ABSTRACT
Human papillomaviruses (HPV) are causative agents of human cancers including those of the cervix and also of the head and neck; HPV16 is the most commonly found type in these diseases. The viral E2 protein regulates transcription from the viral genome by interacting with DNA-binding sequences in the HPV transcriptional control region; it also regulates replication by interacting with and recruiting the HPV replication factor E1 to the viral origin. Therefore, E2 is essential for the viral life cycle. The E2 protein interacts with several proteins involved in the cellular response to DNA damage including p53, TopBP1, and PARP. We therefore set out to establish whether DNA-damaging agents can regulate E2 activity. Here we show that UVB irradiation downregulates transcriptional activity of both HPV16 and HPV8 E2, while hydroxyurea and etoposide do not. This downregulation of E2 activity is independent of p53 function as it occurs in p53 wild type and null cell types as well as in the presence of functional HPV16 E6 that degrades p53. Using stable cell lines expressing E2 we show that this downregulation of E2 function by UVB is due to a reduction of the E2 protein half-life. The identification of the pathway(s) through which UVB downregulates E2 transcriptional activity and protein levels will present a novel target for the treatment of HPV-related diseases.
Subject(s)
DNA-Binding Proteins , Oncogene Proteins, Viral/metabolism , Transcriptional Activation/radiation effects , Ultraviolet Rays , Antineoplastic Agents/pharmacology , DNA Damage , Down-Regulation/radiation effects , Half-Life , HeLa Cells , Humans , Leupeptins/pharmacology , Oncogene Proteins, Viral/drug effects , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/radiation effects , Trans-Activators/drug effects , Trans-Activators/metabolism , Trans-Activators/radiation effects , Tumor Cells, Cultured , Tumor Suppressor Protein p53/drug effects , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/radiation effectsABSTRACT
The human papilloma virus-type 16 (HPV-16) E6 and E7 proteins interact with the p53 and pRb tumor suppressor proteins, respectively. The effect of E6 or E7 expression on UV irradiation (5 and 20 J/m2)-induced genotoxic injury of confluent primary murine astrocytes was determined. Retroviral vectors were used to overexpress E6 and E7. Astrocytes expressing E7 showed increased vulnerability to UV-induced apoptosis while E6 over expressing astrocytes were protected from the same insults. Cell death in the E7 overexpressing cells was apoptotic because it showed DNA ladders, activation of caspase-3, formation of apoptotic bodies and decreased DNA content to less than the G0 level. After UV-irradiation the level of E2F1 in E7-expressing astrocytes was higher than E6-, LXSN- or mock-infected cells, and caspase-3 was activated to a greater extent. E7-expressing astrocytes showed the highest levels of Bax under normal growth conditions. The mitochondrial membrane potential of E7-expressing astrocytes was depolarized by 90% after UV-irradiation while the depolarization in control cells was about 50%. E6 overexpression decreased while E7 overexpression increased UV-induced astrocyte apoptosis.