M. Dolenga, P. Hechtman
May 1, 1992
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Journal
In Vitro Cellular & Developmental Biology - Animal
Abstract
Dear Editor: Carbobenzoxylation of amino acids and peptides is widely used to prepare derivatives with protected amino-terminal groups. CBZamino acids and peptides have been employed as substrate analogues in the investigation of the function and specificity of the catalytic sites of proteases (Turkiewicz et al., 1986; Urbanek et al., 1985) and as antagonists for receptor-mediated functions. The latter studies have included cholecystokinin-mediated amylase secretion by pancreatic acinar cells (Maton et al., 1985) and fast axonal transport in dorsal root ganglia (Hammerschlag et al., 1989). The use of CBZ-derivatives as probes of specific functions in intact cells raises concerns as to whether such compounds may exert multiple effects on cell growth or viability. CBZ amino acids have not, to our knowledge, been tested for cytostatic or cytotoxic properties. The documentation of such properties would be of interest to investigators employing these compounds in a variety of studies. CBZ-proline is an inhibitor of prolidase (Hui and Lajtha, 1980), an enzyme which hydrolyzes dipeptides containing proline in the C terminal position. We attempted to use this reagent as an inhibitor of prolidase activity in the fetal calf serum component of growth medium and observed that 10 mM CBZ-proline caused cessation of fibrublast growth. Our findings indicate that growth inhibition also occurs with other CBZ amino acids and that the mechanism of inhibition does not appear to be related to protein synthesis or to binding of growth factors. The cell lines employed were human skin fibroblasts which were initiated from foreskins obtained at circumcision and LLCPK cells, a primary proximal tubule cell line obtained from pig kidney. Cultures were grown in 60 mm 2 Falcon plastic petri dishes in minimal essential medium (MEM) supplemented with either 10% fetal calf serum (FCS) or with the serum substitute ITS+ (Collaborative Research, Bedford, MA). Cell number was determined after trypsinization using a Coulter Counter. Each condition was represented by three plates and cell titre in each plate was the average of three determinations. Protein synthesis was measured in confluent cells maintained in serum free MEM. aH-Leucine (specific activity 60.0 Ci/mmol, New England Nuclear) was added to the media after the cells had been starved for leucine by incubation for 1 hour in leucine-free MEM. After 6 and 24 hours cells were washed with phosphate-buffered saline, lysed with 2.5% Triton X-100-3 M NaCI and incorporation of 3H into cellular protein determined by the method of Mans and Novelli (1961). Each point represents the average of four determinations. Protein was estimated by the method of Lowry et al. (1951) using crystalline bovine serum albumin as the protein standard. CBZ-Amino acids were obtained from Sigma (St. Louis, MO), neutralized to pH 7.6 and sterilized prior to addition to growth media.