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1.
Nature ; 619(7969): 363-370, 2023 Jul.
Article in English | MEDLINE | ID: mdl-37407814

ABSTRACT

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle1,2, when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein3-5. Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell's progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.


Subject(s)
Cell Cycle , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , G2 Phase , S Phase , Animals , Cyclin A2/metabolism , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 4/deficiency , Cyclin-Dependent Kinase 4/metabolism , Mitogens/deficiency , Mitogens/metabolism , Mitosis , Phosphorylation , Retinoblastoma Protein/chemistry , Retinoblastoma Protein/metabolism , Cyclin-Dependent Kinase 6/deficiency , Cyclin-Dependent Kinase 6/metabolism , G1 Phase
2.
J Neurosci ; 33(9): 3865-78, 2013 Feb 27.
Article in English | MEDLINE | ID: mdl-23447598

ABSTRACT

During corticogenesis, pituitary adenylate cyclase-activating polypeptide (PACAP; ADCYAP1) may contribute to proliferation control by activating PAC1 receptors of neural precursors in the embryonic ventricular zone. PAC1 receptors, specifically the hop and short isoforms, couple differentially to and activate distinct pathways that produce pro- or anti-mitogenic actions. Previously, we found that PACAP was an anti-mitogenic signal from embryonic day 13.5 (E13.5) onward both in culture and in vivo and activated cAMP signaling through the short isoform. However, we now find that mice deficient in PACAP exhibited a decrease in the BrdU labeling index (LI) in E9.5 cortex, suggesting that PACAP normally promotes proliferation at this stage. To further define mechanisms, we established a novel culture model in which the viability of very early cortical precursors (E9.5 mouse and E10.5 rat) could be maintained. At this stage, we found that PACAP evoked intracellular calcium fluxes and increased phospho-PKC levels, as well as stimulated G1 cyclin mRNAs and proteins, S-phase entry, and proliferation without affecting cell survival. Significantly, expression of hop receptor isoform was 24-fold greater than the short isoform at E10.5, a ratio that was reversed at E14.5 when short expression was 15-fold greater and PACAP inhibited mitogenesis. Enhanced hop isoform expression, elicited by in vitro treatment of E10.5 precursors with retinoic acid, correlated with sustained pro-mitogenic action of PACAP beyond the developmental switch. Conversely, depletion of hop receptor using short-hairpin RNA abolished PACAP mitogenic stimulation at E10.5. These observations suggest that PACAP elicits temporally specific effects on cortical proliferation via developmentally regulated expression of specific receptor isoforms.


Subject(s)
Cerebral Cortex/cytology , Gene Expression Regulation, Developmental/drug effects , Mitogens/pharmacology , Neurons/drug effects , Pituitary Adenylate Cyclase-Activating Polypeptide/pharmacology , RNA Isoforms/metabolism , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide/genetics , Age Factors , Animals , Bromodeoxyuridine/metabolism , Calcium/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Division/drug effects , Cell Division/genetics , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Cerebral Cortex/embryology , Cerebral Ventricles/cytology , Cerebral Ventricles/embryology , Dose-Response Relationship, Drug , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Female , Flow Cytometry , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Humans , In Situ Nick-End Labeling , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogens/deficiency , Mitogens/genetics , Nerve Tissue Proteins/metabolism , Neurogenesis/drug effects , Neurogenesis/genetics , Neurons/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide/deficiency , Pituitary Adenylate Cyclase-Activating Polypeptide/genetics , Pregnancy , Protein Kinase C/metabolism , Pyridines/pharmacology , Pyrrolidinones/pharmacology , RNA Isoforms/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism , Tretinoin/pharmacology
3.
Asian Pac J Allergy Immunol ; 30(2): 114-22, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22830290

ABSTRACT

BACKGROUND: The hybridoma technique is the standard method for production of monoclonal antibodies of interest. However, the newly formed hybridomas and cells at low density often grow poorly or die. This is the major obstacle to production of monoclonal antibodies. OBJECTIVE: The aim of this study was to establish a method for preparation of conditioned medium in the absence of mitogen for promoting the growth of hybridomas after cell fusion and during single cell cloning. METHODS: Culture supernatants were obtained from the cultures of BW5147 mouse thymoma cells without mitogen stimulation. Novel conditioned mediums were investigated for their ability to support hybridoma single-cell cloning and hybridoma production. RESULTS: We demonstrated that these conditioned mediums could support hybridoma single-cell growth, both for stable and newly generated hybridomas, at a level equal to the commercial conditioned medium BM-Condimed H1. The conditioned medium was most effective at a final concentration of 20% with the basal medium supplemented with 10% fetal calf serum. The novel conditioned medium could also be effectively employed for generation of hybridomas secreting various monoclonal antibodies. Interestingly, fibroblast overgrowth in the post-fusion wells was markedly reduced when using the novel conditioned medium, as compared to the commercial BM-Condimed H1, likely due to the absence of mitogen in the conditioned medium. CONCLUSION: We describe the method for production of a novel conditioned medium for hybridoma technology. This method is simple, needing no special technology or sophisticated equipment. The novel conditioned medium is, therefore, recommended for use in place of expensive commercial conditioned medium for hybridoma technology, especially in resource-limited countries.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Culture Media, Conditioned/pharmacology , Hybridomas/cytology , Animals , Antibodies, Monoclonal/immunology , Cell Fusion , Clone Cells/cytology , Clone Cells/drug effects , Clone Cells/immunology , Culture Media, Conditioned/chemistry , Fibroblasts/cytology , Fibroblasts/drug effects , Hybridomas/immunology , Mice , Mitogens/deficiency , Single-Cell Analysis , Thymoma/metabolism , Thymoma/pathology , Thymus Neoplasms/metabolism , Thymus Neoplasms/pathology
4.
J Immunol ; 172(5): 2962-9, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14978099

ABSTRACT

Oct-2, a transcription factor expressed in the B lymphocyte lineage and in the developing CNS, functions through of a number of discrete protein domains. These include a DNA-binding POU homeodomain flanked by two transcriptional activation domains. In vitro studies have shown that the C-terminal activation domain, a serine-, threonine- and proline-rich sequence, possesses unique qualities, including the ability to activate transcription from a distance in a B cell-specific manner. In this study, we describe mice in which the endogenous oct-2 gene has been modified through gene targeting to create a mutated allele, oct-2DeltaC, which encodes Oct-2 protein isoforms that lack all sequence C-terminal to the DNA-binding domain. Surprisingly, despite the retention of the DNA-binding domain and the glutamine-rich N-terminal activation domain, the truncated protein(s) encoded by the oct-2DeltaC allele are unable to rescue any of the previously described defects exhibited by oct-2 null mice. Homozygous oct-2DeltaC/DeltaC mice die shortly after birth, and B cell maturation, B-1 cell self renewal, serum Ig levels, and B lymphocyte responses to in vitro stimulation are all reduced or absent, to a degree equivalent to that seen in oct-2 null mice. We conclude that the C-terminal activation domain of Oct-2 is required to mediate the unique and indispensable functions of the Oct-2 transcription factor in vivo.


Subject(s)
DNA-Binding Proteins/physiology , Peptide Fragments/physiology , Trans-Activators/physiology , Transcription Factors/physiology , Amino Acid Sequence , Animals , Animals, Newborn , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , CD36 Antigens/genetics , CD36 Antigens/metabolism , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Line , Cells, Cultured , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Targeting , Immunoglobulins/blood , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogens/deficiency , Mitogens/genetics , Mitogens/physiology , Molecular Sequence Data , Mutagenesis, Insertional , Octamer Transcription Factor-2 , Peptide Fragments/deficiency , Peptide Fragments/genetics , Protein Structure, Tertiary/genetics , Survival Analysis , Trans-Activators/deficiency , Trans-Activators/genetics , Transcription Factors/deficiency , Transcription Factors/genetics
5.
EMBO J ; 19(9): 1998-2007, 2000 May 02.
Article in English | MEDLINE | ID: mdl-10790366

ABSTRACT

An intracellular timer is thought to help control the timing of oligodendrocyte differentiation. We show here that the expression of the helix-loop-helix gene Id4 in oligodendrocyte precursor cells decreases in vivo and in vitro with a time course expected if Id4 is part of the timer. We also show that Id4 expression decreases prematurely when the precursor cells are induced to differentiate by mitogen withdrawal. Both Id4 mRNA and protein decrease together under all of these conditions, suggesting that the control of Id4 expression is transcriptional. Finally, we show that enforced expression of Id4 stimulates cell proliferation and blocks differentiation induced by either mitogen withdrawal or treatment with thyroid hormone. These findings suggest that a progressive fall in Id4 transcription is part of the intracellular timer that helps determine when oligodendrocyte precursor cells withdraw from the cell cycle and differentiate.


Subject(s)
Cell Differentiation , DNA-Binding Proteins , Helix-Loop-Helix Motifs , Oligodendroglia/cytology , Proteins/chemistry , Proteins/metabolism , Repressor Proteins , Animals , Biological Clocks/genetics , Biological Clocks/physiology , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Fluorescent Antibody Technique , Gene Expression , Inhibitor of Differentiation Protein 1 , Inhibitor of Differentiation Proteins , Kinetics , Mitogens/deficiency , Mitogens/pharmacology , Oligodendroglia/drug effects , Oligodendroglia/metabolism , Platelet-Derived Growth Factor/deficiency , Platelet-Derived Growth Factor/pharmacology , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/metabolism , Temperature , Thyroid Hormones/pharmacology , Time Factors , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection
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