RESUMO
Human papillomaviruses (HPVs) are the causative factor for >90% of cervical cancers and 25% of head and neck cancers. The incidence of HPV positive (+) head and neck squamous cell carcinomas has greatly increased in the last 30 years. E6 and E7 are the two key viral oncoproteins that induce and propagate cellular transformation. An immune response generated during cisplatin/radiation therapy improves tumor clearance of HPV(+) cancers. Augmenting this induced response during therapy with an adenoviral HPV16 E6/E7 vaccine improves long-term survival in pre-clinical models. Here, we describe the generation of an HPV16 E6/E7 construct, which contains mutations that render E6/E7 non-oncogenic, while preserving antigenicity. These mutations do not allow E6/E7 to degrade p53, pRb, PTPN13, or activate telomerase. Non-oncogenic E6/E7 (E6(Δ)/E7(Δ)) expressed as a stable integrant, or in the [E1-, E2b-] adenovirus, lacks the ability to transform human cells while retaining the ability to induce an HPV-specific immune response. Moreover, E6(Δ)/E7(Δ) plus chemotherapy/radiation statistically enhances clearance of established HPV(+) cancer in vivo.
Assuntos
Adenocarcinoma/terapia , Adenocarcinoma/virologia , Vacinas Anticâncer/farmacologia , Neoplasias de Cabeça e Pescoço/terapia , Neoplasias de Cabeça e Pescoço/virologia , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Vacinas contra Papillomavirus/farmacologia , Proteínas Repressoras/imunologia , Adenocarcinoma/metabolismo , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/terapia , Vacinas contra Papillomavirus/genética , Proteínas Repressoras/genéticaRESUMO
Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.
