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.

Poly(fumaric-co-sebacic anhydride). A degradation study as evaluated by FTIR, DSC, GPC and X-ray diffraction.

The degradation of three poly(fumaric-co-sebacic anhydride) [P(FA:SA)] copolymers is examined in a composition of microspheres made by the hot melt encapsulation process. The emergence of low molecular weight oligomers occurs during degradation of the copolymer microspheres, as evidenced by a variety of characterization methods. Characterization was conducted to determine the extent of degradation of the polyanhydride microspheres using Fourier-transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and X-ray diffraction. It is demonstrated that degradation of P(FA:SA) is greatly accelerated at basic pH, yet there is little difference between degradation in neutral and acidic buffers. A good correlation exists between the results of each characterization method, which allows a better understanding of the degradation process and the resulting formation of low molecular weight oligomers in poly(fumaric-co-sebacic anhydride).

Mice that lack the angiogenesis inhibitor, thrombospondin 2, mount an altered foreign body reaction characterized by increased vascularity.

Disruption of the thrombospondin 2 gene (Thbs2) in mice results in a complex phenotype characterized chiefly by abnormalities in fibroblasts, connective tissues, and blood vessels. Consideration of this phenotype suggested to us that the foreign body reaction (FBR) might be altered in thrombospondin 2 (TSP2)-null mice. To investigate the participation of TSP2 in the FBR, polydimethylsiloxane (PDMS) and oxidized PDMS (ox-PDMS) disks were implanted in TSP2-null and control mice. Growth of TSP2-null and control skin fibroblasts in vitro also was evaluated on both types of disks. Normal fibroblasts grew as a monolayer on both surfaces, but attachment of the cells to ox-PDMS was weak and sensitive to movement. TSP2-null fibroblasts grew as aggregates on both surfaces, and their attachment was further compromised on ox-PDMS. After a 4-week implantation period, both types of PDMS elicited a similar FBR with a collagenous capsule in both TSP2-null and control mice. However, strikingly, the collagenous capsule that formed in TSP2-null mice was highly vascularized and thicker than that formed in normal mice. In addition, abnormally shaped collagen fibers were observed in capsules from mutant mice. These observations indicate that the presence or absence of an extracellular matrix component, TSP2, can influence the nature of the FBR, in particular its vascularity. The expression of TSP2 therefore could represent a molecular target for local inhibitory measures when vascularization of the tissue surrounding an implanted device is desired.

Degradation of double-walled polymer microspheres of PLLA and P(CPP:SA)20:80. II. In vivo degradation.

Double-walled (DW) polymer microspheres with a core of poly(1,3-bis-(p-carboxyphenoxy propane)-co-(sebacic anhydride)20:80 (P(CPP:SA)20:80) and an external coat of poly(L-lactic acid) (PLLA) were implanted subcutaneously and intramuscularly and were allowed to degrade for up to 6 months. The tissue surrounding the implant was fixed and sectioned for histological evaluation by light and scanning electron microscopy. The remaining polymer was extracted with chloroform and analyzed by gel permeation chromatography (G PC), Fourier-transform infrared (FTIR) spectroscopy, and differential scanning calorimetry (DSC). The inner core of the more hydrolytically labile P(CPP:SA)20:80 degraded first, becoming more granular over time. The PLLA coating showed no changes morphologically. The DW microspheres induced a stronger inflammatory response during the first week than did the SW PLLA microspheres, but this resolved by the second week. The SW PLLA microspheres showed no changes by SEM or optical microscopy. No differences in degradation were seen between samples implanted subcutaneously and those implanted intramuscularly. In vivo degradation was correlated to the results of an in vitro degradation study published as the first paper in this two paper series.

Degradation of double-walled polymer microspheres of PLLA and P(CPP:SA)20:80. I. In vitro degradation.

Double-walled (DW) polymer microspheres with a core of poly(1,3-bis-(p-carboxyphenoxy propane)-co-(sebacic anhydride)20:80 (P(CPP:SA)20:80) and an external coat of poly(L-lactic acid) (PLLA) were degraded for up to 6 months in vitro. The effects of hydrolytic degradation on the polymers were studied by Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), gel permeation chromatography (GPC) and scanning electron microscopy (SEM). The inner core of the polyanhydride copolymer (P(CPP:SA)20:80) degraded first, breaking down into oligomers during the first week and then into monomers which were trapped by the outer shell of PLLA, crystallized, and remained in the core for the duration of the study. As expected, the PLLA coat degraded at a slower rate due to the ester bonds between lactic acid monomers being more hydrolytically resistant than the anhydride bonds. The PLLA in the DW microspheres decreased from its original molecular weight (Mw) of 24,000 g mol(-1) to approximately 5,000 g mol(-1) over the 6 month study. The melting temperature of the PLLA decreased more than 30 degrees C during the study, but no changes were observed in the FTIR spectra. The 6 month samples were very brittle, showing the concomitant drop in mechanical strength with the decrease in Mw.

Automated genotyping in diagnosis.

At present automated genotyping in diagnosis involves the detection, digitrzation, and analysis of labeled DNA using computer software. This chapter describes the use of the Applied Biosystems (Foster City, CA) 373 DNA Sequencer and Genescan 672 software for sizing fluorescently labeled PCR products in a diagnostic molecular genetics laboratory. The Applied Biosystems Genotyper software is not covered since this is not used at present in this laboratory. An outline of the steps involved in automated genotyping, from polymerase chain reaction (PCR) to archiving data, is shown in Fig. 1. Fig. 1. Overview of the procedure. Labeled PCR products are produced by either incorporation of fluorescent dNTPs or labeled primers. A polyacrylamide gel is cast, scanned, and prerun, and the Genescan collection and analysis files are set up. The PCR products are mixed with a size standard, denatured, and loaded onto the prerun gel. After electrophoresrs the collected data is transferred to another Macintosh for analysis. A results file is generated and the PCR products are scored and checked. The results file is then archived.