The goal of this work is to elucidate the mechanism by which antisense oligodeoxynucleotides inhibit proliferation of cells transformed by human c-myc and cHaras proto-oncogneses. Human cmyc codes for a conserved nuclear protein, p65, which may be involved in regulation of gene expression, and c-Ha-ras codes for a conserved inner plasma membrane protein, p21, which may be involved in transducing growth factor signals. Cell transformation may be caused by inappropriate expression or response of oncogene products during cell proliferation, so that reducing the level of cmyc and cHa-ras expression may in principle be sufficient to reverse transformation. Calculated secondary structures of cmyc and cHaras mRNAs predict many loops and bulges between the cap and the initiation codon, susceptible to antisense oligodeoynucleotide hybridization arrest. Addition of an anti-cmyc pentadecanucleotide to HL60 cells in culture inhibits proliferation and stimulates differentiation in a dose dependent and sequence specific manner and an antic- Haras pentadecanucleotide inhibits proliferation of T24transformed NIH 3T3 cells. In this project, the dose dependent effects of antisense oligodeoxynucleotides on cmyc and cHa-ras mRNA transcription, stability, translation, DNA replication, and cell morphology will be determined in normal and transformed cells. Oligodeoxynucleotide uptake, compartmentalization and degradation will be studied in the same cell lines. Oligodeoxynucleotides, however, are not stable enough for intravenous or oral administration. Oligodeoxynucleoside methylphosphonates are uncharged and nuclease resistant, enter animal cells and protect them from viral challenge, but are unexpectedly less effective in vitro than normal oligodeoxynucleotides. R and S stereoisomers occur at each phosphorus atom, but only the S form has the same conformation as a normal oligodeoxyonucleotide. Stereospecific allS oligodeoxynucleoside methylphosphonates will be synthesized and tested for their efficacy in hybridization arrest.
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