scholarly journals Different responses to cell crowding determine the clonal fitness of p53 and Notch inhibiting mutations in squamous epithelia

2020 ◽  
Author(s):  
Vasiliki Kostiou ◽  
Michael WJ Hall ◽  
Philip H Jones ◽  
Benjamin A Hall
Keyword(s):  
Areal Density ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Downstream Target ◽  
Normal Tissues ◽  
Fitness Advantage ◽  
Naturally Occurring ◽  
Type Population ◽  
Mutant Cells

AbstractThe growth and competition of cells in epithelial tissues plays an important role in both tissue homeostasis and the robustness of normal tissues to pre-cancer mutation. Whilst wild-type cells compete neutrally for dominance in the un-mutated tissue, naturally occurring mutations in individual cells may lend them a fitness advantage that can allow tissue colonisation. In mouse oesophageal epithelia, the growth of p53 mutants and a dominant negative mutant of the Notch downstream target Maml1 (DN_Maml1) have been shown to have different colonisation properties despite strong quantitative similarities in the growth of individual clones. Here we show that in order to recapitulate these behaviours whilst maintaining tissue turnover models need to take account of the response of cells to increased areal density in the tissue colonised by mutant cells. We demonstrate that p53 mutant clone growth approximates a logistic curve, but that without including limitations on mutation induced expansion the overall proliferation rate of the tissue drops due to space restrictions. In contrast, the ability of DN_Maml1 mutations to displace the wild-type population reflects a feedback that effects both mutant and wild-type cells equally. We go on to show how these distinct feedbacks are consistent with the distribution of mutations observed in human datasets.

Physical and physiological components of the graviresponses of wild-type and mutant Paramecium Tetraurelia

2000 ◽  
Vol 203 (6) ◽  
pp. 1059-1070 ◽  
Author(s):  
U. Nagel ◽  
H. Machemer
Keyword(s):  
Swimming Speed ◽  
Sedimentation Rates ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Centre Of Gravity ◽  
In The Wild ◽  
Type Population ◽  
G Value ◽  
Mutant Cells

Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia showed improved orientation away from the centre of gravity (negative gravitaxis) when accelerations were increased from 1 to 7 g. Gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g value suggests a physical basis for gravitaxis. Transiently enhanced rates of reversal of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results provide evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent swimming reversals. Gravity bimodally altered propulsion rates of wild-type P. tetraurelia so that sedimentation was partly antagonized in upward and downward swimmers (negative gravikinesis). In the mutant, only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type population. Observed swimming speed and sedimentation rates in the wild-type and mutant cells were linearly related to acceleration, allowing the determination of gravikinesis as a linear (and so far non-saturating) function of gravity.

PAX5/TEL Exhibits a Dominant-Negative Role by Inhibiting the Expression of Genes Involved in BCR Signalling and Suppressing the Binding of Wild Type PAX5 to Its Direct Target Gene CD79A (mb-1) in a human Pre-B ALL Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3455-3455
Author(s):  
Gabriela B. Iwanski ◽  
Nils Heinrich Thoennissen ◽  
PohYeen Lor ◽  
Norihiko Kawamata ◽  
Daniel Nowak ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Fusion Gene ◽  
Cell Receptor ◽  
Dominant Negative ◽  
Fusion Product ◽  
Wild Type ◽  
Rt Pcr ◽  
Downstream Target

Abstract Abstract 3455 Poster Board III-343 Acute lymphoblastic leukemia (ALL), one of the most common malignancies in childhood, is a heterogeneous disease with individual leukemia subtypes differing in their response to chemotherapy. Recent findings suggest that disruptions of B cell receptor (BCR) signalling pathways may be involved in the development of ALL. The transcription factor PAX5 is essential for the commitment of lymphoid progenitors to the B-lymphocytic lineage. In 30% of childhood B-ALL cases, PAX5 is a frequent target of aberrancies, showing monoallelic loss, point mutations, or chromosomal translocations, whereas the role of these aberrancies is still poorly understood. Using high resolution SNP-chip analysis, we have recently identified several candidate partner genes fused to PAX5 in pediatric ALL, ETV6 (TEL), FOXP1, AUTS2, C20orf112, which bind to PAX5 recognition sequences as strongly as wild-type PAX5 (wt PAX5) suppressing its transcriptional activity in a dominant-negative fashion. In order to study the role of PAX5/TEL in leukemic evolution of B-ALL, we transfected the leukemic BCP cell line Nalm 6, which endogenously expresses PAX5, with a retroviral vector encoding PAX5/TEL and confirmed its expression by Western blotting and RT-PCR. Previously, the fusion gene PAX5/TEL has been cloned into the retroviral vector pMSCV-IRES-GFP (MIGR) from a patient diagnosed with B-cell precursor ALL (BCP) with t(9;12)(q11;p13). This fusion product consists of the 5′-end NH2 terminal region of the PAX5 gene and the almost whole sequence of the TEL gene. PAX5/TEL-MIGR expressing cells were sorted for GFP and analyzed by gene expression profiling on Affymetrix HG-U133 plus 2.0 Array in comparison to cells transfected with vector control (MIGR) and a MIGR vector encoding wt PAX5 (wtPAX5/MIGR). The probes were normalized with the Affymetrix MAS5.0 software. Probes were considered to be differentially expressed with a fold change ≤ 2 or ≥ 2, respectively. We identified a set of about 200 genes that were differentially expressed in the PAX5/TEL expressing cells, most of which were downregulated, compared to the controls. A subset of these genes encodes proteins important for BCR signalling: RAG1, one of two key mediators in the process of V(D)J recombination, VPREB3, which is involved in the early phase of pre-BCR assembly, the Runt domain transcription factor Runx1 (AML1) and FOXP1. The latter two genes are fusion partners of PAX5 in pediatric B-ALL and loss of FOXP1 leads to impaired DH–JH and VH–DJH rearrangement. Additionally, we found BACH2, which plays an important role during B-cell development, as well as protein kinase C-epsilon (PKCe) to be downregulated. PKCe is highly expressed in B cells linking the BCR to the activation of mitogen-activated protein kinases (MAPK). We confirmed the downregulation of the affected genes by RT-PCR. Strikingly, VPREB3 expression showed a significant downregulation of up to 170-fold, and RAG1 up to 90-fold. Loss of the RAG1/2 locus has been found in four precursor B-cell ALL cases, which indicates that defects in this process might contribute to leukemogenesis. We also detected a significant decrease in the expression of wt PAX5 as well as its direct downstream target CD79A (mb-1). CD79A (mb-1) encodes the B cell receptor component Ig-a and its early B cell-specific mb-1 promoter is a target for regulation by early B cell-specific transcription factors like E2A, early B cell factor (EBF), and PAX5. The latter is important for the activation of the mb-1 promoter by recruiting Ets proteins through protein-protein interactions. We investigated the binding efficiency of wt PAX5 to the promoter region of CD79A by chromatin-immunoprecipitation (ChIP). For the ChIP assay, we used a PAX5 antibody detecting the C-terminal region of PAX5 so that the antibody can bind the wt PAX5 but not the fusion product PAX5/TEL of which the C-terminal side is fused to TEL. Binding of wt PAX5 to the promoter region of CD79A was diminished by expression of the PAX5/TEL-fusion protein compared to the controls, leading to repression of CD79A, which we also confirmed by RT-PCR. In conclusion, we show that the expression of PAX5/TEL in a leukemic cell line has a repressor function on the expression of wt PAX5 as well as other genes important in BCR signalling. Also, we demonstrated that PAX5/TEL has a negative impact on the binding affinity of one of the direct downstream target genes of wt PAX5. Our results indicate a repressor role of the fusion gene PAX5/TEL including BCR signalling and point towards its contribution to leukemic transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Rab11 Regulates the Compartmentalization of Early Endosomes Required for Efficient Transport from Early Endosomes to the Trans-Golgi Network

2000 ◽  
Vol 151 (6) ◽  
pp. 1207-1220 ◽  
Author(s):  
Mona Wilcke ◽  
Ludger Johannes ◽  
Thierry Galli ◽  
Véronique Mayau ◽  
Bruno Goud ◽  
...  
Keyword(s):  
Intracellular Transport ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Trans Golgi Network ◽  
Early Endosomes ◽  
Intracellular Compartments ◽  
Golgi Network ◽  
Mutant Forms ◽  
In Cells

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20°C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

scholarly journals Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3698-3706 ◽  
Author(s):  
C L Wu ◽  
M Classon ◽  
N Dyson ◽  
E Harlow
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Functional Domains ◽  
Dominant Negative Mutant ◽  
G1 Arrest ◽  
Wild Type ◽  
Cycle Progression

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant

Development ◽  
1994 ◽  
Vol 120 (4) ◽  
pp. 901-909 ◽  
Author(s):  
E. Levine ◽  
C.H. Lee ◽  
C. Kintner ◽  
B.M. Gumbiner
Keyword(s):  
Early Embryo ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Truncated Form ◽  
Adhesive Properties ◽  
Negative Mutant ◽  
E Cadherin

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.

Oleamide, a Sleep-Inducing Supplement, Upregulates Doublecortin in Hippocampal Progenitor Cells via PPARα

2021 ◽  
pp. 1-16
Author(s):  
Avik Roy ◽  
Madhuchhanda Kundu ◽  
Sudipta Chakrabarti ◽  
Dhruv R. Patel ◽  
Kalipada Pahan
Keyword(s):  
Progenitor Cells ◽  
Neuronal Differentiation ◽  
Chromatin Immunoprecipitation ◽  
Molecular Mechanisms ◽  
Hippocampal Neurogenesis ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Lipid Lowering ◽  
Dominant Negative Mutant ◽  
Wild Type

Background: Doublecortin (DCX), a microtubule associated protein, has emerged as a central biomarker of hippocampal neurogenesis. However, molecular mechanisms by which DCX is regulated are poorly understood. Objective: Since sleep is involved with the acquisition of memory and oleamide or 9-Octadecenamide (OCT) is a sleep-inducing supplement in human, we examined whether OCT could upregulate DCX in hippocampal progenitor cells (HPCs). Methods: We employed real-time PCR, western blot, immunostaining, chromatin immunoprecipitation, lentiviral transduction in HPCs, and the calcium influx assay. Results: OCT directly upregulated the transcription of Dcx in HPCs via activation of peroxisome proliferator-activated receptor α (PPARα), a lipid-lowering transcription factor. We observed that, HPCs of Ppara-null mice displayed significant impairment in DCX expression and neuronal differentiation as compared to that of wild-type mice. Interestingly, treatment with OCT stimulated the differentiation process of HPCs in wild-type, but not Ppara-null mice. Reconstruction of PPARα in mouse Ppara-null HPCs restored the expression of DCX, which was further stimulated with OCT treatment. In contrast, a dominant-negative mutant of PPARα significantly attenuated the stimulatory effect of OCT on DCX expression and suppressed neuronal differentiation of human neural progenitor cells. Furthermore, RNA microarray, STRING, chromatin immunoprecipitation, site-directed mutagenesis, and promoter reporter assay have identified DCX as a new target of PPARα. Conclusion: These results indicate that OCT, a sleep supplement, directly controls the expression of DCX and suggest that OCT may be repurposed for stimulating the hippocampal neurogenesis.

Role of the atypical protein kinase Cζ in regulation of 5′-AMP-activated protein kinase in cardiac and skeletal muscle

2009 ◽  
Vol 297 (2) ◽  
pp. E349-E357 ◽  
Author(s):  
John R. Ussher ◽  
Jagdip S. Jaswal ◽  
Cory S. Wagg ◽  
Heather E. Armstrong ◽  
David G. Lopaschuk ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Muscle Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Minor Role ◽  
Atypical Protein Kinase ◽  
Downstream Target ◽  
Ampk Phosphorylation ◽  
Amp Activated Protein Kinase

During metabolic stress, phosphorylation and activation of 5′-AMP-activated protein kinase (AMPK) becomes a major regulator of cellular energy metabolism in heart and skeletal muscle. Despite this, the upstream regulation of AMPK in both heart and muscle is poorly understood. Recent work has implicated the atypical protein kinase Cζ (PKCζ) as a regulator of AMPK in endothelial cells via phosphorylation of LKB1, an upstream AMPK kinase (AMPKK). Our goal was to determine the potential role PKCζ plays in regulating AMPK in cardiac and skeletal muscle. Cultures of H9c2 myocytes (cardiac) and C2C12 myotubes (skeletal muscle) were pretreated with a selective PKCζ pseudosubstrate peptide inhibitor and treated with various AMPK activating agents to determine whether PKCζ regulates AMPK. PKCζ activity was also examined in isolated working rat hearts subjected to ischemia. We show that PKCζ is not involved in regulating threonine 172 AMPK phosphorylation induced by metformin or phenformin in either cardiac or skeletal muscle cells but is involved in 5-aminoimidazole-4-carboxamine-1-β-d-ribofuranoside (AICAR)-induced AMPK phosphorylation in cardiac muscle cells. Activation of PKCζ with high palmitate concentrations is also insufficient to increase AMPK phosphorylation. Furthermore, we show that the ischemia-induced activation of AMPK is not accompanied by increased PKCζ activity. Finally, we show that PKCζ may actually be a downstream target of AMPK in skeletal muscle, since adenoviral expression of a dominant-negative mutant of AMPK prevented metformin- and AICAR-induced phosphorylation of PKCζ. We conclude that PKCζ plays a very minor role in the regulation of AMPK in cardiac and skeletal muscle and may actually be a downstream target of AMPK in skeletal muscle.

scholarly journals The Naturally Occurring Variant of Estrogen Receptor (ER) ERΔE7 Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ERα and ERβ

Endocrinology ◽  
2003 ◽  
Vol 144 (7) ◽  
pp. 2967-2976 ◽  
Author(s):  
Juana M. García Pedrero ◽  
Pedro Zuazua ◽  
Carlos Martínez-Campa ◽  
Pedro S. Lazo ◽  
Sofía Ramos
Keyword(s):  
Estrogen Receptor ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Activation Function ◽  
Inhibitory Effect ◽  
Dominant Negative ◽  
Wild Type ◽  
Independent Manner ◽  
Naturally Occurring ◽  
Estrogen Response

Abstract We have isolated and functionally characterized the exon 7-skipped variant (ERΔE7) of estrogen receptor (ER)α, which has emerged as the predominant variant expressed in multiple normal and tumoral tissues. However, to date no function has been established for this variant in mammalian cells. ERΔE7 exhibits a negligible ability to bind ligands, insensitivity to allosteric modulation by estrogen and antiestrogens, and loss of estrogen-dependent interaction with p160 coactivators such as SRC-1 and AIB1. ERΔE7 is able to form heterodimers with both ERα and ERβ in a ligand-independent manner. Transient expression experiments in HeLa cells show that increasing amounts of ERΔE7 result in a progressive inhibition of the estrogen-dependent transcriptional activation by both wild-type ERα and ERβ on estrogen response element-driven promoters. The inhibitory effect of ERΔE7 is due to the inhibition of binding of wild-type receptors to their responsive elements. Surprisingly, the activation function (AF)-1-dependent transactivation triggered by epithelial growth factor and phorbol-12-myristate-13-acetate is also abolished in ERΔE7 despite AF1 integrity, suggesting a cross-talk between AF1 and AF2 regions of the receptor. These results indicate that the naturally occurring variant ERΔE7 is a dominant negative receptor that, when expressed at high levels relative to wild-type ERs, might have profound effects on several estrogen-dependent functions.

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