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.

Thyroid hormone regulation of alpha-lactalbumin: differential glycosylation and messenger ribonucleic acid synthesis in mouse mammary glands.

Mouse mammary tissue, when cultured in the presence of insulin, corticoids, PRL, and physiological levels of T3, shows increased synthesis and secretion of alpha-lactalbumin. Tissue cultured in the presence of insulin, hydrocortisone, PRL, and T3 synthesizes two distinct forms of alpha-lactalbumin, but secretes only one form. Tissue cultured in the absence of T3 synthesizes and secretes only one form. To address the question of whether these two electrophoretically distinct forms arose by differential glycosylation of the same polypeptide or by synthesis of two different polypeptide precursor chains, mammary tissue was cultured in the presence of insulin, corticoids, and PRL with or without T3, and the mRNA and alpha-lactalbumins were isolated. Northern blot analyses indicated that mammary gland tissue cultured in the presence of T3 contained 2.46 times more alpha-lactalbumin mRNA than tissue cultured only in the presence of insulin, hydrocortisone, and PRL. This enhanced mRNA level was confirmed by in vitro translation experiments where tissue cultured in the presence of insulin, hydrocortisone, PRL, and T3 produced mRNA that resulted in 2.1 times as much radiolabeled alpha-lactalbumin as tissue cultured in the absence of T3. Sodium dodecyl sulfate-polyacrylamide gel analysis of the in vitro translation products revealed only one band, suggesting the presence of only one message. Endoglycosidase digestion of the two forms of alpha-lactalbumin produced in the presence of T3 resolved them into a single band on sodium dodecyl sulfate-polyacrylamide gels. Thus, the electrophoretic differences between the two forms synthesized in the presence of T3 appear to be due to differential N-linked glycosylation of the same polypeptide chain and not to synthesis of two different polypeptide precursor chains.