Oligonucleotides antisense to catalytic subunit of cyclic AMP-dependent protein kinase inhibit mouse mammary epithelial cell DNA synthesis.
Mouse mammary epithelial cells were plated onto 24-well culture plates (50,000 per well), allowed to attach and serum starved for 24 h. Following serum starvation, DNA synthesis was induced by the addition of 10% fetal calf serum and determined by a 1-h pulse with [3H]thymidine from 17 to 18 h after serum addition. Addition of oligonucleotides antisense to the translation start region of cyclic AMP-dependent protein kinase (kinase A) mRNA inhibited thymidine incorporation into DNA (total or percentage of cells incorporating thymidine, as measured by autoradiography). This inhibition was apparent whether compared to controls with no oligonucleotide addition, sense oligonucleotides, or mismatch oligonucleotides. Enzymatic assays indicated that the antisense oligonucleotides lowered kinase A activity in cells. Time course studies indicated that the inhibition in DNA synthesis was not an artifact of the time at which DNA synthesis was estimated. Long-term (4 day) cultures indicated that effects on induction of DNA synthesis were reflected in long-term cell proliferation.
Sheffield
L G
LG
Dairy Science Department, University of Wisconsin, Madison 53706.
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004261:DNA Replication / Q000502:physiology*; D004848:Epithelium / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*
D016376:Oligonucleotides, Antisense; D011494:Protein Kinases