Regulation Of Cell Cycle Events Research Articles

Nkx2\u20135 Second Heart Field Target Gene Ccdc117 Regulates DNA Metabolism and Proliferation

The cardiac transcription factor Nkx2-5 is essential for normal outflow tract (OFT) and right ventricle (RV) development. Nkx2-5−/− null mouse embryos display severe OFT and RV hypoplasia and a single ventricle phenotype due to decreased proliferation of Second Heart Field (SHF) cells, a pool of cardiac progenitors present in anterior pharyngeal arch mesoderm at mid-gestation. However, definition of the precise role of Nkx2-5 in facilitating SHF expansion is incomplete. We have found that Nkx2-5 positively and directly regulates a novel target gene, Ccdc117, in cells of the SHF at these stages. The nuclear/mitotic spindle associated protein Ccdc117 interacts with the MIP18/MMS19 cytoplasmic iron-sulfur (FeS) cluster assembly (CIA) complex, which transfers critical FeS clusters to several key enzymes with functions in DNA repair and replication. Loss of cellular Ccdc117 expression results in reduced proliferation rates associated with a delay at the G1-S transition, decreased rates of DNA synthesis, and unresolved DNA damage. These results implicate a novel role for Nkx2-5 in the regulation of cell cycle events in the developing heart, through Ccdc117′s interaction with elements of the CIA pathway and the facilitation of DNA replication during SHF expansion.

DNA Replication Inhibitor Geminin and Retinoic Acid Signaling Participate in Complex Interactions Associated With Pluripotency.

Several links between DNA replication, pluripotency and development have been recently identified. The involvement of miRNA in the regulation of cell cycle events and pluripotency factors has also gained attention. In the present study, we used the g:Profiler platform to analyze transcription factor binding sites, miRNA networks and protein-protein interactions to identify novel links among the aforementioned processes. A complex circuitry between retinoic acid signaling, SWI/SNF components, pluripotency factors including Oct4, Sox2 and Nanog and cell cycle regulators was identified. It is suggested that the DNA replication inhibitor geminin plays a central role in this circuitry.

Stable Regulation of Cell Cycle Events in Mycobacteria: Insights From Inherently Heterogeneous Bacterial Populations.

Model bacteria, such as E. coli and B. subtilis, tightly regulate cell cycle progression to achieve consistent cell size distributions and replication dynamics. Many of the hallmark features of these model bacteria, including lateral cell wall elongation and symmetric growth and division, do not occur in mycobacteria. Instead, mycobacterial growth is characterized by asymmetric polar growth and division. This innate asymmetry creates unequal birth sizes and growth rates for daughter cells with each division, generating a phenotypically heterogeneous population. Although the asymmetric growth patterns of mycobacteria lead to a larger variation in birth size than typically seen in model bacterial populations, the cell size distribution is stable over time. Here, we review the cellular mechanisms of growth, division, and cell cycle progression in mycobacteria in the face of asymmetry and inherent heterogeneity. These processes coalesce to control cell size. Although Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) utilize a novel model of cell size control, they are similar to previously studied bacteria in that initiation of DNA replication is a key checkpoint for cell division. We compare the regulation of DNA replication initiation and strategies used for cell size homeostasis in mycobacteria and model bacteria. Finally, we review the importance of cellular organization and chromosome segregation relating to the physiology of mycobacteria and consider how new frameworks could be applied across the wide spectrum of bacterial diversity.

A comparative study of phosphopeptide-selective techniques for a sub-proteome of a complex biological sample

Phosphorylation of proteins is important for controlling cellular signaling and cell cycle regulatory events. The process is reversible and phosphoproteins normally constitute a minor part of the global proteome in a cell. Thus, sample preparation techniques tailored for phosphoproteome studies are continuously invented and evaluated. This paper aims at evaluating the performances of the most popular techniques for phospho-enrichments in sub-proteome analysis, such as viral proteomes expressed in human cells during infection. A two-species sample of Adenovirus type 2 infected human cells was used, and in-solution digestion, strong cation exchange (SCX), and electrostatic repulsion hydrophilic interaction chromatography (ERLIC) fractionation, and subsequent enrichment by TiO2, were compared with SDS-PAGE fractionation and in-gel digestion. Evaluation was focused on phosphopeptide detection in the sub-proteome. The results showed that the SCX+TiO2 or ERLIC+TiO2 combinations had the highest enrichment efficiencies, but SDS-PAGE fractionation and in-gel digestion resulted in the highest number of identified proteins and phosphopeptides. Furthermore, the study demonstrates the usefulness of applying as many orthogonal techniques as possible in deep phosphoproteome analysis, since the overlap between approaches was low.

Enhanced cellular uptake of a TAT-conjugated peptide inhibitor targeting the polo-box domain of polo-like kinase 1.

In the last decade, drug delivery systems using biologically active molecules for cellular uptake of therapeutic targets have been studied for application and testing in clinical trials. For instance, the transactivator of transcription (TAT) peptide, or cell-penetrating peptide, was shown to deliver a variety of cargoes, including proteins, peptides, and nucleic acids. Polo-like kinase 1 (Plk1) plays key roles in the regulation of cell cycle events (e.g., mitotic progression). Plk1 was also shown to be activated and highly expressed in proliferating cells such as tumor cells. Amongst these phosphopeptides, Pro-Leu-His-Ser-p-Thr (PLHSpT), which is the minimal sequence for polo-box domain (PBD) binding, was shown to have an inhibitory effect and to induce apoptotic cell death. However, the phosphopeptide showed low cell membrane penetration. Thus, in our study, we synthesized Plk1 inhibitor TAT-PLHSpT to improve agent internalization into cells. TAT-PLHSpT was shown to internalize into the nucleus. The conjugation of TAT with PLHSpT inhibited cancer cell growth and survival. Moreover, it showed an increase in cellular uptake and inhibition of Plk1 kinase activity. Further studies are needed for biological evaluation of the new peptide in tumor-bearing animal models (in vivo). Our results prove that TAT-PLHSpT is a good candidate for specific PBD binding of Plk1 as a therapeutic agent for humans.

Novel Primate miRNAs Coevolved with Ancient Target Genes in Germinal Zone-Specific Expression Patterns

Major nonprimate-primate differences in cortico-genesis include the dimensions, precursor lineages, and developmental timing of the germinal zones (GZs). microRNAs (miRNAs) of laser-dissected GZ compartments and cortical plate (CP) from embryonic E80 macaque visual cortex were deep sequenced. The CP and the GZ including ventricular zone (VZ) and outer and inner subcompartments of the outer subventricular zone (OSVZ) in area 17 displayed unique miRNA profiles. miRNAs present in primate, but absent in rodent, contributed disproportionately to the differential expression between GZ subregions. Prominent among the validated targets of these miRNAs were cell-cycle and neurogenesis regulators. Coevolution between the emergent miRNAs and their targets suggested that novel miRNAs became integrated into ancient gene circuitry to exert additional control over proliferation. We conclude that multiple cell-cycle regulatory events contribute to the emergence of primate-specific cortical features, including the OSVZ, generated enlarged supragranular layers, largely responsible for the increased primate cortex computational abilities.

Tandem ChoRE and CCAAT Motifs and Associated Factors Regulate Txnip Expression in Response to Glucose or Adenosine-Containing Molecules

BackgroundThioredoxin interacting protein (Txnip) is a multifunctional protein involved in regulation of cell cycle events and cellular metabolism. The expression of Txnip is known to be induced by glucose, adenosine-containing molecules, and other physiological cues; however, the underlying regulatory mechanisms remain elusive.Methodology/Principal FindingsIn this study, using promoter reporter, electrophoresis mobility shift (EMSA), and chromatin immuno-precipitation (ChIP) assays, we have identified an additional carbohydrate response element (ChoRE) on the promoter of Txnip gene, which functions cooperatively with the earlier identified ChoRE to mediate optimal Txnip expression. However, these two ChoREs are not sufficient to mediate the induction of Txnip expression by glucose or adenosine-containing molecules; and two CCAAT boxes, both of which can recruit nuclear factor Y (NF-Y) to the Txnip promoter, are also required for the induction. Accordingly, we have found that the function of ChoREs and associated factors is contingent on tandem CCAAT boxes, in that occupancy of the Txnip promoter by NF-Y is a prerequisite for efficacious recruitment of Mondo/MLX to ChoREs under glucose stimulation.Conclusions/SignificanceOur findings suggest a synergy between the tandem CCAAT and ChoRE motifs and associated NF-Y and Mondo/MLX transcription factors in enhancing transcription from the Txnip promoter. This piece of information will be helpful for future dissection of molecular mechanisms governing the transcriptional regulation of Txnip, a glucose responsive gene.

Abstract B100: The expression of thioredoxin-interacting protein (Txnip): Modulators, mechanisms and functions

Abstract Thioredoxin interacting protein (Txnip) is a multifunctional protein involved in regulation of cell cycle events and cellular metabolism; the expression of the Txnip gene is down-regulated in a large range of cancer cells and up-regulated in diabetic and pre-diabetic patients. Therefore, Txnip might be a link between metabolism and cancer development, and the study of regulatory mechanism(s) that governs Txnip expression has important clinical relevance. The expression of Txnip is induced by glucose, which is mediated by an earlier defined carbohydrate response element (ChoRE) on Txnip promoter and its associated transcription factors, MondoA (or its homolog, ChREBP) and Max-like protein X (MLX). Here, we show that the transcription of the Txnip gene is induced by an array of adenosine-containing molecules, of which an intact adenosine moiety is necessary and sufficient. When cells are treated with adenosine-containing molecules, the glucose uptake is inhibited and cell cycle progression is retarded, probably attributed to enhanced Txnip expression. The induction of Txnip expression by adenosine-containing molecules is in a glucose-dependent manner, and MondoA and MLX are known to convey stimulatory signals from extracellular molecules to the Txnip promoter. Therefore, the regulatory role of adenosine-containing molecules is exerted via amplifying glucose signaling, hence suggesting that these molecules may modulate the kinetics of glucose homeostasis. To gain more knowledge about the regulation of Txnip expression, we have also studied the underlying regulatory mechanisms of glucose and adenosine-containing molecules on Txnip expression in details. An additional ChoRE on the promoter of Txnip gene has been identified, and this ChoRE is able to recruit MondoA and MLX in a similar fashion as the previously identified ChoRE in vitro and in vivo. Both ChoREs function cooperatively to mediate optimal Txnip expression under glucose or adenosine-containing molecules treatment. However, these two ChoREs are not sufficient to mediate the induction of Txnip expression by glucose or adenosine-containing molecules, and two CCAAT boxes, both can recruit nuclear factor Y (NF-Y) to the Txnip promoter, are also required for the induction. We also found that the function of ChoREs and associated factors is contingent on tandem CCAAT boxes, in that the occupancy of the Txnip promoter by the CCAAT box-associated NF-Y is a prerequisite for efficacious recruitment of MondoA/MLX to ChoREs under glucose stimulation. Such a strategy suggests a synergy between NF-Y and MondoA/MLX in enhancing Txnip expression, which leads us to propose a model for the glucose-dependent Txnip expression mediated by dynamic chromatin changes in response to diverse physiological inducers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B100.

Dynamic Histone H1 Isotype 4 Methylation and Demethylation by Histone Lysine Methyltransferase G9a/KMT1C and the Jumonji Domain-containing JMJD2/KDM4 Proteins

The linker histone H1 generally participates in the establishment of chromatin structure. However, of the seven somatic H1 isotypes in humans some are also implicated in the regulation of local gene expression. Histone H1 isotype 4 (H1.4) represses transcription, and its lysine residue 26 (Lys26) was found to be important in this aspect. H1.4K26 is known to be methylated and acetylated in vivo, but the enzymes responsible for these post-translational modifications and the regulatory cues that promote H1.4 residence on chromatin are poorly characterized. Here we report that the euchromatic histone lysine methyltransferase G9a/KMT1C mediates H1.4K26 mono- and dimethylation in vitro and in vivo and thereby provides a recognition surface for the chromatin-binding proteins HP1 and L3MBTL1. Moreover, we show evidence that G9a promotes H1 deposition and is required for retention of H1 on chromatin. We also identify members of the JMJD2/KDM4 subfamily of jumonji-C type histone demethylases as being responsible for the removal of H1.4K26 methylation.

The oncogene c-Myc coordinates regulation of metabolic networks to enable rapid cell cycle entry

The c-myc proto-oncogene is rapidly activated by serum and regulates genes involved in metabolism and cell cycle progression. This gene is thereby uniquely poised to coordinate both the metabolic and cell cycle regulatory events required for cell cycle entry. However, this function of Myc has not been evaluated. Using a rat fibroblast model of isogenic cell lines, myc-/-, myc+/-, myc+/+ and myc-/- cells with an inducible c-myc transgene (mycER), we show that the Myc protein programs cells to utilize both oxidative phosphorylation and glycolysis to drive cell cycle progression. We demonstrate this coordinate regulation of metabolic networks is essential, as specific inhibitors of these pathways block Myc-induced proliferation. Metabolic events temporally correlated with cell cycle entry include increased oxygen consumption, mitochondrial function, pyruvate and lactate production, and ATP generation. Treatment of normal cells with inhibitors of oxidative phosphorylation recapitulates the myc-/- phenotype, resulting in impaired cell cycle entry and reduced metabolism. Combined with a kinetic expression profiling analysis of genes linked to mitochondrial function, our study indicates that Myc’s ability to coordinately regulate the mitochondrial metabolic network transcriptome is required for rapid cell cycle entry. This function of Myc may underlie the pervasive presence of Myc in many human cancers.

Xenopus importin beta validates human importin beta as a cell cycle negative regulator

BackgroundHuman importin beta has been used in all Xenopus laevis in vitro nuclear assembly and spindle assembly studies. This disconnect between species raised the question for us as to whether importin beta was an authentic negative regulator of cell cycle events, or a dominant negative regulator due to a difference between the human and Xenopus importin beta sequences. No Xenopus importin beta gene was yet identified at the time of those studies. Thus, we first cloned, identified, and tested the Xenopus importin beta gene to address this important mechanistic difference. If human importin beta is an authentic negative regulator then we would expect human and Xenopus importin beta to have identical negative regulatory effects on nuclear membrane fusion and pore assembly. If human importin beta acts instead as a dominant negative mutant inhibitor, we should then see no inhibitory effect when we added the Xenopus homologue.ResultsWe found that Xenopus importin beta acts identically to its human counterpart. It negatively regulates both nuclear membrane fusion and pore assembly. Human importin beta inhibition was previously found to be reversible by Ran for mitotic spindle assembly and nuclear membrane fusion, but not nuclear pore assembly. During the present study, we observed that this differing reversibility varied depending on the presence or absence of a tag on importin beta. Indeed, when untagged importin beta, either human or Xenopus, was used, inhibition of nuclear pore assembly proved to be Ran-reversible.ConclusionWe conclude that importin beta, human or Xenopus, is an authentic negative regulator of nuclear assembly and, presumably, spindle assembly. A difference in the Ran sensitivity between tagged and untagged importin beta in pore assembly gives us mechanistic insight into nuclear pore formation.

Nuclear accumulation of cyclin D1 during S phase inhibits Cul4-dependent Cdt1 proteolysis and triggers p53-dependent DNA rereplication

Deregulation of cyclin D1 occurs in numerous human cancers through mutations, alternative splicing, and gene amplification. Although cancer-derived cyclin D1 mutants are potent oncogenes in vitro and in vivo, the mechanisms whereby they contribute to neoplasia are poorly understood. We now provide evidence derived from both mouse models and human cancer-derived cells revealing that nuclear accumulation of catalytically active mutant cyclin D1/CDK4 complexes triggers DNA rereplication, resulting from Cdt1 stabilization, which in turn triggers the DNA damage checkpoint and p53-dependent apoptosis. Loss of p53 through mutations or targeted deletion results in increased genomic instability and neoplastic growth. Collectively, the data presented reveal mechanistic insights into how uncoupling of critical cell cycle regulatory events will perturb DNA replication fidelity, thereby contributing to neoplastic transformation.

A Confederacy of Kinases: Cdk2 and Cdk4 Conspire to Control Embryonic Cell Proliferation

The mouse embryo is surprisingly resistant to loss of individual cyclins or cyclin-dependent kinases. In a recent issue of Developmental Cell, Berthet et al. (2006) describe collaboration of Cdk2 and Cdk4 in embryogenesis that is revealed only upon their simultaneous loss, resulting in inappropriate activation of the retinoblastoma protein and embryonic lethality.

Living and Dying for Sex

A mechanistic understanding of aging has yet to be described; this paper puts forth a new theory that has the potential to explain aging in all sexually reproductive life forms. The theory also puts forth a new definition of aging – any change in an organism over time. This definition includes not only the changes associated with the loss of function (i.e. senescence, the commonly accepted definition of aging), but also the changes associated with the gain of function (growth and development). Using this definition, the rate of aging would be synonymous with the rate of change. The rate of change/aging is most rapid during the fetal period when organisms develop from a single cell at conception to a multicellular organism at birth. Therefore, ‘fetal aging’ would be determined by factors regulating the rate of mitogenesis, differentiation, and cell death. We suggest that these factors also are responsible for regulating aging throughout life. Thus, whatever controls mitogenesis, differentiation and cell death must also control aging. Since life-extending modalities consistently affect reproduction, and reproductive hormones are known to regulate mitogenesis and differentiation, we propose that aging is primarily regulated by the hormones that control reproduction (hence, the Reproductive-Cell Cycle Theory of Aging). In mammals, reproduction is controlled by the hypothalamic-pituitary-gonadal (HPG) axis hormones. Longevity inducing interventions, including caloric restriction, decrease fertility by suppressing HPG axis hormones and HPG hormones are known to affect signaling through the well-documented longevity regulating GH/IGF-1/PI3K/Akt/Forkhead pathway. This is exemplified by genetic alterations in Caenorhabditis elegans where homologues of the HPG axis pathways, as well as the daf-2 and daf-9 pathways, all converge on daf-16, the homologue of human Forkhead that functions in the regulation of cell cycle events. In summary, we propose that the hormones that regulate reproduction act in an antagonistic pleiotrophic manner to control aging via cell cycle signaling; promoting growth and development early in life in order to achieve reproduction, but later in life, in a futile attempt to maintain reproduction, become dysregulated and drive senescence.

2,3,7,8-Tetrachlorodibenzo-p-dioxin blocks androgen-dependent cell proliferation of LNCaP cells through modulation of pRB phosphorylation.

Cell-cycle regulatory events associated with inhibition of androgen-dependent cell proliferation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied in the human-derived LNCaP cell line. TCDD blocked the G(1) to S transition of LNCaP cells synchronized in G(0)/G(1) when these cells were induced to reinitiate cell-cycle progression by dihydrotestosterone (DHT). Western blot analyses of these cells revealed altered expression levels of G(1) regulatory proteins, including increases in hypophosphorylated retinoblastoma protein and concomitant decreases in cyclin D1. p21(WAF1/CIP1), which is involved in the assembly of cyclin D1/cyclin-dependent kinase-4 complexes, was increased by DHT or TCDD when each compound was administered singly but was reduced to background levels in cells simultaneously treated with DHT and TCDD. Reporter gene assays revealed the presence of several Ah receptor response-element motifs in the promoter and first intron of the p21(WAF1/CIP1) gene that respond to TCDD-mediated Ah receptor activation independently of p53. Transcription studies showed that activation of aryl hydrocarbon receptor blocks androgen-dependent gene induction in LNCaP cells as well as in African green monkey CV-1 cells. These data point to at least two mechanisms whereby TCDD blocks androgen receptor function: 1) by blocking androgen-induced cell proliferation through modulation of the expression and activities of regulatory proteins controlling cell-cycle progression; and 2) by squelching androgen receptor-mediated gene transcription through receptor cross-talk, possibly involving competition for coregulators or by direct protein interaction.

Modulations of critical cell cycle regulatory events during chemoprevention of ultraviolet B-mediated responses by resveratrol in SKH-1 hairless mouse skin.

Multiple exposures to solar ultraviolet (UV) radiation cause critical damages that may lead to the development of several cutaneous disorders including skin cancer, the most frequently diagnosed malignancy in the USA. Therefore, efforts are needed to: (i) study the mechanism(s) of UV-mediated cutaneous damages, and (ii) design novel approaches for the management of skin cancer. 'Chemoprevention' via plant-based agents may be a useful approach for the management of neoplasia. Here, we evaluated the involvement of cell cycle regulatory molecules during resveratrol-mediated protection from multiple exposures of UVB (180 mJ/cm(2); on alternate days x 7 exposures) radiations in the SKH-1 hairless mouse skin. Resveratrol was topically applied on the skin of SKH-1 hairless mice at a dose of 10 micromol/mouse (in 0.2 ml acetone; 30 min prior to each UVB exposure). Studies were performed at 24 h following the last UVB exposure. Topical application of resveratrol resulted in significant decrease in UVB-induced bi-fold skin thickness, hyperplasia, and infiltration of leukocytes. The data from immunoblot and/or immunohistochemical analyses revealed that multiple exposure to UVB radiations causes significant upregulation in: (i) proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation, and (ii) cyclin-dependent kinase (cdk)-2, -4 and -6, cyclin-D1, and cyclin-D2. Resveratrol treatment resulted in significant downregulation in UV-mediated increases in these critical cell cycle regulatory proteins. An interesting observation of this study was that resveratrol treatment resulted in a further stimulation of UVB-mediated increases in cyclin kinase inhibitor WAF1/p21 and tumor suppressor p53. Further, resveratrol was also found to cause significant decreases in UVB-mediated upregulation of: (i) the mitogen-activated protein kinase kinase, and (ii) the 42 kDa isotype of mitogen-activated protein kinase (MAPK). Thus, our data suggested that the antiproliferative effects of resveratrol might be mediated via modulation in the expression and function of cell cycle regulatory proteins cyclin-D1 and -D2, cdk-2, -4 and -6, and WAF1/p21. Our data further suggest that the modulation of cki-cyclin-cdk network by resveratrol may be associated with inhibition of the MAPK pathway. We suggest that resveratrol may be useful for the prevention of UVB-mediated cutaneous damages including skin cancer.

Ago1 and Dcr1, Two Core Components of the RNA Interference Pathway, Functionally Diverge from Rdp1 in Regulating Cell Cycle Events inSchizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1+, dcr1+, and rdp1+, have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1+ and dcr1+ but not rdp1+, exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1+ and dcr1+ are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1+ is dispensable for this process, the functions of ago1+ and dcr1+ in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1+ is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1+ function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.

Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitor adaphostin proceeds through a RAF-1/MEK/ERK- and AKT-dependent process.

Effects of the tyrphostin tyrosine kinase inhibitor adaphostin (NSC 680410) have been examined in human leukemia cells (Jurkat, U937) in relation to mitochondrial events, apoptosis, and perturbations in signaling and cell cycle regulatory events. Exposure of cells to adaphostin concentrations > or =0.75 microM for intervals > or =6 h resulted in a pronounced release of cytochrome c and AIF, activation of caspase-9, -8, and -3, and apoptosis. These events were accompanied by the caspase-independent downregulation of Raf-1, inactivation of MEK1/2, ERK, Akt, p70S6K, dephosphorylation of GSK-3, and activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK. Adaphostin also induced cleavage and dephosphorylation of pRb on CDK2- and CDK4-specific sites, as well as the caspase-dependent downregulation of cyclin D1. Inducible expression of a constitutively active MEK1 construct markedly diminished adaphostin-induced cytochrome c and AIF release, JNK activation, and apoptosis in Jurkat cells. Ectopic expression of Raf-1 or constitutively activated (myristolated) Akt also significantly attenuated adaphostin-induced apoptosis, but protection was less than that conferred by enforced activation of MEK. Lastly, antioxidants (e.g., L-N-acetylcysteine; L-NAC) opposed adaphostin-mediated mitochondrial dysfunction, Raf-1/MEK/ERK downregulation, JNK activation, and apoptosis. However, in contrast to L-NAC, enforced activation of MEK failed to block adaphostin-mediated ROS generation. Together, these findings demonstrate that the tyrphostin adaphostin induces multiple perturbations in signal transduction pathways in human leukemia cells, particularly inactivation of the cytoprotective Raf-1/MEK/ERK and Akt cascades, that culminate in mitochondrial injury, caspase activation, and apoptosis. They also suggest that adaphostin-related oxidative stress acts upstream of perturbations in these signaling pathways to trigger the cell death process.

The dynamics of cell cycle regulation.

Major events of the cell cycle--DNA synthesis, mitosis and cell division-are regulated by a complex network of protein interactions that control the activities of cyclin-dependent kinases. The network can be modeled by a set of nonlinear differential equations and its behavior predicted by numerical simulation. Computer simulations are necessary for detailed quantitative comparisons between theory and experiment, but they give little insight into the qualitative dynamics of the control system and how molecular interactions determine the fundamental physiological properties of cell replication. To that end, bifurcation diagrams are a useful analytical tool, providing new views of the dynamical organization of the cell cycle, the role of checkpoints in assuring the integrity of the genome, and the abnormal regulation of cell cycle events in mutants. These claims are demonstrated by an analysis of cell cycle regulation in fission yeast.

UCN-01 (7-Hydroxystauorsporine) Blocks PMA-Induced Maturation and Reciprocally Promotes Apoptosis in Human Myelomonocytic Leukemia Cells (U937)

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21CIP1 induction, p27KIP1 cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21CIP1 mRNA antisense construct, suggesting that p21CIP1 expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program. Key Words:PMA, UCN-01, Differentiation, Apoptosis