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F. Bai, J. Xi, T.P. Fleming, E.F. Wawrousek, U.P. Andley; Loss of p53-dependent Checkpoint Function in Lens Epithelial Cells Derived from B-Crystallin Knockout Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1246.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To analyze the changes in cell cycle proteins that may contribute to the hyperproliferation and genomic instability of αB-/- lens epithelial cells, previously demonstrated in our laboratory. Methods: Primary lens epithelial cell cultures were derived from wild type or αB-/- mice. We examined the expression of p53 in these cells. Lens epithelial cells from p53-/- mice were used as a negative control for p53. The H1299 cell line was used as a positive control for the expression of mutant p53. The level of p53 in lens epithelial cells was detected by immunoblotting with p53 antibodies. Immunofluorescence and confocal microscopy were used to detect the expression of αB-crystallin, and examine the microtubules in cells. Fluorescence in situ hybridization (FISH) was used to detect ploidy changes. To assess cell cycle checkpoint function, cells were γ-irradiated (40 Gy) and then either labeled with BrdU to detect S-phase re-entry, or treated with nocodazole to arrest them in mitosis. Results: Hyperproliferative αB-/- cells entered the S phase and underwent mitosis after γ-irradiation, unlike wild type cells. A high level of p53 in these cells was detected by immunoblotting. However, re-entry of cells into S phase and mitosis after DNA damage by γ-irradiation is consistent with the loss of p53 checkpoint function. This apparent conflict is explained by the presence of inactive mutant forms of p53 in these cells. These results show that loss of p53 function in αB-/- lens cells is associated with their hyperproliferation and genomic instability. The hyperproliferative αB-/- cells contained a significant proportion of cells arrested in cytokinesis, as well as binucleated cells in which nuclear division had occurred without cell division. FISH analysis using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative αB-/- cells were 25% diploid and 75% tetraploid whereas wild type cells were 90% diploid. Wild type and αB-/- cells were infected with Ad12-SV40 hybrid virus. SV40 T antigen induced cell death in confluent αB-/- cells, but not wild type, indicating that T antigen does not always increase proliferation, but may sensitize apoptotic death of αB-/- lens epithelial cells because of their intrinsic genomic instability. Conclusions: These results suggest that the hyperproliferation of αB-/- lens epithelial cells is associated with an increase in the expression of mutant p53. αB may act at the checkpoint that controls the exit of cells from mitosis.
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