Consequences of the inhibition of Hdm2 expression in human osteosarcoma cells using antisense oligonucleotides

The present study was performed to identify a potent and sequence-specific antisense oligonucleotide (ASO), to inhibit Hdm2 expression in human cancer cell lines and to study the downstream consequences. Ten chimeric 2'-O-met hoxyethyl (MoE)-modified hemimers were synthesized that targeted various re gions from the 5'- to the 3'-end of Hdm2 mRNA. The IC50 of the most potent ASO, NCH-4401, was subsequently determined and compared to the IC50 of a 2' -MoE-modified. ASO, with a complete phosphorothioate backbone (NCH-4668), a nd to a 3 bp mismatched ASO (NCH-4529). NCH-4401 inhibited Hdm2 expression in SJSA-1 cells with an IC50 of 120 nM, whereas NCH-4668 was less potent wi th an IC50 of 180 nM. The mismatched control ASO was completely inactive, i ndicating a sequence-dependent mechanism of action of NCH-4401. NCH-4401 wa s subsequently used to study the consequences of inhibiting Hdm2 expression in human osteosarcoma cells. NCH-4401 completely inhibited Hdm2 protein ex pression in SJSA-1 cells at a concentration of 300 nM, already 4 h after st art of ASO treatment. At an ASO concentration of 300 nM, p53 protein was in duced 12.5-fold and p21 was induced 8-fold over background levels, 24 h aft er start of ASO treatment. The dramatic induction of p53 in SJSA-1 cells pr ompted us to investigate whether the accumulation of p53 in these cells was followed by induction of apoptosis. However, no signs for apoptosis were d etected in SJSA-1 cells, following induction of wild-type p53 using the Yop ro method and the induction of caspase-3 activity. SJSA-1 cells were subseq uently treated with NCH-4401 at different concentrations in combination wit h two well-known DNA-damaging agents, i.e. carboplatin and mitomycin C. Apo ptosis induction following treatment of cells with DNA-damaging agents and NCH-4401 was determined in parallel by measuring caspase-3 activation and u ptake of the DNA dye Yopro. Carboplatin and mitomycin C together only sligh tly induced apoptosis in SJSA-1 cells to a factor of similar to2-fold, as m easured by the induction of caspase-3 activity. The downregulation of Hdm2 expression by NCH-4401 did not induce apoptosis on its own and did not pote ntiate the mitomycin C/carboplatin-induced programmed cell death.