Plasmid insertional mutation may confer glucocorticoid responsiveness of cell growth.
Culture of CHO.K1, a Chinese hamster ovary cell line, requires no particular care about the glucocorticoid level in media. Cell growth of CHO.K1 is little affected by dexamethasone at concentrations up to 3 microM. A clone of CHO.K1 stably transfected with an expression vector displayed a favored growth in dexamethasone-containing media. Ironically, dexamethasone was used to trigger the expression of antisense PCNA from the expression vector to impede the cell growth. Proliferating cell nuclear antigen (PCNA), an auxiliary factor of DNA polymerase delta, is required for cell proliferation. The stable cell clone, designated as B11 had a retarded growth rate as compared to its parental cell. However, the B11 cell growth rate and the cell cycle progression were increased by dexamethasone. The glucocorticoid produced no similar effect on the parental cell or other stable transfectants of the same plasmid. Thus, the stimulatory effect of dexamethasone on B11 has little connection with the expression of antisense PCNA and possibly involves a relevant gene in the B11 genome that was mutated due to the random plasmid insertion. A preliminary effort in identifying the targeted gene was made by using plasmid rescue method, and two plasmids were obtained. The rescued DNA of both plasmids specifically hybridized genomic DNA of the parental cells, and one of these plasmids detected a cellular transcript that was absent in B11 cells, suggesting its potential for further investigation.
Chen
G S
GS
Institute of Life Science, National Tsing-Hua University, Hsin-Chu, Taiwan.
Lee
C H
CH
Lee
Y C
YC
Chi
C W
CW
Liu
Y C
YC
D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects / Q000235:genetics*; D002999:Clone Cells / Q000166:cytology / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics; D016373:DNA, Antisense / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D006064:Gonadotropins, Equine / Q000235:genetics; D016254:Mutagenesis, Insertional; D009693:Nucleic Acid Hybridization; D010957:Plasmids / Q000235:genetics*; D018809:Proliferating Cell Nuclear Antigen / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection
D016373:DNA, Antisense; D004274:DNA, Recombinant; D006064:Gonadotropins, Equine; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D003907:Dexamethasone