Response to anti-DKK1 therapy in uterine carcinosarcoma: A case report.

Targeted therapies are being increasingly used in clinical practice and trials. However, tumor heterogeneity among sites of metastatic disease can occur creating a conundrum when utilizing biomarker directed therapies. Here we demonstrate a patient with recurrent uterine carcinosarcoma whose local recurrence and metastatic recurrence had a varied response to paclitaxel in combination with DKN-01, a monoclonal antibody against DKK1, a modulator of Wnt/ß-catenin and PI3K/AKT signaling pathways. This may be explained by differences in mutational profile found between the two sites. Our findings highlight the importance of analyzing tissue from the primary tumor as well as metastatic lesions, especially if there is a discrepancy in their response to treatment.

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism.

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.

p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101.

Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101-/- embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101-/- embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21(WAF-1/CIP-1), was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101-/- embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.

Targeted disruption in murine cells reveals variable requirement for Smad4 in transforming growth factor beta-related signaling.

The tumor suppressor gene Smad4 has been proposed to be a common mediator of transforming growth factor beta (TGFbeta)-related signaling pathways. We investigated the role of Smad4 in TGFbeta-related pathways by targeted disruption of its locus in murine cell lines. TGFbeta responses, including growth arrest, induction of the endogenous PAI-1 gene, and other extracellular matrix components, were normal in Smad4-deficient fibroblasts. Assembly of a TGFbeta-induced DNA-binding complex on one of two regulatory regions in the human plasminogen activator inhibitor (PAI)-1 promoter did not require Smad4 but was, instead, dependent on a TFE-3 binding site. In contrast, Smad4 was required for activation of the Xenopus Mix.2 promoter in response to TGFbeta/activin. Smad4 was also involved in the regulation of the Msx homeobox protein family members in response to bone morphogenetic protein (BMP). Interestingly, the expression of the endogenous Msx-2 was reduced, whereas that of Msx-3 was activated in differentiating Smad4(-/-) ES cells relative to wild-type cells. Moreover, reporter assays of the Msx-2 promoter revealed an absolute requirement for Smad4 in fibroblasts and ES cells for activation. Our results indicate that Smad4 is dispensable for critical TGFbeta-induced responses but is required for others in murine fibroblasts. We have identified transcriptional targets for Smad4 in the BMP signaling pathway, which may contribute to the genetic defect observed in the Smad4-deficient embryos.

The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo.

Mutations in the SMAD4/DPC4 tumor suppressor gene, a key signal transducer in most TGFbeta-related pathways, are involved in 50% of pancreatic cancers. Homozygous Smad4 mutant mice die before day 7.5 of embryogenesis. Mutant embryos have reduced size, fail to gastrulate or express a mesodermal marker, and show abnormal visceral endoderm development. Growth retardation of the Smad4-deficient embryos results from reduced cell proliferation rather than increased apoptosis. Aggregation of mutant Smad4 ES cells with wild-type tetraploid morulae rescues the gastrulation defect. These results indicate that Smad4 is initially required for the differentiation of the visceral endoderm and that the gastrulation defect in the epiblast is secondary and non-cell autonomous. Rescued embryos show severe anterior truncations, indicating a second important role for Smad4 in anterior patterning during embryogenesis.

The interferon regulatory transcription factor IRF-1 controls positive and negative selection of CD8+ thymocytes.

Little is known about the molecular mechanisms and transcriptional regulation that govern T cell selection processes and the differentiation of CD4+ and CD8+ T cells. Mice lacking the interferon regulatory transcription factor-1 (IRF-1) have reduced numbers of mature CD8+ cells within the thymus and peripheral lymphatic organs. Here we show that positive and negative T cell selection of two MHC class I-restricted TCR alphabeta transgenes, H-Y and P14, are impaired in IRF-1-/- mice. The absence of IRF-1 resulted in decreased expression of LMP2, TAP1, and MHC class I on thymic stromal cells. Despite decreased MHC class I expression on IRF-1-/- thymic stromal cells, the defect in CD8+ T cells development did not reside in the thymic environment, and IRF-1-/- stromal cells can fully support development of CD8+ thymocytes in in vivo bone marrow chimeras and in vitro reaggregation cultures. Moreover, IRF-1-/- thymocytes displayed impaired TCR-mediated signal transduction, and the induction of negative selection in TCR Tg thymocytes from IRF-1-/- mice required a 1000-fold increase in selecting peptide. We also provide evidence that IRF-1 is mainly expressed in mature, but not immature, thymocytes and that expression of IRF-1 in immature thymocytes is induced after peptide-specific TCR activation. These results indicate that IRF-1 regulates gene expression in developing thymocytes required for lineage commitment and selection of CD8+ thymocytes.

The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse.

Mutations of the BRCA1 gone in humans are associated with predisposition to breast and ovarian cancers. We show here that Brca1+/- mice are normal and fertile and lack tumors by age eleven months. Homozygous Brca1(5-6) mutant mice die before day 7.5 of embryogenesis. Mutant embryos are poorly developed, with no evidence of mesoderm formation. The extraembryonic region is abnormal, but aggregation with wild-type tetraploid embryos does not rescue the lethality. In vivo, mutant embryos do not exhibit increased apoptosis but show reduced cell proliferation accompanied by decreased expression of cyclin E and mdm-2, a regulator of p53 activity. The expression of cyclin-dependent kinase inhibitor p21 is dramatically increased in the mutant embryos. Buttressing these in vivo observations is the fact that mutant blastocyst growth is grossly impaired in vitro. Thus, the death of Brca1(5-6) mutant embryos prior to gastrulation may be due to a failure of the proliferative burst required for the development of the different germ layers.

Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis.

Progress in understanding the abnormal regulation of hematopoiesis in chronic myelogenous leukemia (CML) would be facilitated if neoplastic cells, at all stages of the disease, could be studied in an animal model. In this report, we show that irradiated severe combined immunodeficient (SCID) mice can be transplanted with both normal (Philadelphia chromosome [Ph]-negative) and neoplastic (Ph+) cells from CML patients with either chronic or blast phase disease. Mice transplanted with peripheral blood (PB) or bone marrow (BM) cells from 9 of 12 chronic phase CML patients were well engrafted with human cells including multilineage colony-forming progenitors and CD34+ cells for at least 90 days posttransplantation. Repeated posttransplant injections of cytokines did not enhance the number of engrafted human cells. Interestingly, approximately 70% of the human progenitors found in the engrafted SCID BM were Ph-, suggesting that the growth of primitive normal cells is favored in this in vivo transplant model. A similar number of normal cells were found in mice transplanted with either PB or BM cells, suggesting that elevated numbers of primitive normal cells are present in CML PB. When cells from patients with CML in either myeloid or lymphoid blast crisis were transplanted into SCID mice, the BM of these mice was more rapidly repopulated and to a higher level than that observed with transplants of chronic phase cells. Moreover, all human colony-forming progenitors present in the BM of mice transplanted with blast crisis cells were Ph+, and the majority of cells showed the same morphological features of the blast crisis cells originally transplanted. These experiments provide a starting point for the creation of an animal model of CML and establish the feasibility of using this model for the future characterization of transplantable CML stem cells during disease progression.

Expression of bcr-abl abrogates factor-dependent growth of human hematopoietic M07E cells by an autocrine mechanism.

The introduction of a retrovirus vector expressing p210bcr-abl (P210) into the human factor-dependent cell line M07E resulted in the rapid outgrowth of factor-independent cells. Early after infection, four factor-independent clones were isolated and analyzed in greater detail along with mass populations obtained from separate infections. High levels of P210 tyrosine kinase activity were measured in the factor-independent cells. The mass populations and three of the four clones remained responsive to exogenous growth factors. Concentrated conditioned media isolated from the factor-independent populations and from all clones contained biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-3 (IL-3) was detected at low levels in the mass population and in two of the clones. Neutralizing antibodies to IL-3, GM-CSF, and mast cell growth factor inhibited proliferation of the factor responsive clones by 60% to 90%. These results indicate that the growth autonomy of the P210-expressing M07E cells was acquired via an autocrine mechanism. In addition to factor-independent growth, P210-expressing M07E cells readily acquired a more mature megakaryocytic phenotype compared with control M07E cells. These data provide experimental evidence that expression of P210 tyrosine kinase in human hematopoietic cells induced growth factor secretion resulting in a pleiotropic effect on growth factor dependence and differentiation.

A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.

Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.

Murine models of normal and neoplastic human haematopoiesis.

The ability to transplant human haematopoietic cells into immune deficient mice provides a unique opportunity for studying the organization and regulation of the human stem cell developmental program. One challenge for the future will be to reconstitute mice with functional cells of all lineages. The creation of animal models of many haematopoietic diseases should revolutionize the development and testing of novel therapeutic strategies. Significant progress has been made in establishing models of human neoplastic diseases such as leukaemia and lymphoma. Although the number of patients examined is still small, it appears that there may be a correlation between growth in immune deficient mice and clinical outcome. Future studies should examine the range of diseases that grow in mice and whether in vivo assays have prognostic value clinically. These leukaemia models, in conjunction with high efficiency gene transfer techniques, offer a powerful approach to examine the biological consequences of expressing oncogenes or other key regulatory genes on human leukaemic transformation and progression.

A model of human acute lymphoblastic leukemia in immune-deficient SCID mice.

A human acute lymphoblastic leukemia (ALL) cell line that was transplanted into immune-deficient SCID mice proliferated in the hematopoietic tissues, invaded various organs, and led to the death of the mice. The distribution of leukemic cells in SCID mice was similar to the course of the disease in children. A-1 cells marked with a retrovirus vector showed clonal evolution after the transplant. SCID mice that were injected with bone marrow from three patients with non-T ALL had leukemic cells in their bone marrow and spleen. This in vivo model of human leukemia is an approach to understanding leukemic growth and progression and is a novel system for testing new treatment strategies.

The Escherichia coli cell division mutation ftsM1 is in serU.

The ftsM1 mutation is believed to be in a gene implicated in the regulation of cell division in Escherichia coli because it displayed the lon mutation phenotypes. In this study, we show that this mutation is located in serU, a gene which codes for tRNA(Ser)2, and has the phenotypes of the serU allele supH. Both ftsM1 and supH suppressed the leuB6 and ilvD145 missense mutations, and both conferred temperature and UV light irradiation sensitivity to the harboring cells. Cells which carried the ftsM1 mutation or the supH suppressor had very low colony-forming abilities on salt-free L agar, and this phenotype was almost completely abolished by the presence of plasmids bearing the ftsZ+ gene. Furthermore, sensitivity of the mutant cells to UV irradiation was also markedly diminished when they carried a ftsZ+-bearing plasmid. These results suggest that supH-containing cells have reduced FtsZ activities, in accordance with their displaying the phenotypes of the lon mutant cells. The possibility that ftsM1 (supH) is functionally involved in the biosynthesis of a specific protein which affects cell division is discussed.