scholarly journals Molecular imaging of nuclear factor-Y transcriptional activity maps proliferation sites in live animals

2012 ◽  
Vol 23 (8) ◽  
pp. 1467-1474 ◽  
Author(s):  
Frauke Goeman ◽  
Isabella Manni ◽  
Simona Artuso ◽  
Balaji Ramachandran ◽  
Gabriele Toietta ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Genetically Engineered ◽  
Hepatocyte Proliferation ◽  
Luciferase Expression ◽  
Nuclear Factor ◽  
Proliferating Cells ◽  
Nuclear Factor Y ◽  
Reporter Mice

In vivo imaging involving the use of genetically engineered animals is an innovative powerful tool for the noninvasive assessment of the molecular and cellular events that are often targets of therapy. On the basis of the knowledge that the activity of the nuclear factor-Y (NF-Y) transcription factor is restricted in vitro to proliferating cells, we have generated a transgenic reporter mouse, called MITO-Luc (for mitosis-luciferase), in which an NF-Y–dependent promoter controls luciferase expression. In these mice, bioluminescence imaging of NF-Y activity visualizes areas of physiological cell proliferation and regeneration during response to injury. Using this tool, we highlight for the first time a role of NF-Y activity on hepatocyte proliferation during liver regeneration. MITO-Luc reporter mice should facilitate investigations into the involvement of genes in cell proliferation and provide a useful model for studying aberrant proliferation in disease pathogenesis. They should be also useful in the development of new anti/proproliferative drugs and assessment of their efficacy and side effects on nontarget tissues.

scholarly journals Mouse models for BRAF-induced cancers

2007 ◽  
Vol 35 (5) ◽  
pp. 1329-1333 ◽  
Author(s):  
C. Pritchard ◽  
L. Carragher ◽  
V. Aldridge ◽  
S. Giblett ◽  
H. Jin ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mouse Models ◽  
Human Cancer ◽  
Cre Recombinase ◽  
Genetically Engineered ◽  
Mitogen Activated Protein Kinase ◽  
Braf Mutations ◽  
Single Nucleotide Change

Oncogenic mutations in the BRAF gene are detected in ∼7% of human cancer samples with a particularly high frequency of mutation in malignant melanomas. Over 40 different missense BRAF mutations have been found, but the vast majority (>90%) represent a single nucleotide change resulting in a valine→glutamate mutation at residue 600 (V600EBRAF). In cells cultured in vitro, V600EBRAF is able to stimulate endogenous MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] and ERK phosphorylation leading to an increase in cell proliferation, cell survival, transformation, tumorigenicity, invasion and vascular development. Many of these hallmarks of cancer can be reversed by treatment of cells with siRNA (small interfering RNA) to BRAF or by inhibiting MEK, indicating that BRAF and MEK are attractive therapeutic targets in cancer samples with BRAF mutations. In order to fully understand the role of oncogenic BRAF in cancer development in vivo as well as to test the in vivo efficacy of anti-BRAF or anti-MEK therapies, GEMMs (genetically engineered mouse models) have been generated in which expression of oncogenic BRaf is conditionally dependent on the Cre recombinase. The delivery/activation of the Cre recombinase can be regulated in both a temporal and spatial manner and therefore these mouse models can be used to recapitulate the somatic mutation of BRAF that occurs in different tissues in the development of human cancer. The data so far obtained following Cre-mediated activation in haemopoietic tissue and the lung indicate that V600EBRAF mutation can drive tumour initiation and that its primary effect is to induce high levels of cyclin D1-mediated cell proliferation. However, hallmarks of OIS (oncogene-induced senescence) are evident that restrain further development of the tumour.

scholarly journals Raf-1 Physically Interacts with Rb and Regulates Its Function: a Link between Mitogenic Signaling and Cell Cycle Regulation

1998 ◽  
Vol 18 (12) ◽  
pp. 7487-7498 ◽  
Author(s):  
Sheng Wang ◽  
Richik N. Ghosh ◽  
Srikumar P. Chellappan
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Physical Interaction ◽  
Proliferating Cells

ABSTRACT Cells initiate proliferation in response to growth factor stimulation, but the biochemical mechanisms linking signals received at the cell surface receptors to the cell cycle regulatory molecules are not yet clear. In this study, we show that the signaling molecule Raf-1 can physically interact with Rb and p130 proteins in vitro and in vivo and that this interaction can be detected in mammalian cells without overexpressing any component. The binding of Raf-1 to Rb occurs subsequent to mitogen stimulation, and this interaction can be detected only in proliferating cells. Raf-1 can inactivate Rb function and can reverse Rb-mediated repression of E2F1 transcription and cell proliferation efficiently. The region of Raf-1 involved in Rb binding spanned residues 1 to 28 at the N terminus, and functional inactivation of Rb required a direct interaction. Serum stimulation of quiescent human fibroblast HSF8 cells led to a partial translocation of Raf-1 into the nucleus, where it colocalized with Rb. Further, Raf-1 was able to phosphorylate Rb in vitro quite efficiently. We believe that the physical interaction of Raf-1 with Rb is a vital step in the growth factor-mediated induction of cell proliferation and that Raf-1 acts as a direct link between cell surface signaling cascades and the cell cycle machinery.

scholarly journals A Simple Method to AssessIn VivoProliferation in Lung Vasculature with EdU: The Case of MMC-Induced PVOD in Rat

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Antigny Fabrice ◽  
Ranchoux Benoît ◽  
Nadeau Valérie ◽  
Edmund Lau ◽  
Bonnet Sébastien ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pulmonary Veins ◽  
Endothelial Cell Proliferation ◽  
Pulmonary Vasculature ◽  
Microvascular Endothelial Cell ◽  
Proliferating Cells ◽  
Simple Method ◽  
Gold Standard Method

5-Ethynyl-2′-deoxyuridine (EdU) incorporation is becoming the gold standard method forin vitroandin vivovisualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a “terminal” state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD.In vivopulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.

scholarly journals An Engineered Arginase FC Protein Inhibits Tumor GrowthIn VitroandIn Vivo

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Lihua Li ◽  
Yan Wang ◽  
Jun Chen ◽  
Bi Cheng ◽  
Jiehua Hu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Fusion Protein ◽  
Genetically Engineered ◽  
Arginine Deiminase ◽  
Cellular Migration ◽  
Fc Fusion Protein

Arginine is a semiessential amino acid required for the growth of melanoma and hepatocellular carcinoma, and the enzymatic removal of arginine by pegylated arginine deiminase (ADI) or arginase is being tested clinically. Here, we report a genetically engineered arginase FC fusion protein exhibiting a prolonged half-life and enhanced efficacy. The use of this enzyme to treat different tumor lines both inhibited cell proliferation and impaired cellular migrationin vitroandin vivo. Our data reinforce the hypothesis that nutritional depletion is a key strategy for cancer treatment.

Uncoupling between proliferation and differentiation of ovine granulosa cells in vitro

1994 ◽  
Vol 142 (3) ◽  
pp. 497-510 ◽  
Author(s):  
D Monniaux ◽  
C Pisselet ◽  
J Fontaine
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Growth ◽  
Proliferating Cells ◽  
Progesterone Secretion ◽  
Proliferation And Differentiation ◽  
Igf I

Abstract Granulosa cells of ovarian follicles both proliferate and undergo differentiation. In vivo, an inverse relationship between proliferation and steroidogenesis is observed. However, both processes can be enhanced by insulin-like growth factor-I (IGF-I) in vitro. Studies were undertaken in the ewe to understand the mechanisms controlling the balance between proliferation and differentiation in cultured granulosa cells from antral follicles better. For this purpose, granulosa cells from ovine small follicles (1–3 mm in diameter) and large follicles (5–7 mm in diameter) were compared for progesterone secretion, cytochrome P450 side-chain cleavage (P450scc) expression and their proportions of non-proliferating (G0) cells, in response to IGF-I and FSH stimulation in vitro. IGF-I mainly enhanced the proliferation of granulosa cells from small follicles but it strongly increased progesterone secretion and P450scc expression in granulosa cells from large follicles, in synergy with FSH. Blocking granulosa cell proliferation by the administration of colcemid or aphidicolin had no effect or a weak stimulating effect on progesterone secretion. At the beginning of the culture period, the proportion of non-proliferating cells, estimated by continuous [3H]thymidine labelling experiments, was clearly higher in large than in small follicles (91% vs 30%, P<0·001). For both cell types, treatment with IGF-I in vitro reduced the proportion of non-proliferating cells at 72 h of culture (40% vs 70% respectively in IGF-I-stimulated and unstimulated cells from large follicles, P<0·001, and 17% vs 30% respectively in IGF-I-stimulated and unstimulated cells from small follicles, P<0·001). Treatment with FSH had no effect on the proportion of non-proliferating cells. As revealed by immunohistochemistry experiments, IGF-I, in synergy with FSH, clearly increased the percentage of cells expressing P450scc enzyme and the intensity of staining in granulosa cells from large follicles. Unexpectedly, heavily stained cells in mitosis were observed in IGF-I-stimulated cells from large follicles after 96 h of culture, suggesting that dividing cells might also produce progesterone. Overall, these results support the hypothesis that the growth-promoting and the cytodifferentiative effects of IGF-I are clearly distinct. Moreover, they suggest that uncoupling between proliferation and steroidogenesis may occur in cultured ovine granulosa cells. The loss of proliferative activity accompanying terminal follicular growth in vivo could be reversed in vitro. During terminal follicular growth in vivo, the existence of an active mechanism inhibiting granulosa cell proliferation, and unrelated to terminal differentiation, is therefore strongly suspected. Journal of Endocrinology (1994) 142, 497–510

scholarly journals α1-Adrenergic Receptor Antagonists: Novel Therapy for Pituitary Adenomas

2005 ◽  
Vol 19 (12) ◽  
pp. 3085-3096 ◽  
Author(s):  
Manory A. Fernando ◽  
Anthony P. Heaney
Keyword(s):  
Cell Proliferation ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Pituitary Tumor ◽  
Adrenergic Receptor ◽  
Pituitary Tumors ◽  
Nuclear Factor Κb ◽  
Nuclear Factor

Abstract Pituitary tumors are common and cause considerable morbidity due to local invasion and altered hormone secretion. Doxazosin (dox), a selective α1-adrenergic receptor antagonist, used to treat hypertension, also inhibits prostate cancer cell proliferation. We examined the effects of dox on murine and human pituitary tumor cell proliferation in vitro and in vivo. dox treatment inhibited proliferation of murine pituitary tumor cells, induced G0-G1 cell cycle arrest, and reduced phosphorylated retinoblastoma levels. In addition, increased annexin-fluorescein isothiocyanate immunoreactivity and cleaved caspase-3 levels, in keeping with dox-mediated apoptosis, were observed in the human and murine pituitary tumor cells, and dox administration to mice, harboring corticotroph tumors, decreased tumor growth and reduced plasma ACTH levels. dox-mediated antiproliferative and proapoptotic actions were not confined to α-adrenergic receptor-expressing pituitary tumor cells and were unaffected by cotreatment with the α-adrenergic receptor blocker, phenoxybenzamine. dox treatment led to reduced phosphorylated inhibitory κB (IκB)-α expression, and nuclear factor-κB transcription and decreased basal and TNFα-induced proopiomelanocortin transcriptional activation. These results demonstrate that the selective α1-adrenergic receptor antagonist dox inhibits pituitary tumor cell growth in vitro and in vivo by mechanisms that are in part independent of its α-adrenergic receptor-blocking actions and involve down-regulation of nuclear factor-κB signaling. dox is proposed as a possible novel medical therapy for pituitary tumors.

scholarly journals Cell-type specific circadian bioluminescence rhythms recorded from Dbp reporter mice reveal circadian oscillator misalignment

2021 ◽  
Author(s):  
Ciearra B. Smith ◽  
Vincent van der Vinne ◽  
Eleanor McCartney ◽  
Adam C. Stowie ◽  
Tanya L. Leise ◽  
...  
Keyword(s):  
Locomotor Activity ◽  
Circadian Rhythms ◽  
Luciferase Expression ◽  
Dependent Manner ◽  
Cell Type ◽  
Reporter Mice ◽  
Mouse Tissues ◽  
Cell Type Specific

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vitro and In vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei in vitro. In vivo studies show stable Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ liver reporter mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies provide clear evidence for circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

scholarly journals Major Histocompatibility Complex Class II Transcriptional Platform: Assembly of Nuclear Factor Y and Regulatory Factor X (RFX) on DNA Requires RFX5 Dimers

2002 ◽  
Vol 22 (15) ◽  
pp. 5616-5625 ◽  
Author(s):  
Nabila Jabrane-Ferrat ◽  
Nada Nekrep ◽  
Giovanna Tosi ◽  
Laura J. Esserman ◽  
B. Matija Peterlin
Keyword(s):  
Class Ii ◽  
Nuclear Factor ◽  
Factor X ◽  
Complex Class ◽  
Regulatory Factor ◽  
Mhc Ii ◽  
Nuclear Factor Y ◽  
Histocompatibility Complex

ABSTRACT Major histocompatibility complex class II (MHC-II) genes are regulated in a B-cell-specific and gamma interferon-inducible manner. Conserved upstream sequences (CUS) in their compact promoters bind nuclear factor Y (NFY) and regulatory factor X (RFX) complexes. These DNA-bound proteins form a platform that attracts the class II transactivator, which initiates and elongates MHC-II transcription. In this report, we analyzed the complex assembly of these DNA-bound proteins. First, we found that NFY can interact with RFX in cells. In particular, NFYA and NFYC bound RFXANK/B in vitro. Next, RFX5 formed dimers in vivo and in vitro. Within a leucine-rich stretch N-terminal to the DNA-binding domain in RFX5, the leucine at position 66 was found to be critical for this self-association. Mutant RFX5 proteins that could not form dimers also did not support the formation of higher-order DNA-protein complexes on CUS in vitro or MHC-II transcription in vivo. We conclude that the MHC-II transcriptional platform begins to assemble off CUS and then binds DNA via multiple, spatially constrained interactions. These findings offer one explanation of why in the Bare Lymphocyte Syndrome, which is a congenital severe combined immunodeficiency, MHC-II promoters are bare when any subunit of RFX is mutated or missing.

scholarly journals Death and Proliferation of Lymphocytes in Immune Response and Tumor Cells Are Controlled by pH Balance

2021 ◽  
Author(s):  
Wei-ping Zeng
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Unified Theory ◽  
Biological Processes ◽  
Proliferating Cells ◽  
Proliferation Of Lymphocytes ◽  
The Warburg Effect ◽  
Acid Treatments

Many biological processes are controlled by cell death and proliferation. Previous evidence suggests that cell proliferation and death by apoptosis are regulated by separate pathways. The present study found that cellular pH was positively correlated with proliferation but negatively with cell death. Alkaline treatments enhanced lymphocyte proliferation in response to antigen challenge in vivo and in in vitro cultures, whereas acid treatments induced cell death. Low pH was incompatible with the survival of highly proliferating cells, and the susceptibility to the acid-induced death was determined in part by the proliferative status of the lymphocytes. Likewise, alkaline treatments maintained tumor cell proliferation whereas acid treatments induced death. These data support a unified theory for the regulation of cell death and proliferation where a cellular pH balance controls both events, and the mitochondria as proton generators act as pH-stats. Thus, the Warburg effect is viewed as necessary for proliferating cells to have a high cellular pH environment to both survive and accelerate proliferation.

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