Menin molecular interactions: insights into normal functions and tumorigenesis.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease predisposed by heterozygous germline mutations in the MEN1 tumor suppressor gene. Biallelic loss of MEN1 resulting from small mutation and/or loss of heterozygosity occurs in a large tissue spectrum of MEN1 tumors or non-hereditary tumors. Mouse models of MEN1 underexpression or overexpression have also supported the tumor-suppressor effect of the MEN1 gene. Menin, the 610-amino-acid protein encoded by MEN1, is expressed ubiquitously and found predominantly in the nucleus. Sequence analyses do not reveal motifs of known function other than two nuclear localization sequences. Menin has been found to partner in vitro with a variety of proteins that comprise transcription factors, DNA processing factors, DNA repair proteins, and cytoskeletal proteins. The diverse functions of menin interactors suggest roles for menin in multiple biological pathways. Inactivation of menin switches its JunD partner from a downstream action of growth suppression to growth promotion. This is a plausible mechanism for menin tumorigenesis.

Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice.

In an effort to create a conventional knockout mouse model for multiple endocrine neoplasia type 1 (MEN1), we targeted disruption of the mouse Men1 gene through homologous recombination in ES cells. Men1 exons 2-4 were replaced by a PGK-neomycin cassette inserted in the opposite direction of Men1 transcription (Men1(MSK/+)). Unexpectedly, the Men1 conventional knockout was lethal in heterozygous, chimeric animals. Analysis of embryos revealed late gestational lethality with some embryos showing omphalocele. This was a very surprising phenotype, given that humans and mice that are heterozygotes for loss of function mutations in MEN1 are phenotypically normal except for a risk of endocrine tumors. Northern analysis of Men1(MSK/+) embryonic stem cell RNA revealed the presence of an abundant, novel transcript of 2.1 kb, in addition to the expected wild-type transcripts of 2.7 kb and 3.1 kb. RT-PCR analysis identified this aberrant transcript as arising from the antisense strand of the PGK promoter. We hypothesize that this transcript is producing either a toxic effect at the RNA level, or a dominant negative effect through the production of an amino-terminal truncated protein product. This example serves as a cautionary reminder that mouse knockouts using PGK-neo may sometimes display phenotypes that reflect more than just the loss of function of the targeted gene.

Design of modified oligodeoxyribonucleotide probes to detect telomere repeat sequences in FISH assays.

A series of dye-labeled oligonucleotide probes containing base and sugar modifications were tested for the ability to detect telomeric repeat sequences in FISH assays. These modified oligonucleotides, all 18 nt in length, were complementary to either the cytidine-rich (C(3)TA(2))(n)or guanosine-rich (T(2)AG(3))(n)telomere target sequences. Oligonucleotides were modified to either increase target affinity by enhancing duplex stability [2'-OMe ribose sugars and 5-(1-propynyl)pyrimidine residues] or inhibit the formation of inter- or intramolecular structures (7-deazaguanosine and 6-thioguanosine residues), which might interfere with binding to the target. Several dye-labeled oligonucleotide probes were found that could effectively stain the telomeric repeat sequences of either cytidine- or guanosine-rich strands in a specific manner. Such probes could be used as an alternative to peptide nucleic acids for investigating the dynamics of telomere length and maintenance. In principle, these relatively inexpensive and readily synthesized modified oligonucleotides could be used for other FISH-related assays.

Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite and single-copy DNA sequences.

A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human beta-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence in situ hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.

Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes.

The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood. Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells. To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells. Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described. Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH). Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome. Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact. Northern analysis showed full-length, human CFTR mRNA. Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR. Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR. This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression. These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.

Synthesis and characterization of pseudopeptide bradykinin B2 receptor antagonists containing the 1,3,8-triazaspiro[4.5]decan-4-one ring system.

A series of pseudopeptides containing alkyl-, cycloalkyl-, aryl-, and aralkyl-substituted 1,3,8-triazaspiro[4.5]decan-4-one-3-acetic acids as amino acid surrogates to replace the Pro2-Pro3-Gly4-Phe5 section of the peptide bradykinin B2 receptor antagonist [Pro3, Phe5]HOE 140 (D-Arg0-Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-D-Tic7+ ++-Oic8-Arg9) were prepared. These psuedopeptides were examined in vitro for their B2 receptor affinities as well as for their ability to block bradykinin mediated actions in vivo. Two compounds in particular, NPC 18521 (I) and NPC 18688 (V) were quite potent in these latter assays, indicating that a significant portion of this prototypical second generation decapeptide antagonist can be replaced with a more compact nonpeptide molecule.

Mutation of aspartate residues in the third extracellular loop of the rat B2 bradykinin receptor decreases affinity for bradykinin.

Two aspartates in the third extracellular loop of the rat B2 bradykinin (BK) receptor have been implicated as important residues for agonist binding. Asp268 and Asp286 were mutated to alanine residues and changes in agonist and antagonist binding affinity were examined. The IC50 value for BK as a competitor of [3H] NPC 17731 binding to the rat wild type receptor was 1.1 nM, while the Ala268 and Ala286 receptor mutants exhibited IC50 values of 19 nM and 28 nM, respectively. The Ala268Ala268 receptor mutant exhibited an IC50 for BK of 500 nM. These mutations had little effect on binding affinity when NPC 17761, a BK antagonist, was used to compete [3H] NPC 17731 binding. Electrophysiological examination of Xenopus oocytes expressing wild type or Ala268 Ala286 receptors confirmed the importance of the Asp268 and Asp286 residues for BK recognition. BK activated the mutant receptor with comparable efficacy relative to the wild type receptor, but a 1750-fold reduction in potency was observed.

GABAergic cells and signals appear together in the early post-mitotic period of telencephalic and striatal development.

Single cell suspensions derived from embryonic telencephala taken from embryos of gestational day 13 (E13) as well as rat striatal tissue from E14, 15 and 17 were prepared by tissue digestion with papain. Cell suspensions were analyzed by flow cytometry or plated onto poly-D-lysine-coated culture dishes for either nuclear staining or immunocytochemistry. Experiments on functional Na+ channels and GABAA receptor expression were carried out using a fluorescence-activated cell sorter (FACS) and a negatively charged fluorescent indicator dye (oxonol). FACS analysis of embryonic cell suspensions at E13-17 consistently revealed one major subpopulation accounting for 85-90% of the events and one minor subpopulation (10-15% of the total). When sorted, the major subpopulation consisted of phase-bright cells of 5-7 microns diameter some of which had neurites. The minor population consisted of phase-dark cells and resealed membranes of 0.5-4 microns diameter as well as debris. Almost all the cells obtained in the high FALS (forward-angle light scatter) subpopulation at E17 expressed 200-kDa neurofilament and tetanus toxin antigens while the small diameter cells seldom expressed tetanus toxin and particles never did. A small number of GABA-containing neurons were detected in the telencephalon at E13 (3%) and in the developing striatum at E14 (6%). All of the GABA-containing neurons expressed neurofilament. In the embryonic rat striatum, nanomolar concentrations of muscimol (GABAA agonist) induced depolarizing responses. A small number of cells in the high FALS subpopulation were responsive to muscimol starting at embryonic day 14, and the number of responsive cells increased at E15.(ABSTRACT TRUNCATED AT 250 WORDS)

Two functionally different glutamate receptors of the kainate subtype in embryonic rat mesencephalic cells.

The amino acid glutamate is a widespread excitatory neurotransmitter in the brain. It activates cation-selective channels expressed by nearly every neuron and by glial cells; also various second messenger cascades. Little is known about the ontogeny of glutamate neurotransmission during neurogenesis. We have analyzed the development and differentiation of excitatory amino acid responses and Na+ channels in cells dissociated from embryonic rat ventral mesencephalon and striatum as well as cortex and cerebellum using fluorescent voltage-sensitive oxonol dyes and flow cytometry. Analysis of fluorescence distribution revealed complex profiles under resting conditions which changed in a characteristic manner over the period studied (Embryonic (E) Days 12-20). The response to the Na+ channel agonist veratridine appeared at E12/13 in the mesencephalon. At E13 L-glutamate and kainate evoked changes in membrane potential interpreted as cellular hyperpolarization. At E15 some cells still responded by hyperpolarizing but an equal number began to depolarize. By E18 most cells depolarized. Both hyper- and depolarizations were eliminated by a specific antagonist at kainate receptors (6-cyano-7-nitroquinoxyline-2,3-dione) and by resuspending the cells in Na(+)-free medium. Both responses exhibited a concentration dependency with higher doses evoking stronger effects. In contrast, there was little effect of veratridine in the striatum at E15-E16, and the response to kainate or L-glutamate was predominantly depolarizing during the same embryonic period, with little or no effect until E18. These data show that in the developing CNS, sodium channel responses as well as excitatory aminoacid neurotransmitter responses first become functional in the mesencephalon and subsequently in the striatum, thus suggesting an anatomical gradient of expression. Our results also show that glutamate receptor-coupled functions vary with embryonic age and with regional distribution, suggesting possible roles of glutamate in early CNS embryogenesis, as morphogens or modulators of synaptic plasticity.

Muscarinic acetylcholine receptor subtypes as agonist-dependent oncogenes.

We have evaluated the muscarinic acetylcholine family of G protein-coupled receptors (mAChRs) for their oncogenic potential. These receptors are preferentially expressed in postmitotic cells, transducing signals specified by their endogenous agonist, the neurotransmitter acetylcholine. Cells transfected with individual human mAChR genes were morphologically indistinguishable from parental NIH 3T3 cells in the absence of agonist. In contrast, when cultures were supplemented with carbachol, a stable analog of acetylcholine, foci of transformation readily appeared in m1, m3, or m5 but not in m2 or m4 mAChRs transfectants. Receptor expression was verified by ligand binding and was similar for each transfected culture. Transformation was dose-dependent and required only low levels of receptor expression. In transformation-competent cells, agonist induced phosphatidylinositol hydrolysis, whereas in m2 or m4 transfectants, receptors were coupled to the inhibition of adenylyl cyclase. These findings demonstrate that mAChRs linked to phosphatidylinositol hydrolysis can act as conditional oncogenes when expressed in cells capable of proliferation.

Electrical and chemical excitability appear one week before birth in the embryonic rat spinal cord.

Embryonic rat spinal cord cells were acutely dissociated with the enzyme papain, stained with a voltage-sensitive oxonol dye and incubated with various pharmacological agents. Changes in the fluorescence intensity and, by inference, membrane potential of the cells were analyzed in a flow cytometer. Veratridine caused depolarization of the cells in a TTX-sensitive manner from as early as embryonic day 13. Depolarizing responses to muscimol and kainate appeared slightly later, at embryonic days 14 and 15, and were blocked by the antagonists bicuculline and CNQX, respectively. Responses to veratridine and kainate did not occur in sodium-free medium. The emergence of these excitable membrane properties coincides with postmitotic differentiation and synaptic development in the embryonic spinal cord.

Embryonic and early postnatal hippocampal cells respond to nanomolar concentrations of muscimol.

Embryonic and early postnatal tissue taken from rat hippocampi were papain digested in order to obtain cell suspensions suitable for analysis in a fluorescence-activated cell sorter (FACS). Cell suspensions consisted of two major peaks of forward-angle light scatter (FALS). FACS analysis showed that the population which stained intensely with the vital dye Acridine orange (AO) scattered significant levels of light (high FALS) and amounted to 85% of the total events collected in embryonic cell suspensions and 65% in postnatal (PN) samples. Two minor populations were weakly stained with AO and scattered little light. Oxonol, a voltage-sensitive indicator dye, was used to detect membrane polarization changes. The AO and oxonol staining patterns were very similar. All the events exposed to media containing 50 mM KCl were depolarized (increase in intensity of oxonol fluorescence). The depolarizing effect of veratridine, a sodium channel activator, was more pronounced in the high FALS subpopulation. In embryonic hippocampal cell suspensions nanomolar concentrations of GABAA agonists depolarized the high FALS subpopulation in a dose-dependent manner. This effect was prevented by preincubation with bicuculline or picrotoxin. In hippocampal cell suspensions obtained from 5-7-day-old rat pups (PN5-7), GABAA agonists depolarized one cell subpopulation and hyperpolarized another. Our results indicate that physiological responses can be resolved in subpopulations of hippocampal cell suspensions by FACS analysis. This technique seems to be a sensitive assay to measure physiological responses (changes in membrane potential) as a parameter of receptor expression. GABAA agonists induced pure depolarizing responses in embryonic and early postnatal hippocampus when active neurogenesis is taking place. The response become hyperpolarizing-depolarizing ones after inhibitory synapses appear.

Growth and differentiation properties of O-2A progenitors purified from rat cerebral hemispheres.

We have used the monoclonal antibody A2B5 (which binds to subclasses of surface gangliosides) to select glial precursor cells from postnatal rat brain and compare their properties in culture with those of the bipotential O-2A progenitor cells of newborn optic nerve. Two methods, fluorescence-activated cell sorting (FACS) and differential adhesion, resulted in greater than 90% enrichment in A2B5-positive bipolar cells and multipolar cells with short processes. These cells expressed vimentin and reacted with yet another antibody (NSP4), which binds to O-2A progenitor cells of optic nerve. The 2-10% of the remaining cells consisted of type 1 astrocytes and/or microglial cells. When maintained in defined medium for 3 days, 28-40% of A2B5-positive cells incorporated thymidine, while most other cells became differentiated into galactocerebroside-positive oligodendrocytes. In the presence of 10% fetal calf serum for 3 days, over 50% of the cells developed a stellate phenotype and expressed GFAP, characteristic of type 2 astrocytes. This phenotypic plasticity of the A2B5 positive cells was also observed in clones derived from single cells grown on a layer of type 1 astrocytes. Thus, A2B5-positive cells from cerebrum are O-2A progenitors that can generate O-2A lineage cells. The effects of the two growth factors, insulin and platelet derived growth factor (PDGF) (which is synthesized by type 1 astrocytes), were tested on cerebrum O-2A progenitors. PDGF induced a doubling of the percentage of A2B5-positive cells incorporating thymidine during a 20-hr pulse and a large increase (up to 40-fold) of the progenitor population over 3 days. The largest number of O-2A lineage cells was obtained when purified progenitors were grown in the presence of PDGF and insulin. Thus, A2B5-positive glial cells from cerebrum overall behave as the O-2A progenitors of optic nerve, but they more readily divide than differentiate, as if they were at an earlier stage along the O-2A lineage pathway.

Flow cytometric analysis of membrane potential in embryonic rat spinal cord cells.

Flow cytometric analysis of membrane potential in suspensions of embryonic rat spinal cord cells was carried out in a fluorescence-activated cell sorter (FACS) using anionic voltage-sensitive, fluorescent dyes (oxonols). The FACS or flow cytometer is an analytical instrument that measures optical properties of large cell populations at a single cell level of resolution. The incorporation of oxonol allows relative measurements of membrane potential, since the partition of oxonol within the plasmalemma is directly related to the degree of cell depolarization. Incubation of cells in elevated K+ concentrations or with the Na+ channel agonist batrachotoxin (BTX) changed the fluorescence intensity distribution pattern of the live-cell population; these changes were consistent with the depolarizing effects of these manipulations. Fluorescence shifts were either undetectable or minimal in the dead-cell population. The BTX-induced shift was blocked by tetrodotoxin (TTX) and was reversed in Na+-free medium, indicating embryonic expression of functional Na+ channels. Fluorescence microscopy of sorted cells showed that live cells typically exhibited circumferential ring-like patterns, whose intensities were enhanced under depolarizing conditions. The results show that flow cytometry combined with oxonol dyes can be used to measure the relative membrane potential of large numbers of individual central nervous system cells. The analysis of the changes in the distributions of these membrane potentials can be used to reveal the development of functional ion conductance mechanisms.

Dopaminergic neurons from embryonic mouse mesencephalon are enriched in culture through immunoreaction with monoclonal antibody to neural specific protein 4 and flow cytometry.

Dopaminergic neurons represent a rare neurotransmitter phenotype within the mammalian central nervous system. The mesencephalic dopaminergic neurons form the ascending dopaminergic pathways in mammals and are involved in motor and limbic functions. Here we report that about 30% of all developing mouse mesencephalic cells, including virtually all of the dopaminergic phenotype, express surface membrane determinant(s) recognized by a monoclonal antibody to neural specific protein 4 (NSP4). We have been able to isolate and culture neurons from the mesencephalon according to their expression of NSP4, using the anti-NSP4 immunoreaction in conjunction with fluorescence-activated cell sorting. Cultures of NSP4+-sorted cells showed a significant enrichment in three morphologically distinct putative dopaminergic phenotypes when compared to unsorted mesencephalic cultures, whereas the cultures of NSP4--sorted cells were virtually devoid of dopaminergic neurons. This flow cytometric enrichment in dopaminergic neurons should provide the necessary cells for multidisciplinary study of dopaminergic phenotype differentiation.

Thymosin alpha 1-induced modulation of cellular responses and functional T-cell subsets in mice with experimental autoimmune thyroiditis.

The effects of Ta-1, a peptide constituent of thymosin fraction 5, were studied on murine autoimmune thyroiditis using two congenic strains of mice, B10.Br (Br) and B10.D2 (D2), which are sensitive and resistant to experimental autoimmune thyroiditis (EAT) induction, respectively. EAT was induced by either 2 weekly iv injections of mouse thyroglobulin with adjuvant lipopolysaccharide (LPS) or intradermal injection of thyroglobulin mixed with complete Freund's adjuvant (CFA). The criteria for induction and intensity of thyroiditis were the level of lymphoid infiltration in the thyroid gland and the titer of anti-thyroglobulin antibodies. Ta-1 was given in 5 or 10 daily sc injections in doses ranging from 0.0001 to 0.1 microgram/injection. The injections were commenced at varying intervals from the 1st to the 4th week after immunization. T-Cell subsets in the spleens were determined 2 weeks after the first antigen injection and thyroid infiltration was determined 3 weeks later. Treatment with Ta-1 between the two antigen injections increased the level of thyroiditis in resistant mice, but had no effect in sensitive mice. Treatment for the first 2 weeks had similar effects in resistant mice, but also suppressed thyroiditis in the sensitive strain. Later treatments, during the 3rd and 4th weeks after immunization also revealed immunomodulating properties of Ta-1, with a suppressing effect on thyroiditis in sensitive mice and an enhancing effect in the resistant strain. Both effects of Ta-1 were dose dependent. The effects of Ta-1 on the individual phenotypes were also dose dependent. The dose of 0.01 microgram greatly lowered the percentages of Lyt-2+3+ cells in D2 mice and mildly increased the percentages in Br mice, but did not change the Lyt-1+ cell level in either strain. On the other hand, the dose of 0.001 microgram greatly increased the percentage of Lyt-1+ cells in D2 mice and mildly decreased it in the Br strain, but did not alter the Lyt-2+3+ cell subset in either strain. Thus, both doses of Ta-1 modulated Lyt-1+/2+3+ ratios, with each dose affecting a different T-cell subset. The changes in the response to thyroglobulin are apparently exerted through the regulation of the functional T-cell subset balance.

Analysis of thymocyte subpopulations following treatment with sex hormones.

Sex steroids were found to affect both murine and avian immune systems. Female and male (NZB X NZW)F1 mice were castrated at 2 weeks of age and given Silastic implants containing either dihydrotestosterone or estradiol. Four weeks following treatment, the thymuses were studied for cell cycle kinetics and for the presence of various cell surface antigens using fluorescein-conjugated antisera and flow cytometric techniques. Estradiol therapy resulted in an increase in mature thymocytes, that is, thymocytes that had decreased peanut agglutinin receptors and decreased Thy 1 antigens on their surfaces. Additional studies with anti-Lyt 1 and 2 indicated that these mature thymocytes were of the "helper" rather than the "suppressor" phenotype. Estradiol therapy resulted in an increase in the percentages of proliferating cells in the spleen and a decrease in the percentages of proliferating cells in the thymus. In contrast, in the avian system, estradiol had little effect on proliferation in immunological organs. Dihydrotestosterone was a potent inhibitor of proliferation in the avian bursa. These results indicate that sex hormones have specific effects on different immune cell subpopulations. In the murine system the male is the heterogametic sex but in the avian system the female is the heterogametic sex. Based upon the present and previous studies, we suggest that the primary modulating hormone for immunological sex effects in the mammal is estrogen, whereas in the bird it is androgen.

The human autologous mixed lymphocyte reaction. III. Immune circuits.

The human autologous mixed lymphocyte reaction represents the proliferative response of T cells to determinants presented on autologous non-T cells. Purified T4+ cells vigorously proliferate in response to stimulation by either (B + null) cells or M phi, whereas purified T8+ cells proliferate very little without a source of help. Such help can be provided by mitomycin C-treated T4+ cells, which indicates that proliferation of T4+ cells is not necessary for the help. M phi suppress the human AMLR, as measured by the response of T cells to stimulation by (B + null) cells. The target of this M phi-induced suppression was found to be the T4+ inducer cell. In contrast to the suppressive effects of M phi on T4+ cells, M phi did not suppress T8+ cells. The available data suggest that the human AMLR is a two-part inducer circuit. One part can be stimulated preferentially by M phi and the other by (B + null) cells; however, M phi prevent activation of the (B + null) cell part of the circuit. These results may provide an explanation for the differential activities of different regulatory circuits without and also with antigenic stimulation.

Genetic studies in NZB mice. V. Recombinant inbred lines demonstrate that separate genes control autoimmune phenotype.

The genetic basis for autoimmunity in NZB mice has been investigated through analysis of recombinant inbred lines produced by mating NZB mice with two different non-autoimmune strains. Several genes (at least six) were found to be necessary for the production of eight traits characteristic of the NZB mice that were studied. No fundamental genetic defect (an "autoimmunity gene") was identified that could give rise to the various autoimmune traits studied. This study strongly suggests that NZB disease results from the actions of several separate genes that together result in the characteristic manifestations of autoimmunity.