E6 and E7 oncoproteins from high risk human papillomaviruses (HPVs) transform cells in tissue culture and induce tumors in vivo. Both E6, which inhibits p53 functions, and E7, which inhibits pRb, can also abrogate growth arrest induced by DNA-damaging agents in cultured cells. In this study, we have used transgenic mice that express HPV-16 E6 or E7 in the epidermis to determine how these two proteins modulate DNA damage responses in vivo. Our results demonstrate that both E6 and E7 abrogate the inhibition of DNA synthesis in the epidermis after treatment with ionizing radiation. Increases in the levels of p53 and p21 proteins after irradiation were suppressed by E6 but not by E7. Through the study of p53-null mice, we found that radiation-induced growth arrest in the epidermis is mediated through both p53-dependent and p53-independent pathways. The abrogation of radiation responses in both E6 and E7 transgenic mice was more complete than was seen in the p53-null epidermis. We conclude that E6 and E7 each have the capacity to modulate p53-dependent as well as p53-independent cellular responses to radiation. Additionally, we found that the conserved region (CR) 1 and CR2 domains in E7 protein, which are involved in the inactivation of pRb function and required for E7's transforming function, were also required for E7 to modulate DNA damage responses in vivo. Thus pRb and͞or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.Human papillomaviruses (HPVs) are the causative agents of warts. A subset of HPVs, referred to as high risk HPVs, are associated with human anogenital cancers such as cervical cancers in women and penile cancers in men (1). Two genes of these high risk HPVs, E6 and E7, are expressed in the cells derived from HPV-associated cancers (2, 3). E6 and E7 cooperate with each other (4, 5) and with other oncogenes (6-9) in the immortalization or transformation of cells. The transforming activities of E6 and E7 correlate, at least in part, with their inactivation of two cellular tumor suppressor gene products, p53 and pRb, which regulate the processes of division, differentiation, and͞or death in cells (10-12). E6 binds to p53 (13) and mediates degradation of p53 through the ubiquitin-proteasomal degradation pathway (14). E7 binds to pRb (15), inhibits pRb's function (16), and promotes degradation of pRb (17,18). In vivo experiments using mice transgenic for E6 and E7 have shown that E6 and E7 together can induce tumors in the targeted tissues (19-21). Our laboratory has generated transgenic mice in which the expression of HPV-16 E6 (S.S. and P.F.L. unpublished work) or E7 (22) singly was directed to mouse squamous epithelial cells by the human keratin 14 (K14) promoter. These mice develop papillomas and carcinomas in the late stage of their life, indicating that E6 and E7 each is sufficient to induce tumors. Because of the long latent period of tumor induction, it is proposed that other genetic changes must contribute to the carcinogenesis induced by E...

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.

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Stability measurements of SCA 04-01/212 (anti-ssDNA) which possesses a groove-shaped active site were performed by Gdn-HCl-induced unfolding, analyzed assuming a simple two-state equilibrium, and expressed as the free energy of unfolding, delta Gn-u. A delta Gn-u of 1.44 +/- 0.13 kcal/mol was determined experimentally for SCA 04-01/212. In addition, the conformational stabilities of HCDR transplants, hybrid antibody molecules resulting from the transplantation of HCDRs from SCA 4-4-20 (anti-fluorescein) into the corresponding regions of 04-01 in all combinations, were determined using the identical protocol applied to SCA 04-01. On the basis of the results of these stability experiments, the HCDR transplants were categorized into three groups, representing low, intermediate, and high stability. Data were discussed in terms of the relationships between structure-function and conformational stability pertaining to the groove-shaped antibody active site of SCA 04-01/212 and the pocket-shaped active site of SCA 4-4-20/212.

The ability of antibodies to specifically select and stabilize through binding one or more isomers of highly dynamic ligands remains a relatively unexplored immunochemical problem. The experimental strategy employed in this study was to elicit homogeneous antibodies to polyaromatic fluorescein which exists in one isomeric form. The binding properties of a monoclonal rat antifluorescein antibody specific to a given isomer were quantitatively studied to determine the capacity to bind dynamic analogues of fluorescein which exists in multiple isomers. To generate monoclonal anti-fluorescein antibodies that reacted with specific dynamic analogues of fluorescein possessing unconjugated aromatic ring systems, immune spleenocytes from Lou/M rats immunized with FITC(I)-KLH were fused with Balb/c SP2/0-Ag14 murine myeloma cells forming rat-mouse hybridomas. Cell line P2A12-1-C8 was selected for further characterization from the original 23 stable rat hybrids, since it produced a monoclonal antibody with a binding affinity 2.0 x 10(10)/M for fluorescein based on dissociation rate measurements. P2A12-1-C8 exhibited significant reactivity with HPF and phenol red, which are dynamic structural analogues of the homologous fluorescein ligand. No reactivity was demonstrated with phenolphthalein, which based on relative chemical structures was expected to be more reactive than phenol red. Computer-based molecular modeling and energy minimization studies of fluorescein, HPF, phenol red, and phenolphthalein showed that in terms of the most energetically favorable orientation of the three aromatic rings, phenol red more closely simulated fluorescein than phenolphthalein. The results were analyzed in terms of the mechanisms of dynamic ligand stabilization and binding involving accommodation of specific ligand isomers by energetically permissible conformational states exhibited by an antibody active site. Thus, antibody reactivity of an anti-fluorescein antibody with phenol red and phenolphthalein was dictated more by ligand dynamics and aromatic orientation than by chemical structure similarities.