The intracellular mechanism of action of exogeneously administered antisense oligonucleotides is not known. Two mechanisms have been suggested based on studies in cell fire systems. These are: 1) inhibition of ribosome attachment or movement along MRNA; and, 2) creating a substrate for RNAse H degradation of targeted MRNA. RNAse H is an enzyme which destroys the RNA portion of an RNA/DNA hybrid complex and is present in the cytoplasm of all proliferating cells. Its natural function in cells is unknown. We have obtained a full-length CDNA coding for bacterial RNAse H. We have succeeded in constructing a mammalian episomally replicating expression vector which contains the bacterial RNAse H gene in a sense orientation. We have demonstrated by in situ gel assay that the bacterial RNAse H gene is expressed at high levels in the transfected cells and that this activity can be induced with cadmium. Cells transfected with this construct and sham-transfected cells are being compared for their responsiveness to antisense oligonucleotide addition. We are utilizing a model system which makes use of the fact that U937 cells apparently possess an autocrine growth loop regulated by IL-6. If IL-6 antisense is added to these cells, their growth is markedly inhibited. If RNAse H is involved in the intracellular functioning of antisense oligos, then cells with high levels of RNAse H should respond better to addition of these compounds.
