Lappalainen K, Pirilä L, Jääskeläinen I, Syrjänen K, Syrjänen S
MediCity Research Laboratory, Faculty of Medicine, University of Turku, Finland.
Anticancer Res. 1996 Sep-Oct;16(5A):2485-92.
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.
尽管已知人乳头瘤病毒（HPV）感染与宫颈癌有关，但目前尚无针对HPV感染的特异性抗病毒治疗方法。反义寡脱氧核苷酸（AS-ODNs）可能提供一种有效的治疗HPV感染的方法，因为使用修饰的ODNs或载体系统已提高了其稳定性和递送效率。在本研究中，我们研究了脂质体AS-ODNs（0.1、1和5 microM）对CaSki细胞中HPV 16 E7 mRNA和蛋白水平的影响。我们使用了含有溴化二甲基二辛基铵（DDAB）或2,3-二油酰氧基-N-[2（精胺甲酰胺基）乙基]-N，N-二甲基-1-丙胺三氟乙酸盐（DOSPA）的阳离子脂质体（10 microM）。这两种脂质体均以二油酰磷脂酰乙醇胺（DOPE）作为辅助脂质。AS-ODNs的靶标是E7蛋白，因为它是HPV 16的两种癌蛋白之一。只有靶向E7起始密码子的脂质体AS-ODNs对E7 mRNA表达有影响；检测到两个较短的转录本，表明RNase H降解被激活。靶向E7起始位点下游的脂质体随机ODN或脂质体ODN不影响mRNA模式。然而，通过免疫沉淀检测到的E7蛋白水平没有变化。进一步的研究表明，AS-ODNs在兔网织红细胞裂解物测定中抑制了E7 mRNA的翻译。该数据与mRNA水平的变化一起证明AS-ODNs到达了靶mRNA。CaSki细胞中E7蛋白水平未改变的一个可能解释可能是免疫沉淀对检测蛋白水平的微小变化不够敏感。然而，在能够避免非序列特异性效应之前，载体系统和AS-ODNs修饰领域仍需要进一步进展。
