Human papillomavirus type 16 E6 and E7 oncogenes abrogate radiation-induced DNA damage responses in vivo through p53-dependent and p53-independent pathways.

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 and/or pRb-like proteins likely mediate both p53-dependent and p53-independent responses to radiation.

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice.

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.

High-affinity rat anti-fluorescein monoclonal antibody with unique fine specificity properties including differential recognition of dynamic ligand analogues.

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.

Relative conformational stabilities of single-chain pocket and groove-shaped antibody active sites including HCDR transplant intermediates.

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.

Effect of transplantation of antibody heavy chain complementarity determining regions on ligand binding.

Active site structure-function analyses of anti-fluorescein single chain antibody 4-4-20 and anti-single-stranded DNA single chain antibody 04-01 were conducted studying the ligand binding properties of hybrid antibodies resulting from systematic transplantation of heavy chain complementarity regions (HCDRs) from monoclonal antibody 4-4-20 into 04-01. Two prototype monoclonal antibodies were chosen because the primary structures of their respective light chains were nearly identical but the specificities and shape of their active sites were distinctly different. Based on nearly identical light chains, the diverse active site conformations (i.e. 4-4-20 pocket and 04-01 cleft) and subsequent specificities of the two antibodies were likely dictated by heavy chain properties. As a result, specificities of each HCDR transplant were analyzed in terms of binding reactivity with either fluorescein or (dT)8. Results of binding studies, together with idiotypic and secondary structure analyses, were used to determine the relative contribution of each HCDR to the active site conformations and ligand specificities of monoclonal antibodies 4-4-20 and 04-01. Collectively the various analyses led to the conclusion that the intradomain conformational dynamics and cooperativity necessary for the structural integrity of the low affinity cleft-shaped 04-01 anti-single-stranded DNA active site are probably less stringent than those of a high affinity pocket-shaped anti-fluorescein active site.

Conversion of an anti-single-stranded DNA active site to an anti-fluorescein active site through heavy chain complementarity determining region transplantation.

Complementarity determining region (CDR) transplant studies were conducted between two monoclonal antibodies of distinctly different specificities (anti-fluorescein monoclonal antibody (mAb) 4-4-20 and anti-single-stranded DNA (ssDNA) mAb 04-01) which possessed nearly identical light chains but dissimilar heavy chains. The variations in binding specificities between the two immunoglobulins suggested that the active-site features of anti-fluorescein antibodies were dictated by characteristics intrinsic to the heavy chain (H-chain). To identify specific regions of the H-chain which influence the structure and function of an anti-fluorescein active site, CDR transplantation was systematically employed to convert the anti-ssDNA 04-01 antibody active site to an active site with anti-fluorescein activity. Each mAb 4-4-20 H-chain CDR (HCDR) was transplanted into the H-chain of a single-chain derivative of the 04-01 molecule. A fluorescence polarization ligand binding assay was utilized to determine the equilibrium dissociation constant, Kd, of hybrid transplant single-chain antibody HCDR1-HCDR2-HCDR3(4-4-20) for fluorescein (3.8 x 10(-7) M, indicating successful conversion of an anti-ssDNA active site to an anti-fluorescein binding site. A similar Kd (6.3 x 10(-7) M) was determined using a fluorescein fluorescence quenching assay. The transplantation results are discussed in terms of the relative contribution of each HCDR to a successful conversion in antibody specificity.

Mutational analysis of active site contact residues in anti-fluorescein monoclonal antibody 4-4-20.

The contribution to high affinity Fl binding by each crystallographically defined Mab 4-4-20 (Ka = 1.7 x 10(10) M-1; Qmax = 90%) ligand contact residue (L27dHis, L32Tyr, L34Arg, L91Ser, L96Trp and H33Trp) has been determined by site-specific mutagenesis studies. All six antigen contact residues were changed to Ala in the single-chain derivative of Mab 4-4-20 and following expression in E. coli, denaturation, refolding and purification, each SCA mutant was characterized in terms of Fl binding affinity, Qmax, lambda max and idiotype. Results demonstrated that Ala substitutions at each ligand contact residue reduced the binding affinities and quenching maxima for all residues except L27d which retained wild type characteristics. The SCA TyrL32Ala, SerL91Ala and TrpH33Ala mutants exhibited binding affinities that were approximately 1000-fold lower than the wild type value and greatly reduced Qmax values. Additionally, other amino acid substitutions were performed at three of the six antigen contact residues (L91Ser, L96Trp and H33Trp) to further evaluate the role of each in Fl binding. Therefore, the following mutations were constructed and characterized: SerL91Asn, TrpL96Tyr, TrpL96Phe, TrpL96Leu, TrpH33Tyr and TrpH33Phe. Results of site-specific mutagenesis studies are discussed in terms of Mab active site structure and suggest that L32Tyr, L91Ser and H33Trp are important for high affinity Fl binding and efficient Fl quenching.