The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6. This ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. These findings show that RNAi provides a powerful molecular strategy to inactivate intracellular E6 function efficiently. Moreover, they define E6 as a most promising therapeutic target to eliminate HPV-positive tumour cells specifically by RNAi. Thus, by sequence-specific targeting of antiapoptotic genes, siRNAs may be developed into novel therapeutics that can efficiently correct the apoptosis deficiency of cancer cells.

No abstract

Increased resistance to apoptosis is a hallmark of many tumor cells. The functional inhibition of specific antiapoptotic factors may provide a rational basis for the development of novel therapeutic strategies. We investigated here whether the RNA interference (RNAi) technology could be used to increase the apoptotic susceptibility of cancer cells. As a molecular target, we chose the antiapoptotic livin (ML-IAP, KIAP) gene, which is expressed in a subset of human tumors. We identified vector-borne small interfering (si)RNAs, which could efficiently block endogenous livin gene expression. Silencing of livin was associated with caspase-3 activation and a strongly increased apoptotic rate in response to different proapoptotic stimuli, such as doxorubicin, UVirradiation, or TNFa. The effects were specific for Livinexpressing tumor cells. Our results (i) provide direct evidence that the intracellular interference with livin gene expression resensitizes human tumor cells to apoptosis, (ii) define the livin gene as a promising molecular target for therapeutic inhibition, and (iii) show that the livin gene is susceptible to efficient and specific silencing by the siRNA technology.

The four Rep proteins encoded by adeno-associated virus type 2 (AAV-2) inhibit transcription of their own promoters and of several heterologous promoters. To gain insight into the molecular mechanism of Repmediated transcription repression, we studied the effects of the four Rep proteins on the accumulation of mRNA transcribed from the human papillomavirus type 18 upstream regulatory region HPV18 URR, the human immunodeficiency virus long terminal repeat, and the AAV-2 p5 and p19 promoters by transient transfection experiments in HeLa cells. We observed a distinct contribution of the C-and N-terminal sequences in which the four Rep proteins (Rep78, Rep68, Rep52, and Rep40) differ from each other. While Rep78 showed a more than 10-fold inhibition of the four promoters studied, transcriptional repression mediated by Rep68 and Rep52 was reduced and nearly completely abolished for Rep40. The contribution of the C terminus of Rep78 was reduced with respect to the inhibition of the AAV-2 p5 and p19 promoters. Point mutations and deletions showed that a C-terminal zinc binding motif is required for zinc binding in vitro but plays no obvious role in the inhibition of homologous and heterologous promoters. Overall, inhibition of the four different promoters was dependent on the identical Rep protein domains with the exception of the AAV-2 p5 promoter. Expression of the AAV-2 p5 promoter was inhibited by a Rep78 protein with a mutation in the nucleotide binding motif, whereas expression of the AAV-2 p19 promoter, the human immunodeficiency virus long terminal repeat, and the HPV18 URR was not. Mutational analysis of the HPV18 URR showed that several, but not a single, cis regulatory elements are involved in the inhibition process. This finding suggests that transcriptional repression is mediated by protein-protein interactions of the Rep proteins either with multiple transcription factors or with target proteins of sequence-specific transcription factors of the basal transcription machinery. Adeno-associated virus type 2 (AAV-2) is a human parvovirus with a single-stranded genome approximately 4.7 kb in length (8, 58). Two 145-bp terminal repeats at the ends of the linear genome serve as origins of DNA replication. Promoters located at map positions 5, 19, and 40 express four overlapping nonstructural proteins (Rep78, Rep68, Rep52, and Rep40) and three overlapping structural proteins (VP1, VP2, and VP3). The nonstructural proteins are needed for DNA replication and regulation of the AAV-2 promoters p5, p19, and p40. Rep78 and Rep68 are expressed from the p5 promoter and contain a different C terminus as a result of translation of Rep68 from a spliced mRNA. Rep52 and Rep40 are N-terminally truncated forms of Rep78 and Rep68, respectively, that are transcribed from the p19 promoter. It is not clear whether additional forms of Rep68 and Rep40 are expressed from alternatively spliced mRNAs (64). The role of the Rep proteins in DNA replication has been elucidated in more detail. The two large Rep proteins Rep78 and Rep68 are ne...

The malignant transformation potential of high-risk human papillomaviruses (HPVs) is closely linked to the expression of the viral E6 and E7 genes. To elucidate the molecular mechanisms resulting in HPV oncogene expression, a systematic analysis of the cis-regulatory elements within the HPV type 18 (HPV18) upstream regulatory region (URR) which regulate the activity of the E6/E7 promoter was performed. As the functional behavior of a given cis-regulatory element can be strongly influenced by the overall composition of a transcriptional control region, individual elements were inactivated by site-directed mutagenesis in the physiological context of the complete HPV18 URR. Subsequently, the effects of these mutations on the activity of the E6/E7 promoter were assessed by transient transfection assays. We found that the transcriptional stimulation of the E6/E7 promoter largely depends on the integrity of cis-regulatory elements bound by API, Spl, and in certain epithelial cells, KRF-1. In contrast to previous reports implying a key role for NF1 and Oct-i recognition motifs in the stimulation of papillomavirus oncogene expression, the inactivation of these elements in the context of the HPV18 URR did not strongly affect the transcriptional activity of the E6/E7 promoter. Mutation of a promoter-proximal glucocorticoid response element completely abolished dexamethasone inducibility of the HPV18 E6/E7 promoter and resulted in an increase of its basal activity. Functional dissection of the HPV18 constitutive enhancer region indicates that its transcriptional activity is largely generated by functional synergism between a centrally located AP1 module and thus far undetected cis-active elements present in the 5' flank of the enhancer. Furthermore, comparative analyses using homologous and heterologous promoters show that the transcriptional activity of HPV18 enhancer elements is influenced by the nature of the test promoter in a cell-type-specific manner.

No abstract

The p21WAF1/CIP1/SDI1 gene is an important regulator of crucial cellular processes, including cell cycle control, cellular dierentiation, and the response to genotoxic stress. Induction of p21 gene expression upon DNA damage is widely believed to be p53-dependent. In the present study we analysed the expression of p21 following genotoxic stress, using dierent DNA-damaging agents and cellular systems. We found that the p21 response markedly varied between dierent cell lines and also for dierent genotoxic agents within the same cell line. Genotoxic induction of p21 mRNA expression can occur in the presence of p53-antagonists, such as overexpressed mdm-2 or human papillomavirus (HPV) E6, and in cells harbouring mutated p53 genes. Moreover, upon genotoxic stress, p21 mRNA and protein expression were found to be uncoupled in several cell lines. Thus, transcriptional and postranscriptional changes in p21 expression following DNA damage are not necessarily linked to the intracellular p53 status but strongly depend on the individual cellular background and the type of DNA-damaging agent. Our ®ndings indicate that p21 expression following genotoxic stress underlies a complex control and can be substantially modulated on the posttranscriptional level in a cellspeci®c manner.

A substantial proportion of the worldwide liver cancer incidence is associated with chronic hepatitis B virus (HBV) infection. The therapeutic management of HBV infections is still problematic and novel antiviral strategies are urgently required. Using the peptide aptamer screening system, we aimed to isolate new molecules, which can block viral replication by interfering with capsid formation. Eight peptide aptamers were isolated from a randomized expression library, which speci®cally bound to the HBV core protein under intracellular conditions. One of them, named C1-1, eciently inhibited viral capsid formation and, consequently, HBV replication and virion production. Hence, C1-1 is a novel model compound for inhibiting HBV replication by blocking capsid formation and provides a new basis for the development of therapeutic molecules with speci®c antiviral potential against HBV infections. Oncogene (2001) 20, 6579 ± 6586.