4C, top left)

4C, top left). of the length of treatment, p53-null cells arrest in G2, but ultimately adapt and proceed into mitosis. Interestingly, they fail to undergo cytokinesis, become multinucleated, and then die from apoptosis. Upon transient treatment with DNA damaging agents, wild-type p53 cells reversibly arrest and repair the damage, whereas p53-null cells fail to do so and die. These data indicate that p53 can promote cell survival by inducing reversible cell cycle arrest, Ethoxyquin thereby allowing for DNA repair. Thus, transient treatments may exploit differences between wild-type p53 and p53-null cells. were CSF2RA examined by phase contrast microscopy. repression (22), no change in either protein was observed in control cells containing normal p53 levels (Fig. 4A, left panel and data not shown). In order to investigate the long-term outcome of sustained exposure to chemotherapeutic agents, clone 1 and clone 7 cells were treated with doxorubicin for 3 weeks and proliferation was compared to untreated cells by Giemsa staining (Fig. 4B) and light microscopy (Fig. 4C). In the absence of DNA damage, both clone 1 and clone 7 cells grew to confluency (Fig. 4B, left). In contrast, neither cell type proliferated in the continued presence of doxorubicin (Fig. 4B, right). Closer observation of doxorubicin-treated cells microscopically demonstrates that, although they do not proliferate, clone 1 cells persist throughout the duration of treatment (Fig. 4C, top left). Higher power magnification of these cells reveals two predominating morphologies. One group of cells has a flattened, fried egg appearance, resembling the appearance of Ethoxyquin senescent cells (Fig. 4C, bottom left), and the other group has an elongated, spindle-like morphology (Fig. 4C, bottom right). Microscopic examination of doxorubicin-treated clone 7 cells fails to reveal any remaining cells at 3 weeks (Fig. 4C, top right), suggesting that all cells have undergone cell death by apoptosis. In order to investigate the possibility that the clone 1 cells with the fried egg morphology represent senescent cells, senescent-associated -galactosidase (-gal) staining was performed on cells following no treatment or continuous exposure to doxorubicin (0.05 g/ml) for 7 days (Fig. 4D). In contrast to untreated clone 1 cells, those undergoing doxorubicin treatment exhibited a high degree of -gal staining at 7 days. No -gal positivity was observed in clone 7 cells before or after doxorubicin exposure. Taken together, these data indicate that cells expressing p53 respond to prolonged DNA damage by stably arresting with a 4N DNA content, expressing cell cycle markers consistent with G1, and become senescent. p53-expressing tumor cells recover from short-term chemotherapeutic treatment whereas p53- ablated tumor cells do not The above experiments addressed the role of p53 in the response to continuous exposure to chemotherapeutic drugs. In order to investigate the role of p53 in the cellular response to transient DNA damage, the U2OS-derived shRNA clones were pulsed with 0.05 g/ml doxorubicin for 6 hours followed by drug wash-out and analyzed by flow cytometry (Fig. 5A and 5B). After 6 hours of doxorubicin treatment, clone 1 and clone 7 cells had similar cell cycle profiles, and one day following wash-out of drug, both cell types were cell cycle arrested. However, following an observation period of seven days, the p53-replete control cells resumed cycling and had a Ethoxyquin cell cycle profile resembling untreated cells. In contrast, the majority of p53-ablated cells had a hypodiploid DNA content, consistent with apoptosis. The percentage of hypodiploid cells at each time point is summarized in Fig. 5B. The presence of micronuclei following transient exposure to doxorubicin was also analyzed (Supplemental Fig. S5). Following treatment with 0.05 g/ml doxorubicin for 6 hours followed by drug wash-out, p53-ablated clone 7 cells were observed to contain multiple nuclei at high rates by two days after treatment, and this phenomenon was observed throughout the observation period. In contrast, multinucleation was a rare event in p53-expressing clone 1 cells. Open in a separate window Figure 5 p53-expressing cells recover.