Hyperphosphorylated tau aggregation and cytotoxicity modulators screen identified prescription drugs linked to Alzheimer's disease and cognitive functions.

The neurodegenerative Alzheimer's disease (AD) affects more than 30 million people worldwide. There is thus far no cure or prevention for AD. Aggregation of hyperphosphorylated tau in the brain correlates with the cognitive decline of patients of AD and other neurodegenerative tauopathies. Intracerebral injection of tau aggregates isolated from tauopathy brains causes similar pathology in the recipient mice, demonstrating the pathogenic role of abnormally phosphorylated tau. Compounds controlling the aggregation of hyperphosphorylated tau therefore are probable modulators for the disease. Here we report the use of recombinant hyperphosphorylated tau (p-tau) to identify potential tauopathy therapeutics and risk factors. Hyperphosphorylation renders tau prone to aggregate and to impair cell viability. Taking advantage of these two characters of p-tau, we performed a screen of a 1280-compound library, and tested a selective group of prescription drugs in p-tau aggregation and cytotoxicity assays. R-(-)-apomorphine and raloxifene were found to be p-tau aggregation inhibitors that protected p-tau-treated cells. In contrast, a subset of benzodiazepines exacerbated p-tau cytotoxicity apparently via enhancing p-tau aggregation. R-(-)apomorphine and raloxifene have been shown to improve cognition in animals or in humans, whereas benzodiazepines were linked to increased risks of dementia. Our results demonstrate the feasibility and potential of using hyperphosphorylated tau-based assays for AD drug discovery and risk factor identification.

Hyperphosphorylation Renders Tau Prone to Aggregate and to Cause Cell Death.

Alzheimer's disease (AD) is a neurodegenerative disorder without a cure or prevention to date. Hyperphosphorylated tau forms the neurofibrillary tangles (NFTs) that correlate well with the progression of cognitive impairments. Animal studies demonstrated the pathogenic role of hyperphosphorylated tau. Understanding how abnormal phosphorylation renders a normal tau prone to form toxic fibrils is key to delineating molecular pathology and to developing efficacious drugs for AD. Production of a tau bearing the disease-relevant hyperphosphorylation and molecular characters is a pivotal step. Here, we report the preparation and characterization of a recombinant hyperphosphorylated tau (p-tau) with strong relevance to disease. P-tau generated by the PIMAX approach resulted in phosphorylation at multiple epitopes linked to the progression of AD neuropathology. In stark contrast to unmodified tau that required an aggregation inducer, and which had minimal effects on cell functions, p-tau formed inducer-free fibrils that triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. P-tau-induced apoptosis was suppressed by inhibitors for reactive oxygen species. Hyperphosphorylation apparently caused rapid formation of a disease-related conformation. In both aggregation and cytotoxicity, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with an increased propensity for fibrillization. These characters of p-tau are consistent with the emerging view that hyperphosphorylation causes tau to become an aggregation-prone and cytotoxic species that underlies diffusible pathology in AD and other tauopathies. Our results further suggest that p-tau affords a feasible tool for Alzheimer's disease mechanistic and drug discovery studies.

Cytosolic Ku70 regulates Bax-mediated cell death.

The first known function of Ku70 is as a DNA repair factor in the nucleus. Using neuronal neuroblastoma cells as a model, we have established that cytosolic Ku70 binds to the pro-apoptotic protein Bax in the cytosol and blocks Bax's cell death activity. Ku70-Bax binding is regulated by Ku70 acetylation in that when Ku70 is acetylated Bax dissociates from Ku70, triggering cell death. We propose that Ku70 may act as a survival factor in these cells such that Ku70 depletion triggers Bax-dependent cell death. Here, we addressed two fundamental questions about this model: (1) Does all Bax, which is a cytosolic protein, bind to all cytosolic Ku70? and (2) Is Ku70 a survival factor in cells types other than neuronal neuroblastoma cells? We show here that, in neuronal neuroblastoma cells, only a small fraction of Ku70 binds to a small fraction of Bax; most Bax is monomeric. Interestingly, there is no free or monomeric Ku70 in the cytosol; most cytosolic Ku70 is in complex with other factors forming several high molecular weight complexes. A fraction of cytosolic Ku70 also binds to cytosolic Ku80, Ku70's binding partner in the nucleus. Ku70 may not be a survival factor in some cell types (Ku70-depletion less sensitive) because Ku70 depletion does not affect survival of these cells. These results indicate that, in addition to Ku70 acetylation, other factors may be involved in regulating Ku70-Bax binding in the Ku70-depletion less sensitive cells because Ku70 acetylation in these cells is not sufficient to dissociate Bax from Ku70 or to activate Bax.

Regulation of ku70-bax complex in cells.

Ku70, a DNA repair factor in the nucleus, also regulates cell death by binding to the apoptotic protein Bax in the cytoplasm. Acetylation of Ku70 triggers Bax release resulting in Bax dependent cell death. Thus dissociating Bax from Ku70, either by inhibiting histone deacetylase 6 (HDAC6) that deacetylates Ku70 or by increasing Ku70 acetylation induces cell death. Our results showed that in neuroblastoma cells, the depletion of Ku70 results in Bax-dependent cell death. This model provides a rationale for screening Ku70 acetylation modulators that can be tested in clinical trials, either alone or in combination with radiotherapy or DNA-damaging agents for the treatment of cancer.

CREB-binding protein regulates Ku70 acetylation in response to ionization radiation in neuroblastoma.

Ku70 was originally described as an autoantigen, but it also functions as a DNA repair protein in the nucleus and as an antiapoptotic protein by binding to Bax in the cytoplasm, blocking Bax-mediated cell death. In neuroblastoma (NB) cells, Ku70's binding with Bax is regulated by Ku70 acetylation such that increasing Ku70 acetylation results in Bax release, triggering cell death. Although regulating cytoplasmic Ku70 acetylation is important for cell survival, the role of nuclear Ku70 acetylation in DNA repair is unclear. Here, we showed that Ku70 acetylation in the nucleus is regulated by the CREB-binding protein (CBP), and that Ku70 acetylation plays an important role in DNA repair in NB cells. We treated NB cells with ionization radiation and measured DNA repair activity as well as Ku70 acetylation status. Cytoplasmic and nuclear Ku70 were acetylated after ionization radiation in NB cells. Interestingly, cytoplasmic Ku70 was redistributed to the nucleus following irradiation. Depleting CBP in NB cells results in reducing Ku70 acetylation and enhancing DNA repair activity in NB cells, suggesting nuclear Ku70 acetylation may have an inhibitory role in DNA repair. These results provide support for the hypothesis that enhancing Ku70 acetylation, through deacetylase inhibition, may potentiate the effect of ionization radiation in NB cells.

HDAC6 deacetylates Ku70 and regulates Ku70-Bax binding in neuroblastoma.

Ku70 was first characterized as a nuclear factor that binds DNA double-strand breaks in nonhomolog end-joining DNA repair. However, recent studies have shown that Ku70 is also found in the cytoplasm and binds Bax, preventing Bax-induced cell death. We have shown that, in neuroblastoma cells, the binding between Ku70 and Bax depends on the acetylation status of Ku70, such that, when Ku70 is acetylated, Bax is released from Ku70, triggering cell death. Thus, to survive, in neuroblastoma cells, cytoplasmic Ku70 acetylation status is carefully regulated such that Ku70 is maintained in a deacetylated state, keeping Bax complexed with Ku70. We have shown that overexpression of CREB-binding protein (CBP), a known acetyltransferase that acetylates Ku70, releases Bax from Ku70, triggering apoptosis. Although we have shown that blocking deacetylase activity using non-type-specific inhibitors also triggers Ku70 acetylation and Bax-dependent cell death, the targets of these deacetylase inhibitors in neuroblastoma cells remain unknown. Here, we demonstrate that, in neuroblastoma cells, histone deacetylase 6 (HDAC6) binds Ku70 and Bax in the cytoplasm and that knocking down HDAC6 or using an HDAC6-specific inhibitor triggers Bax-dependent cell death. Our results show that HDAC6 regulates the interaction between Ku70 and Bax in neuroblastoma cells and may be a therapeutic target in this pediatric solid tumor.

Glucose deprivation activates AMPK and induces cell death through modulation of Akt in ovarian cancer cells.

OBJECTIVES: Upregulation of glycolysis has been demonstrated in multiple tumor types. Glucose deprivation results in diminished intracellular ATP; this is counteracted by AMPK activation during energy deficiency to restore ATP levels. We sought to determine whether glucose deprivation could induce cytotoxicity in ovarian cancer cells through activation of AMPK, and whether AMPK activators could mimic glucose deprivation induced cytotoxicity. METHODS: Sensitivity to 2DG induced cytotoxicity and glucose deprivation was determined in a panel of ovarian cancer cells. Cellular growth rate, rate of glucose uptake, and response to glucose deprivation were determined. Expression of Glut-1, HIF1-α, AMPK and Akt was determined by immunoblotting. RESULTS: Incubation of ovarian cancer cells with glucose-free media, 2-DG and AMPK activators resulted in cell death. The glycolytic phenotype of ovarian cancer cells was present in both normoxic and hypoxic conditions, and did not correlate with HIF1-α expression levels. Sensitivity to glucose deprivation was independent of growth rate, rate of glucose uptake, and appeared to be dependent upon constitutive activation of Akt. Glucose deprivation resulted in activation of AMPK and inhibition of Akt phosphorylation. Treatment with AMPK activators resulted in AMPK activation, Akt inhibition, and induced cell death in ovarian cancer cells. CONCLUSIONS: Ovarian cancer cells are glycolytic as compared to normal, untransformed cells, and are sensitive to glucose deprivation. Because ovarian cancer cells are dependent upon glucose for growth and survival, treatment with AMPK activators that mimic glucose deprivation may result in broad clinical benefits to ovarian cancer patients.

DEK in the synovium of patients with juvenile idiopathic arthritis: characterization of DEK antibodies and posttranslational modification of the DEK autoantigen.

OBJECTIVE: DEK is a nuclear phosphoprotein and autoantigen in a subset of children with juvenile idiopathic arthritis (JIA). Autoantibodies to DEK are also found in a broad spectrum of disorders associated with abnormal immune activation. We previously demonstrated that DEK is secreted by macrophages, is released by apoptotic T cells, and attracts leukocytes. Since DEK has been identified in the synovial fluid (SF) of patients with JIA, this study was undertaken to investigate how DEK protein and/or autoantibodies may contribute to the pathogenesis of JIA. METHODS: DEK autoantibodies, immune complexes (ICs), and synovial macrophages were purified from the SF of patients with JIA. DEK autoantibodies and ICs were purified by affinity-column chromatography and analyzed by 2-dimensional gel electrophoresis, immunoblotting, and enzyme-linked immunosorbent assay. DEK in supernatants and exosomes was purified by serial centrifugation and immunoprecipitation with magnetic beads, and posttranslational modifications of DEK were identified by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS). RESULTS: DEK autoantibodies and protein were found in the SF of patients with JIA. Secretion of DEK by synovial macrophages was observed both in a free form and via exosomes. DEK autoantibodies (IgG2) may activate the complement cascade, primarily recognize the C-terminal portion of DEK protein, and exhibit higher affinity for acetylated DEK. Consistent with these observations, DEK underwent acetylation on an unprecedented number of lysine residues, as demonstrated by nano-LC-MS/MS. CONCLUSION: These results indicate that DEK can contribute directly to joint inflammation in JIA by generating ICs through high-affinity interaction between DEK and DEK autoantibodies, a process enhanced by acetylation of DEK in the inflamed joint.

Trichostatin A restores Apaf-1 function in chemoresistant ovarian cancer cells.

BACKGROUND: Chemoresistance is the major factor limiting long-term treatment success in patients with epithelial ovarian cancers. Most cytotoxic drugs kill cells through apoptosis; therefore, defective execution of apoptotic pathways results in a drug-resistant phenotype in many tumor types. METHODS: A panel of ovarian cancer cell lines was screened for expression and function of the apoptosome components Apaf-1 and caspase-9. Expression levels were analyzed by immunohistochemistry and immunoblotting; Apaf-1 function was determined by assessing the ability of endogenous Apaf-1 to cleave caspase-9 in the presence or absence of cytochrome c. The effect of the histone deacetylase inhibitor trichostatin A on Apaf-1 expression and function was evaluated. RESULTS: The authors report here that the resistance of ovarian cancer cells to the proapoptotic effects of chemotherapy is due in part to deficient Apaf-1 activity. Although Apaf-1 is expressed in most ovarian cancers, the functional activity is impaired, as Apaf-1 has a diminished ability to recruit and activate caspase-9. Treatment of ovarian cancer cells with trichostatin A results in restoration of Apaf-1 function independent of alterations in Apaf-1 expression. Furthermore, treating chemoresistant cells with sublethal doses of trichostatin A restores Apaf-1 function and sensitizes cells to cisplatin-induced apoptosis. CONCLUSIONS: Targeting intrinsic pathway defects for therapeutic intervention may result in sensitizing tumors to standard chemotherapy or triggering apoptosis in the absence of other apoptotic signals. The identification of drugs that can use Apaf-1 when it is present, yet can overcome its functional inactivation, may be an important clinical advance.

CLU blocks HDACI-mediated killing of neuroblastoma.

Clusterin is a ubiquitously expressed glycoprotein with multiple binding partners including IL-6, Ku70, and Bax. Clusterin blocks apoptosis by binding to activated Bax and sequestering it in the cytoplasm, thereby preventing Bax from entering mitochondria, releasing cytochrome c, and triggering apoptosis. Because increased clusterin expression correlates with aggressive behavior in tumors, clusterin inhibition might be beneficial in cancer treatment. Our recent findings indicated that, in neuroblastoma cells, cytoplasmic Bax also binds to Ku70; when Ku70 is acetylated, Bax is released and can initiate cell death. Therefore, increasing Ku70 acetylation, such as by using histone deacetylase inhibitors, may be therapeutically useful in promoting cell death in neuroblastoma tumors. Since clusterin, Bax, and Ku70 form a complex, it seemed likely that clusterin would mediate its anti-apoptotic effects by inhibiting Ku70 acetylation and blocking Bax release. Our results, however, demonstrate that while clusterin level does indeed determine the sensitivity of neuroblastoma cells to histone deacetylase inhibitor-induced cell death, it does so without affecting histone deacetylase-inhibitor-induced Ku70 acetylation. Our results suggest that in neuroblastoma, clusterin exerts its anti-apoptotic effects downstream of Ku70 acetylation, likely by directly blocking Bax activation.

Ku70 acetylation in neuroblastoma pathogenesis and therapy.

Neuroblastoma is a cancer that occurs in children. It develops from stem cells that normally give rise to parts of the peripheral nervous system and adrenal glands. Although most children with localized neuroblastoma are cured, children with wide-spread disease have a small chance of survival even after surgery, chemotherapy, radiation and bone marrow transplantation. Ten to fifteen percent of patients die from treatment complications, and long-term survival is less than 30%. Although contemporary molecular tumor marker discoveries have improved prognostication, few have led to new therapeutic approaches. To solve this problem, we are working to understand which molecules in the stem cells from which this cancer arises malfunction to cause neuroblastoma and apply this information to develop new models to treat this disease. Our efforts have focused on the functional regulation of a protein called Ku70, which coordinately regulates DNA repair and cell death. We propose that the incorrect balance between these two activities underlies this cancer's development, and that re-balancing with drug therapy offers a way to treat this disease.

Bcl6 is expressed in neuroblastoma: tumor cell type-specific expression predicts outcome.

Neuroblastoma (NB) is the most common extracranial solid neoplasm of infancy and childhood. Whereas most low-risk patients do well, children with high-risk tumors often fail intensive treatment. Identification of novel biomarkers is critical to improve prognostication, tailor therapy, and develop new therapeutic targets. Differential RNA-level expression between tumor cells with neuroblastic (N-type) and Schwannian stromal (S-type) phenotypes was used to identify genes of potential interest based on tumor cell type-specific regulation. Gene expression microarray analysis revealed marked differences between N-type and S-type cells in their levels of BCL6 messenger RNA, a transcriptional regulator overexpressed in a variety of hematopoietic malignancies. S-type cells express higher levels of Bcl6 RNA and protein than N-type, and protein levels are significantly limited by proteasome function. An NB tumor tissue microarray linked to clinicopathologic data was immunohistochemically stained to measure Bcl6 protein levels. Bcl6 was detected in both the neuroblastic and Schwannian stromal regions, as distinguished histologically, and correlated with outcome. We found that expression in neuroblastic regions differentiates outcomes, in that Bcl6 expression in neuroblastic regions is associated with increased time to relapse and increased overall survival compared with absent expression in neuroblastic regions, regardless of Schwannian stromal expression. Thus, our findings suggest that Bcl6 may be useful as a prognostic marker and might represent a potential therapeutic target for high-risk NB.

Acetylation and deacetylation regulate CCAAT/enhancer binding protein beta at K39 in mediating gene transcription.

The transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) contains multiple acetylation sites, including lysine (K) 39. Mutation of C/EBPbeta at K39, an acetylation site in the transcriptional activation domain, impairs transcription of C/EBPbeta target genes in a dominant-negative fashion. Further, K39 of C/EBPbeta can be deacetylated by HDAC1, and HDAC1 may decrease C/EBPbeta-mediated transcription, suggesting that acetylation of C/EBPbeta at K39 is dynamically regulated in mediating gene transcription. Acetylation of endogenous C/EBPbeta at K39 is detected in adipose tissue, and also occurs in 3T3-L1 cells undergoing adipocyte conversion. In addition, mutation of K39 in C/EBPbeta impairs activation of its target genes encoding C/EBPalpha and PPARgamma, essential mediators of adipogenesis, as well as adipocyte genes for leptin and Glut4. These findings suggest that acetylation of C/EBPbeta at K39 is an important and dynamic regulatory event that contributes to its ability to transactivate target genes, including those associated with adipogenesis and adipocyte function.

Cooperative regulation of endogenous cAMP-response element binding protein and CCAAT/enhancer-binding protein beta in GH-stimulated c-fos expression.

GH activates the c-fos promoter by regulating multiple transcription factors. This study adds to our understanding of GH-regulated transcription by demonstrating that GH regulates the c-fos cAMP-response element (CRE) and its binding protein, CREB. Activation of the c-fos promoter by GH is impaired by expression of dominant-negative A-CREB. GH stimulates rapid and transient phosphorylation of CREB at Ser 133 (P-CREB), a critical site for transactivation by CREB, in 3T3-F442A preadipocytes. Mutation of this residue impairs GH-induced c-fos expression, suggesting that phosphorylation of CREB at Ser 133 contributes to GH-induced c-fos activation. The MEK inhibitor UO126 impaired the phosphorylation of CREB and that of C/EBPbeta, suggesting that ERKs mediate the phosphorylation of both proteins. UO126, but not the protein kinase A inhibitor H89, blocked GH-induced c-fos mRNA expression. A combination of CREB and C/EBPbeta enhanced c-fos promoter activation, and mutation of the CRE impaired the enhancement, as well as GH-stimulated c-fos activation. GH treatment increased the occupancy of both endogenous phospho-CREB and phospho-C/EBPbeta on the c-fos promoter. The increases were impaired by UO126. The active P-CREB and P-C/EBPbeta are induced by GH to occupy the same c-fos promoter DNA, suggesting that they may participate in a GH-regulated complex on c-fos. These findings suggest that coordinated phosphorylation of CREB and C/EBPbeta in response to GH is mediated by ERK1/2, and that the phosphorylated proteins are part of a regulatory complex that occupies c-fos in vivo to regulate c-fos transcription cooperatively in response to GH.

CREB-binding protein is a mediator of neuroblastoma cell death induced by the histone deacetylase inhibitor trichostatin A.

The cytotoxic mechanism of the histone deacetylase inhibitor (HDACI) Trichostatin A (TSA) was explored in a neuroblastoma (NB) model. TSA induces cell death in neuroblastic-type NB cells by increasing the acetylation of Ku70, a Bax-binding protein. Ku70 acetylation causes Bax release and activation, triggering cell death. This response to TSA depends on the CREB-binding protein (CBP) acetylating Ku70. TSA-induced cell death response correlates with CBP expression. In stromaltype NB cell lines with low levels of CBP and relative resistance to TSA, increasing CBP expression disrupts Bax-Ku70 binding and sensitizes them to TSA. Reducing CBP expression in neuroblastic cell types causes resistance. Cytotoxic response to TSA is Bax-dependent. Interestingly, depleting NB cells of Ku70 also triggers Bax-dependent cell death, suggesting that conditions that leave Bax unbound to Ku70 result in cell death. We also show that CBP, Ku70, and Bax are expressed in human NB tumors and that CBP expression varies across cell types comprising these tumors, with the highest expression observed in neuroblastic elements. Together, these results demonstrate that CBP, Bax, and Ku70 contribute to a therapeutic response to TSA against NB and identify the possibility of using these proteins to predict clinical responsiveness to HDACI treatment.

Multiple mechanisms of growth hormone-regulated gene transcription.

Diverse physiological actions of growth hormone (GH) are mediated by changes in gene transcription. Transcription can be regulated at several levels, including post-translational modification of transcription factors, and formation of multiprotein complexes involving transcription factors, co-regulators and additional nuclear proteins; these serve as targets for regulation by hormones and signaling pathways. Evidence that GH regulates transcription at multiple levels is exemplified by analysis of the proto-oncogene c-fos. Among the GH-regulated transcription factors on c-fos, C/EBPbeta appears to be key, since depletion of C/EBPbeta by RNA interference blocks the stimulation of c-fos by GH. The phosphorylation state of C/EBPbeta and its ability to activate transcription are regulated by GH through MAPK and PI3K/Akt-mediated signaling cascades. The acetylation of C/EBPbeta also contributes to its ability to activate c-fos transcription. These and other post-translational modifications of C/EBPbeta appear to be integrated for regulation of transcription by GH. The formation of nuclear proteins into complexes associated with DNA-bound transcription factors is also regulated by GH. Both C/EBPbeta and the co-activator p300 are recruited to c-fos in response to GH, altering c-fos promoter activation. In addition, GH rapidly induces spatio-temporal re-localization of C/EBPbeta within the nucleus. Thus, GH-regulated gene transcription mediated by C/EBPbeta reflects the integration of diverse mechanisms including post-translational modifications, modulation of protein complexes associated with DNA and re-localization of gene regulatory proteins. Similar integration involving other transcription factors, including Stats, appears to be a feature of regulation by GH of other gene targets.

CCAAT/enhancer-binding protein (C/EBP) beta is acetylated at multiple lysines: acetylation of C/EBPbeta at lysine 39 modulates its ability to activate transcription.

Transcription factor function can be modulated by post-translational modifications. Because the transcription factor CCAAT/enhancer-binding protein (C/EBP) beta associates with the nuclear coactivator p300, which contains acetyltransferase activity, acetylation of C/EBPbeta was examined to understand its regulation and function. C/EBPbeta is acetylated by acetyltransferases p300 and p300/CREB-binding protein associated factor. Endogenous C/EBPbeta in 3T3-F442A preadipocytes is also recognized by an acetyl-lysine-specific antibody. Analysis of truncations of C/EBPbeta and peptides based on C/EBPbeta sequences identified multiple lysines within C/EBPbeta that can be acetylated. Among these, a novel acetylation site at lysine 39 of C/EBPbeta was identified. Mutation of Lys-39 to arginine or alanine impairs its acetylation and the ability of C/EBPbeta to activate transcription at the promoters for C/EBPalpha and c-fos. Different C/EBPbeta-responsive promoters require different patterns of acetylated lysines in C/EBPbeta for transcription activation. Furthermore, C/EBPbeta acetylation was increased by growth hormone, and mutation of Lys-39 impaired growth hormone-stimulated c-fos promoter activation. These data suggest that acetylation of Lys-39 of C/EBPbeta, alone or in combination with acetylation at other lysines, may play a role in C/EBPbeta-mediated transcriptional activation.

Signaling from p53 to NF-kappaB determines the chemotherapy responsiveness of neuroblastoma.

Neuroblastic (N) type neuroblastoma (NB) is the predominant cell type in NB tumors. Previously, we determined that activated nuclear factor kappaB (NF-kappaB) is required for doxorubicin and etoposide to kill N-type NB cells. This study was undertaken to determine how NF-kappaB is activated by these agents. The results show that p53 protein levels increase within 15 to 30 minutes of treatment. This increase occurs before the degradation of inhibitor of NF-kappaB (I-kappaB) alpha and the NF-kappaB-dependent activation of gene transcription. Moreover, p53 is necessary for NF-kappaB activation because cells with inactive p53 were resistant to NF-kappaB-mediated cell death. This pathway was further defined to show that p53 leads to the activation of MAPK/ERK activity kinase (MEK) 1 through a process that depends on protein synthesis and H-Ras. MEK1, in turn, mediates I-kappaB kinase activation. Together, these results demonstrate for the first time how NF-kappaB is activated in NB cells in response to conventional drugs. Furthermore, these findings provide an explanation as to why H-Ras expression correlates with a favorable prognosis in NB and identify intermediary signaling molecules that are targets for discovering treatments for NB that is resistant to conventional agents.

Distribution of co-activators CBP and p300 during mouse oocyte and embryo development.

cAMP response element binding protein (CREB)-binding protein (CBP) and p300 are two structurally related transcriptional co-activators that activate expression of many eukaryotic genes. Current dogma would suggest that these transcriptional co-activators have similar mechanisms of transcription regulation. Studies of CBP or p300 homozygotic mouse mutants indicate that normal embryogenesis requires the existence of both factors. However, whether this is indicative of a dosage effect of these two proteins, or whether these proteins play different roles in mouse embryo development is not clear. Here we demonstrated that both factors are first found in the cytoplasm of oocytes within primordial follicles, and that they enter into the oocyte nucleus at different stages of oocyte growth, suggesting that they may play different roles in gene expression during oocyte growth and development. Consistent with this model, in the pre-implantation mouse embryos, from the two-cell stage to the blastocyst stage, the localizations of CBP and p300 are different, at times opposite, indicating that CBP and p300 also have different functions in early mouse embryogenesis.

Signaling from p53 to NF-kappa B determines the chemotherapy responsiveness of neuroblastoma.

Neuroblastic (N) type neuroblastoma (NB) is the predominant cell type in NB tumors. Previously, we determined that activated nuclear factor kappaB (NF-kappaB) is required for doxorubicin and etoposide to kill N-type NB cells. This study was undertaken to determine how NF-kappaB is activated by these agents. The results show that p53 protein levels increase within 15 to 30 minutes of treatment. This increase occurs before the degradation of inhibitor of NF-kappaB (I-KB) alpha and the NF-kappaB-dependent activation of gene transcription. Moreover, p53 is necessary for NF-kappaB activation because cells with inactive p53 were resistant to NF-kappaB-mediated cell death. This pathway was further defined to show that p53 leads to the activation of MAPK/ERK activity kinase (MEK) 1 through a process that depends on protein synthesis and H-Ras. MEK1, in turn, mediates I-kappaB kinase activation. Together, these results demonstrate for the first time how NF-kappaB is activated in NB cells in response to conventional drugs. Furthermore, these findings provide an explanation as to why H-Ras expression correlates with a favorable prognosis in NB and identify intermediary signaling molecules that are targets for discovering treatments for NB that is resistant to conventional agents.