Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia.

BACKGROUND AND PURPOSE: gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. EXPERIMENTAL APPROACH: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. KEY RESULTS: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

Trafficking of nuclear receptors in living cells.

Most of the steroid receptor family, with the exception of the estrogen receptor, are classically viewed as 'translocating receptors'. That is, they move from an exclusively, or principally, cytoplasmic distribution in the absence of hormone to a predominately nuclear localization in hormone stimulated cells. The estrogen receptor and the nuclear receptor family are found exclusively in the nucleus, both in hormone stimulated and hormone free cells. This behavior has now been studied with GFP-fusions in living cells, and has in general been confirmed. However, there are important exceptions, and new findings, particularly with regard to sub-nuclear localization. We propose that the intracellular distribution of both receptor classes is dependent not only on subcellular localization signals directly encoded in the receptors, but also on the nature and composition of the large, macromolecular complexes formed by each receptor. Furthermore, we find that most members of the receptor superfamily form focal accumulations within the nucleus in response to ligand, and suggest that these structures may participate in the biological life cycle of the receptors. Finally, we propose that receptor movement in the nucleus is highly dynamic, with the receptors undergoing constant exchange between genomic regulatory elements, multi-protein complexes with other transcription factor partners, and subnuclear structures that are as yet poorly defined.

Inducer-dependent transcriptional activation of the P4501A2 gene in vivo and in isolated hepatocytes.

In vitro nuclear run-on transcription analysis using probes directed against different regions of CYP1A2 revealed that the 70-100-fold induction of CYP1A2 mRNA by polycyclic aromatic compounds is associated with a corresponding increase in the transcriptional activation of this gene in rat liver. Probes from regions of the 1st, 2nd, and 4th introns detected approximately 50-100-fold higher CYP1A2 run-on transcription in liver nuclei from inducer-treated animals than in nuclei from untreated animals. The run-on signals from untreated rats were 3-5-fold above background signals. Additional experiments using single-stranded DNA probes and a probe from a region 5' to the CYP1A2 transcription start site revealed that the inducer-dependent transcripts were colinear with the CYP1A2 mRNA and that they did not result from read through of an initiation event 5' to CYP1A2. Run-on transcription analyses were also carried out with nuclei from isolated hepatocytes using the same series of probes spanning CYP1A2. These analyses indicated that the inducer-dependent accumulation of CYP1A2 mRNA in hepatocytes is associated with at least a 20-fold increase in CYP1A2 transcription. In contrast to liver and hepatocytes, these probes failed to detect run-on transcripts from kidney nuclei, indicating that the lack of CYP1A2 mRNA in this tissue is due to the lack of transcriptional activation of this gene by polycyclic aromatic compounds.