Effect of over-expression of bacterial ribonuclease H on the utility of antisense MYC oligodeoxynucleotides in the monocytic leukemia cell line U937.
RNase H has been clearly implicated in vitro in mediating some antisense effects. In vivo evidence is limited to experiments performed in Xenopus oocytes in which antisense oligonucleotides are microinjected. In other mammalian cell systems scant data have been obtained to support or deny a role for RNase H as an antisense mediator in vivo. These experiments were designed to test the hypothesis that RNase H mediates the MYC antisense-induced reduction in MYC protein observed in the human monocytic leukemia cell line U937. A bacterial RNase H-containing episomal replicon was constructed and stable transfectants were obtained which expressed E coli RNase H in their cytoplasm at a 10-fold higher level than endogenous RNase H. These cells failed to demonstrate heightened sensitivity to MYC antisense (phosphorothioate, end capped and phosphodiester) compared with untransfected or E coli RNase H antisense transfected cells. PCR analysis of each transfectant treated and untreated with MYC antisense failed to demonstrate the appearance of truncated MYC mRNA. These results do not support a role for RNase H in the mediation of MYC antisense-induced MYC protein reduction and growth inhibition in U937 cells.
Rosolen
A
A
Clinical Pharmacology Branch, NCI, NIH, Bethesda, MD 20892.
Kyle
E
E
Chavany
C
C
Bergan
R
R
Kalman
E T
ET
Crouch
R
R
Neckers
L
L
D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D016259:Genes, myc; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016914:Ribonuclease H / Q000096:biosynthesis*; D013347:Subcellular Fractions / Q000201:enzymology
D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D016914:Ribonuclease H