Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 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, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. 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 G1 arrest, not suprabasal quiescence or transforming growth factor beta-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.

The E6 protein of human papillomavirus type 16 functions as a transcriptional repressor in a mechanism independent of the tumor suppressor protein, p53.

The E6 protein of human papillomavirus (HPV) type 16 displays a number of activities when transfected into cultured cells, including transcriptional activation of several viral promoters and targeting of p53 for degradation. HPV 16E6 was found to function as a transcriptional repressor of the moloney murine leukemia virus long terminal repeat and the cytomegalovirus immediate early promoter. Although the degree of transcriptional repression was low, a dose-dependent two- to threefold decrease in promoter activity was consistently seen in cells expressing 16E6. HPV 16E6-dependent transcriptional repression was observed in C33a cells, which express mutant p53, and in Saos-2 cells, which lack p53. These results indicate that 16E6-dependent repression of promoter activity is unlikely to be mediated by p53.

The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest.

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.

Regulation of bradykinin receptor level by cholera toxin, pertussis toxin and forskolin in cultured human fibroblasts.

1. The effect of bacterial toxins on bradykinin-triggered release of arachidonic acid was studied in serum-deprived human foreskin (HSWP) fibroblasts prelabelled with [3H]-arachidonic acid. An 18-h exposure of HSWP cells to cholera toxin, pertussis toxin, or forskolin enhanced the bradykinin-stimulated release of arachidonic acid and metabolites. 2. Prolonged treatment of HSWP cells with these agents also caused a 3 to 4 fold rise in cell surface [3H]-bradykinin binding. The rise was inhibited by concurrent incubation with cycloheximide or actinomycin D. In addition, cholera toxin and foreskolin increased [3H]-bradykinin binding in wildtype PC12 cells, but not in mutant PC12 cells with reduced cyclic AMP-dependent protein kinase type II activity. 3. In conclusion, cholera toxin, pertussis toxin and forskolin enhanced arachidonic acid release in response to bradykinin, and increased the number of bradykinin receptors in HSWP fibroblasts. A cyclic AMP-dependent mechanism appears to mediate the actions of the toxins and forskolin.

Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation.

Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.

Loss of bradykinin receptors and TPA-stimulated Na+ influx in SV40-transformed WI-38 cells.

Mitogenic stimulation of Na(+)-H+ exchange activity, as defined by the level of 5-(N,N-hexamethylene)amiloride (HMA)-sensitive Na+ influx, was compared in WI-38 and SV40 virus-transformed WI-38 fibroblasts. Serum or bradykinin dramatically stimulated HMA-sensitive Na+ influx in WI-38 cells, whereas in SV40-transformed WI-38 cells, serum, but not bradykinin, produced a large increase in HMA-sensitive Na+ influx. This lack of a bradykinin response was traced to a dramatic reduction in the number of bradykinin receptors, from 470 fmol/mg protein in WI-38 cells to 29 fmol/mg protein in the SV40-transformed WI-38 cells. Transformation of WI-38 cells with SV40 virus also altered the mechanism by which HMA-sensitive Na+ influx is stimulated. In WI-38 cells, 12-O-tetradecanoylphorbol 13-acetate (TPA) dramatically stimulated HMA-sensitive Na+ influx. In SV40-transformed WI-38 cells, TPA alone had no effect on HMA-sensitive influx and inhibited serum-stimulated HMA-sensitive Na+ influx. Down-regulation of protein kinase C activity decreased serum- and TPA-stimulated HMA-sensitive Na+ influx in the WI-38 cells and relieved the TPA inhibition of serum-stimulated HMA-sensitive Na+ influx in the SV40-transformed WI-38 cells.

Coupling of bradykinin receptors to phospholipase C in cultured fibroblasts is mediated by a G-protein.

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.

Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts.

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)