbeta complexes and intact immunoregulatory tyrosine activation motifs (ITAM), thereby establishing a correlation between these processes. We believe that these studies are the first to directly demonstrate biologically relevant basal signaling through the BCR where the ability to interact with both conventional as well as nonconventional extracellular ligands is eliminated.

p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl).

p62(dok) has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210(bcr-abl) oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62(dok) in normal cell signaling as well as in p210(bcr-abl) leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62(dok)-(/)- mice, that the loss of p62(dok) results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62(dok)-(/)- cells after the removal of growth factor. However, p62(dok) inactivation does not affect DNA damage and growth factor deprivation-induced apoptosis. Furthermore, p62(dok) inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210(bcr-abl) in bone marrow cells. These data indicate that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62(dok) can oppose leukemogenesis by p210(bcr-abl).

Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles.

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Separation of Notch1 promoted lineage commitment and expansion/transformation in developing T cells.

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.

Essential roles for ankyrin repeat and transactivation domains in induction of T-cell leukemia by notch1.

Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types, including lymphoid cells. Signaling is normally initiated through one or more ligand-mediated proteolytic cleavages that permit nuclear translocation of the intracellular portion of the Notch receptor (ICN), which then binds and activates transcription factors of the Su(H)/CBF1 family. Several mammalian Notch receptors are oncogenic when constitutively active, including Notch1, a gene initially identified based on its involvement in a (7;9) chromosomal translocation found in sporadic T-cell lymphoblastic leukemias and lymphomas (T-ALL). To investigate which portions of ICN1 contribute to transformation, we performed a structure-transformation analysis using a robust murine bone marrow reconstitution assay. Both the ankyrin repeat and C-terminal transactivation domains were required for T-cell leukemogenesis, whereas the N-terminal RAM domain and a C-terminal domain that includes a PEST sequence were nonessential. Induction of T-ALL correlated with the transactivation activity of each Notch1 polypeptide when fused to the DNA-binding domain of GAL4, with the exception of polypeptides deleted of the ankyrin repeats, which lacked transforming activity while retaining strong transactivation activity. Transforming polypeptides also demonstrated moderate to strong activation of the Su(H)/CBF1-sensitive HES-1 promoter, while polypeptides with weak or absent activity on this promoter failed to cause leukemia. These experiments define a minimal transforming region for Notch1 in T-cell progenitors and suggest that leukemogenic signaling involves recruitment of transcriptional coactivators to ICN1 nuclear complexes.

6;7 chromosomal translocation in spontaneously arising rat immunocytomas: evidence for c-myc breakpoint clustering and correlation between isotypic expression and the c-myc target.

Our previous studies have shown that spontaneously arising immunocytomas in the LOU/Ws1 strain of rats contain a t(6;7) chromosomal translocation in all seven tumors studied (F. M. Babonits, J. Spira, G. Klein, and H. Bazin, Int. J. Cancer 29:431-437, 1982). We have also shown that the c-myc is located on chromosome 7 (J. Sümegi, J. Spira, H. Bazin, J. Szpirer, G. Levan, and G. Klein, Nature (London) 306:497-499, 1983) and the immunoglobulin H cluster on chromosome 6 (W.S. Pear, G. Wahlström, J. Szpirer, G. Levan, G. Klein, and J. Sümegi, Immunogenetics 23:393-395, 1986). We now report a detailed cytogenetic and molecular analysis of nine additional rat immunocytomas. The t(6;7) chromosomal translocation is found in all tumors. Mapping of the c-myc breakpoints showed that in 10 of 14 tumors, the c-myc breakpoints are clustered in a 1.5-kilobase region upstream of exon 1. In contrast with sporadic Burkitt's lymphoma and mouse plasmacytoma, only 1 of 14 tumors contains the c-myc breakpoints in either exon 1 or intron 1. Analysis of the sequences juxtaposed to the c-myc show that immunoglobulin H switch regions are the targets in at least five tumors and that there is a strong correlation between the secreted immunoglobulin and the c-myc target. Unlike sporadic Burkitt's lymphoma and mouse plasmacytoma, at least two rat immunocytomas show recombination of the c-myc with sequences distinct from immunoglobulin switch regions.

Aberrant class switching juxtaposes c-myc with a middle repetitive element (LINE) and an IgH intron in two spontaneously arising rat immunocytomas.

Our previous studies of spontaneously arising rat immunocytomas of the Lou/Wsl strain have shown that Ig switch regions are frequently the targets for c-myc recombination. In several tumors, however, we were unable to show recombination of the c-myc with Ig switch regions. We have cloned the rearranged c-myc fragments from 2 of these tumors, IR209 and IR223, and found that the c-myc recombines with a LINE region in the IR209 and with intron 1 of the epsilon locus in the IR223. Although switch regions are not found at the breakpoints, the sequences at the breakpoints share limited homology with Ig switch recognition sequences. This suggests that the switch recombinase enzymes are able to recognize sequences in addition to the defined switch recombination sites. At the same time, both the LINE and epsilon intron 1 sequences are located within the Ig cluster, providing further evidence for the selection of c-myc activation by Ig sequences in the pathogenesis of rat immunocytoma, mouse plasmacytoma, and Burkitt's lymphoma.

Generation of high-titer, helper-free retroviruses by transient transfection.

Retroviral gene transfer is presently one of the most powerful techniques for introducing stably heritable genetic material into mammalian cells (reviewed in ref. 1). One serious drawback of this technique, however, has been the difficulty in readily producing high-titer recombinant retroviruses. For many applications, such as infecting rare target cells or the majority of cells in tissue culture, the recombinant virus titer must be at least 10(6) infectious units/mL. Although one can usually obtain high-titer mixtures of recombinant and replication-competent retroviruses in a relatively short time, many applications such as cell marking studies or studying genes in vivo demand freedom from replication-competent virus.

Notch signaling in mammalian hematopoietic stem cells.

Notch is a crucial cell signaling pathway in metazoan development. By means of cell-cell interactions, Notch signaling regulates cellular identity, proliferation, differentiation and apoptosis. Within the last decade, numerous studies have shown an important role for this pathway in the development and homeostasis of mammalian stem cell populations. Hematopoietic stem cells (HSCs) constitute a well-defined population that shows self-renewal and multi-lineage differentiation potential, with the clinically relevant capacity to repopulate the hematopoietic system of an adult organism. Here, we review the emergence, development and maintenance of HSCs during mammalian embryogenesis and adulthood, with respect to the role of Notch signaling in hematopoietic biology.

Notch signaling in leukemia.

Mammalian Notch homologs were first identified from the involvement of Notch1 in a recurrent chromosomal translocation in a subset of human T-cell leukemias. The effect of the translocation was twofold: Notch expression was placed under the control of a T-cell-specific element, and Notch was truncated, resulting in a constitutively active protein. Subsequent work has shown that Notch1 is required for T cell commitment and is exclusively oncotropic for T cells. During the past year, several murine models have been used to dissect the function of Notch signaling in lymphoid development and leukemia. These models show that Notch1 drives the earliest stages of T cell commitment and that Notch signaling must be downregulated by the double positive stage for proper T cell development to occur. Constitutive Notch signaling mediated by Notch1, Notch2, or Notch3 predisposes to T-cell leukemia. Future studies are expected to elucidate the mechanisms by which Notch leads to transformation. Identification of the transcriptional targets of Notch signaling is likely to yield important insights.

Transcriptional deregulation of myc in IgH/myc 6;7 translocation carrying rat immunocytomas.

We have previously shown that the reciprocal translocation t(6;7) associated with the spontaneous immunocytoma of the Louvain rat (RIC) leads to the juxtaposition of myc to the IgH cluster. In 10 of 14 tumors investigated the breakpoints on the myc carrying chromosome were clustered in a 1.5 kb region 5' of the intact gene, proximal to the myc promoters. In this paper we describe the effect of the translocation on myc transcription in the RIC system. Run-on analysis showed transcriptional attenuation in the normal rat myc gene, similar to the situation in mice and humans. The attenuation was almost completely abrogated in the three immunocytomas studied. Sequence analysis of two tumors failed to reveal any structural changes within exon 1, as found by others in Burkitt's lymphoma. We also show that the transcriptional initiation of myc mRNA is changed in the RICs. In an established line of rat fibroblasts (Rat-2), the more distal myc promoter (P2) is the preferred site of initiation. In RIC, however, only 30% of transcripts were initiated from P2. We found that 40% of the transcripts were initiated from P1 and 30% from a novel promoter, designated P1a, located between P1 and P2.

Localization of the rat immunoglobulin heavy chain locus to chromosome 6.

We have previously used rat/mouse somatic cell hybrids to localize the rat c-myc gene to chromosome 7 (Sümegi et al. 1983) and the rat immunoglobulin kappa locus to chromosome 4 (Perlmann et al. 1985). We now report that by utilizing rat/mouse somatic cell hybrids, we have localized the rat immunoglobulin heavy chain locus to chromosome 6.

Production of high-titer helper-free retroviruses by transient transfection.

The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.

The SH2 domain of bcr-Abl is not required to induce a murine myeloproliferative disease; however, SH2 signaling influences disease latency and phenotype.

Bcr-Abl plays a critical role in the pathogenesis of chronic myelogenous leukemia (CML). It was previously shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human CML. This in vivo experimental system allows the direct determination of the effect of specific domains of Bcr-Abl, or specific signaling pathways, on the complex in vivo pathogenesis of CML. In this report, the function of the SH2 domain of Bcr-Abl in the pathogenesis of CML is examined using this murine model. It was found that the Bcr-Abl SH2 mutants retain the ability to induce a fatal MPD but with an extended latency compared with wild type (wt) Bcr-Abl. Interestingly, in contrast to wt Bcr-Abl-induced disease, which is rapid and monophasic, the disease caused by the Bcr-Abl SH2 mutants is biphasic, consisting of an initial B-lymphocyte expansion followed by a fatal myeloid proliferation. The B-lymphoid expansion was diminished in mixing experiments with bcr-abl/DeltaSH2 and wt bcr-abl cells, suggesting that the Bcr-Abl-induced MPD suppresses B-lymphoid expansion.

Cutting edge: protective effects of notch-1 on TCR-induced apoptosis.

The Notch receptor protein was originally identified in Drosophila and is known to mediate cell to cell communication and influence cell fate decisions. Members of this family have been isolated from invertebrates as well as vertebrates. We isolated mouse Notch-1 in a yeast two-hybrid screen with Nur77, which is a protein that has been shown previously to be required for apoptosis in T cell lines. The data presented below indicate that Notch-1 expression provides significant protection to T cell lines from TCR-mediated apoptosis. These data demonstrate a new antiapoptotic role for Notch-1, providing evidence that, in addition to regulating cell fate decisions, Notch-1 can play a critical role in controlling levels of cell death in T cells.

Localization of the rat immunoglobulin lambda light chain locus to chromosome 11.

Previous experiments using rat/mouse somatic cell hybrids have localized the rat c-myc gene to chromosome 7 (Sümegi et al. 1983), the rat immunoglobulin kappa locus to chromosome 4 (Perlmann et al. 1985), and the rat immunoglobulin heavy chain locus to chromosome 6 (Pear et al. 1986). Using a similar approach, we now report the localization of the rat immunoglobulin lambda light chain locus to chromosome 11.