Functional p53 protein is associated with the ability of cells to arrest in G1 after DNA damage. The E6 protein of cancer-associated human papillomavirus type 16 (HPV-16) binds to p53 and targets its degradation through the ubiquitin pathway. To determine whether the ability of E6 to interact with p53 leads to a disruption of cell cycle control, mutated E6 proteins were tested for p53 binding and p53 degradation targeting in vitro, the ability to reduce intracellular p53 levels in vivo, and the ability to abrogate actinomycin D-induced growth arrest in human keratinocytes. Mutations scattered throughout the amino terminus, either zinc finger or the central region but not the carboxy terminus, severely reduced the ability of E6 to interact with p53. Expression of HPV-16 E6 or mutated E6 proteins that bound and targeted p53 for degradation in vitro sharply reduced the level of intracellular p53 induced by actinomycin D in human keratinocytes. A perfect correlation between the ability of E6 proteins to reduce the level of intracellular p53 and their ability to block actinomycin D-induced cellular growth arrest was observed. These results suggest that interaction with p53 is important for the ability of HPV E6 proteins to circumvent growth arrest.

Cells arrest in the G 1 or G 0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G 1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor ␤. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G 1 arrest, not suprabasal quiescence or transforming growth factor ␤-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.

In cultured foreskin fibroblasts, bradykinin stimulates inositol phosphate generation, arachidonic acid release, and Na+/H+ exchange, with doses of 1-3 nM yielding half-maximal stimulation. Binding of 3H-bradykinin to these cells demonstrates a single receptor site with a Kd of 2.0 nM and a Bmax of 91 fmoles/mg protein. Bradykinin analogs of the B2 type inhibit this binding. GTP synergizes with bradykinin to stimulate phosphatidylinositol turnover in permeabilized fibroblasts and GTP-gamma-S decreases the Bmax of bradykinin binding to fibroblast membranes, indicating that a G-protein couples the receptor to phospholipase C. Pretreatment of fibroblasts with either cholera or pertussis toxin enhances bradykinin stimulation of inositol phosphate accumulation.

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