Checkpoint Control Pathways Research Articles

Use of distinct molecular signatures of appendiceal cancer subtypes to assess biomarker development.

189 Background: Appendiceal Neoplasms are diverse entities which have a variety of clinical presentations, histologic subtypes, biologic behavior, and patient outcomes. Advanced disease at initial diagnosis is not uncommon and therapeutic options are limited. This study aims to identify potential targets to develop epigenetics-based therapeutics/biomarkers. Methods: Archival (FFPE) specimens were collected from 25 patients with histologically confirmed and subtyped appendiceal neoplasia and 16 age-matched non-neoplastic controls. Four appendiceal neoplastic processes identified: Low grade appendiceal neoplasm (LAMN), pseudomyxoma peritonei (intra-peritoneal spread of LAMN), conventional-type adenocarcinoma and goblet cell adenocarcinoma. Gene expression profiling was performed using the HTG EdgeSeq Oncology Biomarker panel. Differentially expressed genes (DEGs) were selected with cut-off threshold FDR<0.05 and analyzed using Qlucore Explorer and functional analyses via Ingenuity Pathway Analysis (IPA). Results: Four appendiceal neoplastic processes identified: pseudomyxoma peritonei (20%), low-grade appendiceal mucinous neoplasm (24%), adenocarcinoma (24%), and goblet cell adenocarcinoma (32%). Unsupervised hierarchical clustering identified 498 DEGs (424 up, 74 down) between all appendiceal cancer versus age-matched controls that showed enrichment in B-cell inflammatory signaling, senescence, cell cycle regulation, PI3K/AKT signaling, cell cycle checkpoint control pathways, and sets relating to NANOG in stem cell pluripotency (all p0.001). G1/S checkpoint, apoptosis, ATM, and PTEN signaling are downregulated (p0.001). TP53, E2F1, IL6, IFNG, TNF, and HGF (p0.001) regulated overlapping genes. Four-subgroups comparison found 67 DEGs involved in T-helper cell differentiation, immune-mediated apoptosis, T-cell fatigue, and PD1/PDL1 signaling to be dysregulated (p0.001). IFNG, FGF2, POU5F1, TNF, and MYC (p0.001) were upstream regulators. Conclusions: In this study, downregulated gene expressions in appendiceal cancers are related to cell proliferation and death control mechanisms, with a trend towards overexpression of inflammatory and cell cycle pathways. It is postulated that genes involved in these pathways are hypermethylated. More research is being undertaken on epigenetics-based biomarkers for diagnosis and prognosis. Given PI’s decades of experience in biomarker research, we aim to develop a "single blood test" to expand bedside monitoring alternatives that align with the National Cancer Moonshot Initiative to discover cancer early, when the likelihood of a cure is highest.

Microarray analysis identifies coding and non-coding RNA markers of liver injury in whole body irradiated mice

Radiation injury from medical, accidental, or intentional sources can induce acute and long-term hepatic dysregulation, fibrosis, and cancer. This long-term hepatic dysregulation decreases quality of life and may lead to death. Our goal in this study is to determine acute changes in biological pathways and discover potential RNA biomarkers predictive of radiation injury. We performed whole transcriptome microarray analysis of mouse liver tissue (C57BL/6 J) 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray to identify significant expression changes in mRNAs, lncRNAs, and miRNAs, We also validated changes in specific RNAs through qRT-PCR. We used Ingenuity Pathway Analysis (IPA) to identify pathways associated with gene expression changes. We observed significant dysregulation of multiple mRNAs across all doses. In contrast, miRNA dysregulation was observed upwards of 2 Gray. The most significantly upregulated mRNAs function as tumor suppressors: Cdkn1a, Phlda3, and Eda2r. The most significantly downregulated mRNAs were involved in hemoglobin synthesis, inflammation, and mitochondrial function including multiple members of Hbb and Hba. The most significantly upregulated miRNA included: miR-34a-5p, miR-3102-5p, and miR-3960, while miR-342-3p, miR-142a-3p, and miR-223-3p were most significantly downregulated. IPA predicted activation of cell cycle checkpoint control pathways and inhibition of pathways relevant to inflammation and erythropoietin. Clarifying expression of mRNA, miRNA and lncRNA at a short time point (48 h) offers insight into potential biomarkers, including radiation markers shared across organs and animal models. This information, once validated in human models, can aid in development of bio-dosimetry biomarkers, and furthers our understanding of acute pathway dysregulation.

Subtype of Neuroblastoma Cells with High KIT Expression Are Dependent on KIT and Its Knockdown Induces Compensatory Activation of Pro-Survival Signaling.

Neuroblastoma (NB) is a pediatric cancer with high clinical and molecular heterogeneity, and patients with high-risk tumors have limited treatment options. Receptor tyrosine kinase KIT has been identified as a potential marker of high-risk NB and a promising target for NB treatment. We investigated 19,145 tumor RNA expression and molecular pathway activation profiles for 20 cancer types and detected relatively high levels of KIT expression in NB. Increased KIT expression was associated with activation of cell survival pathways, downregulated apoptosis induction, and cell cycle checkpoint control pathways. KIT knockdown with shRNA encoded by lentiviral vectors in SH-SY5Y cells led to reduced cell proliferation and apoptosis induction up to 50%. Our data suggest that apoptosis induction was caused by mitotic catastrophe, and there was a 2-fold decrease in percentage of G2-M cell cycle phase after KIT knockdown. We found that KIT knockdown in NB cells leads to strong upregulation of other pro-survival growth factor signaling cascades such as EPO, NGF, IL-6, and IGF-1 pathways. NGF, IGF-1 and EPO were able to increase cell proliferation in KIT-depleted cells in an ERK1/2-dependent manner. Overall, we show that KIT is a promising therapeutic target in NB, although such therapy efficiency could be impeded by growth factor signaling activation.

Abstract 256: Ablation of 14-3-3 Protein Exacerbates Doxorubicin Induced Cardiomyopathy: Role of Apoptosis Signal Regulating Kinase-1

Background: 14-3-3η family members are dimeric phosphoserine-binding proteins that participate in signal transduction and checkpoint control pathways. Anthracycline anticancer drug doxorubicin (Dox) can induce cardiotoxicity, which is believed to be based on inflammatory or oxidative injury. However, the role of 14-3-3η is not clear in Dox induced cardiac injury. We examined the role of 14-3-3η protein and apoptosis signal-regulating kinase-1 (Ask1) and inflammatory signaling by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3η protein mutant (DN 14-3-3) in Dox induced cardiac injury. Methods: Cardiac dysfunction was induced by a single injection of Dox into wild-type (WT) and DN 14-3-3η mice. By the end of the study, echocardiography was performed to assess the cardiac function. The heart tissues were used for histopathology and western blotting. Results: Left ventricular (LV) fractional shortening and ejection fraction were dramatically decreased in DN 14-3-3η mice, when compared to WT mice after Dox injection. Inactivation of 14-3-3η protein significantly increased Dox induced mortality. Significant Ask1 activation in DN 14-3-3η after Dox injection was evidenced by pronounced de-phosphorylation at Ser-967 and intense immunofluorescence observed LV sections. Marked increase in myocardial apoptosis, cardiac hypertrophy, and fibrosis were observed with a corresponding up-regulation of proinflammatory factors and cytokine expression in DN 14-3-3η mice after Dox injection. Furthermore cardiac expression of high mobility group box (HMGB)1 and its cascade protein expressions were significantly up-regulated in DN 14-3-3η mice compared to WT mice after Dox injection. Conclusion: Taken together, these findings suggest that depletion of 14-3-3η protein causes reduce survival rate in mice with cardiac dysfunction, presumably via activation of downstream Ask1 signaling pathways. This may provide a novel therapeutic strategy against Dox-induced cardiac injury by regulating Ask1 signaling.

Single-Cell Transcriptomics of the Human Endocrine Pancreas.

Human pancreatic islets consist of multiple endocrine cell types. To facilitate the detection of rare cellular states and uncover population heterogeneity, we performed single-cell RNA sequencing (RNA-seq) on islets from multiple deceased organ donors, including children, healthy adults, and individuals with type 1 or type 2 diabetes. We developed a robust computational biology framework for cell type annotation. Using this framework, we show that α- and β-cells from children exhibit less well-defined gene signatures than those in adults. Remarkably, α- and β-cells from donors with type 2 diabetes have expression profiles with features seen in children, indicating a partial dedifferentiation process. We also examined a naturally proliferating α-cell from a healthy adult, for which pathway analysis indicated activation of the cell cycle and repression of checkpoint control pathways. Importantly, this replicating α-cell exhibited activated Sonic hedgehog signaling, a pathway not previously known to contribute to human α-cell proliferation. Our study highlights the power of single-cell RNA-seq and provides a stepping stone for future explorations of cellular heterogeneity in pancreatic endocrine cells.

MYBL2 haploinsufficiency increases susceptibility to age-related haematopoietic neoplasia

The haematopoietic system is prone to age-related disorders ranging from deficits in functional blood cells to the development of neoplastic states. Such neoplasms often involve recurrent cytogenetic abnormalities, among which a deletion in the long arm of chromosome 20 (del20q) is common in myeloid malignancies. The del20q minimum deleted region contains nine genes, including MYBL2, which encodes a key protein involved in the maintenance of genome integrity. Here, we show that mice expressing half the normal levels of Mybl2 (Mybl2+/Δ) develop a variety of myeloid disorders upon ageing. These include myeloproliferative neoplasms, myelodysplasia (MDS) and myeloid leukaemia, mirroring the human conditions associated with del20q. Moreover, analysis of gene expression profiles from patients with MDS demonstrated reduced levels of MYBL2, regardless of del20q status and demonstrated a strong correlation between low levels of MYBL2 RNA and reduced expression of a subset of genes related to DNA replication and checkpoint control pathways. Paralleling the human data, we found that these pathways are also disturbed in our Mybl2+/Δ mice. This novel mouse model, therefore, represents a valuable tool for studying the initiation and progression of haematological malignancies during ageing, and may provide a platform for preclinical testing of therapeutic approaches.

S-phase-coupled apoptosis in tumor suppression.

DNA replication is essential for accurate transmission of genomic information from parental to daughter cells. DNA replication is licensed once per cell division cycle. This process is highly regulated by both positive and negative regulators. Over-replication, under-replication, as well as DNA damage in a cell all induce the activation of checkpoint control pathways such as ATM/ATR, CHK kinases, and the tumor suppressor protein p53, which provide "damage controls" via either DNA repairs or apoptosis. This review focuses on accumulating evidence, with the emphasis on recently discovered Killin, that S-phase checkpoint control is crucial for a mammalian cell to make a life and death decision in order to safeguard genome integrity.

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints

In this review, I stress the importance of direct data and accurate terminology when formulating and communicating conclusions on how the G2/M and metaphase/anaphase transitions are regulated. I argue that entry into mitosis (i.e., the G2/M transition) is guarded by several checkpoint control pathways that lose their ability to delay or stop further cell cycle progression once the cell becomes committed to divide, which in vertebrates occurs in the late stages of chromosome condensation. After this commitment, progress through mitosis is then mediated by a single Mad/Bub-based checkpoint that delays chromatid separation, and exit from mitosis (i.e., completion of the cell cycle) in the presence of unattached kinetochores. When cells cannot satisfy the mitotic checkpoint, e.g., when in concentrations of spindle poisons that prohibit the stable attachment of all kinetochores, they are delayed in mitosis for many hours. In normal cells, the duration of this delay depends on the organism and ranges from ∼4h in rodents to ∼22h in humans. Recent live cell studies reveal that under this condition, many cancer cells (including HeLa and U2OS) die in mitosis by apoptosis within ∼24h, which implies that biochemical studies on cancer cell populations harvested in mitosis after a prolonged mitotic arrest are contaminated with dead or dying cells.

Abstract 657: Identification and characterization of genes that sensitize pancreatic cancer cells to gemcitabine

Abstract Pancreatic cancer is ranked the fourth most lethal cancer as less than 5% of diagnosed patients have 5 year survival or better, with median survival of only 3 to 6 months. This poor survival has been attributed, in part, to the late stage of diagnosis. Presently, several classes of drugs (anti-proliferative agents, platanating agents, fluoropyrimidines, and taxanes) are used in the treatment of this deadly disease, but patient response is poor when compared to other cancers. Therefore, there is an urgent need to improve early diagnosis as well as to identify novel molecular targets that sensitize pancreatic cancer cells to chemotherapeutic agents. Gemcitabine (an inhibitor of DNA replication) is of particular interest to us because it remains the drug of choice in the treatment of pancreatic cancer. To our knowledge, we have recently completed the first genome-wide siRNA screen of pancreatic cancer cells to identify genes and pathways that sensitize cell killing by gemcitabine. Bioinformatics and biostatistical analysis allowed us to identify 122 strong candidate genes that we propose to validate in secondary screens. The candidates are grouped into biological pathways or networks that include cell cycle and checkpoint control, and signaling and survival pathways. In addition to the use of viability assays (CellTiterGlo) to validate our candidates, we will use high resolution time-lapse microscopy along with FACs to directly track the fates of cells. Our preliminary time-lapse studies using cell lines stably expressing gfpH2B (to visualize nuclei and chromosomes), showed that gemcitabine arrests cells in S phase for >48 hours. The prolonged checkpoint delay may allow tumor cells in patients to survive as gemcitabine has a relatively short half-life (less than 24 hours) once it is infused into the patient. Using inhibitors that abrogate the S phase checkpoint (Chk1 inhibitors) as well as Chk1 siRNA treatment following gemcitabine addition, we saw cells prematurely enter mitosis with unreplicated genomes and die. More detailed studies showed that the centromeres of all the chromosomes were physically ripped away from the bulk chromatin. Cells delay in mitosis and die, or exit mitosis with acentric chromosomes and die. This specific mitotic defect will be used as one of the assays to categorize the candidates from our siRNA screen. Our goal is to categorize candidates that kill cells through this pathway as well as candidates that kill cells through other pathways. This information will allow us to develop mechanisms by which pancreatic cancer cells can be sensitized to killing by gemcitabine. We intend to translate our discoveries towards identifying new targets for drug development as well as genetic markers to profile patient tumors to improve treatment strategies and responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 657.

Chk2 down-regulation by promoter hypermethylation in human bulk gliomas

Aims Gliomas account for 80% of malignant brain tumors. DNA damage response and subsequent checkpoint control pathways could maintain the integrity of the genome and thus defend tumorigenesis. Four kinases, ATM, ATR, ChK1 and Chk2 are the damage sensors and the early effectors in DNA damage responses. Given their importance, we investigated the transcriptional regulation of these four genes. Main methods Tissues from ten normal brains and thirty human gliomas were utilized for mRNA analysis via real-time PCR. Another twelve normal brain tissues and forty gliomas were used for confirmation. Methylation-specific PCR (MSP) was used to determine the methylation status of the Chk2 promoter. Quantitative chromatin immunoprecipitation (ChIP) was used to measure the influence of methylation on Sp1 binding. Key findings We found that the expression of ATR, ChK1 and Chk2 in gliomas was significantly down-regulated relative to the normal brain tissues. The most significant reduction of expression was of the Chk2 gene, whose expression was ∼ 10-fold decreased in gliomas ( P < 0.0001). Down-regulation of Chk2 was validated in the second real-time PCR analysis. This reduction in expression was partially due to promoter methylation. The Chk2 proximal promoter recruited Sp1 for transcriptional activation. We found that hypermethylation of the Chk2 promoter undermined the binding of the transcriptional factor Sp1. Significance Our results indicate that Chk2 methylation could be involved in glioma carcinogenesis and Chk2 expression may potentially be used for the diagnosis of glioma.

Phosphorylation-Dependent Interaction with 14-3-3 in the Regulation of Bad Trafficking in Retinal Ganglion Cells

To focus on the proteomic analysis of 14-3-3 proteins and to determine their cellular localization and functional role during glaucomatous neurodegeneration. Complementary proteomic approaches were used to identify phosphorylated proteins in a chronic pressure-induced rat model of glaucoma. To detect interacting proteins, specific protein complexes were eluted using coimmunoprecipitation and recombinant protein-based affinity pull-down for subsequent mass spectrometric analysis. Western blot analysis was performed for validation of the proteomic findings, and immunohistochemical analysis of rat eyes and human donor eyes determined the cellular localization of 14-3-3 proteins. In addition, in vivo treatment experiments were conducted using JNK and protein phosphatase inhibitors. Findings of mass spectrometry, Western blotting, and tissue immunolabeling revealed the presence of different 14-3-3 isotopes in RGCs and their up-regulation and phosphorylation during glaucomatous neurodegeneration. Consecutive experiments through proteomic analysis identified various proteins interacting with 14-3-3, which included calmodulin and a proapoptotic member of the Bcl-2 family, Bad; 14-3-3 was found to keep phospho-Bad sequestered in the cytoplasm. However, this association was disrupted in ocular hypertensive eyes in correlation with Bad dephosphorylation and 14-3-3 phosphorylation, thereby leading to mitochondrial translocation of Bad for apoptotic function. Inhibition of JNK activity and of protein phosphatase activity complementarily secured the 14-3-3-scaffold of Bad in the cytoplasm and preserved optic nerve axons in ocular hypertensive eyes. Findings of this in vivo study identify that an important protein family associated with checkpoint control pathways, 14-3-3, is involved in cellular signaling during glaucomatous neurodegeneration in a phosphorylation-dependent manner.

Role of Claspin in regulation of nucleotide excision repair factor DDB2

The replication checkpoint protein Claspin is important for maintenance of genomic stability and is required for cells to overcome genotoxic stress. Upon UV-induced DNA damage, Claspin is required for activation of the ATR-mediated DNA damage checkpoint response, leading to arrest of DNA replication and inhibition of cell cycle progression. Located at the DNA replication fork, Claspin is also suggested to monitor replication and sense damage. Our present studies in HeLa cells demonstrate associations between the Claspin/ATR-related DNA damage checkpoint response and the global genomic nucleotide excision repair pathway. siRNA-mediated knockdown of Claspin abolishes the UV-induced degradation of DDB2 and impairs the co-localization of DDB2 to DNA damage sites. Thus, the presence of Claspin is required for the total turnover of DNA damage binding protein DDB2, as well as for its functionality in DNA damage recognition. Claspin, however, seems not to be required for maintaining the cellular level of the NER factor XPC and its UV-induced post-translational modifications. Co-localization of XPC with DNA lesions is also intact in the absence of Claspin as is the repair of the UV-induced lesions CPD and 6-4PP. Claspin itself may be directly responsible for physical interaction between the two pathways since Claspin is able to associate with DDB1, DDB2 and XPC. Taken together, these findings reveal physical and functional interplay between Claspin and NER-related proteins and demonstrate crosstalk between the DNA damage checkpoint control and DNA damage repair pathways.

Swimming stress in DN 14-3-3 mice triggers maladaptive cardiac remodeling: role of p38 MAPK

It is generally believed that a mechanical signal initiates a cascade of biological events leading to coordinated cardiac remodeling. 14-3-3 family members are dimeric phosphoserine-binding proteins that regulate signal transduction, apoptotic, and checkpoint control pathways. To evaluate the molecular mechanism underlying swimming stress-induced cardiac remodeling, we examined the role of 14-3-3 protein and MAPK pathway by pharmacological and genetic means using transgenic mice with cardiac-specific expression of dominant-negative (DN) mutants of 14-3-3 (DN 14-3-3/TG) and p38alpha/beta MAPK (DNp38alpha and DNp38beta) mice. p38 MAPK activation was earlier, more marked, and longer in the myocardium of the TG group compared with that of the nontransgenic (NTG) group after swimming stress, whereas JNK activation was detected on day 5 and decreased afterward. In contrast, ERK1/2 was not activated after swimming stress in either group. Cardiomyocyte apoptosis, cardiac hypertrophy, and fibrosis were greatly increased in the TG group compared with those in the NTG group. Moreover, we found a significant correlation between p38 MAPK activation and apoptosis in the TG group. Furthermore, DN 14-3-3 hearts showed enhanced atrial natriuretic peptide expression. In contrast, DNp38alpha and DNp38beta mice exhibited reduced mortality and increased resistance to cardiac remodeling after 28 days of swimming stress compared with TG and NTG mice. Besides, treatment with a p38 MAPK inhibitor, FR-167653, resulted in regression of cardiac hypertrophy and fibrosis and improvement in the survival rate in the TG group. These results indicate for the first time that 14-3-3 protein along with p38 MAPK plays a crucial role in left ventricular remodeling associated with swimming stress.

Differential role of 14‐3‐3 family members in Xenopus development

The 14-3-3 proteins are intracellular dimeric phosphoserine/threonine binding molecules that participate in signal transduction, checkpoint control, nutrient sensing, and cell survival pathways. Previous work established that 14-3-3 proteins are required in early Xenopus laevis development by modulating fibroblast growth factor signaling. Although this general requirement for 14-3-3 proteins in Xenopus early embryogenesis is established, there is no information about the specific role of individual 14-3-3 genes. Botanical studies previously demonstrated functional specificity among 14-3-3 genes during plant development. In this study, an antisense morpholino oligo microinjection approach was used to characterize the requirement for six specific 14-3-3 family members in Xenopus embryogenesis. Microinjection experiments followed by Western blot analysis showed that morpholinos reduced specific 14-3-3 protein levels. Embryos lacking specific 14-3-3 isoforms displayed unique phenotypic defects. In particular, reduction of 14-3-3 tau (tau) protein, and to a lesser extent, 14-3-3 epsilon (epsilon), resulted in embryos with prominent gastrulation and axial patterning defects and reduced mesodermal marker gene expression. In contrast, reduction of 14-3-3 zeta (zeta) protein caused no obvious phenotypic abnormalities. Reduction of 14-3-3 gamma (gamma) protein resulted in eye defects without gastrulation abnormalities. Therefore, individual 14-3-3 genes have separable functions in vertebrate embryonic development.

Inhibitory effect of 14-3-3 proteins on serum-induced proliferation of cardiac fibroblasts

Proliferation in cardiac fibroblasts (CFs) can be induced by a wide variety of growth factors that recruit multiple signal transduction pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C. As a family of dimeric phophoserine-binding proteins, 14-3-3s are associated with a multitude of proteins that regulate signal transduction, apoptosis and checkpoint control pathways. However, it remains unknown whether the 14-3-3 proteins play an active role in cardiac proliferation and alter their expression patterns in response to growth factors in CFs. R18 peptide, an isoform-independent 14-3-3 inhibitor, was used to disrupt 14-3-3 function by adenovirus-mediated transfer of R18-EYFP (AdR18). Our results demonstrate that the 14-3-3 isoforms gamma, zeta and epsilon were highly expressed in CFs and the expression of 14-3-3 epsilon was elevated following serum stimulation. Inhibition of 14-3-3 proteins by AdR18 potentiated mitogen-induced DNA synthesis in CFs. This potentiation was presumably due to the increased inactivated glycogen synthase kinase-3 beta by Ser9 phosphorylation and nuclear factor of activated T-cell nuclear accumulation. However, AdR18 had no effect on extracellular signal-regulated kinase phosphorylation and reduced p70 S6 kinase (p70S6K) phosphorylation upon mitogenic stimulation. Furthermore, though R18 can block 14-3-3 binding abilities, it did not affect the serum-induced upregulation of 14-3-3 epsilon protein. Collectively, these findings reveal that the expression of 14-3-3 epsilon can be upregulated by serum in CFs and 14-3-3s may exert an inhibitory effect on serum-induced proliferation.

Inactivation of 14-3-3 Protein Exacerbates Cardiac Hypertrophy and Fibrosis through Enhanced Expression of Protein Kinase C .BETA.2 in Experimental Diabetes

Diabetic cardiomyopathy is associated with cardiac hypertrophy and fibrosis. Activation of protein kinase C (PKC) has been implicated in the diabetes-induced cardiovascular complications. PKCbeta2 isoform is preferentially found to be activated in the diabetic myocardium. However, the role of PKCbeta2 in diabetic cardiomyopathy is not clear. 14-3-3 family members are dimeric phosphoserine-binding proteins that regulate signal transduction, apoptotic and checkpoint control pathways, and have been shown to bind with PKC isozymes and negatively regulate their enzymatic activities. The present study tests whether 14-3-3 protein regulates cardiac hypertrophy and fibrosis in streptozotocin (STZ)-induced diabetic mice, using transgenic mice with cardiac specific over-expression of dominant negative (DN) 14-3-3 protein. In addition, we examined the relationship between 14-3-3 protein and PKCbeta2 in the diabetic myocardium. Cardiac myocyte diameter, content of cardiac fibrosis, left ventricular tissue expressions of atrial natriuretic peptide, transforming growth factor beta1, collagen III and PKCbeta2 were significantly elevated 28 and 56 d after STZ injection in transgenic DN-14-3-3 mice, when compared to their non-transgenic counterparts. These results clearly demonstrate that the functional inactivation of 14-3-3 protein in DN-14-3-3 mice exacerbates diabetes-induced cardiac hypertrophy and fibrosis. The exacerbations of cardiac hypertrophy and fibrosis were significantly and positively correlated with the enhanced expression of PKCbeta2 in DN-14-3-3 mice. Our results indicate for the first time that 14-3-3 protein negatively regulates cardiac hypertrophy and fibrosis, possibly through controlling the expression of PKCbeta2 in the diabetic myocardium.

Dominant negative 14-3-3 promotes cardiomyocyte apoptosis in early stage of type I diabetes mellitus through activation of JNK

14-3-3 family members are dimeric, phosphoserine binding proteins that regulate signal transduction, apoptotic, and checkpoint control pathways. Recently, cardiomyocyte apoptosis has been characterized in type I diabetes mellitus. In order to study the molecular mechanism underlying diabetes-induced cardiomyocyte apoptosis, we examined the role of 14-3-3 protein and MAPK pathways in transgenic mice with cardiac specific expression of dominant negative 14-3-3η (DN-14-3-3). p38 MAPK was highly activated 1, 28, and 56 days after diabetes induction by streptozotocin, whereas peak JNK activation was found on day 3 and decreased afterwards. In contrast, ERK1/2 were not activated in diabetic myocardium. Cardiomyocyte apoptosis was peaked on day 3 and decreased on 7, 28, and 56 days. p38 MAPK and JNK activation as well as cardiomyocyte apoptosis were greatly increased in DN-14-3-3 mice relative to non-transgenic mice. Moreover, we found a significant correlation between JNK activation and apoptosis in diabetic myocardium. These results indicate for the first time that 14-3-3 protein plays a critical anti-apoptotic role in diabetic myocardium by inhibiting the JNK pathway.

Aurora A, Meiosis and Mitosis

The Aurora family kinases are pivotal to the successful execution of cell division. Together they ensure the formation of a bipolar mitotic spindle, accurate segregation of chromosomes and the completion of cytokinesis. They are also attractive drug targets, being frequently deregulated in cancer and able to transform cells in vitro. In this review, we summarize current knowledge about the three family members, Aur-A, Aur-B and Aur-C. We then focus on Aur-A, its roles in mitotic progression, and its emerging roles in checkpoint control pathways. Aur-A activity can be controlled at several levels, including phosphorylation, ubiquitin-dependent proteolysis and interaction with both positive regulators, such as TPX2, and negative ones, like the tumor suppressor protein p53. In addition, work in Xenopus oocytes and early embryos has revealed a second role for Aur-A, directing the polyadenylation-dependent translation of specific mRNAs important for cell cycle progression. This function extends to post-mitotic neurons, and perhaps even to cycling somatic cells.

Role of 14-3-3-Mediated p38 Mitogen-Activated Protein Kinase Inhibition in Cardiac Myocyte Survival

14-3-3 family members are dimeric phosphoserine-binding proteins that regulate signal transduction, apoptotic, and checkpoint control pathways. Targeted expression of dominant-negative 14-3-3eta (DN-14-3-3) to murine postnatal cardiac tissue potentiates Ask1, c-jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activation. DN-14-3-3 mice are unable to compensate for pressure overload, which results in increased mortality, dilated cardiomyopathy, and cardiac myocyte apoptosis. To evaluate the relative role of p38 MAPK activity in the DN-14-3-3 phenotype, we inhibited cardiac p38 MAPK activity by pharmacological and genetic methods. Intraperitoneal injection of SB202190, an inhibitor of p38alpha and p38beta MAPK activity, markedly increased the ability of DN-14-3-3 mice to compensate for pressure overload, with decreased mortality. DN-14-3-3 mice were bred with transgenic mice in which dominant-negative p38alpha (DN-p38alpha) or dominant-negative p38beta (DN-p38beta) MAPK expression was targeted to the heart. Compound transgenic DN-14-3-3/DN-p38beta mice, and to a lesser extent compound transgenic DN-14-3-3/DN-p38alpha mice, exhibited reduced mortality and cardiac myocyte apoptosis in response to pressure overload, demonstrating that DN-14-3-3 promotes cardiac apoptosis due to stimulation of p38 MAPK activity.

A plant cyclin B2 is degraded early in mitosis and its ectopic expression shortens G2-phase and alleviates the DNA-damage checkpoint.

Mitotic progression is timely regulated by the accumulation and degradation of A- and B-type cyclins. In plants, there are three classes of A-, and two classes of B-type cyclins, but their specific roles are not known. We have generated transgenic tobacco plants in which the ectopic expression of a plant cyclin B2 gene is under the control of a tetracycline-inducible promoter. We show that the induction of cyclin B2 expression in cultured cells during G2 phase accelerates the entry into mitosis and allows cells to override the replication checkpoint induced by hydroxyurea in the simultaneous presence of caffeine or okadaic acid, drugs that are known to alleviate checkpoint control. These results indicate that in plants, a B2-type cyclin is a rate-limiting regulator for the entry into mitosis and a cyclin B2-CDK complex might be a target for checkpoint control pathways. The cyclin B2 localization and the timing of its degradation during mitosis corroborate these conclusions: cyclin B2 protein is confined to the nucleus and during mitosis it is only present during a short time window until mid prophase, but it is effectively degraded from this timepoint onwards. Although cyclin B2 is not present in cells arrested by the spindle checkpoint in metaphase, cyclin B1 is accumulating in these cells. Ectopic expression of cyclin B2 in developing plants interferes with differentiation events and specifically blocks root regeneration, indicating the importance of control mechanisms at the G2- to M-phase transition during plant developmental processes.