There is little known about the regulation of gene expression during TCR-mediated differentiation of immature CD4+8+ (double positive) thymocytes into mature T cells. Using the DPK CD4+8+thymocyte precursor cell line, we demonstrate that the early growth response-1 gene (Erg-1), encoding a zinc finger transcription factor, is rapidly upregulated after TCR stimulation. We also report that Egr-1 is expressed by a subset of normal double positive thymocytes in the thymic cortex, as well by a majority of medullary single positive thymocytes. Expression of Egr-1 is dramatically reduced in the thymus of major histocompatibility complex knockout mice, but can be induced by anti-CD3 antibody stimulation of isolated thymocytes from these animals. These and other data suggest that high level expression of Egr-1 in the thymus is a consequence of selection. A similar pattern of expression is found for family members Egr-2 and Egr-3. Using the DPK cell line, we also demonstrate that expression of Egr-1, 2, and 3 is dependent upon ras activation, as is the initiation of differentiation to a single positive cell. In contrast, the calcineurin inhibitor cyclosporin A, which inhibits DPK cell differentiation as well as positive selection, inhibits expression of Egr-2 and Egr-3, but not Egr-1. The identification of the Egr family in this context represents the first report of a link between the two known signaling pathways involved in positive selection and downstream transcriptional regulators.
It has been reported that the progression of osteosarcoma was closely associated with the aberrant activation of canonical Wnt signaling. Wnt inhibitory factor-1 (WIF-1) is a secreted Wnt inhibitor whose role in human osteosarcoma remains unknown. In this study, WIF-1 expression in NHOst and osteosarcoma cell lines was determined by real-time reverse transcription-PCR, methylation-specific PCR, and Western blotting analysis. In addition, tissue array from patient samples was examined for WIF-1 expression by immunohistochemistry. Compared with normal human osteoblasts, WIF-1 mRNA and protein levels were significantly downregulated in several osteosarcoma cell lines. The downregulation of WIF-1 mRNA expression is associated with its promoter hypermethylation in these tested cell lines. Importantly, WIF-1 expression was also downregulated in 76% of examined osteosarcoma cases. These results suggest that the downregulation of WIF-1 expression plays a role in osteosarcoma progression. To further study the potential tumor suppressor function of WIF-1 in osteosarcoma, we established stable 143B cell lines overexpressing WIF-1. WIF-1 overexpression significantly decreased tumor growth rate in nude mice as examined by the s.c. injection of 143B cells stably transfected with WIF-1 and vector control. WIF-1 overexpression also markedly reduced the number of lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Together, these data suggest that WIF-1 exerts potent antiosteosarcoma effect in vivo in mouse models. Therefore, the reexpression of WIF-1 in WIF-1-deficient osteosarcoma represents a potential novel treatment and preventive strategy. Mol Cancer Ther; 9(3); 731-41. ©2010 AACR.
Haemophilia patients with inhibitors can develop bleeding episodes, which are refractory to monotherapy with either recombinant factor VIIa (rFVIIa) or activated prothrombin complex concentrates (APCC). Management of such bleeds is often difficult. We previously reported the safety of using a combination of rFVIIa and APCC given in sequential fashion. In this report, we update our experience with sequential therapy. A retrospective review of medical records was conducted including all reports of sequential therapy defined as receiving both rFVIIa and APCC within 6 h. Data extracted included demographic data, treatment prior to and following hospital admission, clinical data including type and location of bleed, response to therapy, physical examination and laboratory data. In addition, for some patients, thromboelastography was performed to document the effect of sequential therapy on clot formation characteristics. Four patients comprising 35 admissions, 209 hospital days and 115 days of sequential therapy were included in the updated dataset. No patient developed thrombosis or overt disseminated intravascular coagulation (DIC) although elevations in the D-dimer above 5 microg mL(-1) were noted in 42% of the courses that lasted >3 days. Efficacy is suggested by the fact that patients had resolution of their bleeds after a median of 3 days of sequential therapy after failing to respond to a median of 3 days of monotherapy. Thromboelastography demonstrated an additive effect. Sequential therapy is a safe, potentially efficacious approach in the management of refractory bleeding episodes in patients with haemophilia and inhibitors.
Osteosarcoma is the most common primary bone malignancy, with a high propensity for local invasion, early metastasis and relapse. While the molecular mechanisms behind osteosarcoma development and metastasis have not yet been fully elucidated, research has highlighted an important role for Wnt signaling. Several Wnt ligands, receptors and coreceptors are highly expressed in osteosarcoma cell lines, while Wnt inhibitors are downregulated. As a result, research has begun to identify mechanisms with which to inhibit Wnt signaling. The use of Wnt pathway inhibitors and the targeting of c-Met, a Wnt regulated proto-oncogene, may be two possible mechanisms for treatment of osteosarcoma. In addition, as the Wnt signaling pathway is a regulator of stem cells, reagents that function as Wnt inhibitors are currently under investigation as inhibitors of cancer stem cell proliferation. Research involving the Wnt signaling pathway and cancer stem cells holds promise for novel treatment options in the future.
Osteosarcoma (OS) is a primary malignant bone tumor with a high propensity for local recurrence and distant metastasis. We previously showed a secreted, dominant-negative LRP5 receptor (DNLRP5) suppressed in vitro migration and invasion of the OS cell line SaOS-2. Therefore, we hypothesized DNLRP5 also has in vivo antitumor activity against OS. We used the 143B cell line as a model to study the effect of DNLRP5 by stable transfection. Inhibition of Wnt signaling by DNLRP5 was verified by a reduction in TOPFLASH luciferase activity. In soft agar, DNLRP5-transfected 143B cells formed fewer and smaller colonies than control transfected cells. DNLRP5 transfection reduced in vivo tumor growth of 143B cells in nude mice. DNLRP5 also decreased in vitro cellular motility in a scratch wound assay. In a spontaneous pulmonary metastasis model, DNLRP5 reduced both the size and number of lung metastatic nodules. The reduction in cellular invasiveness by DNLRP5 was associated with decreased expression of matrix metalloproteinase-2, N-cadherin, and Snail. Our data suggest canonical Wnt/LRP5 signaling reflects an important underlying mechanism of OS progression. Therefore, strategies to suppress LRP5-mediated signaling in OS cells may lead to a reduction in local or systemic disease burden.
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