Human fibroblasts in culture exhibit a low sensitivity against cyclosporin A treatment.
The nephrotoxicity of cyclosporin A (CSA) after chronic treatment is well known and includes in later stages tubular atrophy associated with interstitial fibrosis. In order to examine whether interstitial fibrosis due to CSA treatment in vivo is related to a hyperproliferative activity of fibroblasts, the effects of CSA on the growth characteristics of cultured human skin fibroblasts (HUSF) were investigated at CSA concentrations ranging from 10 ng/ml to 50 micrograms/ml. We found that CSA at concentrations higher than 7.5 micrograms/ml inhibited cell proliferation (p < 0.05 at concentrations above 5 micrograms/ml; n = 3) and cloning efficiency (p < 0.05 at concentrations above 5 micrograms/ml; n = 3) in a dose-dependent manner and caused a promotion of cell attachment at concentrations above 10 micrograms/ml (p < 0.05; n = 4), but did not influence cell spreading. At lower concentrations CSA-treated HUSF did not differ in their growth characteristics from the corresponding controls. A 50% inhibition of proliferation was calculated by extrapolation for a CSA concentration of 70 micrograms/ml for HUSF. The inhibition of HUSF proliferation was reversible even at the highest CSA concentration of 50 micrograms/ml. Under the same experimental conditions, a 50% inhibition of proliferation was observed for Madin-Darby canine kidney (MDCK) cells to be 5.5 micrograms/ml, e.g. at a 15-fold lower CSA concentration. Moreover, CSA caused a dose-dependent and reversible cell elongation of HUSF and a significant increase in the average cell diameter from 19.2 +/- 0.3 microns (control; mean +/- SEM, n = 4) to 22.2 +/- 0.2 microns for 50 micrograms/ml CSA (mean +/- SEM, n = 4) and in median cell volume from 4,210 +/- 160 fl (control; mean +/- SEM, n = 4) to 7,020 +/- 190 fl for 50 micrograms/ml CSA (mean +/- SEM; n = 4). These alterations described above were not correlated with a cytotoxic effect as checked by a fluorescent staining for cell vitality. Alterations in the organization of cytoskeletal components such as stress fibers, intermediate-sized filaments and microtubules directly due to CSA treatment were not observed. In contrast, the amount of fibronectin present on the cell surface was considerably increased by CSA. Although HUSF in culture do not respond to CSA treatment by an increased proliferative activity, they are much less affected by CSA than other cell types (i.e MDCK cells).(ABSTRACT TRUNCATED AT 400 WORDS)
Dartsch
P C
PC
Institute of Physiology I, University of Tübingen, FRG.
Schmid
H
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D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D012867:Skin / Q000166:cytology / Q000187:drug effects
D005353:Fibronectins; D016572:Cyclosporine