Destabilization of the retinoblastoma tumor suppressor by human papillomavirus type 16 E7 is not sufficient to overcome cell cycle arrest in human keratinocytes.

The E7 oncoprotein of human papillomavirus type 16 promotes cell proliferation in the presence of antiproliferative signals. Mutagenesis of E7 has revealed that this activity requires three regions, conserved regions 1 and 2 and a C-terminal zinc finger. Binding to the retinoblastoma tumor repressor (Rb) through an LxCxE motif in conserved region 2 is necessary, but not sufficient, for E7 to induce proliferation. We tested the hypothesis that binding to Rb is not sufficient because conserved region 1 and/or the C terminus are required for E7 to functionally inactivate Rb and thus induce proliferation. One mechanism proposed for how E7 inactivates Rb is by blocking Rb-E2F binding. Either conserved region 1 or the C terminus was necessary, in combination with the LxCxE motif, for E7 to block Rb-E2F binding in vitro. While all full-length E7 proteins with mutations outside of the LxCxE motif inhibited Rb-E2F binding, some failed to abrogate cell cycle arrest, demonstrating that blocking Rb-E2F binding is not sufficient for abrogating antiproliferative signals. Another mechanism proposed for how E7 inactivates Rb is by promoting the destabilization of Rb protein. Mutations in conserved region 1 or the LxCxE motif prevented E7 from reducing the half-life of Rb. Though no specific C-terminal residues of E7 were essential for destabilizing Rb, a novel class of mutations that uncouple the destabilization of Rb from the deregulation of keratinocyte proliferation was discovered. Destabilization of Rb correlated with the abrogation of Rb-induced quiescence but was not sufficient for overriding DNA damage-induced cell cycle arrest or for increasing keratinocyte life span. Finally, the same regions of E7 required for destabilizing Rb were required for reducing p107 and p130 levels. Together, these results suggest that inactivation of all three Rb family members is not sufficient to deregulate keratinocyte cell cycle control.

Radiation inactivation of human prostate cancer cells: the role of apoptosis.

Radiation-induced apoptosis detected by gel electrophoresis was measured in cells of three human prostate carcinoma cell lines (TSU, PC-3 and DU-145) and compared to their intrinsic radiosensitivities as measured by clonogenic assays. The intrinsic radiosensitivities of each cell line were defined by their alpha and beta coefficients and their surviving fraction at 2 Gy, derived from complete survival curves. The temporal expression and kinetics of radiation-induced apoptosis for DU-145 cells, the human prostate carcinoma cell line which expressed the highest rate of radiation-induced apoptosis, was characterized further by differential sedimentation and the immunofluorescence assay (Apoptag) which was specific for 3'-OH ends in cellular DNA. Cell viability was measured microscopically with trypan blue staining. Cell survival after various doses was computer-fitted to either a simple linear or a linear-quadratic equation. Twenty-four hours after a 10-Gy dose of 137Cs gamma rays, DNA fragmentation to nucleosome multimers was strongly expressed in only DU-145 cells. In this cell line, when centrifugation at 12,000g for 10 min was used to separate fragmented from large molecular weight DNA, the proportion of DNA in the supernatant increased to a maximum of approximately 17% of the total by 10-12 h after radiation treatment. Cell death 24 h after irradiation measured by trypan blue exclusion assays followed single-hit kinetics up to 80 Gy. The proportion of cells which were labeled with Apoptag displayed single-hit kinetics and yielded the same inactivation coefficient as measured by trypan blue. Together, these data indicate that the rapid (24 h) inactivation of irradiated DU-145 cells results from apoptosis and accounts for about 5% of the single-hit killing measured by clonogenic assay. Temporal studies of radiation-induced killing of DU-145 cells distinguished this rapid mechanism of cell death from the major mechanism (72-144 h). These may correlate with apoptosis and proliferative cell death, respectively. Of the three prostate cancer cell lines investigated, only DU-145 cells displayed significant levels of radiation-induced DNA fragmentation and rapid cell death, with characteristics of apoptosis. This mechanism of cell death was complete by 24 h after irradiation and was well separated in time from the death of cells by the major mechanisms which occurred after 72 h, and accounted for about 5% of cell inactivation by a single-hit mechanism.

A sensitive assay for taxol and other microtubule-stabilizing agent.

The ability of taxol to protect microtubules in cultured human ovarian carcinoma cells from drug- and cold-induced depolymerization was characterized as a functional assay for microtubule stabilizing agents. Treatment of the cells with concentrations of vinblastine or colchicine of 50 nM or greater, or incubation at 4 degrees C resulted in complete depolymerization of cytoplasmic microtubules. Pretreatment with taxol for 3 h enabled the cells to maintain substantial numbers of microtubules following the application of vinblastine or colchicine. This protective effect was easily observed at 50 nM taxol, whereas taxol-induced microtubule bundling was observed only at concentrations of 500 nM or greater. Concentrations of taxol as low as 10 nM stabilized microtubules against cold-induced depolymerization. Therefore, protection of microtubules from drug- and cold-induced depolymerization provides a sensitive functional assay for taxol. These systems should be similarly effective in identifying novel compounds which stabilize microtubules.