Establishment of a Chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses A23187 induction of both GRP78 and GRP94.
GRP78, a 78,000 dalton protein residing in the endoplasmic reticulum, is postulated to play important roles in protein folding and cell survival during calcium and other physiological stress. Here we describe the construction of an eukaryotic expression vector for the constitutive expression of grp78 antisense RNA and the creation of a CHO cell line, 78WO, which expresses high levels of the grp78 antisense RNA through amplification of the stably transfected antisense vector. We observed that whereas 78WO maintains a basal level of GRP78 similar to that of control cells, GRP78 is no longer inducible by A23187. The 78WO cells have undergone a compensatory increase in grp78 transcription such that the effects of antisense are cancelled out at the protein level under nonstressed conditions. In these same cells, GRP94, a 94,00 dalton ER protein, is also rendered noninducible by A23187. This provides the first evidence that the regulation of two ER proteins might be coupled such that the failure to induce GRP78 results in the down-regulation of GRP94. The 78WO cell line grows with a doubling time of about 26 hr and exhibits decreased tolerance to A23187, suggesting the GRPs contribute to cell viability under calcium stress. The establishment of this cell line, which can be stably maintained, will provide a useful tool for testing whether the induction of the GRPs is important for protein folding or transport and whether their enhanced synthesis is the cause or consequence of a variety of physiological adaptations.
Li
L J
LJ
Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
Li
X
X
Ferrario
A
A
Rucker
N
N
Liu
E S
ES
Wong
S
S
Gomer
C J
CJ
Lee
A S
AS
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism*; D000001:Calcimycin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D002455:Cell Division; D002461:Cell Line, Transformed; D006224:Cricetinae; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D018832:Molecular Chaperones; D014176:Protein Biosynthesis; D016372:RNA, Antisense / Q000378:metabolism*; D014162:Transfection
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D016372:RNA, Antisense; C018551:glucose-regulated proteins; D000001:Calcimycin