p73 and MDM2 confer the resistance of epidermoid carcinoma to cisplatin by blocking p53

p73 responds to DNA damage and exerts its pro-apoptotic function. However, p73 might contribute to the development of drug-resistance in certain tumor cells. In this study, we found that p73 and MDM2 correlate with cisplatin-resistant phenotype of human epidermoid carcinoma-derived cells. p73 and MDM2 were kept at low levels in the cisplatin-sensitive KB-3-1 cells, whereas p53 was induced to be phosphorylated at Ser-15 in response to cisplatin. In contrast, p73 and MDM2 were expressed at higher levels, and cisplatin-mediated p53 phosphorylation was undetectable in the cisplatin-resistant KCP-4 cells. Enforced expression of p73 in KB-3-1 cells caused an accumulation of unphosphorylated form of p53 and MDM2, and conferred the cisplatin resistance. Collectively, our results suggest that a loss of the cisplatin sensitivity is at least in part due to a lack of cisplatin-induced p53 phosphorylation, and p73 might cooperate with MDM2 to be involved in this process.     

Nitric oxide activates p21ras and leads to the inhibition of endothelial NO synthase by protein nitration

Recent data has indicated that exogenous nitric oxide (NO) has the ability to decrease endogenous NO production by inhibiting the enzyme responsible for its generation, NO synthase (NOS). Our previous studies have indicated that increased generation of reactive oxygen species (ROS) play an important role in the inhibitory event. However, the mechanisms for these effects remain unclear. Previous studies have suggested that NO can activate p21ras. Thus, the objective of this study was to determine whether NO-mediated activation of p21ras is involved in the inhibitory process, and to further elucidate the involvement of ROS. Using primary cultures of ovine pulmonary arterial endothelial cells we demonstrated that the NO donor SpermineNONOate, increased p21ras activity by 2.3-fold compared to untreated cells, and that the farnesyl-transferase inhibitor, alpha-hydroxyfarnesylphosphonic acid, reduced p21ras activity and significantly reduced inhibition of eNOS. The overexpression of p21ras increased, while the overexpression of an NO unresponsive mutant of p21ras (p21ras C118S) reduced, the inhibition of eNOS by NO. Further, we identified an increase in the level of superoxide and peroxynitrite in endothelial cells exposed to NO that was reduced by p21ras C118S transient transfection. Conversely, levels of superoxide and peroxynitrite could be increased by the over expression of wild type p21ras. Similarly, eNOS nitration induced by NO exposure was reduced by p21ras C118S transient transfection, and increased by the overexpression of wild-type p21ras. Finally, results also demonstrated that eNOS itself was a significant producer of superoxide, and that this appeared to be related to a p21ras-dependent increase in phosphorylation of Ser1177. Our results implicate a signaling pathway involving p21ras activation, superoxide generation, and peroxynitrite formation as being important in the NO-mediated inhibition of eNOS.     

Ribosomal protein L23 activates p53 by inhibiting MDM2 function in response to ribosomal perturbation but not to translation inhibition

The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G(1) arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.     

MDM2--master regulator of the p53 tumor suppressor protein

MDM2 is an oncogene that mainly functions to modulate p53 tumor suppressor activity. In normal cells the MDM2 protein binds to the p53 protein and maintains p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteosome. Immediately after the application of cellular stress, the ability of MDM2 to bind to p53 is blocked or altered in a fashion that prevents MDM2-mediated degradation. As a result, p53 levels rise, causing cell cycle arrest or apoptosis. In this review, we present evidence for the existence of three highly conserved regions (CRs) shared by MDM2 proteins and MDMX proteins of different species. These highly conserved regions encompass residues 42-94 (CR1), 301-329 (CR2), and 444-483 (CR3) on human MDM2. These three domains are respectively important for binding p53, for binding the retinoblastoma protein, and for transferring ubiquitin to p53. This review discusses the major milestones uncovered in MDM2 research during the past 12 years and potential uses of this knowledge in the fight against cancer.     

Scrape-loaded p21ras down-regulates agonist-stimulated inositol phosphate production by a mechanism involving protein kinase C.

The effect of scrape-loaded [Val-12]p21ras on agonist-stimulated phosphatidylinositol 4,5-bisphosphate (PIP2) turnover in Swiss-3T3 cells was studied. Previously [Morris, Price, Lloyd, Marshall & Hall (1989) Oncogene 4, 27-31] we demonstrated that [Val-12]p21ras activates protein kinase C within 10 min of scrape loading. Here, we show that [Val-12]p21ras inhibits bombesin and platelet-derived growth factor-stimulated PIP2 breakdown 1.5-4 h after scrape loading. This effect persisted for at least 18 h and could be mimicked in control cells by activation of protein kinase C with 12-O-tetradecanoyl 13-acetate (TPA) 15 min prior to ligand stimulation. When protein kinase C was down-regulated by chronic TPA treatment, [Val-12]p21ras was no longer able to inhibit agonist-stimulated inositol phosphate production. These results indicate that changes in inositol phosphate levels caused by ras protein are probably due to activation of protein kinase C and not to an interaction of ras with phospholipase C.     

The Regulation of the p53-mediated Stress Response by MDM2 and MDM4

Exquisite control of the activity of p53 is necessary for mammalian survival. Too much p53 is lethal, whereas too little permits tumorigenesis. MDM2 and MDM4 are structurally related proteins critical for the control of p53 activity during development, homeostasis, and the response to stress. These two essential proteins regulate both the activation of p53 in response to stress and the recovery of cells following resolution of the damage, yet both are oncogenic when overexpressed. Thus, multiple regulatory circuits ensure that their activities are fine-tuned to promote tumor-free survival. Numerous diverse stressors activate p53, and much research has gone into trying to find commonalities between them that would explain the mechanism by which p53 becomes active. It is now clear that although these diverse stressors activate p53 by different biochemical pathways, one common feature is the effort they direct, through a variety of means, toward disrupting the functions of both MDM2 and MDM4. This article provides an overview of the relationship between MDM2 and MDM4, features the various biochemical mechanisms by which p53 is activated through inhibition of their functions, and proposes some emerging areas for investigation of the p53-mediated stress response.Regulation of the p53-mediated stress response by the essential inhibitory proteins MDM2 and MDM4 is critical for survival. In response to stressors such as ionizing radiation, p53 induces a number of potentially lethal but tumor-suppressive processes, including cell cycle arrest, senescence, and apoptosis (reviewed by Horn and Vousden 2007). Both MDM2 and MDM4 are critical to surviving the p53-mediated stress response to whole body ionizing irradiation as mice with reduced levels of either protein undergo p53-dependent death after exposure to doses of radiation that are sublethal to wild-type mice (Mendrysa et al. 2003; Terzian et al. 2007). MDM2 and MDM4 are also required to control p53 function during development, as shown by the early embryonic death of mice lacking either MDM2 or MDM4, unless they also lack p53 (Jones et al. 1995; Montes de Oca Luna et al. 1995; Parant et al. 2001; Migliorini et al. 2002).Although both MDM2 and MDM4 are essential for development, they are detrimental to long-term survival when in excess, because both are oncogenic. Both MDM2 and MDM4 confer the tumorigenic phenotype on cultured cells when experimentally overexpressed (Fakharzadeh et al. 1991; Danovi et al. 2004). In addition, targeted expression of MDM2 in the mammary gland results in tumorigenesis (Lundgren et al. 1997). In people, single nucleotide polymorphisms that reduce expression of either of the orthologs of MDM2 or MDM4 (also referred to as Hdm2 and Hdm4) correlate with increased risk for breast cancer (Bond et al. 2004; Atwal et al. 2009). Approximately 10% of human tumors have been found to overexpress either MDM2 or MDM4 and many of these express wild-type p53 (reviewed in Toledo and Wahl 2006). Because the majority of human cancers express mutant forms of p53, overexpression of MDM2 and MDM4 in the subset of tumors expressing wild-type p53 supports the notion that excessive MDM2 and MDM4 promote tumorigenesis, at least in part, by blocking p53 function. Thus, limiting the activities of MDM2 and MDM4 is important to prevent cancer.     

Fibroblast growth factor-2 interacts with free ribosomal protein S19.

Exogenous FGF-2 added to cells is internalized and part of it translocates to the nucleus of the cells. To get a better understanding of the FGF-2-induced signaling pathway, we looked for proteins associated with FGF-2 in the cytoplasm of the target cells. We first used the GST-FGF-2 to isolate cytoplasmic proteins complexes containing FGF-2 from S100 extract (supernatant 100,000g). Among the retrieved proteins, we focused our studies on RPS19, a protein of the 40S small ribosomal subunit. We showed that FGF-2 interacts directly with RPS19 in vitro. Second, we coimmunoprecipitated RPS19 and FGF-2 from a S240 extract (240,000g supernatant) prepared from FGF-2-stimulated cells and devoid of 40S ribosomal subunit. The result of these experiments suggest that a pool of free RPS19 exists in cells and that FGF-2 interacts in vivo with free RPS19.     

A novel nucleolar protein interacts with ribosomal protein S19

The gene encoding ribosomal protein S19 (RPS19) is mutated in approximately 25% of patients with Diamond-Blackfan anemia (DBA), which is a rare congenital erythroblastopenia. DBA patients have a variety of clinical characteristics, and the role of the RPS19 gene in the pathogenesis of the disease is presently unknown. To investigate a possible role for RPS19 in erythropoiesis, we looked for proteins associated with mouse RPS19 using a yeast two-hybrid system and identified a novel protein, which we named S19 binding protein (S19BP). The deduced amino acid sequence of S19BP derived from cDNA defines a calculated mass of 15,849 and an isoelectric point of 11.3. No known functional motifs were found in S19BP except a short polylysine tract embedded in a putative nucleolar localization signal. Immunolocalization experiments revealed that S19BP was highly concentrated in nucleoli after 6 h of transfection in Cos-7 cells. S19BP was expressed ubiquitously at a basal level but a significantly high level of expression was observed in some tissues.     

The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21

The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated. NF1 GRD also down-regulated RAS function in S. cerevisiae. The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21. However, its specific activity was about 30 times lower. These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations.