P53-Suppression by Overexpression of the HPV16 E6 Gene in Lymphoblastoid TK6 Cells Causes a Change in Cell-Death Mode but not Radiosensitivity

Abstract

The human lymphoblastoid cell line TK6E6 with suppressed expression of tumor suppressor protein p53 shows similar high radiosensitivity as p53-wildtype TK6 cells with a linear survival curve but reduced/delayed apoptosis compared with TK6. By contrast, the related cell line WTK1 expresses high levels of mutated p53, is radioresistant with a shouldered survival curve and shows little apoptosis. The purpose was to test if the differences in radiosensitivity might be related to differences in cell-cycle progression and cell-death mode. The related human lymphoblastoid cell lines TK6 (p53-wildtype), TK6E6 (overexpressing the E6 gene of human papilloma virus 16), and WTK1 (p53-mutant) were irradiated with 6 MV X-rays. Cell survival was determined by the colony formation assay and apoptosis quantified by the sub-G1 fraction using flow cytometry. Cell-cycle analysis was performed by flow cytometry on BrdU pulse-labeled cells. The ratio of p-PLK1 to p-CHK1, which controls the release from the G2 phase into mitosis, was quantified by Western blotting using phospho-specific antibodies. After irradiation (IR) with a dose of 3 Gy, progression of BrdU-negative cells from G1 into S was arrested in TK6 showing an intact G1/S checkpoint which was absent in TK6E6 and WTK1. Normal cell-cycle progression was slower in TK6E6 and WTK1 (cell doubling time: 11.6h) than in TK6 (10.3h). However, progression of BrdU-positive cells from S into G2 showed a larger delay relative to unirradiated controls in TK6E6 (4h) compared with TK6 and WTK1 (2h). Notably, following irradiation of cultures enriched in S-phase, only 50% (1 Gy) to 30% (3 Gy) of the BrdU-labelled TK6E6 cells leaving G2 after release of the G2/M block entered G1 compared with 100% and 60% for TK6 cells. Together with the reduced levels of apoptosis in TK6E6 this strongly suggests a shift in cell death mode from apoptotic towards mitotic death. All three cell lines showed an intact G2/M checkpoint (blockage). A 15-25% decrease in the ratio p-PLK1p-CHK1 was seen after irradiation of TK6 and TK6E6 cells, respectively, which were restored to the original levels 7h and 12h after irradiation. By contrast, p-PLK1/p-CHK1 in unirradiated, normally cycling cells was 45% higher in WTK1 cells than in TK6 but continued to decrease up to 24h after irradiation although by this time WTK1 cells had started to progress through M into G1. Suppression of p53 had little influence on survival rates but caused a shift from apoptotic to mitotic cell death. The radioresistance of p53-mutated WTK1 cells was not associated with an inefficient G2/M block or premature release into mitosis but the cells progressed through G1 and S at the same rate as unirradiated cells. Since WTK1 are known to show genomic instability, their radioresistance might be due to tolerance to some type(s) damage that inactivate p53-wildtype cells, possibly mediated by a gain-of-function mutation in p53 or by a change in a different gene.