Jean-Dominique Guitton

Blockage of urokinase receptor reduces in vitro the motility and the deformability of endothelial cells

The binding of urokinase (u‐PA) to its cell surface receptor (u‐PAR) is critical for tumor cell invasion. Here, we report that the disruption of this binding by an u‐PAR antagonist ATF‐HSA inhibits in vitro the motility of endothelial cells in a dose‐dependent manner. This inhibition was also observed when the cells were first stimulated with potent angiogenic factors, including bFGF or VEGF. [3H]thymidine incorporation assay demonstrated that ATF‐HSA did not affect the cell proliferation. ATF‐HSA was more potent than plasmin inhibitors, suggesting that it exerts its effects not solely by inhibiting the remodeling of the extracellular matrix. In fact, analysis of the cell shape change during migration revealed for the first time that its effect is related to a decrease in cell deformability. These results suggest that u‐PAR antagonist may be a new approach to control angiogenesis.

Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form

A synthetic gene coding for human angiogenin was synthesized by solid support phosphoramidite chemistry as eight long oligodeoxynucleotides which were subsequently assembled and cloned in Escherichia coli. The gene was designed to use codons found in highly expressed E. coli proteins. A pBR322-derived expression vector was constructed containing the E. coli trp promoter, the ribosome-binding site of the bacteriophage lambda cII gene, the angiogenin coding sequence, and the transcription terminator region of the E. coli rrnB operon. Under tryptophan deprivation, angiogenin was strongly expressed in E. coli cells at a yield of 5-10% of total protein. The eukaryotic protein was found to be insoluble but could be easily renatured and purified. The purified angiogenin was demonstrated to be active as an angiogenic factor and exhibited a characteristic RNase activity.

Blockage of the urokinase receptor on the cell surface: Construction and characterization of a hybrid protein consisting of the N‐terminal fragment of human urokinase and human albumin

Receptor‐bound urokinase is likely to be a crucial determinant in both tumor invasion and angiogenesis. We report here that a yeast‐derived genetic conjugate between human serum albumin and the 1–135 N‐terminal residues of urokinase (u‐PA) competitively inhibits the binding of exogenous and endogenous u‐PA to its cell‐anchored receptor (u‐PAR). This hybrid molecule (ATF‐HSA) also inhibits in vitro pro‐urokinase‐dependent plasminogen activation in the presence of u‐PAR bearing cells. These effects are probably responsible for the observed in vitro inhibition of tumor cell invasion in a reconstituted basement membrane extract (Matrigel).

Constrained analogs of KCVFM with improved inhibitory properties against farnesyl transferase

Abstract Constrained analogs of KCVFM, reported thus far as one of the most active peptidic inhibitors of farnesyl transferase, have been synthesized. Replacement of Val-Phe with Val-Tic and (N-Me)Val-Tic led to dramatically more active analogs possessing favored extended conformations. Based on molecular modelling studies the design and synthesis of various conformational probes to be substituted for Val and Phe led to a good correlation between the ratio of extended conformers and biological activity.

From Random Screening of Chemical Libraries to the Optimization of FPP-Competitive Inhibitors of Farnesyltransferase

Together with gene alterations of the p53 tumor suppressor gene, mutations of the ras genes represent the most frequent gene modification umancancers. Ras mutations are found in at least 90% of pancreas, 50% of colon, and 30% of both lung and thyroid cancers (1,2).