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.

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.

Suppression of Ras-induced transformation of NIH 3T3 cells by activated G alpha s.

Conversion of external signals into proliferative responses may be mediated by interactions between signaling pathways that control cell proliferation. Interactions between G alpha s, the alpha subunit of the heterotrimeric guanine nucleotide binding protein that stimulates adenylyl cyclase, and Ras, an important element in growth factor signaling, were studied. Expression of activated G alpha s in NIH 3T3 cells increased intracellular concentrations of adenosine 3',5'-monophosphate (cAMP) and inhibited H-Ras-stimulated DNA synthesis and mitogen-activated protein kinase activity. Activated G alpha s and 8-Br-cAMP suppressed H-Ras-induced transformation of NIH 3T3 cells. Apparently, G alpha s inhibits proliferative signals from Ras by stimulating cAMP production and activating protein kinase A.

Emergent properties of networks of biological signaling pathways.

Many distinct signaling pathways allow the cell to receive, process, and respond to information. Often, components of different pathways interact, resulting in signaling networks. Biochemical signaling networks were constructed with experimentally obtained constants and analyzed by computational methods to understand their role in complex biological processes. These networks exhibit emergent properties such as integration of signals across multiple time scales, generation of distinct outputs depending on input strength and duration, and self-sustaining feedback loops. Feedback can result in bistable behavior with discrete steady-state activities, well-defined input thresholds for transition between states and prolonged signal output, and signal modulation in response to transient stimuli. These properties of signaling networks raise the possibility that information for "learned behavior" of biological systems may be stored within intracellular biochemical reactions that comprise signaling pathways.

Stat3-mediated transformation of NIH-3T3 cells by the constitutively active Q205L Galphao protein.

Expression of Q205L Galphao (Galphao*), an alpha subunit of heterotrimeric guanine nucleotide-binding proteins (G proteins) that lacks guanosine triphosphatase (GTPase) activity in NIH-3T3 cells, results in transformation. Expression of Galphao* in NIH-3T3 cells activated signal transducer and activator of transcription 3 (Stat3) but not mitogen-activated protein (MAP) kinases 1 or 2. Coexpression of dominant negative Stat3 inhibited Galphao*-induced transformation of NIH-3T3 cells and activation of endogenous Stat3. Furthermore, Galphao* expression increased activity of the tyrosine kinase c-Src, and the Galphao*-induced activation of Stat3 was blocked by expression of Csk (carboxyl-terminal Src kinase), which inactivates c-Src. The results indicate that Stat3 can function as a downstream effector for Galphao* and mediate its biological effects.

Complexity in biological signaling systems.

Biological signaling pathways interact with one another to form complex networks. Complexity arises from the large number of components, many with isoforms that have partially overlapping functions; from the connections among components; and from the spatial relationship between components. The origins of the complex behavior of signaling networks and analytical approaches to deal with the emergent complexity are discussed here.

Resolution of a signal transfer region from a general binding domain in gbeta for stimulation of phospholipase C-beta2.

Signaling by guanine nucleotide-binding proteins (G proteins) involves sequential protein-protein interactions. G protein-betagamma subunit (Gbetagamma) interactions with phospholipase C-beta2 (PLC-beta2) were studied to determine if all Gbeta contacts are required for signaling. A peptide encoding Gbeta amino acid residues 86 to 105 stimulated PLC-beta2. Six residues (96 to 101) within this sequence could transfer signals and thus constitute a core signal transfer region. Another peptide, encoding Gbeta amino acid residues 115 to 135, did not substantially stimulate PLC-beta2 by itself but inhibited Gbetagamma stimulation, indicating that residues 115 to 135 constitute a general binding domain. Resolution of signal transfer regions from general binding domains indicates that all protein-protein contacts are not required for signal transfer and that it may be feasible to synthesize agonists and antagonists that regulate intracellular signal flow.

Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP.

Long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse involves interacting signaling components, including calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) pathways. Postsynaptic injection of thiophosphorylated inhibitor-1 protein, a specific inhibitor of protein phosphatase-1 (PP1), substituted for cAMP pathway activation in LTP. Stimulation that induced LTP triggered cAMP-dependent phosphorylation of endogenous inhibitor-1 and a decrease in PP1 activity. This stimulation also increased phosphorylation of CaMKII at Thr286 and Ca2+-independent CaMKII activity in a cAMP-dependent manner. The blockade of LTP by a CaMKII inhibitor was not overcome by thiophosphorylated inhibitor-1. Thus, the cAMP pathway uses PP1 to gate CaMKII signaling in LTP.

Molecular basis for interactions of G protein betagamma subunits with effectors.

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.

Postsynaptic cAMP pathway gates early LTP in hippocampal CA1 region.

The role of the cAMP pathway in LTP was studied in the CA1 region of hippocampus. Widely spaced trains of high frequency stimulation generated cAMP postsynaptically via NMDA receptors and calmodulin, consistent with the Ca2+/calmodulin-mediated stimulation of postsynaptic adenylyl cyclase. The early phase of LTP produced by the same pattern of high frequency stimulation was dependent on postsynaptic cAMP. However, synaptic transmission was not increased by postsynaptic application of cAMP. Early LTP became cAMP-independent when protein phosphatase inhibitors were injected postsynaptically. These observations indicate that in early LTP the cAMP signaling pathway, instead of transmitting signals for the generation of LTP, gates LTP through postsynaptic protein phosphatases.

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.

Signal recognition and integration by Gs-stimulated adenylyl cyclases.

Six Gs-stimulated adenylyl cyclases have been cloned. Two additional forms have been identified as partial cDNAs. These adenylyl cyclases have distinct functional properties and are differentially regulated by protein kinases. The adenylyl cyclases have distinct patterns of distribution in peripheral tissues and various brain regions. The unique functional characteristics of the members of this effector family may allow each cell type and/or brain region to customize the signal-recognition and integrative properties of its cAMP-generation system by varying the ratios of the various adenylyl cyclases.

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.

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.

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.

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.

Long-term potentiation induced by theta frequency stimulation is regulated by a protein phosphatase-1-operated gate.

Long-term potentiation (LTP) can be induced in the Schaffer collateral-->CA1 synapse of hippocampus by stimulation in the theta frequency range (5-12 Hz), an effect that depends on activation of the cAMP pathway. We investigated the mechanisms of the cAMP contribution to this form of LTP in the rat hippocampal slice preparation. theta pulse stimulation (TPS; 150 stimuli at 10 Hz) by itself did not induce LTP, but the addition of either the beta-adrenergic agonist isoproterenol or the cAMP analog 8-bromo-cAMP (8-Br-cAMP) enabled TPS-induced LTP. The isoproterenol effect was blocked by postsynaptic inhibition of cAMP-dependent protein kinase. Several lines of evidence indicated that cAMP enabled LTP by blocking postsynaptic protein phosphatase-1 (PP1). Activators of the cAMP pathway reduced PP1 activity in the CA1 region and increased the active form of inhibitor-1, an endogenous inhibitor of PP1. Postsynaptic injection of activated inhibitor-1 mimicked the LTP-enabling effect of cAMP pathway stimulation. TPS evoked complex spiking when isoproterenol was present. However, complex spiking was not sufficient to enable TPS-induced LTP, which additionally required the inhibition of postsynaptic PP1. PP1 inhibition seems to promote the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII), because (1) a CaMKII inhibitor blocked the induction of LTP by TPS paired with either isoproterenol or activated inhibitor-1 and (2) CaMKII in area CA1 was activated by the combination of TPS and 8-Br-cAMP but not by either stimulus alone. These results indicate that the cAMP pathway enables TPS-induced LTP by inhibiting PP1, thereby enhancing Ca(2+)-independent CaMKII activity.

Heterologous desensitization of the liver adenylyl cyclase: analysis of the role of G-proteins.

Glucagon treatment of chick hepatocytes in primary culture results in a rapid 20-30% decrease in NaF-stimulated adenylyl cyclase activity. This heterologous desensitization is rapidly reversible upon removal of hormone from the culture medium. This decrease in NaF-stimulated adenylyl cyclase activity agrees well with the decrease in Gs activity, as assessed by reconstitution of the cyc- S49 cell adenylyl cyclase. Recovery from heterologous desensitization within 30 min results in restoration of Gs activity to control levels. No changes in Gs-alpha protein levels could be determined by immunoblotting using an alpha s-specific antiserum during heterologous desensitization. Similarly, no change in the beta gamma-subunits or Gi-alpha-subunits were detected by immunoblotting analysis using subunit-specific antisera. Pretreatment of cells with pertussis toxin did not block the onset of either heterologous or homologous desensitization, indicating the lack of involvement of Gi in glucagon-induced desensitization. cAMP-dependent phosphorylation of G-proteins does not account for heterologous desensitization, since none of the G-protein subunits appears to be phosphorylated even in vitro. However, Gs as the locus of change in heterologous desensitization was confirmed by the observation that addition of purified Gs to desensitized cell membranes results in enhancement of NaF-stimulated adenylyl cyclase activity. From these data we conclude that postreceptor heterologous desensitization in chick hepatocytes results from a reversible decrease in Gs activity in the cell surface membrane.