Transcription-independent Pathway Research Articles

The leucine-rich repeat receptor kinase QSK1 regulates PRR-RBOHD complexes targeted by the bacterial effector HopF2Pto.

Plants detect pathogens using cell-surface pattern recognition receptors (PRRs) such as ELONGATION Factor-TU (EF-TU) RECEPTOR (EFR) and FLAGELLIN SENSING 2 (FLS2), which recognize bacterial EF-Tu and flagellin, respectively. These PRRs belong to the leucine-rich repeat receptor kinase (LRR-RK) family and activate the production of reactive oxygen species via the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). The PRR-RBOHD complex is tightly regulated to prevent unwarranted or exaggerated immune responses. However, certain pathogen effectors can subvert these regulatory mechanisms, thereby suppressing plant immunity. To elucidate the intricate dynamics of the PRR-RBOHD complex, we conducted a comparative coimmunoprecipitation analysis using EFR, FLS2, and RBOHD in Arabidopsis thaliana. We identified QIAN SHOU KINASE 1 (QSK1), an LRR-RK, as a PRR-RBOHD complex-associated protein. QSK1 downregulated FLS2 and EFR abundance, functioning as a negative regulator of PRR-triggered immunity (PTI). QSK1 was targeted by the bacterial effector HopF2Pto, a mono-ADP ribosyltransferase, reducing FLS2 and EFR levels through both transcriptional and transcription-independent pathways, thereby inhibiting PTI. Furthermore, HopF2Pto transcriptionally downregulated PROSCOOP genes encoding important stress-regulated phytocytokines and their receptor MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2. Importantly, HopF2Pto requires QSK1 for its accumulation and virulence functions within plants. In summary, our results provide insights into the mechanism by which HopF2Pto employs QSK1 to desensitize plants to pathogen attack.

Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2.

Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a DDI2 protease. Here, we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before transcription of proteasomal genes is upregulated but requires protein translation. Thus, mammalian cells possess an additional DDI2 and transcription-independent pathway for the rapid recovery of proteasome activity after proteasome inhibition.

Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a DDI2 protease. Here, we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before transcription of proteasomal genes is upregulated but requires protein translation. Thus, mammalian cells possess an additional DDI2 and transcription-independent pathway for the rapid recovery of proteasome activity after proteasome inhibition.

The Ciliary Protein Arl13b Functions Outside of the Primary Cilium in Shh-Mediated Axon Guidance

SUMMARYThe small GTPase Arl13b is enriched in primary cilia and regulates Sonic hedgehog (Shh) signaling. During neural development, Shh controls patterning and proliferation through a canonical, transcription-dependent pathway that requires the primary cilium. Additionally, Shh controls axon guidance through a non-canonical, transcription-independent pathway whose connection to the primary cilium is unknown. Here we show that inactivation of Arl13b results in defective commissural axon guidance in vivo. In vitro, we demonstrate that Arl13b functions autonomously in neurons for their Shh-dependent guidance response. We detect Arl13b protein in axons and growth cones, far from its well-established ciliary enrichment. To test whether Arl13b plays a non-ciliary function, we used an engineered, cilia-localization-deficient Arl13b variant and found that it was sufficient to mediate Shh axon guidance in vitro and in vivo. Together, these results indicate that, in addition to its ciliary role in canonical Shh signaling, Arl13b plays a cilia-independent role in Shh-mediated axon guidance.

Pifithrin-Alpha Reduces Methamphetamine Neurotoxicity in Cultured Dopaminergic Neurons.

Methamphetamine (Meth) is a widely abused stimulant. High-dose Meth induces degeneration of dopaminergic neurons through p53-mediated apoptosis. A recent study indicated that treatment with the p53 inhibitor, pifithrin-alpha (PFT-α), antagonized Meth-mediated behavioral deficits in mice. The mechanisms underpinning the protective action of PFT-α against Meth have not been identified, and hence, their investigation is the focus of this study. Primary dopaminergic neuronal cultures were prepared from rat embryonic ventral mesencephalic tissue. High-dose Meth challenge reduced tyrosine hydroxylase immunoreactivity and increased terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling. PFT-α significantly antagonized these responses. PFT-α also reduced Meth-activated translocation of p53 to the nucleus, an initial step before transcription. Previous studies have indicated that p53 can also activate cell death through transcription-independent pathways. We found that PFT-α attenuated endoplasmic reticulum (ER) stressor thapsigargin (Tg)-mediated loss of dopaminergic neurons. ER stress was further monitored through the release of Gaussia luciferase (GLuc) from SH-SY5Y cells overexpressing GLuc-based Secreted ER Calcium-Modulated Protein (GLuc-SERCaMP). Meth or Tg significantly increased GLuc release in to the media, with PFT-α significantly reducing GLuc release. Additionally, PFT-α significantly attenuated Meth-induced CHOP expression. In conclusion, our data indicate that PFT-α is neuroprotective against Meth-mediated neurodegeneration via transcription-dependent nuclear and -independent cytosolic ER stress pathways.

Human Serum Albumin and the p53-Derived Peptide Fusion Protein Promotes Cytotoxicity Irrespective of p53 Status in Cancer Cells.

Human serum albumin (HSA) fusion protein is a viable and effective approach to target and inhibit essential intracellular pathways. It has previously been shown that an HSA fusion protein containing a p53-reactivating peptide (rHSA-p53i) retains the binding activity to MDM2 and MDMX, resulting in p53 transcription-dependent apoptosis. Here, we demonstrate that rHSA-p53i is able to bind and neutralize anti-apoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. This interaction displaces pro-apoptotic Bak and subsequently leads to intrinsic apoptosis via mimicking a p53 transcription-independent pathway. Cytotoxicity induced by rHSA-p53i, via p53 transcription dependent and independent apoptotic pathways, is irrespective of the p53 status in MDA-MB-231, HeLa, and SJSA-1 cells possessing either mutant, deficient, or wild-type p53. The therapeutic potential is also confirmed by treating SJSA-1 and MDA-MB-231 xenograft mouse tumors with rHSA-p53i. These data reveal that rHSA-p53i interferes with at least four intracellular targets, making it a viable therapeutic protein for the treatment of a variety of cancers, as well as a carrier to deliver fatty acid-modified chemotherapeutics.

Abstract LB-263: P53 promotes replication genome stability by epigenetics-enabled MRE11 restart

Abstract DNA damage activates p53, the most frequently mutated tumor suppressor, to guard the genome and control gene transcription, epigenetics and apoptosis. Yet, how p53 suppresses genome instability remains ill-defined and is clouded by its genome-wide alterations. Here, enabled by our kPOND technology for single-cell nascent DNA/protein interactions and single-molecule DNA spreading, we define p53’s role in promoting genomic stability by enabling replication restart of stalled replication forks. Mechanistically, p53 enables MRE11 nuclease recruitment for stalled replication fork restart via control of epigenetic factor. We find an apoptosis-proficient and tumor promoting p53 mutant allele to be replication restart defective, while an apoptosis defective and poorly tumor-promoting murine p53 allele remains mostly restart proficient, revealing an improved correlation of restart functions with tumorigenesis compared to apoptosis functions. Importantly, both null and p53 gain-of-function (GOF) mutant cells show restart defects resulting in replication dependent genomic instability, uncovering a common function defect aside gene transcription. Both mutant and p53 null cells show increased recruitment of error-prone repair factors and DNA polymerases, providing a mechanism for genomic instability observed in these cells by pathway homeostasis imbalance. Collective results unveil a common transcription-independent pathway defect for both p53 null and GOF mutants laying groundwork for new targeting strategies of p53 defects in cancer. Note: This abstract was not presented at the meeting. Citation Format: Katharina Schlacher, Sunetra Roy, Karl-Heinz Tomaszowski. P53 promotes replication genome stability by epigenetics-enabled MRE11 restart [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2017-LB-263

Sodium orthovanadate (vanadate), a potent mitigator of radiation-induced damage to the hematopoietic system in mice.

Previous in vitro and in vivo studies have shown that sodium orthovanadate (vanadate), an inorganic vanadium compound, could effectively suppress radiation-induced p53-mediated apoptosis via both transcription-dependent and transcription-independent pathways. As a potent radiation protector administered at a dose of 20 mg/kg body weight (20 mg/kg) prior to total body irradiation (TBI) by intra-peritoneal (ip) injection, it completely protected mice from hematopoietic syndrome and partially from gastrointestinal syndrome. In the present study, radiation mitigation effects from vanadate were investigated by ip injection of vanadate after TBI in mice. Results showed that a single administration of vanadate at a dose of 20 mg/kg markedly improved the 30-day survival rate and the peripheral blood hemogram, relieved bone marrow aplasia and decreased occurrence of the bone marrow micronucleated erythrocytes in the surviving animals. The dose reduction factor was 1.2 when a single dose of 20 mg/kg was administered 15 min after TBI in mice using the 30-day survival test as the endpoint. Results also showed that either doubling the vanadate dose (40 mg/kg) in a single administration or continuing the vanadate treatment (after a single administration at 20 mg/kg) from the following day at a dose of 5 mg/kg per day for 4 consecutive days further significantly improved the efficacy for rescuing bone marrow failure in the 30-day survival test. Taken together, these findings indicate that vanadate would be a potent mitigator suppressing the acute lethality (hematopoietic syndrome) and minimizing the detrimental effects (anhematopoiesis and delayed genotoxic effects) induced by TBI in mice.

Cytosolic flagellin receptor NLRC4 protects mice against mucosal and systemic challenges.

Bacterial flagellin is a dominant innate immune activator of the intestine. Therefore, we examined the role of the intracellular flagellin receptor, NLRC4, in protecting the gut and/or driving inflammation. In accord with NLRC4 acting via transcription-independent pathways, loss of NLRC4 did not reduce the rapid robust changes in intestinal gene expression induced by flagellin administration. Loss of NLRC4 did not alter basal intestinal homeostasis nor predispose mice to development of colitis upon administration of an anti-IL-10R monoclonal antibody. However, epithelial injury induced by dextran sulfate sodium (DSS) in mice lacking NLRC4 resulted in more severe disease indicating a role for NLRC4 in protecting the gut. Moreover, loss of NLRC4 resulted in increased mortality in response to flagellate, but not aflagellate Salmonella infection. Thus, despite not being involved in rapid intestinal gene remodeling upon detection of flagellin, NLRC4-mediated inflammasome activation results in production of IL-1β and IL-18, two cytokines that protect mice from mucosal and systemic challenges.

Solamargine induces apoptosis associated with p53 transcription-dependent and transcription-independent pathways in human osteosarcoma U2OS cells

Aims Solamargine, a steroidal glycoalkaloid isolated from S. incanum, has been shown to induce apoptosis in several cancer cell lines. In this study, the involvement of p53 in the pro-apoptotic action of solamargine was investigated in human osteosarcoma U2OS cells. Main methods The cytotoxicity of solamargine was evaluated by MTT assay. Solamargine-induced apoptosis was evidenced by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine on the extracellular surface as revealed by DAPI nuclear staining, transmission electron microscopy and flow cytometry, respectively. mRNA expressions of p53 and Bax were investigated using real time-PCR. Western blot was used to examine the changes in the expression levels of p53, Bax, Bcl-2, caspase-3, caspase-9 and cytochrome c. Subcellular localization of p53 was verified by immunofluorescence staining and cell fractionation. Key findings Solamargine substantially reduced cell viability and induced apoptosis in osteosarcoma U2OS cells. In this connection, solamargine increased the mRNA and protein expressions of p53 and Bax (a pro-apoptotic protein downstream to p53). The expression of Bcl-2 (an anti-apoptotic protein) was also reduced. Furthermore, solamargine induced mitochondrial translocation of p53, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. p53-specific transcriptional inhibitor pifithrin-α or mitochondrial translocation inhibitor pifithrin-μ partially reversed solamargine-induced apoptosis. Significance Solamargine activates the mitochondria-mediated apoptotic pathway in U2OS cells via both p53 transcription-dependent and -independent mechanisms. This compound may merit further investigation as a potential therapeutic agent for the treatment of cancer.

Molecular mechanisms of nutlin-induced apoptosis in multiple myeloma

Multiple myeloma (MM) is an incurable plasma cell malignancy in which p53 is rarely mutated. Thus, activation of the p53 pathway by a small molecule inhibitor of the p53-MDM2 interaction, nutlin, in MM cells retaining wild type p53 is an attractive therapeutic strategy. Recently we reported that nutlin plus velcade (a proteasome inhibitor) displayed a synergistic response in MM. However, the mechanism of the p53-mediated apoptosis in MM has not been fully understood. Our data show that nutlin-induced apoptosis correlated with reduction in cell viability, upregulation of p53, p21, and MDM2 protein levels with a simultaneous increase in pro-apoptotic targets PUMA, Bax and Bak, and down-regulation of anti-apoptotic targets Bcl2 and survivin, and activation of caspase in MM cells harboring wild type p53. Nutlin-induced apoptosis was inhibited when activation of caspase was blocked by the caspase inhibitor. Nutlin caused mitochondrial translocation of p53 where it binds with Bcl2, leading to cytochrome C release. Moreover, blocking the transcriptional arm of p53 by the p53-specific transcriptional inhibitor, pifithrin-α, not only inhibited nutlin-induced upregulation of p53-transcriptional targets but also augmented apoptosis in MM cells, suggesting an association of transcription-independent pathway of apoptosis. However, inhibitor of mitochondrial translocation of p53, PFT-µ, did not prevent nutlin-induced apoptosis, suggesting that the p53 transcription-dependent pathway was also operational in nutlin-induced apoptosis in MM. Our study provides the evidence that nutlin-induced apoptosis in MM cells is mediated by transcription-dependent and -independent pathways and supports further clinical evaluation of nutlin as a novel therapeutic agent in MM.

P53-mediated neuronal cell death in ischemic brain injury

p53 is a key modulator of cellular stress responses. It is activated in the ischemic areas of brain, and contributes to neuronal apoptosis. In various stroke models, p53 deficiency or applications of p53 inhibitors can significantly attenuate brain damage. p53-mediated neuronal apoptosis occurs through various molecular mechanisms. The transcriptional pathway is an important mechanism through which p53 induces neuronal apoptosis by up-regulating the expression of its target gene p21(WAF), Peg3/Pw1 or p53-up-regulated modulator of apoptosis (PUMA). In addition, p53 disrupts NF-kappaB binding to p300 and blocks NF-kappaB-mediated survival signaling. On the other hand, the transcription-independent pathway mechanism is also of great importance. In this pathway, p53 is translocated to mitochondrial and mediates the release of cytochrome c. In both pathways, p53 seems to play a key role in post-ischemic brain damage and has become a therapeutic target against stroke pathology.

Translocation of p53 to Mitochondria Is Regulated by Its Lipid Binding Property to Anionic Phospholipids and It Participates in Cell Death Control

p53, can regulate cell apoptosis in both transcription-dependent and -independent manners. The transcription-independent pathway was demonstrated by the translocation of p53 to mitochondria. Our study showed that p53 mitochondrial translocation was found in mitomycin C (MMC)-treated HepG2. The p53 C-terminal domain is clustered with potential nuclear leading sequences and showed strong electrostatic ion-ion interactions with cardiolipin, phosphatidylglycerol and phosphatidic acid in vitro. Disruption of cardiolipin biosynthesis by phosphatidylglycero-phosphate synthase (PGS) or CDP-diacylglycerol synthase 2 (CDS-2) short hairpin RNA (shRNA) transfection eliminated the MMC-induced translocation of mitochondrial p53. The elimination of mitochondrial p53 translocation also reduced Bcl-xL and Bcl-2 mitochondrial distribution. In HEK 293T models with saturated p53 expression, the mitochondrial partition of p53, Bcl-xL, and Bcl-2 obviously decreased in their PGS shRNA- or CDS-2 shRNA-expressing stable clones. In p53-null H1299 models, both the mitochondrial partitions of Bcl-xL and Bcl-2 were strongly reduced in relation to the HEK 293T models. The Bcl-xL mitochondrial partition was elevated in H1299 models expressing pCEP4-p53wt suggesting the direct carrier role of p53 in transporting Bcl-xL to the mitochondria. We also found that the cytosolic pool of Bcl-xL and Bcl-2 remained unaffected in the low-dose MMC treatment but decreased in the high-dose MMC treatment. The cytosolic pool of Bcl-2 and Bcl-xL directly regulated their amounts in p53-dependent mitochondrial distribution. In the low-dose MMC treatment, the increased mitochondrial p53, Bcl-xL, and Bcl-2 could attenuate apoptosis. However, in the high-dose MMC treatment, only the p53 translocated to the mitochondria and resulted in apoptosis progression. On the basis of this study, we thought mitochondrial p53 might regulate apoptosis in a biphasic manner.

Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) alpha. We compared the effects of vanadate to PFTalpha and PFTmicro, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis.

Molecular Mechanisms Mediating Antimyeloma Activity of An MDM2 Antagonist Nutlin.

Abstract 3841Poster Board III-777 Background and aimsp53 gene mutations or deletions are rare in multiple myeloma (MM). Thus, strategies to induce p53 activation in myeloma cells with wild type (wt) p53 may have therapeutic promise. However, p53 signaling and functional consequences are not clearly understood in MM. In this study we used a small molecule MDM2 antagonist; nutlin, to explore the molecular mechanisms associated with nutlin-mediated activation of the p53 signaling pathway resulting in cell cycle arrest and/or apoptosis. MethodsMM1.S and H929 cell lines harboring wt p53, LP1 and U266 cell lines expressing mutant (mt) p53, and five primary samples from patients with MM were treated with different concentrations of nutlin or DMSO control. Nutlin-induced cells were assessed for cell viability, induction of p53 and its downstream targets, cell cycle analysis, apoptosis assays and gene expression profile at different time periods. ResultsTreatment of MM cells harboring wt p53 with nutlin led to a dose-dependent increase in the expression of p53 and its downstream targets, p21 and MDM2. After 24 hours incubation with 10 μM nutlin, p53 protein levels increased approximately 4 to 6-fold in MM1.S and H929 cell lines and in two primary MM samples. Induction of p53 downstream targets was strictly correlated with induction of p53. In contrast, nutlin did not modulate the expression of these proteins in the cells with mt p53. Proliferation of the cells was also affected by nutlin in MM cells harboring wt p53 but not mt p53. Seventy-two hours after incubation with 10 μM nutlin, the viability of the cells (MM cell lines and primary MM samples) with wt p53 declined to 30% as assessed by MTT assay. Nutlin caused up-regulation of several pro-apoptotic targets, PUMA, Bax, and Bak, and down-regulation of two anti-apoptotic targets, Bcl2 and survivin in MM1.S cells. Furthermore, nutlin effectively arrested cell cycle progression in wt p53 MM cells, depleting the cells in S-phase compartment and increasing the cells in G1 and G2/M phase compartment, indicating G1 and G2 arrest. Annexin-V staining and flow cytometry (FCM) studies showed that there was a dose- and time-dependent increase in annexin-V binding in MM1.S cells but not in LP1 or U266 cells. These results suggest that nutlin-induced apoptosis is p53-dependent. At 72 hours following treatment with 10 μM nutlin, annexin-V binding was increased from 22% (1.0 μM) to 80% in MM1.S cells. Western blot (WB) analysis of nutlin-induced cells revealed activation of both caspase-8 and caspase-9 followed by activation of caspase-3 suggesting the association of both extrinsic and intrinsic pathways of apoptosis. In addition, direct inhibition of endogenous survivin by siRNA further enhanced nutlin-induced apoptosis, as measured by WB analysis for activation of caspase-3 and FCM assay for annexin-V binding. Forty-eight hours after nutlin treatment, 60% of MM1.S cells transfected with survivin siRNA were annexin-V positive, whereas control siRNA-transfected cells were 30% annexin-V positive. Moreover, selective blocking of p53 transcription by a p53 inhibitor, pifithrin-α (25 μM), inhibits nutlin-induced up-regulation of p53-transcriptional target genes p21, PUMA, and Bax in MM1.S cells, suggesting a transcription-dependent apoptosis. Studies by confocal microscopy and WB analysis of the fractionated samples revealed accumulation of p53 in both nuclear and cytoplasmic fractions in nutlin-treated MM1.S cells. Since transcriptional activation of p53-target genes occurs in the nucleus, while cytoplasmic p53 mediates transcription-independent apoptosis, the accumulation of nuclear and cytoplasmic p53 suggests that activated p53 used both transcription-dependent and transcription-independent pathways to induce apoptosis in MM cells. Finally, the consequence of the p53 activation in MM was validated by gene expression profiling of MM1.S cells with or without nutlin stimulation, demonstrating up-regulation of p53 and its downstream targets p21, MDM2, and BAX. ConclusionNongenotoxic activation of the p53 pathway by nutlin sensitized MM cells harboring wt p53 to transcription-dependent and transcription-independent apoptosis. Our studies provide the preclinical framework for the evaluation of nutlin as a novel therapeutic approach in the treatment of MM. Disclosures:No relevant conflicts of interest to declare.

P53's mitochondrial translocation and MOMP action is independent of Puma and Bax and severely disrupts mitochondrial membrane integrity

p53's apoptotic program consists of transcription-dependent and transcription-independent pathways. In the latter, physical interactions between mitochondrial p53 and anti- and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators are central. Using isogenic cell systems with defined deficiencies, we characterize in detail how mitochondrial p53 contributes to mitochondrial permeabilization, to what extent its action depends on other key Bcl2 family members and define its release activity. We show that mitochondrial p53 is highly efficient in inducing the release of soluble and insoluble apoptogenic factors by severely disrupting outer and inner mitochondrial membrane integrity. This action is associated with wild-type p53-induced oligomerization of Bax, Bak and VDAC and the formation of a stress-induced endogenous complex between p53 and cyclophilin D, normally located at the inner membrane. Tumor-derived p53 mutants are deficient in activating the Bax/Bak lipid pore. These actions are independent of Puma and Bax. Importantly, the latter distinguishes the mitochondrial from the cytosolic p53 death pathway.

Contribution of p53-mediated Bax transactivation in theaflavin-induced mammary epithelial carcinoma cell apoptosis

Theaflavins, the bioactive flavonoids of black tea, have been demonstrated to inhibit proliferation and induce apoptosis in a variety of cancer cells. However, the contribution of p53 in mammary epithelial carcinoma cell apoptosis by theaflavins remains unclear. It has been reported that p53 triggers apoptosis by inducing mitochondrial outer membrane permeabilization through transcription-dependent and -independent mechanisms. Using wild-type and mutant p53-expressing as well as p53-null cells we found a strong correlation between p53 status and theaflavin-induced breast cancer cell apoptosis. Apoptogenic effect was more pronounced in functional p53-expressing cells in which theaflavins raised p53 protein levels that harmonized with Bax up-regulation and migration to mitochondria. However, in the same cells, when p53-mediated transactivation was inhibited by pifithrin-alpha, theaflavins not only failed to increase transcription but also to induce apoptosis although p53 up-regulation was not altered. In contrast, Bax over-expression restored back theaflavin-induced apoptosis in pifithrin-alpha-inhibited/dominant-negative p53-expressing cells. Inhibition of Bax by RNA-interference also reduced theaflavin-induced apoptosis. These results not only indicated the requirement of p53-mediated transcriptional activation of Bax but also its role as down-stream effecter in theaflavin-induced apoptosis. Bax up-regulation resulted in mitochondrial transmembrane potential loss and cytochrome c release followed by activation of caspase cascade. In contrast, mitochondrial translocation of p53 and its interaction with Bcl-2 family proteins or activation of caspase-8 could not be traced thereby excluding the involvement of p53-mediated transcription-independent pathways. Together these findings suggest that in breast cancer cells, p53 promotes theaflavin-induced apoptosis in a transcription-dependent manner through mitochondrial death cascade.

Mitogen-Activated Protein Kinase Kinase Inhibition Enhances Nuclear Proapoptotic Function of p53 in Acute Myelogenous Leukemia Cells

Activation of the Raf/MEK/ERK pathway and inactivation of wild-type p53 by Mdm2 overexpression are frequent molecular events in acute myelogenous leukemia (AML). We investigated the interaction of Raf/MEK/ERK and p53 pathways after their simultaneous blockades using a selective small-molecule antagonist of Mdm2, Nutlin-3a, and a pharmacologic MEK-specific inhibitor, PD98059. We found that PD98059, which itself has minimal apoptogenic activity, acts synergistically with Nutlin-3a to induce apoptosis in wild-type p53 AML cell lines OCI-AML-3 and MOLM-13. Interestingly, PD98059 enhanced nuclear proapototic function of p53 in these cells. In accordance with the activation of transcription-dependent apoptosis, PD98059 treatment promoted the translocation of p53 from the cytoplasm to the nucleus in OCI-AML-3 cells, in which p53 primarily initiates transcription-independent apoptosis when cells are treated with Nutlin-3a alone. The critical role of p53 localization in cells with increased p53 levels was supported by enhanced apoptosis induction in cells cotreated with Nutlin-3a and the nuclear export inhibitor leptomycin B. PD98059 prevented p53-mediated induction of p21 at the transcriptional level. The repressed expression of antiapototic p21 also seemed to contribute to synergism between PD98059 and Nutlin-3a because (a) the synergistic apoptogenic effect was preserved in G(1) cells, (b) p53-mediated induction of p21 was preferentially seen in G(1) cells, (c) PD98059 strongly antagonized p21 induction by Nutlin-3a, and (d) cells with high p21 levels were resistant to apoptosis. This is the first report showing that the Raf/MEK/ERK pathway regulates the subcellular localization of p53 and the relative contribution of transcription-dependent and transcription-independent pathways in p53-mediated apoptosis.

Nucleocytoplasmic Shuttling of the Rpb4p and Rpb7p Subunits ofSaccharomyces cerevisiaeRNA Polymerase II by Two Pathways

Rpb4p and Rpb7p are subunits of the RNA polymerase II of Saccharomyces cerevisiae that form a dissociable heterodimeric complex. Whereas the only reported function of Rpb7p is related to transcription, Rpb4p has been found to also act in mRNA export and in the major mRNA decay pathway that operates in the cytoplasm, thus raising the possibility that Rpb4p links between the nuclear and cytoplasmic processes. Here we show that both Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Shuttling kinetics of the two proteins are similar as long as their interaction is possible, suggesting that they shuttle as a heterodimer. Under normal conditions, shuttling of Rpb4p and Rpb7p depends on ongoing transcription. However, during severe stresses of heat shock, ethanol, and starvation, the two proteins shuttle via a transcription-independent pathway. Thus, Rpb4p and Rpb7p shuttle via two pathways, depending on environmental conditions.

A novel histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity

Histone deacetylase (HDAC) inhibitors perturb the cell cycle and have great potential as anti-cancer agents, but their mechanism of action is not well established. HDACs classically function as repressors of gene expression, tethered to sequence-specific transcription factors. Here we report that HDAC3 is a critical, transcription-independent regulator of mitosis. HDAC3 forms a complex with A-Kinase-Anchoring Proteins AKAP95 and HA95, which are targeted to mitotic chromosomes. Deacetylation of H3 in mitosis requires AKAP95/HA95 and HDAC3 and provides a hypoacetylated H3 tail that is the preferred substrate for Aurora B kinase. Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin. This transcription-independent pathway, involving interdependent changes in histone modification and protein association, is required for normal progression through mitosis and is an unexpected target of HDAC inhibitors, a class of drugs currently in clinical trials for treating cancer.