Induction of human high K(M) 5'-nucleotidase in cultured 293 cells.
Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.
Gazziola
C
C
Department of Biology, University of Padova, Padova, 35131, Italy.
Moras
M
M
Ferraro
P
P
Gallinaro
L
L
Verin
R
R
Rampazzo
C
C
Reichard
P
P
Bianchi
V
V
D015720:5'-Nucleotidase / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D017338:Cladribine / Q000494:pharmacology; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000032:analysis / Q000378:metabolism; D018076:DNA, Complementary; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids; D011987:Receptors, Steroid / Q000235:genetics; D014162:Transfection; D014544:Uridine Triphosphate / Q000032:analysis / Q000378:metabolism
D000970:Antineoplastic Agents; D018076:DNA, Complementary; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011987:Receptors, Steroid; C055469:ecdysone receptor; D049452:Green Fluorescent Proteins; C055348:muristerone A; D017338:Cladribine; D004441:Ecdysterone; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D014544:Uridine Triphosphate