Isolation of a Chinese Hamster Ovary Cell Clone Possessing Decreased µ-Calpain Content and a Reduced Proliferative Growth Rate

Abstract

A Chinese hamster ovary cell line (CHOp) was cultured in the presence of benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone (ZLLY-CHN2), to select for resistance to this cell-permeant calpain inhibitor. A clone isolated after several courses of exposure (SHI cells) demonstrated decreased sensitivity to ZLLY-CHN2 toxicity and a decreased growth rate. SHI cells also possessed less mu-calpain isozyme relative to CHOp cells, as determined by activity measurement or by protein immunoblotting. Activities of m-calpain, calpastatin, cathepsin B, cathepsin L, and glycogen phosphorylase were not altered. SHI mu-calpain was partially purified by sequential chromatography on Bio-Gel A-1.5m and DEAE-Sepharose. Its chromatographic behavior in either system was the same as for CHOp mu-calpain. Further studies with the partially purified SHI and CHOp mu-calpain fractions failed to distinguish any difference in Ca2+ requirement or in sensitivity to inhibition by calpastatin or ZLLY-CHN2 for these enzymes. These experiments suggest that SHI cells underproduce a form of mu-calpain which is very similar to, if not identical with, CHOp mu-calpain. SHI cells displayed a population doubling time of 29 h compared with 19 h for CHOp cells. The decreased growth rate of SHI cells was the result of a prolonged G1 phase. Introduction of purified human mu-calpain into SHI cells by electroporation transiently restored the growth rate and also increased toxicity associated with exposure to ZLLY-CHN2. SHI cells should be a valuable model in further studies to delineate the function of mu-calpain in cell proliferative growth.