Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

A reduced model clarifies the role of feedback loops and time delays in the Drosophila circadian oscillator.

Although several detailed models of molecular processes essential for circadian oscillations have been developed, their complexity makes intuitive understanding of the oscillation mechanism difficult. The goal of the present study was to reduce a previously developed, detailed model to a minimal representation of the transcriptional regulation essential for circadian rhythmicity in Drosophila. The reduced model contains only two differential equations, each with time delays. A negative feedback loop is included, in which PER protein represses per transcription by binding the dCLOCK transcription factor. A positive feedback loop is also included, in which dCLOCK indirectly enhances its own formation. The model simulated circadian oscillations, light entrainment, and a phase-response curve with qualitative similarities to experiment. Time delays were found to be essential for simulation of circadian oscillations with this model. To examine the robustness of the simplified model to fluctuations in molecule numbers, a stochastic variant was constructed. Robust circadian oscillations and entrainment to light pulses were simulated with fewer than 80 molecules of each gene product present on average. Circadian oscillations persisted when the positive feedback loop was removed. Moreover, elimination of positive feedback did not decrease the robustness of oscillations to stochastic fluctuations or to variations in parameter values. Such reduced models can aid understanding of the oscillation mechanisms in Drosophila and in other organisms in which feedback regulation of transcription may play an important role.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Interlinked dual-time feedback loops can enhance robustness to stochasticity and persistence of memory.

Multiple interlinked positive feedback loops shape the stimulus responses of various biochemical systems, such as the cell cycle or intracellular Ca2+ release. Recent studies with simplified models have identified two advantages of coupling fast and slow feedback loops. This dual-time structure enables a fast response while enhancing resistances of responses and bistability to stimulus noise. We now find that (1) the dual-time structure similarly confers resistance to internal noise due to molecule number fluctuations, and (2) model variants with altered coupling, which better represent some specific biochemical systems, share all the above advantages. We also develop a similar bistable model with coupling of a fast autoactivation loop to a slow loop. This model's topology was suggested by positive feedback proposed to play a role in long-term synaptic potentiation (LTP). The advantages of fast response and noise resistance are also present in this autoactivation model. Empirically, LTP develops resistance to reversal over approximately 1h . The model suggests this resistance may result from increased amounts of synaptic kinases involved in positive feedback.

Simulation of Drosophila circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and CLK.

A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.

MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

INVESTIGATIONS ON THE INTRACELLULAR LOCALIZATION OF PHOSPHATIDYLSERINE SYNTHASE FROM ESCHERICHIA COLI

Phosphatidylserine synthase catalyzes the committed step in the synthesis of the major lipid of Escherichia coli, phosphatidylethanolamine, and may be involved in regulating the balance of the zwitterionic and anionic phospholipids in the membrane. Unlike the other enzymes involved in the biosynthesis of phospholipids in E. coli, phosphatidylserine synthase is not membrane associated but seems to have a high affinity for the ribosomal fraction of cells broken by various methods. Investigations on the enzyme in cell free extracts using glycerol gradient centrifugation revealed that the binding of the synthase to ribosomes may be prevented by the presence of highly basic compounds such as spermidine and by the presence of detergent-lipid substrate micelles under assay conditions. Thus phosphatidylserine synthase may not be ribosome associated under physiological conditions but associated with its membrane bound substrate (Louie and Dowhan (1980) J. Biol. Chem. 255, 1124).^ In addition homogeneous enzyme shows many of the properties of a membrane associated protein. It binds nonionic detergent such as Triton X-100, which is also required during purification of the enzyme. Optimal catalytic activity is also dependent on micelle or surface bound substrate. Phosphatidylserine synthase has been synthesized in vitro using a coupled transcription-translation system dependent on the presence of the cloned structural gene. The translation product was found to preferentially associate with the ribosomal fraction even in the presence of added E. coli membranes. Preferential membrane binding could be induced if the membranes were supplemented with the lipid substrate CDP-diacylglycerol. Similar effects were obtained with the acidic lipids phosphatidylglycerol and cardiolipin. On the other hand the zwitterionic lipid phosphatidylethanolamine and the lipid product phosphatidylserine did not cause any detectable membrane association. These results are consistent with the enzyme recognizing membrane bound substrate (Carman and Dowhan (1979) J. Biol. Chem. 254, 8391) and with the lipid charge influencing membrane interaction.^ Phosphatidylserine synthase is at a branch point in lipid metabolism, which may determine the distribution of the zwitterionic and anionic phospholipids in the membrane. The results obtained here indicate phosphatidylserine synthase may play a significant role in membrane lipid biosynthesis by maintaining charge balance of the E. coli membrane. In determining the localization of phosphatidylserine synthase in vitro one may have a better understanding of its function and control in vivo and may also have a better understanding of its role in membrane assembly.^

PIM KINASE INHIBITION: SINGLE AGENT AND COMBINATION APPROACHES IN B-CELL LYMPHOMA

Proviral integration site for Moloney murine leukemia virus (Pim) kinases are Ser/Thr/Tyr kinases. They modulate B-cell development but become oncoproteins and promote cancer development once overexpressed. Containing three isoforms, Pim-1, -2 and -3 are known to phosphorylate various substrates that regulate transcription, translation, cell cycle, and survival pathways in both hematological and solid tumors. Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma. Elevated Pim kinase levels are common in MCL, and it negatively correlates with patient outcome. SGI-1776 is a small molecule inhibitor selective for Pim-1/-3. We hypothesize that SGI-1776 treatment in MCL will inhibit Pim kinase function, and inhibition of downstream substrates phosphorylation will disrupt transcriptional, translational, and cell cycle processes while promoting apoptosis. SGI-1776 treatment induced moderate to high levels of apoptosis in four MCL cell lines (JeKo-1, Mino, SP-53 and Granta-519) and peripheral blood mononuclear cells (PBMCs) from MCL patients. Phosphorylation of transcription and translation regulators, c-Myc and 4E-BP1 declined in both model systems. Additionally, levels of short-lived Mcl-1 mRNA and protein also decreased and correlated with decline of global RNA synthesis. Collectively, our investigations highlight Pim kinases as viable drug targets in MCL and emphasize their roles in transcriptional and translational regulation. We further investigated a combination strategy using SGI-1776 with bendamustine, an FDA-approved DNA-damaging alkylating agent for treating non-Hodgkin’s lymphoma. We hypothesized this combination will enhance SGI-1776-induced transcription and translation inhibition, while promoting bendamustine-triggered DNA damage and inducing additive to synergistic cytotoxicity in B-cell lymphoma. Bendamustine alone resulted in moderate levels of apoptosis induction in MCL cell lines (JeKo-1 and Mino), and in MCL and splenic marginal zone lymphoma (a type of B-cell lymphoma) primary cells. An additive effect in cell killing was observed when combined with SGI-1776. Expectedly, SGI-1776 effectively decreased global RNA and protein synthesis levels, while bendamustine significantly inhibited DNA synthesis and generated DNA damage response. In combination, intensified inhibitory effects in DNA, RNA and protein syntheses were observed. Together, these data suggested feasibility of using Pim kinase inhibitor in combination with chemotherapeutic agents such as bendamustine in B-cell lymphoma, and provided foundation of their mechanism of actions in lymphoma cells.

Structure and biosynthesis of a pyruvate -dependent enzyme---phosphatidylserine decarboxylase from {\it Escherichia coli\/}

Phosphatidylserine decarboxylase of E. coli, a cytoplasmic membrane protein, catalyzes the formation of phosphatidylethanolamine, the principal phospholipid of the organism. The activity of the enzyme is dependent on a covalently bound pyruvate (Satre and Kennedy (1978) J. Biol. Chem. 253, 479-483). This study shows that the enzyme consists of two nonidentical subunits, $\alpha$ (Mr = 7,332) and $\beta$ (Mr = 28,579), with the pyruvate prosthetic group in amide linkage to the amino-terminus of the $\alpha$ subunit. Partial protein sequence and DNA sequence analysis reveal that the two subunits are derived from a proenzyme ($\pi$ subunit, Mr = 35,893) through a post-translational event. During the conversion of the proenzyme to the $\alpha$ and $\beta$ subunits, the peptide bond between Gly253-Ser254 is cleaved, and Ser254 is converted to the pyruvate prosthetic group at the amino-terminus of the $\alpha$ subunit (Li and Dowhan (1988) J. Biol. Chem. 263, 11516-11522).^ The proenzyme cannot be detected in cells carrying either single or multiple copies of the gene (psd), but can be observed in a T7 RNA polymerase/promoter and transcription-translation system. The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. Changing of the Ser254 to cysteine (S254C) or threonine (S254T) slows the cleavage rate dramatically and results in mutants with a half-time for processing of around 2-4 h. Change of the Ser254 to alanine (S254A) blocks the cleavage of the proenzyme. The reduced processing rate with the mutations of the proenzyme is consistent with less of the functional enzyme being made. Mutants S254C and S254T produce $\sim$15% and $\sim$1%, respectively, of the activity of the wild-type allele, but can still complement a temperature-sensitive mutant of the psd locus. Neither detectable activity nor complementation is observed by mutant S254A. These results are consistent with the hydroxyl-group of the Ser254 playing a critical role in the cleavage of the peptide bond Gly253-Ser254 of the pro-phosphatidylserine decarboxylase, and support the mechanism proposed by Snell and co-workers (Recsei and Snell (1984) Annu. Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases. ^

The role of post-translation regulation of Twist expression in the tumor progression of squamous cell carcinoma of head and neck

Squamous cell carcinoma of head and neck (SCCHN) is the tenth most common cancer in the world. Unfortunately, the survival of patients with SCCHN has not improved in the last 40 years. Therefore new targets for therapy are needed, and to this end we are studying signaling pathways activated by IL-6 which we have found stimulates cell migration and soft agar growth in SCCHN. Our data show that IL-6 increases TWIST expression in a transcription-independent mechanism in many SCCHN cell lines. Further investigation reveals TWIST can be phosphorylated upon IL-6 treatment. By computation prediction (http://scansite.mit.edu/motifscan_seq.phtml ), we found that TWIST has a putative phosphorylation site for casein kinase 2 (CK2) suggesting that this kinase could serve as a link between IL-6 stimulation and Twist stability. To test this hypothesis, we used a CK2 inhibitor and shRNA to CK2 and found that these interventions inhibited IL-6 stimulation of TWIST stability. In addition, mutation of the putative CK2 phosphorylation site (S18/S20A) in TWIST decreased the amount of phospho-ATP incorporated by TWIST in an in vitro kinase assay, and altered TWIST stability. In Boyd chamber migration assay and wound-healing assay, the CK2 inhibitor, DMAT, was found to decrease the motility of IL-6 stimulated SCCHN cells and over expression of either a wild-type or the hyperphosphorylated mimicking mutant S18/20D –Twist rather than the hypo-phosphorylated mimicking mutant S18/20A-Twist can promote SCCHN cell motility.To our knowledge, this is the first report to identify the importance of IL-6 stimulated CK2 phosphorylation of TWIST in SCCHN. As CK2 inhibitors are currently under phase I clinical trials, our findings indicate that CK2 may be a viable therapeutic target in SCCHN. Therefore, further pre-clinical studies of this inhibitor are underway.

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^