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PubMed1977

The structure of postsynaptic densities isolated from dog cerebral cortex. II. Characterization and arrangement of some of the major proteins within the structure.

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ extractor, ethylene glycol-bis (beta- aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.

Actins↗

PubMed2000

The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics.

A microtubule-associated protein, gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), was previously identified as binding to the intracellular domain of GABA(A) receptors by using the yeast two-hybrid screen. In the present work, immunofluorescent staining and green fluorescent protein-tagged receptor subunits showed that GABARAP is associated with and promotes the clustering of GABA(A) receptors in QT-6 quail fibroblasts. The tubulin-binding motif of GABARAP and the gamma2 subunit of the receptor are required. Disruption of microtubules prevents the clustering in a time-dependent manner. When green fluorescent protein-tagged alpha1 or gamma2 subunit coexpressed with beta2, gamma2L, and GABARAP was used, recordings from visually identified cells revealed that clustered GABA(A) receptor had an EC(50) of about 20 microM, vs. 5.7 microM for the diffuse receptor. Clustered receptors deactivated faster and desensitized slower than the diffuse receptors, because of decrease in the apparent affinity of GABA binding. Different properties for clustered receptors relative to unclustered receptors in heterologous cells suggest that homologous differences between extrasynaptic and synaptic clustered receptors in neurons may be due to the organization of the postsynaptic machinery.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Identification of mRNAs that show modulated expression during colon carcinoma cell differentiation.

To investigate the working hypotheses that stem cells or their early descendants are prime targets for neoplastic transformation, and that the degree to which a neoplasm retains the immature phenotype is an important determinant of tumor aggressiveness, we have identified several mRNAs that are downregulated during the in vitro differentiation of HT29-D4 colon carcinoma cells. These genes include heat-shock cognate protein Hsc70, adenylosuccinate lyase, B23/nucleophosmin, alpha-tubulin, and a novel gene designated DS-1. The DS-1 mRNA has a length of approximately 0.9 kb and is downregulated 4.7-fold upon differentiation. From the DS-1 cDNA, a protein of 206 amino acids with a molecular mass of 24 kDa and an isoelectric point of 10.9 can be deduced. An antiserum directed against a synthetic peptide detected a minor band of the expected size in Western blots, as well as a major band of lower size that may represent a processed form of the protein.

Adenylosuccinate Lyase↗

PubMed2003

Regulation of tension-induced mechanotranscriptional signals by the microtubule network in fibroblasts.

Mechanical loading of connective tissues induces the expression of extracellular matrix and cytoskeletal genes that are involved in matrix remodeling. These processes depend in part on force transmission through beta1 integrins and actin filaments, but the role of microtubules in regulating mechanotranscriptional responses is not well defined. We assessed the involvement of microtubules in the mechanotranscriptional regulation of filamin A, an actin-cross-linking protein that protects cells against force-induced apoptosis by stabilizing cell membranes. Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured fibroblasts at focal adhesions. Force enhanced recruitment of alpha-tubulin and the plus end microtubule-binding protein cytoplasmic linker protein-170 (CLIP-170) at focal adhesions. Immunoprecipitation studies demonstrated no direct binding of tubulin to actin or filamin A, but CLIP-170 interacted with tubulin, filamin A, and beta-actin. The association of CLIP-170 with beta-actin was enhanced by force. Force activated the p38 mitogen-activated protein kinase, increased filamin A expression, and induced the relocation of p38 and filamin A to focal adhesions. Disruption of microtubules with nocodazole, independent of force application, enhanced filamin A expression and Sp1-mediated filamin A promoter activity, while stabilization of microtubules with Taxol inhibited force induction of both filamin A mRNA and protein. We conclude that in response to tensile forces applied through beta1 integrins and actin the microtubule network modulates mechanotranscriptional coupling of filamin A.

Actins↗

PubMed2002

Integration and differentiation of human embryonic stem cells transplanted to the chick embryo.

Human embryonic stem (ES) cells are pluripotent cells that can differentiate into a large array of cell types and, thus, hold promise for advancing our understanding of human embryology and for contributing to transplantation medicine. In this study, differentiation of human ES cells was examined in vivo by in ovo transplantation to organogenesis-stage embryos. Colonies of human ES cells were grafted into or in place of epithelial-stage somites of chick embryos of 1.5 to 2 days of development. The grafted human ES cells survived in the chick host and were identified by vital staining with carboxyfluorescein diacetate or use of a green fluorescent protein-expressing cells. Histologic analysis showed that human ES cells are easily distinguished from host cells by their larger, more intensely staining nuclei. Some grafted cells differentiated en masse into epithelia, whereas others migrated and mingled with host tissues, including the dorsal root ganglion. Colonies grafted directly adjacent to the host neural tube produced primarily structures with the morphology and molecular characteristics of neural rosettes. These structures contain differentiated neurons as shown by beta-3-tubulin and neurofilament expression in axons and cell bodies. Axons derived from the grafted cells penetrate the host nervous system, and host axons enter the structures derived from the graft. Our results show that human ES cells transplanted in ovo survive, divide, differentiate, and integrate with host tissues and that the host embryonic environment may modulate their differentiation. The chick embryo, therefore, may serve as an accessible and unique experimental system for the study of in vivo development of human ES cells.

Animals↗

PubMed2005

The galanin-R2 agonist AR-M1896 reduces glutamate toxicity in primary neural hippocampal cells.

Galanin is a neuropeptide involved in a variety of biological functions, including having a strong anticonvulsant activity. To assess a possible role of galanin in modulation of glutamatergic synapses and excitotoxicity, we studied effects of a galanin receptor 2(3) agonist (AR-M1896) on several molecular events induced by glutamate administration in primary neural hippocampal cells. Exposure of cells, after 5 days in vitro, to glutamate 0.5 mM for 10 min caused morphological alterations, including disaggregation of beta-tubulin and MAP-2 cytoskeletal protein assembly, loss of neurites and cell shrinkage. When present in culture medium together with glutamate, 1 and 10 nM of AR-M1896 reduced these alterations. Moreover, AR-M1896 counteracted glutamate-induced c-fos mRNA and c-Fos protein up-regulation after 30-150 min, and 24 h, respectively. Massive nuclear alterations (Hoechst 33258 staining), observed 24 h after glutamate exposure, were also antagonized by AR-M1896 (0.1-100 nM) in a dose-dependent manner. These findings indicate that galanin, probably mainly through its type 2 receptor, interferes with events associated with glutamate toxicity.

Animals↗

PubMed2002

PKC-zeta prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity.

Using intestinal (Caco-2) monolayers, we reported that inducible nitric oxide synthase (iNOS) activation is key to oxidant-induced barrier disruption and that EGF protects against this injury. PKC-zeta was required for protection. We thus hypothesized that PKC-zeta activation and iNOS inactivation are key in EGF protection. Wild-type (WT) Caco-2 cells were exposed to H(2)O(2) (0.5 mM) +/- EGF or PKC modulators. Other cells were transfected to overexpress PKC-zeta or to inhibit it and then pretreated with EGF or a PKC activator (OAG) before oxidant. Relative to WT cells exposed to oxidant, pretreatment with EGF protected monolayers by 1) increasing PKC-zeta activity; 2) decreasing iNOS activity and protein, NO levels, oxidative stress, tubulin oxidation, and nitration); 3) increasing polymerized tubulin; 4) maintaining the cytoarchitecture of microtubules; and 5) enhancing barrier integrity. Relative to WT cells exposed to oxidant, transfected cells overexpressing PKC-zeta (+2.9-fold) were protected as indicated by decreases in all measures of iNOS-driven pathways and enhanced stability of microtubules and barrier function. Overexpression-induced inhibition of iNOS was OAG independent, but EGF potentiated this protection. Antisense inhibition of PKC-zeta (-95%) prevented all measures of EGF protection against iNOS upregulation. Thus EGF protects against oxidative disruption of the intestinal barrier by stabilizing the cytoskeleton in large part through the activation of PKC-zeta and downregulation of iNOS. Activation of PKC-zeta is by itself required for cellular protection against oxidative stress of iNOS. We have thus discovered novel biologic functions, suppression of the iNOS-driven reactions and cytoskeletal oxidation, among the atypical PKC isoforms.

Caco-2 Cells↗

PubMed1995

Subpopulations of tau interact with microtubules and actin filaments in various cell types.

It has been demonstrated that microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. However, there is no clear evidence on the possible roles of the interactions of MAPs in vivo with other cytoskeletal components in maintaining the integrity of the cell architecture. To address this question we extracted the neuronal cytoskeleton from brain cells and studied the selective dissociation of specific molecular isospecies of tau protein under various experimental conditions. Tau, and in some cases MPA-2, were analysed by the use of anti-idiotypic antibodies that recognize epitopes on their tubulin binding sites. Fractions of microtubule-bound tau isoforms were extracted with 0.35 M NaCl or after the addition of nocodazole to allow microtubule depolymerization. Protein eluted with this inhibitor contained most of the assembled tubulin dimer pool and part of the remaining tau and MAP-2. When the remaining cytoskeletal pellet was treated with cytochalasin D to allow depolymerization of actin filaments, only tau isoforms were extracted. Immunoprecipitation studies along with immunolocalization experiments in cell lines containing tau-like components supported the findings on the roles of tau isospecies as linkers between tubulin in the microtubular structure with actin filaments. Interestingly, in certain types of cells, antibody-reactive tau isospecies were detected by immunofluorescence with a discrete distribution pattern along actin filaments, which was affected by cytochalasin disruption of the actin filament network. These results suggest the possible in vivo roles of subsets of tau protein in modulating the interactions between microtubules and actin filaments.

Actins↗

PubMed2000

Acetylcholine induces neuritic outgrowth in rat primary olfactory bulb cultures.

The rat olfactory bulb is innervated by basal forebrain cholinergic neurons and is endowed with both nicotinic and muscarinic receptors. The development of this centrifugal cholinergic innervation occurs mainly in early postnatal stages. This developmental time-course and the demonstration that acetylcholine can modulate some aspects of neuronal proliferation, differentiation or death, suggests the possible involvement of cholinergic afferents in the morphogenesis and/or plasticity of the olfactory bulb. The purpose of the present work was to assess whether acetylcholine could modulate neuronal morphogenesis in the olfactory bulb. Toward this aim, we developed a primary culture model of rat olfactory bulbs. Three major cell types were identified on the basis of their morphological and immunocytochemical phenotype: neuronal-shaped cells expressing the neuronal markers neuron specific enolase, microtubule associated protein 2, neural cell adhesion molecule and beta-tubulin III; glial-like cells immunoreactive for glial fibrillary acidic protein and flattened cells immunolabelled with antibodies against beta-tubulin III and nestin, most likely neuronal precursors. After three to six days of treatment with 100-microM carbachol, a cholinergic agonist, significant increase in neuritic length was observed in cultured olfactory bulb neurons. The neurite outgrowth effect of carbachol was abolished by co-treatment with 1 microM alpha-bungarotoxin, an alpha 7 subunit nicotinic receptor antagonist, but was not affected by the addition of 10 microM atropine, a general muscarinic antagonist. The effect of carbachol was also mimicked by the nicotinic agonists, nicotine (100 microM) and epibatidine (10 microM). This pharmacological profile suggested the involvement of nicotinic receptors of the alpha 7-like subtype as confirmed using 125I-alpha-bungarotoxin receptor autoradiography.Taken together, these data argue for a role for nicotinic receptors in neuritic outgrowth in the rat olfactory bulb and provide a cellular support to the previously described effects of acetylcholine on olfactory bulb plasticity in vivo.

Acetylcholine↗

PubMed2002

Activation of delta-isoform of protein kinase C is required for oxidant-induced disruption of both the microtubule cytoskeleton and permeability barrier of intestinal epithelia.

Using monolayers of intestinal (Caco-2) cells, we showed that oxidants disassemble the microtubule cytoskeleton and disrupt barrier integrity (permeability) (Banan et al., 2000a). Because exposure of our parental cells to oxidants causes protein kinase C (PKC)-delta to be translocated to particulate fractions, we hypothesized that PKC-delta activation is required for these oxidant effects. Monolayers of parental Caco-2 cells were incubated with oxidant (H(2)O(2)) +/- modulators. Other cells were transfected with an inducible plasmid to stably overexpress PKC-delta or with a dominant negative plasmid to stably inhibit the activity of native PKC-delta. In parental cells, oxidants caused translocation of PKC-delta to the particulate (membrane + cytoskeletal) fractions, activation of PKC-delta isoform, increases in monomeric (S1) tubulin and decreases in polymerized (S2) tubulin, disruption of the microtubule cytoarchitecture, and loss of barrier integrity (hyperpermeability). In transfected cells, induction of PKC-delta overexpression by itself (3.5-fold over its basal level) led to oxidant-like disruptive effects. Disruption induced by PKC-delta overexpression was potentiated by oxidants. Overexpressed PKC-delta resided in particulate fractions, indicating its activation. Stable inhibition of native PKC-delta activity (98%) by dominant negative transfection substantially protected against all measures of oxidative disruption. We conclude that 1) oxidants induce loss of intestinal epithelial barrier integrity by disassembling the microtubules in large part through the activation of the PKC-delta isoform; and 2) overexpression and activation of PKC-delta is by itself a sufficient condition for disruption of these cytoskeleton and permeation pathways. Thus, PKC-delta activation may play a key role in intestinal dysfunction in oxidant-induced diseases such as inflammatory bowel disease.

Aminoglycosides↗

PubMed2006

Multiple histone deacetylases and the CREB-binding protein regulate pre-mRNA 3'-end processing.

Trichostatin A (TSA), a specific inhibitor of histone deacetylases (HDACs), induces acetylation of various non-histone proteins such as p53 and alpha-tubulin. We purified several acetylated proteins by the affinity to an anti-acetylated lysine (AcLys) antibody from cells treated with TSA and identified them by mass spectrometry. Here we report on acetylation of CFIm25, a component of mammalian cleavage factor Im (CF Im), and poly(A) polymerase (PAP), a polyadenylating enzyme for the pre-mRNA 3'-end. The residues acetylated in these proteins were mapped onto the regions required for interaction with each other. Whereas CBP acetylated these proteins, HDAC1, HDAC3, HDAC10, SIRT1, and SIRT2 were involved in in vivo deacetylation. Acetylation of the CFIm25 occurred depending on the cleavage factor complex formation. Importantly, the interaction between PAP and CF Im complex was decreased by acetylation. We also demonstrated that acetylation of PAP inhibited the nuclear localization of PAP by inhibiting the binding to the importin alpha/beta complex. These results suggest that CBP and HDACs regulate the 3'-end processing machinery and modulate the localization of PAP through the acetylation and deacetylation cycle.

3' Untranslated Regions↗

PubMed1999

Cytoskeletal dynamics in dendritic spines: direct modulation by glutamate receptors?

A wide heterogeneity in dendritic-spine morphology is observed and ultrastructural changes can be induced following experimental stimulation of neurons. Morphological adaptation of a given spine might, thus, reflect its history or the current state of synaptic activity. These changes could conceivably result from rearrangements of the cytoskeleton that is subjacent to excitatory synapses. This article dicusses the direct and indirect interactions, between glutamate receptors and the cytoskeletal proteins, which include PDZ-containing proteins, actin and tubulin, as well as associated proteins. In fact, the synaptic-activity-controlled balancing of monomeric, dimeric and polymeric forms of actin and tubulin might underlie the changes in spine shape. These continuous adaptations could be relevant for physiological events, such as learning and the formation of memory.

Actin Cytoskeleton↗

PubMed1990

Calcium-mediated modulation of microtubule assembly in human breast epithelial cells.

Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca2+ medium (1.05 mM). To clarify the mechanism by which Ca2+ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca2+ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca2+ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca2+ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca2(+)-grown cells did not display a network of tubulin fibers, whereas high Ca2(+)-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca2+ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatment of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca2+ medium had no observable effect on cell morphology. These results indicate that high levels of Ca2+ either favor tubulin polymerization or stabilize the polymerized state.

Breast↗

PubMed1998

Bcl-xL is phosphorylated in malignant cells following microtubule disruption.

The oncogenic protein Bcl-2 functions as a potent inhibitor of programmed cell death. This survival activity has been shown in some settings to be influenced by the Bcl-2 phosphorylation state. It has been demonstrated that treatment with microtubule-targeted agents results in phosphorylation of both Raf-1 kinase and Bcl-2. The Bcl-2-related family member Bcl-xL also exhibits a death suppressive activity, but its potential for phosphorylation following exposure to drugs that interact with microtubules has not been evaluated. Several tumor cell lines with low or undetectable levels of Bcl-2 protein expression were found to express Bcl-xL. A more slowly migrating Bcl-xL band was observed on immunoblots after cells were treated with microtubule-targeted agents. The appearance of this band was responsive to dose and was absent when the cell lysates were treated with lambda protein phosphatase. Using a Bcl-xL-specific monoclonal antibody, the phosphorylated form of Bcl-xL was immunoprecipitated from cells treated with paclitaxel and metabolically labeled with 32P-labeled inorganic orthophosphate. Herein, we report that Bcl-xL is phosphorylated in malignant cells after incubation with agents that target tubulin, including paclitaxel, vincristine, vinblastine, colchicine, and nocodazole. Moreover, paclitaxel-resistant ovarian carcinoma cell lines that have mutations in tubulin failed to exhibit phosphorylation of Bcl-xL after paclitaxel exposure, but they did demonstrate Bcl-xL phosphorylation in the presence of other tubulin-targeting agents. As observed for Bcl-2, phosphorylation of Bcl-xL was accompanied by phosphorylation of Raf-1. Interestingly, phosphorylation of these three proteins failed to occur or was much less pronounced when cells grown at high density were challenged with drug. Also, reduced Raf-1 expression, observed after treatment of cells with geldanamycin prior to and during incubation with the microtubule-active drugs, correlated with diminished Bcl-xL phosphorylation. Taken together, these results suggest that Bcl-xL, like Bcl-2, is phosphorylated by agents that disrupt microtubule architecture. By analogy with Bcl-2, this phosphorylation may play a critical role in modulating Bcl-xL function and may be an important determinant of microtubule-directed chemotherapeutic efficacy in human tumors.

Antibiotics, Antineoplastic↗

PubMed1996

Live analysis of free centrosomes in normal and aphidicolin-treated Drosophila embryos.

In a number of embryonic systems, centrosomes that have lost their association with the nuclear envelope and spindle maintain their ability to duplicate and induce astral microtubules. To identify additional activities of free centrosomes, we monitored astral microtubule dynamics by injecting living syncytial Drosophila embryos with fluorescently labeled tubulin. Our recordings follow multiple rounds of free centrosome duplication and separation during the cortical division. The rate and distance of free sister centrosome separation corresponds well with the initial phase of associated centrosome separation. However, the later phase of separation observed for centrosomes associated with a spindle (anaphase B) does not occur. Free centrosome separation regularly occurs on a plane parallel to the plasma membrane. While previous work demonstrated that centrosomes influence cytoskeletal dynamics, this observation suggests that the cortical cytoskeleton regulates the orientation of centrosome separation. Although free centrosomes do not form spindles, they display relatively normal cell cycle-dependent modulations of their astral microtubules. In addition, free centrosome duplication, separation, and modulation of microtubule dynamics often occur in synchrony with neighboring associated centrosomes. These observations suggest that free centrosomes respond normally to local nuclear division signals. Disruption of the cortical nuclear divisions with aphidicolin supports this conclusion; large numbers of abnormal nuclei recede into the interior while their centrosomes remain on the cortex. Following individual free centrosomes through multiple focal planes for 45 min after the injection of aphidicolin reveals that they do not undergo normal modulation of their astral dynamics nor do they undergo multiple rounds of duplication and separation. We conclude that in the absence of normally dividing cortical nuclei many centrosome activities are disrupted and centrosome duplication is extensively delayed. This indicates the presence of a feedback mechanism that creates a dependency relationship between the cortical nuclear cycles and the centrosome cycles.

Animals↗

PubMed2004

Estrogens and anti-estrogens: key mediators of prostate carcinogenesis and new therapeutic candidates.

Despite the historical use of estrogens in the treatment of prostate cancer (PCa) little is known about their direct biological effects on the prostate, their role in carcinogenesis, and what mechanisms mediate their therapeutic effects on PCa. It is now known that estrogens alone, or in synergism with an androgen, are potent inducers of aberrant growth and neoplastic transformation in the prostate. The mechanisms of estrogen carcinogenicity could be mediated via induction of unscheduled cell proliferation or through metabolic activation of estrogens to genotoxic metabolites. Age-related changes and race-/ethnic-based differences in circulating or locally formed estrogens may explain differential PCa risk among different populations. Loss of expression of estrogen receptor (ER)-beta expression during prostate carcinogenesis and prevention of estrogen-mediated oxidative damage could be exploited in future PCa prevention strategies. Re-expression of ER-beta in metastatic PCa cells raises the possibility of using ER-beta-specific ligands in triggering cell death in these malignant cells. A variety of new estrogenic/anti-estrogenic/selective estrogen receptor modulator (SERM)-like compounds, including 2-methoxyestradiol, genistein, resveratrol, licochalcone, Raloxifene, ICI 182,780, and estramustine are being evaluated for their potential in the next generation of PCa therapies. Increasing numbers of patients self-medicate with herbal formulations such as PC-SPES. Some of these compounds are selective ER-beta ligands, while most of them have minimal interaction with ER-alpha. Although many may inhibit testosterone production by blockade of the hypothalamal-pituitary-testis axis, the most effective agents also exhibit direct cytostatic, cytotoxic, or apoptotic action on PCa cells. Some of them are potent in interfering with tubulin polymerization, blocking angiogenesis and cell motility, suppressing DNA synthesis, and inhibiting specific kinase activities. Further discovery of other compounds with potent apoptotic activities but minimal estrogen action should promote development of a new generation of effective PCa preventive or treatment regimens with few or no side-effects due to estrogenicity. Further advancement of our knowledge of the role of estrogens in prostate carcinogenesis through metabolic activation of estrogens and/or ER-mediated pathways will certainly result in better preventive or therapeutic modalities for PCa.

Estrogen Receptor Modulators↗

PubMed2005

Phosphorylated MARCKS: a novel centrosome component that also defines a peripheral subdomain of the cortical actin cap in mouse eggs.

MARCKS (myristoylated alanine-rich C-kinase substrate) is a major substrate for protein kinase C (PKC), a kinase that has multiple functions during oocyte maturation and egg activation, for example, spindle function and cytoskeleton reorganization. We examined temporal and spatial changes in p-MARCKS localization during maturation of mouse oocytes and found that p-MARCKS is a novel centrosome component based its co-localization with pericentrin and gamma-tubulin within microtubule organizing centers (MTOCs). Like pericentrin, p-MARCKS staining at the MI spindle poles was asymmetric. Based on this asymmetry, we found that one end of the spindle was preferentially extruded with the first polar body. At MII, however, the spindle poles had symmetrical p-MARCKS staining. p-MARCKS also was enriched in the periphery of the actin cap overlying the MI or MII spindle to form a ring-shaped subdomain. Because phosphorylation of MARCKS modulates its actin crosslinking function, this localization suggests p-MARCKS functions as part of the contractile apparatus during polar body emission. Our finding that an activator of conventional and novel PKC isoforms did not increase the amount of p-MARCKS suggested that an atypical isoform was responsible for MARCKS phosphorylation. Consistent with this idea, immunostaining revealed that the staining patterns of p-MARCKS and the active form of the atypical PKC zeta/lambda isoform(s) were very similar. These results show that p-MARCKS is a novel centrosome component and also defines a previously unrecognized subdomain of the actin cap overlying the spindle.

Actins↗

PubMed1994

Effect on microtubule dynamics of XMAP230, a microtubule-associated protein present in Xenopus laevis eggs and dividing cells.

The reorganization from a radial [corrected] interphase microtubule (MT) network into a bipolar spindle at the onset of mitosis involves a dramatic change in MT dynamics. Microtubule-associated proteins (MAPs) and other factors are thought to regulate MT dynamics both in interphase and in mitosis. In this study we report the purification and functional in vitro characterization of a 230-KD MAP from Xenopus egg extract (XMAP230). This protein is present in eggs, oocytes, testis and a Xenopus tissue culture cell line. It is apparently absent from non-dividing cells in which an immunologically related 200-kD protein is found. XMAP230 is composed of two isoforms with slightly different molecular masses and pIs. It is localized to interphase MTs, dissociates from MTs at the onset of prophase and specifically binds to spindle MTs during metaphase and anaphase. The dissociation constant of XMAP230 is 500 nM, the stoichiometry of binding to MTs is between 1:8 and 1:4, and the in vivo concentration is approximately 200 nM. Both isoforms are phosphorylated and have reduced affinity for microtubules in mitotic extracts. Analysis of the effect of XMAP230 on MT dynamics by video microscopy shows that it increases the growth rate, decreases the shrinking rate of MTs and strongly suppresses catastrophes. These results suggest that in vivo, XMAP230 participates in the control of the MT elongation rate, stabilizes MTs and locally modulates MT dynamics during mitosis.

Animals↗