The generation of high-titer, helper-free retroviruses by transient transection has been achieved by using the highy transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packing cell line BOSC 23 produces infectious retrovirus at >106 infectious units/ml of supernatant within 72 hr after CaPO4-mediated btansfection. 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 weli as be useful for gene therapy. (B-galactosidase (3-gal) expression is directed by the viral promoter in the long terminal repeat and the neomycin resistance cassette is deleted], pCRIPenv-(6), pCRIPgag-2 (6), MFG-lacZ (7), MFG-tPA [similar to MFGlacZ, but expressing the human tissue plasminogen activator gene in place of (-gal (7)], pZAP (8), pSV2Hgm (9), pGPT2E (10), pGD (11), pGDv-abi (12), and pGD210bcr/abl (11).Enzymatic Assays and Nudeic Acid Prdures. Staining for (3-gal activity in intact cells and spleen was performed as described (13). Reverse transcriptase (RT) activity was assayed in the culture medium ofexponentially growing cells as described by Goff et al. (14). The in vitro abl kinase assays were performed as described by Konopka et al. (15) medium (17). The cells were then returned to the 37°C incubator (5% C02) for 24 hr. Subsequently, the medium was changed to 3 ml offresh 10%o FCS, and 24-48 hr later, the medium was removed and either filtered through a 0.45-,m filter or centrifuged at 500 x g for 5 min in a Sorvall RT6000B centrifuge. In experiments with chloroquine, the medium was changed to 10% FCS at 10 hr posttransfection and changed a second time at 24 hr posttransfection. Infections were performed as described (6). Viral titer was determined as the average number ofblue ((-gal-producing) cells per 10-25 high power fields (40,000-100,000 total cells) multiplied by a factor to account for magnification, plate size, and dilution of the infectious stock. When fluorescence-activated cell sorting (FACS) analysis was performed, the percentage of positive cells was multiplied by the total number of cells on the dish. G418 selection was performed as above except that at 48 hr postinfection the cells were split 1:10 into selective Abbreviations: (-gal, ,-galactosidase; G418R, G418 resistance; RT, reverse transcriptase; FCS, fetal calf serum; FACS, fluorescenceactivated cell sorting; SV40, simian virus 40; gpt, guanine phosphoribosyltransferase.

Notch receptors regulate fate decisions in many cells. One outcome of Notch signaling is differentiation of bipotential precursors into one cell type versus another. To investigate consequences of Notch1 expression in hematolymphoid progenitors, mice were reconstituted with bone marrow (BM) transduced with retroviruses encoding a constitutively active form of Notch1. Although neither granulocyte or monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced BM contained immature CD4+ CD8+ T cells in the BM and exhibited a simultaneous block in early B cell lymphopoiesis. These results suggest that Notch1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions, possibly by influencing lineage commitment from a common lymphoid progenitor cell.

Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1–c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.

SummaryNotch 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 "~ 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 TANl-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Notch receptors influence cellular behavior by participating in a seemingly simple signaling pathway, but outcomes produced by Notch signaling are remarkably varied depending on signal dose and cell context. Here, after briefly reviewing new insights into physiologic mechanisms of Notch signaling in healthy tissues and defects in Notch signaling that contribute to congenital disorders and viral infection, we discuss the varied roles of Notch in cancer, focusing on cell autonomous activities that may be either oncogenic or tumor suppressive.

Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans

The Notch pathway is gaining increasing recognition as a key regulator of developmental choices, differentiation, and function throughout the hematolymphoid system. Notch controls the generation of hematopoietic stem cells during embryonic development and may affect their subsequent homeostasis. Commitment to the T cell lineage and subsequent stages of early thymopoiesis is critically regulated by Notch. Recent data indicate that Notch can also direct the differentiation and activity of peripheral T and B cells. Thus, the full spectrum of Notch effects is just beginning to be understood. In this review, we discuss this explosion of knowledge as well as current controversies and challenges in the field.

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.