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-methoxyethyl (MoE)-modified hemimers were synthesized that targeted various regions 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), and to a 3 bp mismatched ASO (NCH4529). NCH4401 inhibited Hdm2 expression in SJSA-1 cells with an IC50 of 120 nm, whereas NCH-4668 was less potent with an IC50 of 180 nm. The mismatched control ASO was completely inactive, indicating a sequence-dependent mechanism of action of NCH-4401. NCH4401 was subsequently used to study the consequences of inhibiting Hdm2 expression in human osteosarcoma cells. NCH-4401 completely inhibited Hdm2 protein expression in SJSA-1 cells at a concentration of 300 nm, already 4 h after start of ASO treatment. At an ASO concentration of 300 nM, p53 protein was induced 12.5-fold and p21 was induced 8-fold over background levels, 24 h after start of ASO treatment. The dramatic induction of p53 in SJSA-1 cells prompted us to investigate whether the accumulation of p53 in these cells was followed by induction of apoptosis. However, no signs for apoptosis were detected in SJSA-1 cells, following induction of wild-type p53 using the Yopro method and the induction of caspase-3 activity. SJSA-1 cells were subsequently treated with NCH-4401 at different concentrations in combination with two well-known DNA-damaging agents, i.e. carboplatin and mitomycin C. Apoptosis induction following treatment of cells with DNA-damaging agents and NCH4401 was determined in parallel by measuring caspase-3 activation and uptake of the DNA dye Yopro. Carboplatin and mitomycin C together only slightly induced apoptosis in SJSA-1 cells to a factor of approximately 2-fold, as measured by the induction of caspase-3 activity. The downregulation of Hdm2 expression by NCH4401 did not induce apoptosis on its own and did not potentiate the mitomycin C/carboplatin-induced programmed cell death.
Geiger
T
T
Department of Oncology, Nucleic Acid Sciences, Novartis Pharma Inc, Basel, Switzerland. thomas.geiger@pharma.novartis.com
Hüsken
D
D
Weiler
J
J
Natt
F
F
Woods-Cook
K A
KA
Hall
J
J
Fabbro
D
D
D017209:Apoptosis; D001483:Base Sequence; D002453:Cell Cycle; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D009153:Mutagens / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000235:genetics*; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051736:Proto-Oncogene Proteins c-mdm2; D012333:RNA, Messenger / Q000235:genetics
D009153:Mutagens; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2