Effects of liposomal antisense oligonucleotides on mRNA and protein levels of the HPV 16 E7 oncogene.
Despite the known association of human papillomavirus (HPV) infection with cervical cancer there is no specific antiviral treatment for HPV infection. Antisense oligode-oxynucleotides (AS-ODNs) may offer an effective way to treat HPV infections as the stability and delivery have been improved using modified ODNs or carrier systems. In this study we investigated the effects of liposomal AS-ODNs (0.1, 1 and 5 microM) on HPV 16 E7 mRNA and protein levels in CaSki cells. We used cationic liposomes (10 microM) containing dimethyldioctadecylammonium bromide (DDAB) or 2,3-dioleyloxy-N-[2(sperminecar-boxamido)ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). Both these liposomes had dioleoylphosphatidyl-ethanolamine (DOPE) as a helper lipid. The target of the AS-ODNs was E7 protein because it is the one of the two oncoproteins of HPV 16. Only liposomal AS-ODNs which were targeted to the initiation codon of E7, had an effect on E7 mRNA expression; two shorter transcripts were detected, suggesting that RNase H degradation was activated. Liposomal random ODN or liposomal ODN targeted downstream from the initiation site of E7 did not affect the mRNA pattern. However, no change was found in the E7 protein levels detected by immunoprecipitation. Further studies showed that AS-ODNs inhibited the translation of E7 mRNA in a rabbit reticulocyte lysate assay. This data, together with the changes in mRNA levels, proved that the AS-ODNs reached the target mRNA. One possible explanation for the unchanged protein level of E7 in CaSki cells might be that immunoprecipitation is not sensitive enough to detect minor changes in protein levels. However, further progress is still needed in the field of carrier systems and modifications of AS-ODNs before non-sequence specific effects can be avoided.
Lappalainen
K
K
MediCity Research Laboratory, Faculty of Medicine, University of Turku, Finland.
Pirilä
L
L
Jääskeläinen
I
I
Syrjänen
K
K
Syrjänen
S
S
D000998:Antiviral Agents / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004337:Drug Carriers; D004354:Drug Screening Assays, Antitumor; D005260:Female; D006801:Humans; D008081:Liposomes; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D009856:Oncogene Proteins, Viral / Q000187:drug effects* / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000187:drug effects* / Q000378:metabolism; D012367:RNA, Viral / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms
D000998:Antiviral Agents; D004337:Drug Carriers; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; C059731:oncogene protein E7, Human papillomavirus type 16