was successfully added to your cart.
Category

DNA Repair

Switching p53 states by calcium: dynamics and interaction of stress systems

By | DNA Repair

Switching p53 states by calcium: dynamics and interaction of stress systems

Md. Jahoor Alam, Gurumayum Reenaroy Devi, Ravins, Romana Ishrat, Subhash M. Agarwal and R. K. Brojen Singh

The integration of calcium and a p53–Mdm2 oscillator model is studied using a deterministic as well as a stochastic approach, to investigate the impact of a calcium wave on single cell dynamics and on the inter-oscillator interaction. The high dose of calcium in the system activates the nitric oxide synthase, synthesizing nitric oxide which then downregulates Mdm2 and influences drastically the p53–Mdm2 network regulation, lifting the system from a normal to a stressed state. The increase in calcium level switches the system to different states, as identified by the different behaviours of the p53 temporal dynamics, i.e. oscillation death to sustain the oscillation state via a mixed state of dampened and oscillation death states. Further increase of the calcium dose in the system switches the system from sustained to oscillation death state again, while an excess of calcium shifts the cell to an apoptotic state. Another important property of the calcium ion is its ability to behave as a synchronizing agent among the interacting systems. The time evolution of the p53 dynamics of the two diffusively coupled systems at stress condition via Ca2+ shows synchronization between the two systems. The noise contained in the system interestingly helps the system to maintain its stabilized state (normal condition). However, noise has the tendency to destruct the synchronization effect, which means that it tries to restrict the system from external signals to maintain its normal condition. However, at the stress condition, the synchronization rate is found to be faster.

Read the full articleRead the full article

Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor

By | DNA Repair

Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor

Maria Rosario Fernandez-Fernandez, Dmitry B. Veprintsev, and Alan R. Fersht

S100B protein is elevated in the brains of patients with early stages of Alzheimer’s disease and Down’s syndrome. S100A4 is correlated with the development of metastasis. Both proteins bind to p53 tumor suppressor. We found that both S100B and S100A4 bind to the tetramerization domain of p53 (residues 325–355) only when exposed in lower oligomerization states and so they disrupt the tetramerization of p53. In addition, S100B binds to the negative regulatory and nuclear localization domains, which results in a very tight binding to p53 protein sequences that exposed the tetramer- ization domain in their C terminus. Because the trafficking of p53 depends on its oligomerization state, we suggest that S100B and S100A4 could regulate the subcellular localization of p53. But, the differences in the way these proteins bind to p53 could result in S100B and S1004 having different effects on p53 function in cell-cycle control.

Read the full articleRead the full article

Possible role of NF-kB and p53 in the glutamate-induced pro-apoptotic neuronal pathway

By | DNA Repair

Possible role of NF-kB and p53 in the glutamate-induced pro-apoptotic neuronal pathway

Mariagrazia Grilli and Maurizio Memo

Apoptosis is now recognized as an important component in many progressive and acute neurodegenerative diseases. Extracellular signals and intracellular mechanisms triggering and regulating apoptosis in neuronal cells are still a matter of investigation. Here we review data from our and other laboratories with the aim to elucidate the nature of some proteins which are known to be involved in cell cycle regulation as well as in promoting degeneration and apoptosis of neurons. The following molecules will be taken into consideration: NF-kB, p53, p21 and MSH2. These proteins are activated by neurotoxic experimental conditions which involve the stimulation of selective receptors for the excitatory aminoacid glutamate. Thus, we hypothesize their contribution to an intracellular pathway responsible for the glutamate- induced neuronal death. Identification of such mechanisms could be relevant for understanding the apoptosis associated with various neurodegenerative diseases as well as for developing novel strategies of pharmacological intervention.

Read the full articleRead the full article

PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells and regulates survival and chemoresistance via NF-kB, MAPkinase and p53 pathways

By | DNA Repair

PI3-kinase/Akt is constitutively active in primary acute myeloid leukaemia cells
and regulates survival and chemoresistance via NF-kB, MAPkinase and p53 pathways

VL Grandage, RE Gale, DC Linch and A Khwaja

The phosphoinositide 3-kinase (PI3-kinase) signalling pathway plays a key role in the regulation of cell survival and proliferation. We show that the PI3-kinase/Akt pathway is constitutively active in primary acute myeloid leukaemia (AML) cells and that blockade by the selective inhibitor LY294002 reduces survival of the total blast population (mean 52%). The ERK/MAPK module is also constitutively active and treatment with the MAPKK inhibitor U0126 reduces cell survival by 22%. In 10 of 18 samples, PI3-kinase contributes to MAPK activation as incubation with LY294002 leads to a marked reduction in its phosphorylation. PI3-kinase inhibition reduces survival of the CD34 þ 38 AML progenitor subset by 44%, whereas MAPKK inhibition has little effect. Reporter assays in primary AML cells show that blocking PI3-kinase leads to a marked reduction of constitutive NF-kB activity and promotes p53-mediated transcription. This is associated with a synergis- tic interaction between LY294002 and Ara-C. An inducible activated form of Akt protects normal myeloid cells from Ara- C and etoposide-mediated apoptosis. These results show that blocking PI3-kinase has direct antileukaemic effects and potentiates the response to conventional cytotoxics via a number of targets including NF-kB, p53 and MAPK. Inhibitors of PI3-kinase and Akt may be useful in the treatment of AML.

Read the full articleRead the full article

p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner

By | DNA Repair

p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner

Carlotta Giorgi, Massimo Bonora, Giovanni Sorrentino, Sonia Missiroli, Federica Poletti, Jan M. Suski, Fabian Galindo Ramirez, Rosario Rizzuto, Francesco Di Virgilio, Ester Zito, Pier Paolo Pandolfi, Mariusz R. Wieckowski, Fabio Mammano, Giannino Del Sal, and Paolo Pinton

The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death pro- grams within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains be- tween the ER and mitochondria (mitochondria-associated mem- branes). We demonstrate that, upon stress stimuli, WT p53 accumu- lates at these sites and modulates Ca2+ homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca2+-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca2+ load, followed by an enhanced transfer to mi- tochondria. The consequent mitochondrial Ca2+ overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca2+ signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca2+ signal-dependent apoptosis.

Read the full articleRead the full article

p53: Good Cop/Bad Cop

By | DNA Repair

p53: Good Cop/Bad Cop

Norman E. Sharpless and Ronald A. DePinho

Activation of the p53 transcription factor in response to a variety of cellular stresses, including DNA damage and oncogene activation, initiates a program of gene expression that blocks the proliferative expansion of damaged cells. While the beneficial impact of the anti- cancer function of p53 is well established, several re- cent papers suggest that p53 activation may in some circumstances act in a manner detrimental to the long- term homeostasis of the organism. Here, we discuss the significant participation of p53 in three non-mutu- ally exclusive theories of human aging involving DNA damage, telomere shortening, and oxidative stress. These “good cop/bad cop” functions of p53 appear to place it at the nexus of two opposing forces, cancer and aging. By extension, this relationship implies that therapies aimed to reduce cancer and postpone aging, and thereby increase longevity, will necessarily work either upstream or downstream, but not on the level of, p53.

Read the full articleRead the full article

Transcriptional Cross Talk between NF- B and p53

By | DNA Repair

Transcriptional Cross Talk between NF- B and p53

Gill A. Webster and Neil D. Perkins

Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF- B. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF- B has been shown to promote resistance to programmed cell death. These observations suggest that a regulatory mechanism must exist to integrate these opposing outcomes and coordinate this critical cellular decision-making event. Here we show that both p53 and NF- B inhibit each other’s ability to stimulate gene expression and that this process is controlled by the relative levels of each transcription factor. Expression of either wild-type p53 or the RelA(p65) NF- B subunit suppresses stimulation of transcription by the other factor from a reporter plasmid in vivo. Moreover, endogenous, tumor necrosis factor alpha-activated NF- B will inhibit endogenous wild-type p53 transactivation. Following exposure to UV light, however, the converse is observed, with p53 downregu- lating NF- B-mediated transcriptional activation. Both p53 and RelA(p65) interact with the transcriptional coactivator proteins p300 and CREB-binding protein (CBP), and we demonstrate that these results are consistent with competition for a limiting pool of p300/CBP complexes in vivo. These observations have many implications for regulation of the transcriptional decision-making mechanisms that govern cellular processes such as apoptosis. Furthermore, they suggest a previously unrealized mechanism through which dysregulated NF- B can contribute to tumorigenesis and disease.

Read the full articleRead the full article

Bendable Electro-chemical Lactate Sensor Printed with Silver Nano- particles

By | DNA Repair

Bendable Electro-chemical Lactate Sensor Printed with Silver Nanoparticles

Md Abu Abrar, Yue Dong, Paul Kyuheon Lee & Woo Soo Kim

Here we report a exible amperometric lactate biosensor using silver nanoparticle based conductive electrode. Mechanically bendable cross-serpentine-shaped silver electrode is generated on exible substrate for the mechanical durability such as bending. The biosensor is designed and fabricated by modifying silver electrode with lactate oxidase immobilized by bovine serum albumin. The in- sensor pseudo Ag/AgCl reference electrode is fabricated by chloridization of silver electrode, which evinced its long-term potential stability against a standard commercial Ag/AgCl reference electrode. The amperometric response of the sensor shows linear dependence with lactate concentration of 1~25 mM/L. Anionic selectivity is achieved by using drop-casted Na on coated on silver electrode against anionic interferences such as ascorbate. This non-invasive electrochemical lactate sensor also demonstrates excellent resiliency against mechanical deformation and temperature uctuation which leads the possibility of using it on human epidermis for continuous measurement of lactate from sweat. Near eld communication based wireless data transmission is demonstrated to re ect a practical approach of the sensor to measure lactate concentration portably using human perspiration.

Read the full articleRead the full article

Concerted Regulation of Wild-Type p53 Nuclear Accumulation and Activation by S100B and Calcium-Dependent Protein Kinase C

By | DNA Repair

Concerted Regulation of Wild-Type p53 Nuclear Accumulation and Activation by S100B and Calcium-Dependent Protein Kinase C

Christian Scotto, Christian Delphin, Jean Christophe Deloulme, and Jacques Baudier

The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G1 check- point control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272–4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G1 phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G1 arrest at the nonpermissive temperature (37.5°C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32°C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G1 phase and activation of a p53-dependent G1 checkpoint control.

Read the full articleRead the full article

Glucose Metabolism, Lactate, and Ammonia Production by the Human Placenta In Vitro

By | DNA Repair

Glucose Metabolism, Lactate, and Ammonia Production by the Human Placenta In Vitro

Ian R. Holzman, Anthony F. Philipps, And Frederick C. Battaglia

Fifteen human placentas were obtained at term. Placental frag- ments were incubated in a bicarbonate-buffered Earle’s solution. Additions of glutamate (1 mM) or glutamine (1 or 2 mM) were made. All incubations showed a net utilization of glucose. There was a striking variability among placentas in the net glucose utilization rate (1.27 pmoles/g/hr-11.44 pmoles/g/hr, coefficient of variation = 62%). The intraplacental coefficient of variation in glucose utilization was only 14%. All placental incubations showed a net production of both lactate (mean = 7.5 pmoles/g placental wet weight/hr) and ammonia (mean = 3.5 prnoles/g placental wet weight/hr). There was no correlation between lactate or ammonia production and glucose utilization. The addition of sodium gluta- mate (1 gmole/ml) produced no change in glucose utilization or ammonia production. The addition of glutamine (1 and 2 pmoles/ ml) produced a significant increase in ammonia production over that found in the controls, but no change in glucose utilization. Incubation with 2 pmoles glutamine/ml demonstrated an increase in lactate production. All incubations showed a striking increase in ammonia concentration after 45 min of incubation.

Individual placentas may differ markedly in their ability to utilize glucose in an in vitro system. Ammonia production may be a normal metabolic endproduct in a tissue lacking an active urea cycle or a byproduct of the purine nucleotide cycle

Read the full articleRead the full article