Gastrointestinal differentiation marker Cytokeratin 20 is regulated by homeobox gene CDX1.

CDX1 is a transcription factor that plays a key role in intestinal development and differentiation. However, the downstream targets of CDX1 are less well defined than those of its close homologue, CDX2. We report here the identification of downstream targets of CDX1 using microarray gene-expression analysis and other approaches. Keratin 20 (KRT20), a member of the intermediate filament and a well-known marker of intestinal differentiation, was initially identified as one of the genes likely to be directly regulated by CDX1. CDX1 and KRT20 mRNA expression were significantly correlated in a panel of 38 colorectal cancer cell lines. Deletion and mutation analysis of the KRT20 promoter showed that the minimum regulatory region for the control of KRT20 expression by CDX1 is within 246 bp upstream of the KRT20 transcription start site. ChIP analysis confirmed that CDX1 binds to the predicted CDX elements in this region of the KRT20 promoter in vivo. In addition, immunohistochemistry showed expression of CDX1 parallels that of KRT20 in the normal crypt, which further supports their close relationship. In summary, our observations strongly imply that KRT20 is directly regulated by CDX1, and therefore suggest a role for CDX1 in maintaining differentiation in intestinal epithelial cells. Because a key feature of the development of a cancer is an unbalanced program of proliferation and differentiation, dysregulation of CDX1 may be an advantage for the development of a colorectal carcinoma. This could, therefore, explain the relatively frequent down regulation of CDX1 in colorectal carcinomas by hypermethylation.

Aminoglycosides induce acute cell signaling and chronic cell death in renal cells that express the calcium-sensing receptor.

The aminoglycoside antibiotics (AGAs) are calcium-sensing receptor (CaR) agonists that are toxic to the renal proximal tubule. Proximal tubule-derived opossum kidney (OK) cells express CaR-like proteins and respond to AGAs with intracellular Ca2+ mobilization and extracellular regulated protein kinase (ERK) phosphorylation. To examine the possible cellular basis of AGA toxicity, acute and chronic responses to AGA treatment in OK cells and in CaR stably transfected HEK-293 cells (CaR-HEK) were studied. Changes in cell-fate signaling, proliferation, and cell death were detected by semiquantitative Western blotting, Hoechst staining, cell counting, and FACS analysis. Confocal microscopy was used to study the relative internalization of fluorophore-labeled gentamicin in CaR-transfected and -nontransfected cells. Here it is reported that the AGA neomycin and gentamicin elicit acute, phosphatidylinositol-3 kinase-dependent phosphorylation of Akt, glycogen synthase kinase 3beta, and p38 mitogen-activated protein kinase. After 24 h of gentamicin treatment, OK cell proliferation was observed, whereas after 4 d, the OK cells underwent cell death, an effect that was mimicked by the CaR agonists spermine and polyarginine. Furthermore, gentamicin elicited substantially more cell death in CaR-HEK cells than in nontransfected HEK-293 cells. The pan-specific caspase inhibitor Z-VAD significantly inhibited cell death in both OK and CaR-HEK cells. Finally, the intracellular uptake of Texas Red-labeled gentamicin was equivalent in both CaR-transfected and vector-transfected HEK-293 cells, suggesting that the CaR does not enhance drug uptake. Together, these observations demonstrate that the AGAs induce both acute and chronic cell fate changes in OK cells and CaR-HEK cells and that the proximal tubular CaR is likely to contribute to signaling underlying the renal toxicity of the AGAs.

Genomics and the search for novel biomarkers in toxicology.

The advent of 'genomics' technology, in particular transcript profiling, has already had a measurable impact on the drug discovery process in the areas of target identification and validation. This review is concerned with the potential application of this technology to toxicology and drug safety assessment, with particular emphasis on biomarker discovery and characterization. An advantage (or possibly a drawback!) of transcript profiling is that candidate biomarkers of toxicity can be speedily identified, with the caveat that a significant amount of subsequent experimental and bioinformatic effort needs to be expended in order to evaluate and validate them. Attention is also drawn to the critical need for robust experimental design with studies of this type and to issues associated with the analysis of large data sets. In summary, while genomics technology undoubtedly offers much that can assist drug safety assessment, its potential has yet to be realized fully in this area. However, a large amount of resource continues to be applied to 'toxicogenomics'. Tangible benefits, in terms of new biomarkers of toxicity and reduced numbers of adverse drug effects, remain realistic objectives.