Antisense oligonucleotides to CFTR confer a cystic fibrosis phenotype on B lymphocytes

1992 ◽  
Vol 263 (6) ◽  
pp. C1147-C1151 ◽  
Author(s):  
R. D. Krauss ◽  
G. Berta ◽  
T. A. Rado ◽  
J. K. Bubien
Keyword(s):  
Cell Cycle ◽  
Cystic Fibrosis ◽  
Antisense Oligonucleotides ◽  
T84 Cells ◽  
Cftr Protein ◽  
Low Levels ◽  
B Lymphoid ◽  
Cycle Dependent ◽  
Cl Permeability

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl-permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a "CF phenotype." These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

scholarly journals A U-Rich Element in the 5′ Untranslated Region Is Necessary for the Translation of p27 mRNA

2000 ◽  
Vol 20 (16) ◽  
pp. 5947-5959 ◽  
Author(s):  
S. Sean Millard ◽  
Anxo Vidal ◽  
Maurice Markus ◽  
Andrew Koff
Keyword(s):  
Cell Cycle ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Mrna Processing ◽  
Differentiated Cells ◽  
Specific Subset ◽  
Number Of Factors ◽  
Rnp Complex ◽  
Cycle Dependent

ABSTRACT Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5′UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5′UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.

scholarly journals Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

2004 ◽  
Vol 3 (5) ◽  
pp. 1185-1197 ◽  
Author(s):  
Bidyottam Mittra ◽  
Dan S. Ray
Keyword(s):  
Cell Cycle ◽  
Binding Protein ◽  
High Specificity ◽  
Binding Activity ◽  
Mrna Levels ◽  
Crithidia Fasciculata ◽  
Target Mrna ◽  
Cycle Dependent

ABSTRACT Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

scholarly journals Cell Cycle Synchrony in Giardia intestinalis Cultures Achieved by Using Nocodazole and Aphidicolin

2008 ◽  
Vol 7 (4) ◽  
pp. 569-574 ◽  
Author(s):  
Marianne K. Poxleitner ◽  
Scott C. Dawson ◽  
W. Zacheus Cande
Keyword(s):  
Cell Cycle ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Giardia Intestinalis ◽  
Mitotic Spindles ◽  
Intestinal Protozoan ◽  
Cycle Arrest ◽  
Cycle Dependent ◽  
Dependent Processes

ABSTRACT Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the importance of Giardia as a model organism, research on Giardia has been hampered by an inability to achieve cell cycle synchrony for in vitro cultures. This report details successful methods for attaining cell cycle synchrony in Giardia cultures. The research presented here demonstrates reversible cell cycle arrest in G1/S and G2/M with aphidicolin and nocodazole, respectively. Following synchronization, cells were able to recover completely from drug treatment and remained viable and maintained synchronous growth for 6 h. These techniques were used to synchronize Giardia cultures to increase the percentages of mitotic spindles in the cultures. This method of synchronization will enhance our ability to study cell cycle-dependent processes in G. intestinalis.

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

2011 ◽  
Vol 112 (1) ◽  
pp. 307-317 ◽  
Author(s):  
Maria-Cristina Arcangeletti ◽  
Isabella Rodighiero ◽  
Prisco Mirandola ◽  
Flora De Conto ◽  
Silvia Covan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Cytomegalovirus ◽  
Human Embryo ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Embryo Fibroblasts ◽  
Cycle Dependent

scholarly journals Exogenous Interleukin-2 Administration Corrects the Cell Cycle Perturbation of Lymphocytes from Human Immunodeficiency Virus-Infected Individuals

2001 ◽  
Vol 75 (22) ◽  
pp. 10843-10855 ◽  
Author(s):  
Mirko Paiardini ◽  
Domenico Galati ◽  
Barbara Cervasi ◽  
Giuseppe Cannavo ◽  
Luca Galluzzi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Cell Cycle Control ◽  
Interleukin 2 ◽  
Protein Ubiquitination ◽  
Immunodeficiency Virus ◽  
Cell Cycle Perturbation ◽  
Cycle Dependent

ABSTRACT Human immunodeficiency virus (HIV)-induced immunodeficiency is characterized by progressive loss of CD4+ T cells associated with functional abnormalities of the surviving lymphocytes. Increased susceptibility to apoptosis and loss of proper cell cycle control can be observed in lymphocytes from HIV-infected individuals and may contribute to the lymphocyte dysfunction of AIDS patients. To better understand the relation between T-cell activation, apoptosis, and cell cycle perturbation, we studied the effect of exogenous interleukin-2 (IL-2) administration on the intracellular turnover of phase-dependent proteins. Circulating T cells from HIV-infected patients display a marked discrepancy between a metabolic profile typical of G0 and a pattern of expression of phase-dependent proteins that indicates a more-advanced position within the cell cycle. This discrepancy is enhanced by in vitro activation with ConA and ultimately results in a marked increase of apoptotic events. Conversely, treatment of lymphocytes with IL-2 alone restores the phase-specific pattern of expression of cell cycle-dependent proteins and is associated with low levels of apoptosis. Interestingly, exogenous IL-2 administration normalizes the overall intracellular protein turnover, as measured by protein synthesis, half-life of newly synthesised proteins, and total protein ubiquitination, thus providing a possible explanation for the effect of IL-2 on the intracellular kinetics of cell cycle-dependent proteins. The beneficial effect of IL-2 administration is consistent with the possibility of defective IL-2 function in vivo, which is confirmed by the observation that lymphocytes from HIV-infected patients show abnormal endogenous IL-2 paracrine/autocrine function upon in vitro mitogen stimulation. Overall these results confirm that perturbation of cell cycle control contributes to HIV-related lymphocyte dysfunction and, by showing that IL-2 administration can revert this perturbation, suggest a new mechanism of action of IL-2 therapy in HIV-infected patients.

scholarly journals Subunit composition determines E2F DNA-binding site specificity.

1997 ◽  
Vol 17 (12) ◽  
pp. 6994-7007 ◽  
Author(s):  
Y Tao ◽  
R F Kassatly ◽  
W D Cress ◽  
J M Horowitz
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Binding Sites ◽  
Dna Bending ◽  
Susceptibility Gene ◽  
Specific Sequence ◽  
Cellular Genes ◽  
Cycle Dependent

The product of the retinoblastoma (Rb) susceptibility gene, Rb-1, regulates the activity of a wide variety of transcription factors, such as E2F, in a cell cycle-dependent fashion. E2F is a heterodimeric transcription factor composed of two subunits each encoded by one of two related gene families, denoted E2F and DP. Five E2F genes, E2F-1 through E2F-5, and two DP genes, DP-1 and DP-2, have been isolated from mammals, and heterodimeric complexes of these proteins are expressed in most, if not all, vertebrate cells. It is not yet clear whether E2F/DP complexes regulate overlapping and/or specific cellular genes. Moreover, little is known about whether Rb regulates all or a subset of E2F-dependent genes. Using recombinant E2F, DP, and Rb proteins prepared in baculovirus-infected cells and a repetitive immunoprecipitation-PCR procedure (CASTing), we have identified consensus DNA-binding sites for E2F-1/DP-1, E2F-1/DP-2, E2F-4/DP-1, and E2F-4/DP-2 complexes as well as an Rb/E2F-1/DP-1 trimeric complex. Our data indicate that (i) E2F, DP, and Rb proteins each influence the selection of E2F-binding sites; (ii) E2F sites differ with respect to their intrinsic DNA-bending properties; (iii) E2F/DP complexes induce distinct degrees of DNA bending; and (iv) complex-specific E2F sites selected in vitro function distinctly as regulators of cell cycle-dependent transcription in vivo. These data indicate that the specific sequence of an E2F site may determine its role in transcriptional regulation and suggest that Rb/E2F complexes may regulate subsets of E2F-dependent cellular genes.

scholarly journals The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle

1999 ◽  
Vol 112 (8) ◽  
pp. 1257-1271 ◽  
Author(s):  
Y. Gachet ◽  
S. Tournier ◽  
M. Lee ◽  
A. Lazaris-Karatzas ◽  
T. Poulton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Binding Domain ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Tubulin Binding ◽  
Cycle Dependent ◽  
Almost All

The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100–150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3259-3266 ◽  
Author(s):  
K.T. Jones ◽  
J. Carroll ◽  
J.A. Merriman ◽  
D.G. Whittingham ◽  
T. Kono
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Dependent Manner ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
A Cell ◽  
Mature Mouse ◽  
Cycle Dependent ◽  
Stage 1

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4–5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

Sign in / Sign up

Export Citation Format

Share Document