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This invention provides isolated nucleic acid encoding human MAD2, isolated human MAD2 protein. This invention further provides a method of detecting the presence of MAD2 in a tissue sample, a method of determining whether a tumor is susceptible to treatment with a mitotic spindle inhibitor by detecting the presence of MAD2 in the tumor and a method of suppressing tumor formation in a subject which comprises administering the nucleic acid encoding human MAD2 to the subject in an amount effective to enhance expression of MAD2. This invention also provides a nucleic acid reagent capable of detecting the MAD2 gene or gene product and a method for in situ identification of tumors which may be susceptible to treatment with mitotic spindle inhibitors by detecting the absence of nucleic acid encoding MAD2 in the tumor.

We have used the chemical cleavage reagent methidiumpropyl-EDTA-Fe(II) to determine the location of the nucleosomes along the mouse β-major globin gene in erythroid and nonerythroid cells. In mouse L cells, in which the globin gene is inactive, the nucleosomes are precisely positioned with respect to the underlying DNA sequence from positions −3000 to +1500 relative to the cap site. In uninduced and induced murine erythroleukemia cells, the same phasing persists but is interrupted from positions −200 to +500. This gap in the phased distribution of nucleosomes appears to be protected from MPE-Fe(II) digestion, and is bounded on both sides by hypersensitive sites. These results define at least two structural states for the globin gene: an inactive state in which the gene is covered with a continuous array of phased nucleosomes and an active state in which this array is disrupted over the 5′ half of the structural gene.

Angiogenic defects in Id mutant mice inhibit the growth of tumor xenografts, providing a genetic model for antiangiogenic stress. Our work tests the consequences of such stress on progression of more physiological Pten +/− tumors. While tumor growth occurs despite impaired angiogenesis, disruption of vasculature by Id loss causes tumor cells to experience hypoxia and necrosis, the extent of which is tumor dependent. We show that bone-marrow-derived endothelial precursors contribute functionally to neovasculature of some but not all Pten +/− tumors, partially rescuing Id mutant phenotype. We demonstrate that loss of Id1 in tumor endothelial cells results in downregulation of several proangiogenic genes, including α 6 and β 4 integrins, matrix metalloprotease-2 , and fibroblast growth factor receptor-1 . Inhibition of these factors phenocopies loss of Id in in vivo angiogenesis assays.

The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome–microtubule attachment during metaphase. To determine the function of the mitotic checkpoint protein Mad2 during normal cell division and when mitosis goes awry, we have knocked out Mad2 in mice. We find that E5.5 embryonic cells lacking Mad2, like mad2 yeast, grow normally but are unable to arrest in response to spindle disruption. At E6.5, the cells of the epiblast begin rapid cell division and the absence of a checkpoint results in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survive without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells, and for embryonic viability, even in the absence of spindle damage.

11000 Background: In the past decade, sequencing studies have delineated somatic mutations in human cancers. Given that cancer cells interact with their microenvironment and the presence of somatic...

Neo-vascularization has been implicated in a number of inflammatory diseases as well as tumor growth. Both angiogenesis, the sprouting of resident tissue endothelial cells (ECs), and vasculogenesis, the recruitment of bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs), are thought to participate in neo-vascularization. EPCs have been implicated in tumor growth, however, the biologic significance of EPCs during inflammation is unclear. We studied neo-vascularization and the role of EPCs during inflammation in well-characterized murine models of graft-versus-host disease (GVHD). We found a significantly increased number of donor BM-derived EPCs in peripheral blood and BM in allogeneic bone marrow transplantation (allo-BMT) recipients with GVHD at different time points after BMT. We next quantified neo-vascularization during inflammation in GVHD target organs by immunofluorescence microscopy and by flow cytometry. We found significantly increased numbers of donor-derived ECs in the liver as well as a significantly higher vessel density in the liver, illeum and colon. We adoptively transferred selected GFP+ EPCs and observed incorporation into the neo-vasculature of the inflamed intestines (Fig. 1A) and liver during GVHD. Taken together, these data suggest that neovascularization during GVHD is due to vasculogenesis from donor EPCs. Next we used an antibody (E4G10) against the vascular endothelial adhesion molecule VE-cadherin, which recognizes a terminal epitope that is exposed on circulating EPCs, but is masked in the established vasculature, and found a significant reduction of EPCs in the peripheral blood and BM. We observed that depletion of EPCs was associated with a significant inhibition of donor BM-derived neo-vascularization in the liver, illeum and colon during GVHD. E4G10 treated recipients had significantly better survival and lower clinical GVHD scores in all tested models (B6BALB/c [1×106 T], B6B6D2F1 [1×106 T], B6B6D2F1 [2×106 T], B6B6D2F1 [3×106 T]). We found significantly reduced numbers of allo-reactive donor T cells in secondary lymphoid organs during GVHD, but no changes in the expression of activation markers and homing molecules, as a consequence of E4G10 administration. In blinded histopathological analyses we found significantly less GVHD and reduced numbers of tissue-infiltrating CD3+ T cells in the liver, illeum and colon in E4G10-treated allo-BMT recipients. To better emulate the clinical setting, we first assessed the role of EPCs in tumor growth in allo-BMT recipients. We transferred sorted GFP+ EPCs as well as renal carcinoma (RENCA) cells to BALB/c recipients and found that GFP+ EPCs were recruited to the neo-vasculature of lung metastases. We detected a significant inhibitory effect of E4G10 administration on tumor growth, as determined by in vivo bioluminescence imaging, in both tumors tested (RENCA, A20 lymphoma) as well as a significant survival prolongation in tumor-bearing mice that were treated with E4G10 in the RENCA and C1498 (AML) model. Finally, we performed experiments in which tumor-bearing allo-BMT recipients received allogeneic T cells, which mediate the favorable graft-versus-tumor (GVT) effect but also cause inflammation in GVHD target organs. We found that administration of E4G10 led to a significantly higher rate of tumor-free survival in all models (B6'BALB/c [1×105 B6 T and 2×105 RENCA], B10BR'B6 [1×105 B10BR T and 2×105 C1498], B6'BALB/c [2×105 B6 T and 5×105 A20]), which was due to both attenuation of GVHD as well as inhibition of tumor growth (Fig. 1B). We conclude that depletion of EPCs is a strategy to simultaneously ameliorate inflammatory disease and tumors, providing a new approach to improve therapeutic outcome of allogeneic hematopoietic stem cell transplantation. This study demonstrates the biological significance of EPCs for neo-vascularization during inflammation and identifies the specific targeting of EPCs to disrupt neo-vascularization as a novel therapeutic concept to decrease inflammation. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group. Fig. 1. (A) EPCs are incorporated in neo-vasculature during GVHD. Sorted B6 GFP+EPCs (20,000), B6 GFP-BM and GFP-T cells were transferred at the day of BMT. GFP+EPC derived GFP+ECs are surrounding the luminal (L) space in neo-vasculature of the inflamed colon at day +14 after allo-BMT. (B) Depletion of EPCs leads to improved survival of tumor bearing allo-BMT recipients with GVHD due to simultaneous beneficial effects on inflammation and tumor growth. Lethally irradiated recipients were transplanted with 5×106 donor BM cells, 2.5×105 donor T cells, challenged intravenously with A20 lymphoma at day 0 and treated with 1 mg E4G10 or control antibody i.p. at days 0,2,4,6,8 and 10 after allo-BMT, combined data of 3 experiments are showm, n=28–33 per group.

Up until now, the precise mechanism for endostatin's antiangiogenesis action was not known. In a recent report, Abdollahi et al. (2004) have taken advantage of gene array and proteomic analysis to map the antiangiogenic pathways turned on by endostatin. This study resolves some of the controversies surrounding endostatin's biology, and provides a new direction to help dissect the molecular pathways involved in endostatin's selective tumor antiangiogenic effects.