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Biomedical subjects

C J Marshall

Publications and source records attributed to C J Marshall.

At least 37 records · Page 2Linked to original sources
PubMed1999

An in vitro 96-well plate assay of the mitogen-activated protein kinase cascade.

Mitogen-activated protein (MAP) kinases of the extracellular signal-regulated kinase (ERK) family are activated in response to many growth and differentiation factors as well as some oncogenes. ERK activation follows phosphorylation by a class of specific upstream MAP kinase/ERK kinase (MEK) exemplified by MEK-1. Activated ERKs control many short- and long-term changes in cell function through phosphorylating a number of intracellular target substrates which include stathmin, a phosphoprotein regulating microtubule stability. We report here the development of a simple, 96-well plate, quantitative in vitro assay measuring purified ERK2 catalytic activation by a constitutive MEK-1 mutant (S218E S222E). Enzymatic activity was detected by 33P phosphorylation of purified biotinylated stathmin captured on streptavidin-coated scintillation proximity assay beads which eliminates the need for wash steps. The assay was optimized and the K0.5 value for ATP was found to be 0.9 microM and the Km for stathmin was determined to be 16 microM. The assay was also used to determine IC50 values for the protein kinase inhibitors PD98059 and staurosporine. This simple assay allows several hundred quantitative measurements of MEK1-dependent ERK2 activation to be performed in a day.

Adenosine Triphosphate↗
PubMed1999

Cooperation of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways during granulocyte colony-stimulating factor-induced hemopoietic cell proliferation.

Hemopoietic cytokines such as interleukin-3 and granulocyte colony-stimulating factor (G-CSF) are potent activators of hemopoietic cell growth and strongly induce activation of extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38 mitogen-activated protein (MAP) kinases. However, the role of these kinases is unclear. Using specific chemical inhibitors for MEK and p38, we demonstrate here that both ERK and p38 pathways are critically involved in the transduction of a proliferative signal and cooperate in G-CSF-induced cell proliferation. We show that, like ERK and JNK activation, activation of p38 and its downstream substrate MAP kinase-activated protein kinase 2 by interleukin-3 or G-CSF requires Ras activation. We demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in activation of the p38 pathway: a region within the 100 membrane-proximal amino acids is sufficient to induce low levels of p38 and MAP kinase-activated protein kinase 2 activation, whereas the membrane-distal phosphorylation site Tyr763 mediates strong activation of these kinases. The levels of p38 activation correlate closely with those of Ras activation by G-CSF, suggesting that the degree of Ras activation is a critical determinant for the extent of p38 activation by hemopoietic cytokines.

Animals↗
PubMed1999

Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer.

The definitive long-term repopulating human hematopoietic stem cell, which seeds the adult blood system, was previously thought to derive from the extra-embryonic yolk sac. However, there is now considerable evidence that in both avian and murine systems, yolk sac hematopoietic cells are largely a transient, embryonic population and the definitive stem cell, in fact, derives from a distinct region within the embryonic mesoderm, the aorta-gonad-mesonephros region. In the human embryo, an analogous region has been found to contain a cluster of cells distinct from, but closely associated with, the ventral endothelium of the dorsal aorta, the appearance of which is restricted both spatially and temporally. We have used antibodies recognising hematopoietic regulatory factors to further characterise this region in the human embryo. These studies indicate that all factors examined, including vascular endothelial growth factor and its receptor FLK-1, Flt-3 ligand and its receptor STK-1, and stem cell leukemia transcription factor, are expressed by both hematopoietic cells in the cluster and endothelial cells. However, there is some discontinuity in cells directly underlying the cluster. Furthermore, we have identified a morphologically distinct region of densely-packed, rounded cells in the mesenchyme directly beneath the ventral wall of the dorsal aorta, and running along its entire length. In the preumbilical AGM region, directly underlying the hematopoietic cluster, but not at more rostral and caudal levels, this region of mesenchyme expresses tenascin-C, an extracellular matrix glycoprotein known to facilitate cell-cell interactions and migration. This region of cells may therefore provide the microenvironmental support for the intraembryonic development of definitive hematopoietic stem cells, a process in which tenascin-C may play a pivotal role.

Antigens, CD34↗
PubMed1999

Improved quality-control detection of false-negative Pap smears using the Autopap 300 QC system.

Federally-mandated quality control (QC) in Papanicolaou (Pap) smear testing requires rescreening of 10% of negative smears, to include cases selected randomly as well as smears from patients that may have a higher risk for developing cervical cancer based on clinical information. FDA approval of NeoPath's AutoPap 300 QC system (NeoPath, Inc., Redmond, WA) allows practical QC rescreening of all negatives. We tested the ability of AutoPap to help increase identification of detection errors compared to random 10%/high-risk selection. From March 1-August 30, 1997, we utilized AutoPap/high-risk status to select cases for manual rescreen, and compared the rate of identification of primary screening errors to that for the preceding year using 10% random selection/high-risk status. Of 35,027 smears accessioned, 31,240 (89.1%) were screened as negative and 7,965 were selected for manual rescreen. Of these, 353 were determined to be abnormal. Most abnormals identified by this protocol were classified as atypical squamous or glandular cells of undetermined significance (ASCUS or AGUS). However, 59 low-grade squamous intraepithelial lesions (LSIL) and 13 high-grade squamous intraepithelial lesions (HSIL), many with few abnormal cells, were also identified. These results represented an increase in pickup rate of false negative due to detection errors of 2.3-, 2.8- and 5.6-fold for atypical squamous or glandular cells of undetermined significance, LSIL, and HSIL, respectively, when accounting for the volume differences over the time period measured. Our findings strongly support the conclusions drawn from clinical trials of the AutoPap that false negatives due to detection error can be significantly reduced when using AutoPap as part of a routine quality control program.

False Negative Reactions↗
PubMed1999

Activated MEK stimulates expression of AP-1 components independently of phosphatidylinositol 3-kinase (PI3-kinase) but requires a PI3-kinase signal To stimulate DNA synthesis.

To investigate the contribution that ERK/mitogen-activated protein kinase signalling makes to cell cycle progression and gene expression, we have constructed cell lines to express an inducible version of activated MEK1. Using these cells, we show that activation of MEK leads to the expression of Fra-1 and Fra-2 but not c-Fos. Treatment of Ras-transformed cells with the MEK inhibitor PD098059 blocks expression of Fra-1 and Fra-2, showing that in Ras transformation ERK signalling is responsible for Fra-1 and Fra-2 expression. Activation of MEK1 in growth-arrested cells leads to DNA synthesis; however, ERK activation alone is insufficient because the induction of DNA synthesis is blocked by inhibition of phosphatidylinositol 3-kinase (PI3-kinase). Activation of PI3-kinase is indirect, perhaps through autocrine growth factors, and is required for the induction of cyclin D1.

3T3 Cells↗
PubMed1999

Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals.

Germ line mutations in the breast cancer susceptibility gene BRCA2 predispose to early-onset breast cancer, but the function of the nuclear protein encoded by the gene is ill defined. Using the yeast two-hybrid system with fragments of human BRCA2, we identified an interaction with the human DSS1 (deleted in split hand/split foot) gene. Yeast and mammalian two-hybrid assays showed that DSS1 can associate with BRCA2 in the region of amino acids 2472 to 2957 in the C terminus of the protein. Using coimmunoprecipitation of epitope-tagged BRCA2 and DSS1 cDNA constructs transiently expressed in COS cells, we were able to demonstrate an association. Furthermore, endogenous BRCA2 could be coimmunoprecipitated with endogenous DSS1 in MCF7 cells, demonstrating an in vivo association. Apparent orthologues of the mammalian DSS1 gene were identified in the genome of the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Yeast strains in which these DSS1-like genes were deleted showed a temperature-sensitive growth phenotype, which was analyzed by flow cytometry. This provides evidence for a link between the BRCA2 tumor suppressor gene and a gene required for completion of the cell cycle.

Amino Acid Sequence↗
PubMed1998

Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2.

BACKGROUND: Mitogen-activated protein (MAP) kinases (or extracellular signal regulated kinases; Erks) and stress-activated protein (SAP) kinases mediate cellular responses to a wide variety of signals. In the Erk MAP kinase pathway, activation of MAP kinases takes place in the cytoplasm and the activated enzyme moves to the nucleus. This translocation to the nucleus is essential to MAP kinase signalling because it enables the kinase to phosphorylate transcription factors. Whether components of the pathway mediated by the SAP kinase p38 change their cellular location on activation is not clear; we have therefore studied the cellular localisation of components of this pathway before and after stimulation. RESULTS: The p38 SAP kinase substrate MAP-kinase-activated protein kinase-2 (MAPKAP kinase-2) contains a putative nuclear localisation signal which we show is functional and required for activation by a variety of stimuli. Following phosphorylation of MAPKAP kinase-2, nuclear p38 was exported to the cytoplasm in a complex with MAPKAP kinase-2. Export of MAPKAP kinase-2 required phosphorylation by p38 but did not appear to require the kinase activity of MAPKAP kinase-2. The p38 activators MKK3 and MKK6 were present in both the nucleus and the cytoplasm, consistent with a role in activating p38 in the nucleus. CONCLUSIONS: In the p38 SAP kinase pathway, MAPKAP kinase-2 serves both as an effector of p38 by phosphorylating substrates and as a determinant of cellular localisation of p38. Nuclear export of p38 and MAPKAP kinase-2 may permit them to phosphorylate substrates in the cytoplasm.

Amino Acid Sequence↗
PubMed1998

Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1.

Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation, and the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signalling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signalling pathways to varying degrees. Other signalling pathways could also influence the consequences of Ras signalling. Here we show that when signalling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Cells that lack p21Waf1/Cip1 do not require Rho signalling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signalling is the suppression of p21Waf1/Cip1 induction.

3T3 Cells↗
PubMed1998

Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C.

Receptor tyrosine kinase-mediated activation of the Raf-1 protein kinase is coupled to the small guanosine triphosphate (GTP)-binding protein Ras. By contrast, protein kinase C (PKC)-mediated activation of Raf-1 is thought to be Ras independent. Nevertheless, stimulation of PKC in COS cells led to activation of Ras and formation of Ras-Raf-1 complexes containing active Raf-1. Raf-1 mutations that prevent its association with Ras blocked activation of Raf-1 by PKC. However, the activation of Raf-1 by PKC was not blocked by dominant negative Ras, indicating that PKC activates Ras by a mechanism distinct from that initiated by activation of receptor tyrosine kinases.

Animals↗
PubMed1998

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic.

Bailie et al. [In Vitro Cell Dev. Biol. (1992) 28A, 621-624] reported that primary cultures of rat hepatocytes possess low affinity binding sites for nerve growth factor (NGF). NGF treatment of primary cultures of rat hepatocytes with a maximally effective concentration of NGF (20 ng/ml, 0.8 nM) caused acute phasic activation of Raf-1 and p42(MAPkinase), and a smaller sustained activation of B-Raf. The transient increase in Raf-1 and p42(MAPkinase) activity returned to baseline within approximately 30 min. NGF treatment of hepatocytes did not induce expression of cyclin dependent kinase (cdk) inhibitor proteins, but instead stimulated cdk2 activity and increased [3H]thymidine incorporation into DNA. In contrast to hepatocytes, NGF treatment of PC12 pheochromocytoma cells caused large sustained activations of B-Raf and p42(MAPkinase), and a lower phasic activation of Raf-1. The sustained activations of B-Raf and p42(MAPkinase) were for more than 5 h. Treatment of PC12 cells with NGF increased p21(Cip1/WAF-1) expression, reduced cdk2 activity and inhibited DNA synthesis, the opposite to the effects of NGF treatment of hepatocytes. However when p42(MAPkinase) was chronically activated in hepatocytes, via infection with an inducible oestrogen receptor-Raf-1 fusion protein, expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins increased, cdk2 activity decreased, and DNA synthesis decreased. Equally, treatment of hepatocytes with 50 mM ethanol elevated the basal activity of p42(MAPkinase) and temporally extended the ability of NGF treatment to activate p42(MAPkinase). Ethanol and NGF co-treatment increased expression of p21(Cip-1/WAF1) and p16(INK4a) cdk inhibitor proteins and decreased hepatocyte DNA synthesis. These data demonstrate that NGF can cause either acute/phasic or sustained activation of the MAP kinase cascade in different cell types. Acute activation of the MAP kinase cascade correlated with increased DNA synthesis. In contrast, sustained activation of the MAP kinase cascade correlated with increased expression of cdk inhibitor proteins, a reduction in cdk activity, and an inhibition of DNA synthesis. These data suggest a general mechanism exists where acute activation of the MAP kinase cascade promotes G1 progression/S phase entry and that chronic activation of the MAP kinase cascade inhibits this process.

Animals↗
PubMed1998

Phylogenetic analysis of three lipocalin-like proteins present in the milk of Trichosurus vulpecula (Phalangeridae, Marsupialia).

Three proteins have been identified in the milk of the common brush tail possum. Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include beta-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas beta-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100-110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpecula beta-lactoglobulin, along with two other macropod beta-lactoglobulins, forms a subclass of beta-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents.

Amino Acid Sequence↗
PubMed1998

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes.

Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-alpha inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (alpha, beta, delta, epsilon, eta, and zeta) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes alpha and eta) are potent activators of c-Raf1, atypical PKC-zeta cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-alpha and PKC-eta was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-alpha, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-O-tetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.

3T3 Cells↗
PubMed1997

Nuclear location and cell cycle regulation of the BRCA2 protein.

Women carrying a germ-line mutation in the BRCA1 or BRCA2 genes have a high risk of developing breast cancer, and loss of the wild-type allele in tumors suggests that these genes function as tumor suppressor genes. The BRCA2 gene encodes a 3418-amino acid protein with no significant sequence similarity to any known protein. To begin to elucidate the cellular role of BRCA2, we have raised antibodies to the BRCA2 protein and used these to study its subcellular localization and expression. We show that BRCA2 is a nuclear protein expressed in response to cell proliferation and that BRCA2 expression is initiated before DNA synthesis.

Animals↗
PubMed1997

Lysozyme and alpha-lactalbumin from the milk of a marsupial, the common brush-tailed possum (Trichosurus vulpecula).

Lysozyme and alpha-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14,896 and 13,985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula alpha-lactalbumin, along with other marsupial alpha-lactalbumins, formed a family distinct from the eutherian alpha-lactalbumins and the alpha-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Amino Acid Sequence↗