904 resultados para viral disease immunofluorescence assay
Resumo:
Background Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. Results In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. Conclusion Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease.
Resumo:
BACKGROUND The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mimics PC-progression as it occurs in humans. However, the timing of disease incidence and progression (especially late stage) makes it logistically difficult to conduct experiments synchronously and economically. The development and characterization of androgen depletion independent (ADI) TRAMP sublines are reported. METHODS Sublines were derived from androgen-sensitive TRAMP-C1 and TRAMP-C2 cell lines by androgen deprivation in vitro and in vivo. Epithelial origin (cytokeratin) and expression of late stage biomarkers (E-cadherin and KAI-1) were evaluated using immunohistochemistry. Androgen receptor (AR) status was assessed through quantitative real time PCR, Western blotting, and immunohistochemistry. Coexpression of AR and E-cadherin was also evaluated. Clonogenicity and invasive potential were measured by soft agar and matrigel invasion assays. Proliferation/survival of sublines in response to androgen was assessed by WST-1 assay. In vivo growth of subcutaneous tumors was assessed in castrated and sham-castrated C57BL/6 mice. RESULTS The sublines were epithelial and displayed ADI in vitro and in vivo. Compared to the parental lines, these showed (1) significantly faster growth rates in vitro and in vivo independent of androgen depletion, (2) greater tumorigenic, and invasive potential in vitro. All showed substantial downregulation in expression levels of tumor suppressor, E-cadherin, and metastatis suppressor, KAI-1. Interestingly, the percentage of cells expressing AR with downregulated E-cadherin was higher in ADI cells, suggesting a possible interaction between the two pathways. CONCLUSIONS The TRAMP model now encompasses ADI sublines potentially representing different phenotypes with increased tumorigenicity and invasiveness.
