Influence of Induced Reactive Oxygen Species in p53-Mediated Cell Fate Decisions

Abstract

The p53 tumor suppressor gene can induce either apoptosis or a permanent growth arrest (also termed senescence) phenotype in response to cellular stresses. We show that the increase in intracellular reactive oxygen species (ROS) associated with the magnitude of p53 protein expression correlated with the induction of either senescence or apoptosis in both normal and cancer cells. ROS inhibitors ameliorated both p53-dependent cell fates, implicating ROS accumulation as an effector in each case. The absence of Bax or PUMA strongly inhibited both p53-induced apoptosis and ROS increase, indicating an important role these p53 targets affecting mitochondrial function genes in p53-mediated ROS accumulation. Moreover, physiological p53 levels in combination with an exogenous ROS source were able to convert a p53 senescence response into apoptosis. All of these findings establish a critical role of ROS accumulation and mitochondrial function in p53-dependent cell fates and show that other ROS inducers can collaborate with p53 to influence these fate decisions. Thus, our studies imply that therapeutic agents that generate ROS are more likely to be toxic for normal cells than p53-negative tumor cells and provide a rationale for identifying therapeutic agents that do not complement p53 in ROS generation to ameliorate the cytotoxic side effects in normal cells.The p53 tumor suppressor protein can trigger the onset either of reversible or permanent growth arrest (51, 52) or of apoptosis (27,34). However, the mechanisms involved in the decision between these cellular responses are not well understood. Cell type, the presence of growth factors or oncogenes, the intensity of the stress signal, and the cellular level of p53 have been cited as important factors in determining a specific p53-induced response (7,12,53). Posttranslational modifications of the p53 gene have also been reported to influence the response observed. For example, p53 phosphorylation by different kinases in response to stress can select for arrest or apoptosis, suggesting the involvement of modifiers upstream of the p53 gene (29). Moreover, p53 mutants that can induce growth arrest but not apoptosis, or vice versa, have been identified (12,49,60), which is consistent with the concept that certain p53 gene mutations may cause selective loss of the ability to transactivate certain p53-responsive promoters (35).Several p53-target genes have been reported to be specifically involved in apoptosis. These genes include those encoding KILLER/DR5 (56), Bax (39), IGF-BP3 (6), and, more recently, PIG3 (45), PAG608 (24), PERP (1), Noxa (43), PIDD (33), p53AIP1 (44), APAF-1 (46), ferredoxin reductase (FDXR) (23), and PUMA (41, 57). Some of the genes, like the PIG3 and FDXR genes, are involved in reactive oxygen species (ROS)-related pathways (45). Moreover, apoptosis triggered by p53 has been reported to be dependent on an increase of ROS and the release of apoptotic factors resulting from mitochondrial damage (45).An increase in ROS has independently been implicated in c...

2-Deoxyglucose combined with wild-type p53 overexpression enhances cytotoxicity in human prostate cancer cells via oxidative stress

“…Recent reports also documented that the combination of ionizing radiation and adenoviral p53 gene therapy increased radiosensitivity of both p53-mutant and wild type cancer cells [7;8;35]. In addition several reports have suggested that p53 over expression can induce oxidative stress and alterations in respiration in cancer cells [1,2,9,10,20,36] and that 2DG can enhance cell killing in malignant cells by agents that induce metabolic oxidative stress [3,4,37]. Finally, several investigators have reported that androgenindependent prostate cancer cells have a high frequency of p53 mutations [38;39].…”

“…The biochemical rationale for this combination to enhance cancer cell killing was based on previous results in other human cancer cells suggesting that 2DG would inhibit glucose metabolism leading to a reduction in intracellular pyruvate and NADPH [3,4,37], limiting the capacity of the tumor cells to metabolize hydroperoxides [4,15,25], and p53 would increase metabolic hydroperoxide production [1,2,20,36] leading to enhanced oxidative stress. As shown in our results the combination of 2DG and Adp53 lead to over expression of p53 (Figure 1), increased clonogenic cell killing (Figure 2 and Figure 3), and increased oxidative stress that appeared to be mediated by increased steady-state levels of hydroperoxides including H 2 O 2 (Fig 4-Fig 6) in prostate cancer cells.…”

“…Replication-deficient adenovirus gene therapy vectors, which induce over expression of human recombinant wild-type p53 in human cancer cells, have demonstrated cytotoxic effects that are suggested to involve hydroperoxide-mediated oxidative stress both in vitro and in vivo as well as having been associated with increased mitochondrial respiration [1,2,7,8,9,10]. Moreover, strategies combining adenovirus-mediated wild-type p53 gene transfer with DNA-damaging treatments, such as irradiation and chemotherapeutic agents, have also been explored in glioblastoma, lung, colorectal, ovarian, and head and neck cancers.…”

“…Recently p53-induced apoptosis in cancer cells has been shown to occur through induction of p53-induced genes (PIGs) and the tumor suppressive effects of some PIGs are thought to be the result of increased formation of reactive oxygen species and mitochondrial respiration (ROS) (i.e. O 2 •-and H 2 O 2 ), which in turn lead to oxidative stress [1,2,9,10,20]. Since 2DG would be expected to inhibit the formation of pyruvate and NADPH, which function in the detoxification pathways of H 2 O 2 [3,4,15], we hypothesized that the combination of 2DG and wt p53 over expression would lead to increased steady-state levels of ROS and enhanced cell killing by oxidative stress.…”

“…

AbstractOver expression of the tumor suppressor gene, wild type p53 (wtp53), using adenoviral vectors (Adp53) has been suggested to kill cancer cells by hydroperoxide-mediated oxidative stress [1,2] and nutrient distress induced by the glucose analog, 2-deoxyglucose (2DG), has been suggested to enhance tumor cell killing by agents that induce oxidative stress via disrupting hydroperoxide metabolism [3,4]. In the current study clonogenic cell killing of PC-3 and DU-145 human prostate cancer cells (lacking functional p53) mediated by 4 h exposure to 50 plaque forming units (pfus)/ cell of Adp53 (that caused the enforced over expression of wtp53) was significantly enhanced by treatment with 2DG.

…”