Analysis of protein biomarkers in human clinical tumor samples: critical aspects to success from tissue acquisition to analysis.

There has been increased interest in the analysis of protein biomarkers in clinical tumor tissues in recent years. Tissue-based biomarker assays can add value and aid decision-making at all stages of drug development, as well as being developed for use as predictive biomarkers and for patient stratification and prognostication in the clinic. However, there must be an awareness of the legal and ethical issues related to the sourcing of human tissue samples. This article also discusses the limits of scope and critical aspects on the successful use of the following tissue-based methods: immunohistochemistry, tissue microarrays and automated image analysis. Future advances in standardization of tissue biobanking methods, immunohistochemistry and quantitative image analysis techniques are also discussed.

Endoglin expression in the endothelium is regulated by Fli-1, Erg, and Elf-1 acting on the promoter and a -8-kb enhancer.

Angiogenesis is critical to the growth and regeneration of tissue but is also a key component of tumor growth and chronic inflammatory disorders. Endoglin plays a key role in angiogenesis by modulating cellular responses to transforming growth factor-beta (TGF-beta) signaling and is upregulated in proliferating endothelial cells. To gain insights into the transcriptional hierarchies that govern endoglin expression, we used a combination of comparative genomic, biochemical, and transgenic approaches. Both the promoter and a region 8 kb upstream of exon 1 were active in transfection assays in endothelial cells. In transgenic mice, the promoter directed low-level expression to a subset of endothelial cells. By contrast, inclusion of the -8 enhancer resulted in robust endothelial activity with additional staining in developing ear mesenchyme. Subsequent molecular analysis demonstrated that both the -8 enhancer and the promoter depend on conserved Ets sites, which were bound in endothelial cells in vivo by Fli-1, Erg, and Elf-1. This study therefore establishes the transcriptional framework within which endoglin functions during angiogenesis.

Cyclin A1 protein shows haplo-insufficiency for normal fertility in male mice.

In higher eukaryotes, the cyclins constitute a family of proteins involved in progression through the cell cycle. The cyclin A1 gene (Ccna1) is expressed during meiosis and is required for spermatogenesis. Targeted disruption of the Ccna1 gene with a LacZ reporter gene has allowed us to study the expression pattern of this gene in more detail. We have confirmed expression in mouse pre-meiotic spermatocytes and also detected expression in the accessory olfactory bulb, hippocampus and amygdala of the adult brain. We have also found that the amount of cyclin A1 protein influences the fertility of male mice and its action is modulated by genetic background. On an outbred genetic background (129S6/SvEv x MF1), Ccna1 (tm1Col) -/- animals are sterile due to spermatogenic arrest prior to the first meiotic division while Ccna1 (tm1Col) +/- mice show reduced sperm production and fertility. This is even more pronounced on an inbred genetic background (129S6/SvEv) where Ccna1 (tm1Col) +/- male mice are sterile due to a severe reduction in the total number of sperm.