Cell shape regulates subcellular organelle location to control early Ca2+ signal dynamics in vascular smooth muscle cells.

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cell's functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances, and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLCß pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm, and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

Procedures for risk-stratification of lung cancer using buccal nanocytology.

Lung cancer is the leading cause of cancer deaths in the U.S. with survival dramatically depending on stage at diagnosis. We had earlier reported that nanocytology of buccal cells can accurately risk-stratify smokers for the presence of early and late-stage lung cancer. To translate the technique into clinical practice, standardization of operating procedures is necessary to consistently yield precise and repeatable results. Here, we develop and validate simple, robust, and easily implementable procedures for specimen collection, processing, etc. in addition to a commercially-viable instrument prototype. Results of this work enable translation of the technology from academic lab to physicians' office.

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1.

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H2O2). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H2O2-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.

Computation as the mechanistic bridge between precision medicine and systems therapeutics.

Over the past 50 years, like molecular cell biology, medicine and pharmacology have been driven by a reductionist approach. The focus on individual genes and cellular components as disease loci and drug targets has been a necessary step in understanding the basic mechanisms underlying tissue/organ physiology and drug action. Recent progress in genomics and proteomics, as well as advances in other technologies that enable large-scale data gathering and computational approaches, is providing new knowledge of both normal and disease states. Systems-biology approaches enable integration of knowledge from different types of data for precision medicine and systems therapeutics. In this review, we describe recent studies that contribute to these emerging fields and discuss how together these fields can lead to a mechanism-based therapy for individual patients.

Training in systems pharmacology: predoctoral program in pharmacology and systems biology at Mount Sinai School of Medicine.

Our recently developed predoctoral training program in pharmacology and systems biology prepares students to become experts in systems-level models of disease that identify therapeutic targets and predict adverse effects or new uses of existing therapeutics. Multiple computational modeling modes are introduced throughout a curriculum that integrates basic cell and molecular sciences with the physiology and pathophysiology of disease states. Problem-based learning exercises enable students from different experimental and computational backgrounds to design experiments and interpret data quantitatively.

Validation of the pedigree assessment tool (PAT) in families with BRCA1 and BRCA2 mutations.

BACKGROUND: The lifetime risk of breast cancer (BC) in patients with hereditary breast cancer syndromes is as high as 80%. The Pedigree Assessment Tool (PAT) is a scoring system to aid in identifying these patients. This validation study compares the PAT to BRCA gene mutation probability models in predicting suitability for genetic referral. METHODS: Retrospective review identified subjects undergoing genetic counseling and BRCA testing from 2001 to 2008 at two institutions. PAT score and BRCA mutation probabilities were calculated using Myriad II and Penn II models. Comparisons were made between models in ability to discriminate patients appropriate for genetic evaluation based on accuracy in predicting a positive test result. RESULTS: Records evaluated represent 520 families. BRCA testing revealed 146 mutation-positive and 374 mutation-negative families. c-Statistic analysis was used to compare the discriminating ability of the models to correctly assign families as mutation (+) and (-). Both the PAT and Penn II model outperformed the Myriad II model. Using a threshold PAT score >or=8 and mutation probability >or=10% to assign families as mutation (+) versus (-), sensitivity, specificity, and positive and negative predictive values were calculated for each model. The PAT was more sensitive than the Myriad II model and more specific than the Penn II model. CONCLUSIONS: In overall performance, the PAT is at least comparable to the Myriad II and Penn II models in screening women appropriate for genetic referral. Simplicity and identification of families with non-BRCA hereditary BC syndromes suggest that the PAT is better suited for BC risk screening.

Cell spreading as a hydrodynamic process.

Many cell types have the ability to move themselves by crawling on extra-cellular matrices. Although cell motility is governed by actin and myosin filament assembly, the pattern of the movement follows the physical properties of the network ensemble average. The first step of motility, cell spreading on matrix substrates, involves a transition from round cells in suspension to polarized cells on substrates. Here we show that the spreading dynamics on 2D surfaces can be described as a hydrodynamic process. In particular, we show that the transition from isotropic spreading at early time to anisotropic spreading is reminiscent of the fingering instability observed in many spreading fluids. During cell spreading, the main driving force is the polymerization of actin filaments that push the membrane forward. From the equilibrium between the membrane force and the cytoskeleton, we derive a first order expression of the polymerization stress that reproduces the observed behavior. Our model also allows an interpretation of the effects of pharmacological agents altering the polymerization of actin. In particular we describe the influence of Cytochalasin D on the nucleation of the fingering instability.

Effects of activating NK cell receptor expression and NK cell reconstitution on the outcomes of unrelated donor hematopoietic cell transplantation for hematologic malignancies.

Inhibitory NK cell receptors are recognized as important determinants of NK cell activity in hematopoietic cell transplantation (HCT). The role of activating receptors and their acquisition after HCT is less certain. Therefore, we comprehensively evaluated both inhibitory and activating receptors in 59 patients receiving unrelated donor HCT. NK cell numbers normalized quickly relative to B and T cells; however, the expression of both inhibitory and activating isoforms of killer immunoglobulin-like receptors (KIRs) was delayed. Most NK cells expressed an immature phenotype during the first 6 months post-HCT; however, we found high expression of activating NKp46 and NKp44 natural cytotoxicity receptors (NCRs), and cytotoxicity was preserved. Early reconstituting NK cells from unmanipulated grafts showed lower cytotoxicity than those from T-cell-depleted grafts. Differences in NK cell reconstitution had significant effects on clinical outcomes. Patients whose NK cells reconstituted earlier had better survival and lower relapse rates. The best survival group was recipients who possessed HLA-C2 but their donor lacked the cognate-activating KIR2DS1. Collectively, our data underscore the clinical relevance of reconstituting NK cells and their activating KIRs and NCRs. In addition to NK cell quantification and genotyping, comprehensive assessment of NK cell functions and phenotypes, including activating receptors, is essential.

Proximity of intracellular regulatory networks to monotone systems.

Networks that contain only sign-consistent loops, such as positive feedforward and feedback loops, function as monotone systems. Simulated using differential equations, monotone systems display well-ordered behaviour that excludes the possibility for chaotic dynamics. Perturbations of such systems have unambiguous global effects and a predictability characteristic that confers robustness and adaptability. The authors assess whether the topology of biological regulatory networks is similar to the topology of monotone systems. For this, three intracellular regulatory networks are analysed where links are specified for the directionality and the effects of interactions. These networks were assembled from functional studies in the experimental literature. It is found that the three biological networks contain far more positive 'sign-consistent' feedback and feedforward loops than negative loops. Negative loops can be 'eliminated' from the real networks by the removal of fewer links as compared with the corresponding shuffled networks. The abundance of positive feedforward and feedback loops in real networks emerges from the presence of hubs that are enriched with either negative or positive links. These observations suggest that intracellular regulatory networks are 'close-to-monotone', a characteristic that could contribute to the dynamical stability observed in cellular behaviour.

A large-scale method for the selective depletion of alphabeta T lymphocytes from PBSC for allogeneic transplantation.

BACKGROUND: We sought to develop a method for the clinical large-scale depletion of alphabeta T lymphocytes from mobilized peripheral stem cells, which would allow the allogeneic transplantation of a graft enriched for stem cells, natural killer (NK) cells and gammadelta T lymphocytes. METHODS: Therefore, we obtained mononuclear cells from either mobilized or non-mobilized healthy adult volunteer donors and incubated the cells with a biotinylated anti-alphabeta T-cell Ab and subsequently with an anti-biotin Ab conjugated with magnetic microbeads. The depletion was then performed using a CliniMACS device. RESULTS: The median T-cell depletion was 3.9 log (range 3.5-4.1 log). The recovery of the gammadelta and NK cells was 92% and 80%, respectively. The recovery of CD34+ stem cells from the mobilized donors was 66%. DISCUSSION: This method had no negative influence on the in vitro colony formation of stem cells, and transplantation of alphabeta-depleted cells into NOD-SCID IL-2 common gamma chain knockout (NOD-scid IL2r (null)) mice resulted in a rapid engraftment of human myeloid and lymphoid cells. This method will allow large-scale depletion of alphabeta T cells from mobilized peripheral blood in clinical trials.

Inhibitory KIR-HLA receptor-ligand mismatch in autologous haematopoietic stem cell transplantation for solid tumour and lymphoma.

Genes that encode killer Ig-like receptors (KIRs) and their HLA class I ligands segregate independently; thus, some individuals may express an inhibitory KIR gene but not its cognate ligand. We hypothesised that these patients with KIR-HLA receptor-ligand mismatch have a low risk of relapse after an autologous haematopoietic stem cell transplantation (HCT). Sixteen consecutive patients with lymphoma or solid tumour were enrolled onto a prospective study. They received high-dose busulphan and melphalan followed by autologous CD133(+) HCT. We found that 8 of the 16 patients experienced disease progression after autologous HCT, including 5 of the 6 patients (83%) with no inhibitory KIR-HLA mismatch and 3 of the 6 patients (50%) with 1 mismatched pair; none of the 4 (0%) patients with 2 mismatched pairs experienced disease progression. Survival analyses showed that inhibitory KIR-HLA mismatch was the only significant prognostic factor (P=0.01). The potential applicability of the receptor-ligand mismatch model to autologous HCTs and to patients with lymphoma or solid tumour is clinically significant because of the prevalence of the HCT procedure.

Purification of human natural killer cells using a clinical-scale immunomagnetic method.

BACKGROUND: Infection, graft failure, disease relapse, and GvHD are significant adverse events associated with allogeneic BMT. Although donor leukocyte infusion has been used to prevent or to treat infection, graft failure, and relapse, the potential clinical benefits are often outweighed by the risk of T cell-mediated GvHD. Results from animal studies suggest that donor natural killer (NK) cells may be an ideal cell type for prevention or treatment of these adverse events. We have therefore sought to develop an automated, efficient, and clinical-scale human NK cell-purification method. METHODS: Twelve leukopheresis products were purified for NK cells using a two-step immunomagnetic method. CD3(+) cells were first depleted from the apheresis products. CD56(+) cells were then enriched from the CD3(+) cell-depleted products. RESULTS: The median percentage of CD3(-)CD56(+) NK cells in the final products was 91.0%, and the median recovery was 48.7%. The median depletion for CD3(+)CD56(-) T cells was 5.3 log. Natural cytotoxicity of the purified cells was approximately five-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL2. DISCUSSION: We described a large-scale purification method for automated, efficient, and rapid isolation of human NK cells that yielded minimal contamination with T cells or B cells. These purified NK cells may be expedient for preclinical and clinical uses.

The signal transfer regions of G alpha(s).

The crystal structure of soluble functional fragments of adenylyl cyclase complexed with G alpha(s) and forskolin, shows three regions of G alpha(s) in direct contact with adenylyl cyclase. The functions of these three regions are not known. We tested synthetic peptides encoding these regions of G alpha(s) on the activities of full-length adenylyl cyclases 2 and 6. A peptide encoding the Switch II region (amino acids 222-247) stimulated both adenylyl cyclases 2- to 3-fold. Forskolin synergized the stimulation. Addition of peptides in the presence of activated G alpha(s) partially inhibited G alpha(s) stimulation. Corresponding Switch II region peptides from G alpha(q) and G alpha(i) did not stimulate adenylyl cyclase. A peptide encoding the Switch I region (amino acids 199-216) also stimulated AC2 and AC6. The stimulatory effects of the two peptides at saturating concentrations were non-additive. A peptide encoding the third contact region (amino acids 268-286) located in the alpha 3-beta 5 region, inhibits basal, forskolin, and G alpha(s)-stimulated enzymatic activities. Since this region in G alpha(s) interacts with both the central cytoplasmic loop and C-terminal tail of adenylyl cyclases this peptide may be involved in blocking interactions between these two domains. These functional data in conjunction with the available structural information suggest that G alpha(s) activation of adenylyl cyclase is a complex event where the alpha 3-beta 5 loop of G alpha(s) may bring together the central cytoplasmic loop and C-terminal tail of adenylyl cyclase thus allowing the Switch I and Switch II regions to function as signal transfer regions to activate adenylyl cyclase.

Mitogen-activated protein kinase regulates early phosphorylation and delayed expression of Ca2+/calmodulin-dependent protein kinase II in long-term potentiation.

Activation of mitogen-activated protein kinase (MAPK) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are required for numerous forms of neuronal plasticity, including long-term potentiation (LTP). We induced LTP in rat hippocampal area CA1 using theta-pulse stimulation (TPS) paired with beta-adrenergic receptor activation [isoproterenol (ISO)], a protocol that may be particularly relevant to normal patterns of hippocampal activity during learning. This stimulation resulted in a transient phosphorylation of p42 MAPK, and the resulting LTP was MAPK dependent. In addition, CaMKII was regulated in two, temporally distinct ways after TPS-ISO: a transient rise in the fraction of phosphorylated CaMKII and a subsequent persistent increase in CaMKII expression. The increases in MAPK and CaMKII phosphorylation were strongly colocalized in the dendrites and cell bodies of CA1 pyramidal cells, and both the transient phosphorylation and delayed expression of CaMKII were prevented by inhibiting p42/p44 MAPK. These results establish a novel bimodal regulation of CaMKII by MAPK, which may contribute to both post-translational modification and increased gene expression.

Functional modules in biological signalling networks.

Signalling pathways carry information from the outside of the cell to cellular machinery capable of producing biochemical or physiological responses. Although linear signalling plays an important role in biological regulation, signalling pathways are often interconnected to form networks. We have used computational analysis to study emergent properties of simple networks that consist of up to four pathways, We find that when one pathway gates signal flow through other pathways which produce physiological responses, gating results in signal prolongation such that the signal may be consolidated into a physiological response. When two pathways combine to form a feedback loop such feedback loops can exhibit bistability. Negative regulators of the loop can serve as the locus for flexibility whereby the system has the capability of switching states or functioning as a proportional read-out system. Networks where bistable feedback loops are connected to gates can lead to persistent signal activation at distal locations. These emergent properties indicate system analysis of signalling networks may be useful in understanding higher-order biological functions.

Modular design of Gbeta as the basis for reversible specificity in effector stimulation.

The G protein Gbetagamma subunit complex stimulates effectors by direct interactions utilizing extensive Gbeta regions over the surface of its propeller structure that faces the Galpha subunit. Our previous experiments have shown the resolved functions of signal transfer and general binding for Gbeta regions involved in stimulation of the effector phospholipase C-beta2, PLC-beta2, within the region Gbeta-(86-135), which comprises three beta strands arranged in a structurally contiguous fashion (Buck, E., Li, J., Chen, Y., Weng, G., Sacarlata, S., and Iyengar, R. (1999) Science 283, 1332-1335). This raises an important question as to why mutagenesis studies indicate that an extensive set of sites all over the Gbeta propeller structure and outside the 86-135 region are involved in Gbeta regulation of PLC-beta2. Using peptides to define functions of these Gbeta regions, we find that Gbeta signaling to PLC-beta2 relies on a collection of modular signal transfer and general binding units, each with lower apparent affinity relative to Gbetagamma-PLC interactions. Gbeta-(42-54) functions as a signal transfer region, Gbeta-(228-249) and Gbeta-(321-340) function in general binding, and Gbeta-(64-84) and Gbeta-(300-313) seem to play a structural role rather than a direct contact with the effector. A substitution within the Gbeta-(42-54) signal transfer region that increases the K(act) of this peptide for PLC-beta2 is accompanied by an increase in the observed maximal extent of signal transfer. We conclude that the lower K(act) for individual signal transfer regions may result in a decrease in the maximal effect of signal transfer. The spatial resolution of the signal transfer and general binding regions over a wide surface of Gbeta allow geometrical constraints to achieve specificity even with relatively low affinity interactions.

Use of a modified ornithine decarboxylase promoter to achieve efficient c-MYC- or N-MYC-regulated protein expression.

One of the advantages of viral-directed enzyme prodrug therapy (VDEPT) is its potential for tumor-specific cytotoxicity. However, the viruses used to deliver cDNAs encoding prodrug-activating enzymes transduce normal cells as well as tumor cells, and several approaches to achieve tumor-specific expression of the delivered cDNAs are being investigated. One such approach is to regulate transcription of the prodrug-activating enzyme with a promoter that is preferentially activated by tumor cells. Published data suggest that the most promising transcription factor/promoter/enhancer combinations are those activated by a tumor-specific transcription factor to retain tumor cell specificity but that are equal in strength to nonspecific viral promoters in their ability to up-regulate target cDNAs. This report identifies MYC-responsive, modified ornithine decarboxylase (ODC) promoter/enhancer sequences that up-regulate target protein expression in tumor cells overexpressing either N-MYC or c-MYC protein. The most efficient of the four constructs assessed contained six additional CACGTG MYC binding sites 5' to the endogenous ODC promoter (R6ODC). Reporter assays with this chimeric promoter/enhancer regulating expression of chloramphenicol acetyltransferase demonstrated 50-250-fold more activity in MYC-expressing cells compared with similar assays with promoterless plasmids. The R6ODC regulatory sequence was approximately equivalent to the CMV promoter in inducing expression of the neomycin resistance gene in c-MYC-expressing SW480 and HT-29 colon carcinoma cells and in N-MYC-expressing NB-1691 neuroblastoma cells. The modified ODC promoter may, therefore, be useful in achieving tissue-specific expression of target proteins in tumor cells that overexpress c- or N-MYC.

G protein coupled receptor signaling through the Src and Stat3 pathway: role in proliferation and transformation.

Extracellular signals when routed through signaling pathways that use heterotrimeric G proteins can engage multiple signaling pathways leading to diverse biological consequences. One locus at which signal sorting occurs is at the level of G proteins. G protein alpha-subunits appear to be capable of interacting with different effectors leading to engagement of distinct signaling pathways. Regulation of different pathways in turn leads to different biological outcomes. The process of neoplastic transformation is controlled to a large extent through the activation and inhibition of signaling pathways. Signaling pathways such as the Ras-MAPK, v-Src-Stat3 pathways are activated in the process of transformation. Expression of activated Galpha subunits have been shown to cause transformation of cells. While activation of the MAPK 1,2 pathway by various Galpha subunits has been reported for several years, recent studies show the activation and involvement of Src and Stat3 pathways in Galphao and Galphai mediated transformation of cells. Recent studies also suggest that both Galphai and Galphas may be able to interact with and activate Src. The activation of Src and Stat3 by G proteins has also been demonstrated by ligand-induced activation of G protein receptors. So increasingly it is becoming clear that the Src and Stat3 pathways are potential effectors for G proteins and that they may play a role in G protein function.

Expression of Q227L-Galpha(s) inhibits intimal vessel wall hyperplasia after balloon injury.

Interaction between signaling pathways regulates many cellular functions, including proliferation. The Galpha(s)/cAMP pathway is known to inhibit signal flow from receptor tyrosine kinases to mitogen-activated protein kinase (MAPK)-1,2 and, thus, inhibit proliferation. Elevation of cAMP or adenovirus-directed expression of mutant (Q227L)-Galpha(s) (alpha(s)*) inhibits the proliferation of rat vascular smooth muscle cells (VSMCs) in culture. Platelet-derived growth factor (PDGF) stimulated MAPK activation and DNA synthesis was also blocked by expression of alpha(s)*. However, it is not known whether such mechanisms are operative in vivo. Proliferation of vascular smooth muscle cells in vivo was induced by balloon injury of carotid arteries in the rat. Recombinant adenovirus encoding beta-galactosidase (beta-gal) or alpha(s)* was applied to arterial segments injured by the balloon catheters. The alpha(s)*-treated vessels showed decreased phospho-MAPK staining in the intima as compared with beta-gal-treated vessels. Application of alpha(s)*, but not beta-gal containing adenovirus, inhibited formation of neointima by 50%. No change was observed in total vessel diameter or in the media or adventitia. These results suggest that the interaction between the Galpha(s) and MAPK pathways can regulate proliferation in vivo and that targeted expression of activated Galpha(s) may have therapeutic potential for the treatment of vascular pathophysiologies that arise from intimal hyperplasia.