Inhibition of epidermal growth factor receptor signalling reduces hypercalcaemia induced by human lung squamous-cell carcinoma in athymic mice.

The purpose of this study was to evaluate the role of the epidermal growth factor receptor (EGFR) in parathyroid hormone-related protein (PTHrP) expression and humoral hypercalcaemia of malignancy (HHM), using two different human squamous-cell carcinoma (SCC) xenograft models. A randomised controlled study in which nude mice with RWGT2 and HARA xenografts received either placebo or gefitinib 200 mg kg(-1) for 3 days after developing HHM. Effectiveness of therapy was evaluated by measuring plasma calcium and PTHrP, urine cyclic AMP/creatinine ratios, and tumour volumes. The study end point was at 78 h. The lung SCC lines, RWGT2 and HARA, expressed high levels of PTHrP mRNA as well as abundant EGFR protein, but very little erbB2 or erbB3. Both lines expressed high transcript levels for the EGFR ligand, amphiregulin (AREG), as well as, substantially lower levels of transforming growth factor-alpha (TGF-alpha), and heparin binding-epidermal growth factor (HB-EGF) mRNA. Parathyroid hormone-related protein gene expression in both lines was reduced 40-80% after treatment with 1 muM of EGFR tyrosine kinase inhibitor PD153035 and precipitating antibodies to AREG. Gefitinib treatment of hypercalcaemic mice with RWGT2 and HARA xenografts resulted in a significant reduction of plasma total calcium concentrations by 78 h. Autocrine AREG stimulated the EGFR and increased PTHrP gene expression in the RWGT2 and HARA lung SCC lines. Inhibition of the EGFR pathway in two human SCC models of HHM by an anilinoquinazoline demonstrated that the EGFR tyrosine kinase is a potential target for antihypercalcaemic therapy.

Preparation of an autolysis-resistant interleukin-1 beta converting enzyme mutant.

We describe the expression, purification, and characterization of human interleukin-1 beta converting enzyme (ICE) containing an affinity tag and modified to resist autoproteolysis. The point mutation Asp381 to Glu was added to eliminate the major site of autolytic degradation while maintaining catalytic activity, and an N-terminal polyhistidine tag was added in place of the ICE pro-region to facilitate purification. N-His (D381E) ICE was expressed in Escherichia coli and purified by nickel-chelating Sepharose and size-exclusion chromatography (SEC). The enzyme was stabilized greater than 80-fold against autolytic degradation relative to wild-type N-His ICE. SDS-PAGE analysis with silver-staining revealed no impurities, and 85% of the protein was catalytically active as determined by titration with a novel titrant, PD 163594 (3-[2-(2-benzyloxycarbonylamino-3-methylbutyrylamino)prop ionylamino]-4- oxo-5-(2-oxo-2H-chromen-7-yloxypentanoic acid). An oxidized adduct of ICE with glutathione, formed by disulfide rearrangement with oxidized glutathione to inhibit and stabilize the enzyme during purification, was rapidly reduced upon exposure to 5 mM DTT. One mole of glutathione was released per mole of active enzyme. Of the nine cysteines in ICE, eight were present in their reduced form in the glutathione adduct. N-His (D381E) ICE cleaved Ac-YVAD-Amc with the Michaelis-Menten parameters K(M) = 14 microM and Kcat = 0.7 s-1, values essentially identical to those reported for enzyme from natural sources.