Isolation of a Chinese hamster ovary cell clone possessing decreased mu-calpain content and a reduced proliferative growth rate.
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.
Mellgren
R L
RL
Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo, Ohio 43699-0008 USA.
Lu
Q
Q
Zhang
W
W
Lakkis
M
M
Shaw
E
E
Mericle
M T
MT
D000818:Animals; D016466:CHO Cells / Q000166:cytology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000502:physiology*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002455:Cell Division; D002470:Cell Survival; D006224:Cricetinae; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003978:Diazomethane / Q000031:analogs & derivatives / Q000494:pharmacology; D010450:Endopeptidases; D007527:Isoenzymes / Q000502:physiology; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology; D006005:Phosphorylases / Q000378:metabolism
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; D009842:Oligopeptides; C090218:benzyloxycarbonylleucyl-leucyl-tyrosine diazomethyl ketone; D003978:Diazomethane; C033668:calpastatin; D006005:Phosphorylases; D002403:Cathepsins; D010450:Endopeptidases; D002154:Calpain; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L