5-fluorouracil activation of p53 involves an MDM2-ribosomal protein interaction

5-fluorouracil (5-FU) is a widely used chemotherapeutic drug for the treatment of a variety of solid tumors. The anti-tumor activity of 5-FU has been attributed in part to its ability to induce p53-dependent cell growth arrest and apoptosis. However, the molecular mechanisms underlying p53 activation by 5-FU remain largely obscure. Here we report that 5-FU treatment leads to p53 stabilization and activation by blocking MDM2 feedback inhibition through ribosomal proteins. 5-FU treatment increased the fraction of ribosome-free L5, L11, and L23 ribosomal proteins and their interaction with MDM2, leading to p53 activation and G1/S arrest. Conversely, individual knockdown of these ribosomal proteins by small interfering RNA prevented the 5-FU-induced p53 activation and reversed the 5-FU-induced G1/S arrest. These results demonstrate that 5-FU treatment triggers a ribosomal stress response so that ribosomal proteins L5, L11, and L23 are released from ribosome to activate p53 by ablating the MDM2-p53 feedback circuit.     

Aberrant expression of nucleostemin activates p53 and induces cell cycle arrest via inhibition of MDM2

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G(1) cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G(1) arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.     

Perturbation of 60 S Ribosomal Biogenesis Results in Ribosomal Protein L5- and L11-dependent p53 Activation

Ribosomal proteins play an important role in p53 activation in response to nucleolar stress. Multiple ribosomal proteins, including L5, L11, L23, and S7, have been shown to bind to and inhibit MDM2, leading to p53 activation. However, it is not clear whether ribosomal protein regulation of MDM2 is specific to some, but not all ribosomal proteins. Here we show that L29 and L30, two ribosomal proteins from the 60 S ribosomal subunit, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60 S ribosomal biogenesis by knocking down either L29 or L30 drastically induced the level and activity of p53, leading to p53-depedent cell cycle arrest. This p53 activation was drastically inhibited by knockdown of L11 or L5. Consistently, knockdown of L29 or L30 enhanced the interaction of MDM2 with L11 and L5 and markedly inhibited MDM2-mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis activates p53 via L11- and L5-mediated MDM2 suppression. Mechanistically, knockdown of L30 or L29 significantly increased the NEDDylation and nuclear retention of L11. Knocking down endogenous NEDD8 suppressed p53 activation induced by knockdown of L30. These results demonstrate that NEDDylation of L11 plays a critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis.     

Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress

Ribosomal proteins play a critical role in tightly coordinating p53 signaling with ribosomal biogenesis. Several ribosomal proteins have been shown to induce and activate p53 via inhibition of MDM2. Here, we report that S27a, a small subunit ribosomal protein synthesized as an 80-amino acid ubiquitin C-terminal extension protein (CEP80), functions as a novel regulator of the MDM2-p53 loop. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of S27a significantly attenuates the p53 activation in cells in response to treatment with ribosomal stress-inducing agent actinomycin D or 5-fluorouracil. Interestingly, MDM2 in turn ubiquitinates S27a and promotes proteasomal degradation of S27a in response to actinomycin D treatment, thus forming a mutual-regulatory loop. Altogether, our results reveal that S27a plays a non-redundant role in mediating p53 activation in response to ribosomal stress via interplaying with MDM2.     

Nucleostemin: Another nucleolar “Twister” of the p53-MDM2 loop

Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and downregulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development.Key words: ribosomal biogenesis, nucleolar stress, nucleostemin, p53, MDM2, cell cycle, cell growth     

Perturbation of RNA Polymerase I transcription machinery by ablation of HEATR1 triggers the RPL5/RPL11-MDM2-p53 ribosome biogenesis stress checkpoint pathway in human cells

Ribosome biogenesis is an energy consuming process which takes place mainly in the nucleolus. By producing ribosomes to fuel protein synthesis, it is tightly connected with cell growth and cell cycle control. Perturbation of ribosome biogenesis leads to the activation of p53 tumor suppressor protein promoting processes like cell cycle arrest, apoptosis or senescence. This ribosome biogenesis stress pathway activates p53 through sequestration of MDM2 by a subset of ribosomal proteins (RPs), thereby stabilizing p53. Here, we identify human HEATR1, as a nucleolar protein which positively regulates ribosomal RNA (rRNA) synthesis. Downregulation of HEATR1 resulted in cell cycle arrest in a manner dependent on p53. Moreover, depletion of HEATR1 also caused disruption of nucleolar structure and activated the ribosomal biogenesis stress pathway – RPL5 / RPL11 dependent stabilization and activation of p53. These findings reveal an important role for HEATR1 in ribosome biogenesis and further support the concept that perturbation of ribosome biosynthesis results in p53-dependent cell cycle checkpoint activation, with implications for human pathologies including cancer.     

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 with a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 can constitute a mechanistic link between DNA damage and the ribosomal stress pathway, and a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.     

Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway

The gene encoding p53 mediates a major tumor suppression pathway that is frequently altered in human cancers. p53 function is kept at a low level during normal cell growth and is activated in response to various cellular stresses. The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by either direct repression of p53 transactivation activity in the nucleus or promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults, the two best-characterized p53-dependent checkpoint pathways, both activate p53 through inhibition of MDM2. Here we report that the human homologue of MDM2, HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is distinct from the ARF binding site. We show that the functional consequence of L11-HDM2 association, like that with ARF, results in the prevention of HDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation, accumulating p21 protein levels, and inducing a p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2. Interference with ribosomal biogenesis by a low concentration of actinomycin D is associated with an increased L11-HDM2 interaction and subsequent p53 stabilization. We suggest that L11 functions as a negative regulator of HDM2 and that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal integrity.     

Nucleostemin

Several nucleolar proteins, such as ARF, ribosomal protein (RP) L5, L11, L23 and S7, have been shown to induce p53 activation by inhibiting MDM2 E3 ligase activity and consequently to trigger cell cycle arrest and/or apoptosis. Our recent study revealed another nucleolar protein called nucleostemin (NS), a nucleolar GTP binding protein, as a novel regulator of the p53-MDM2 feedback loop. However, unlike other known nucleolar regulators of this loop, NS surprisingly plays a dual role, as both up and down regulations of its levels could turn on p53 activity. Here, we try to offer some prospective views for this unusual phenomenon by reconciling previously and recently published studies in the field in hoping to better depict the role of NS in linking the p53 pathway with ribosomal biogenesis during cell growth and proliferation as well as to propose NS as another potential molecular target for anti-cancer drug development.     

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.     

Regulation of the MDM2-p53 pathway by ribosomal protein L11 involves a post-ubiquitination mechanism

Inhibition of the MDM2-p53 feedback loop is critical for p53 activation in response to cellular stresses. The ribosomal proteins L5, L11, and L23 can block this loop by inhibiting MDM2-mediated p53 ubiquitination and degradation in response to ribosomal stress. Here, we show that L11, but not L5 and L23, leads to a drastic accumulation of ubiquitinated and native MDM2. This effect is dependent on the ubiquitin ligase activity of MDM2, but not p53, and requires the central MDM2 binding domain (residues 51-108) of L11. We further show that L11 inhibited 26 S proteasome-mediated degradation of ubiquitinated MDM2 in vitro and consistently prolonged the half-life of MDM2 in cells. These results suggest that L11, unlike L5 and L23, differentially regulates the levels of ubiquitinated p53 and MDM2 and inhibits the turnover and activity of MDM2 through a post-ubiquitination mechanism.     

Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (-/-) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity.     

Proteome analysis and morphological studies reveal multiple effects of the immunosuppressive drug mycophenolic acid specifically resulting from guanylic nucleotide depletion

Mycophenolic acid (MPA), one of the most promising immunosuppressive drugs recently developed, is a potent inhibitor of IMP dehydrogenase, the first committed step toward GMP synthesis. We found that all the drug effects on yeast cells were prevented by bypassing GMP synthesis, thus confirming the high specificity of MPA. Although the primary target of MPA is clearly identified, we aimed to further understand how GTP depletion leads to growth arrest and developed a new approach based on proteome analysis combined with overexpression studies. Essential proteins down-expressed in the presence of MPA were identified by protein two-dimensional gel analysis and subsequently overexpressed in yeast. Two such proteins, Cdc37p and Sup45p, when overexpressed allowed partial relief of MPA toxicity, strongly suggesting that their lower amount after MPA treatment significantly contributed to the MPA effect. These conserved proteins involved in cell cycle progression and translation are therefore important secondary targets for MPA. Our data establish that MPA effects occur through inhibition of a unique primary target resulting in guanine nucleotides depletion, thereby affecting multiple cellular processes.     

Potential Role of Mycophenolate Mofetil in the Management of Neuroblastoma Patients

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA) at concentrations (10^? 7–10^? 6 M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up‐regulation of p53, p21 and bax and down‐regulation of bcl‐2 and survivin). MPA‐induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA‐induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 µM) leading to competition on HPRT‐mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 µM (up to 100 µM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol‐mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The apoptotic effects of MPA do not appear to be significantly increased by the UDP‐glucuronosyltransferase inhibitor niflumic acid.     

Potential role of mycophenolate mofetil in the management of neuroblastoma patients

In human neuroblastoma cell lines (LAN5, SHEP and IMR32), mycophenolic acid (MPA) at concentrations (10(-7)-10(-6) M) readily attainable during immunosuppressive therapy with mycophenolate mofetil (Cellcept), induces guanine nucleotide depletion leading to cell cycle arrest and apoptosis through a p53 mediated pathway (up-regulation of p53, p21 and bax and down-regulation of bcl-2 and survivin). MPA-induced apoptosis is also associated to a marked decrease of p27 protein. In the same cell lines MPA, at lower concentrations (50 nM), corresponding to the plasma levels of the active free drug during Cellcept therapy, induces differentiation toward the neuronal phenotype by causing a partial chronic guanine nucleotide depletion. MPA-induced differentiation is not associated to p27 accumulation as occurs using retinoic acid. At a fixed concentration of MPA a higher percentage of apoptotic or differentiated cells is obtained when non dialysed serum substitutes for the dialysed one, due to the higher hypoxanthine concentration in the former (about 10 microM) leading to competition on HPRT-mediated salvage of guanine. At hypoxanthine or oxypurinol concentrations higher than 1 microM (up to 100 microM) no further enhancement of MPA effects was obtained, in agreement with the recently described safety of the allopurinol-mycophenolate mofetil combination in the treatment of hyperuricemia of kidney transplant recipients. The apoptotic effects of MPA do not appear to be significantly increased by the UDP-glucuronosyltransferase inhibitor niflumic acid.