scholarly journals MDM2 Is Required for Suppression of Apoptosis by Activated Akt1 in Salivary Acinar Cells

2006 ◽  
Vol 26 (23) ◽  
pp. 8840-8856 ◽  
Author(s):  
Kirsten H. Limesand ◽  
Kathryn L. Schwertfeger ◽  
Steven M. Anderson
Keyword(s):  
Transgenic Mice ◽  
Gamma Irradiation ◽  
Salivary Glands ◽  
P53 Protein ◽  
Acinar Cells ◽  
Dominant Negative ◽  
Common Side Effect ◽  
Protein Accumulation ◽  
Irreversible Damage

ABSTRACT Chronic damage to the salivary glands is a common side effect following head and neck irradiation. It is hypothesized that irreversible damage to the salivary glands occurs immediately after radiation; however, previous studies with rat models have not shown a causal role for apoptosis in radiation-induced injury. We report that etoposide and gamma irradiation induce apoptosis of salivary acinar cells from FVB control mice in vitro and in vivo; however, apoptosis is reduced in transgenic mice expressing a constitutively activated mutant of Akt1 (myr-Akt1). Expression of myr-Akt1 in the salivary glands results in a significant reduction in phosphorylation of p53 at serine18, total p53 protein accumulation, and p21WAF1 or Bax mRNA following etoposide or gamma irradiation of primary salivary acinar cells. The reduced level of p53 protein in myr-Akt1 salivary glands corresponds with an increase in MDM2 phosphorylation in vivo, suggesting that the Akt/MDM2/p53 pathway is responsible for suppression of apoptosis. Dominant-negative Akt blocked phosphorylation of MDM2 in salivary acinar cells from myr-Akt1 transgenic mice. Reduction of MDM2 levels in myr-Akt1 primary salivary acinar cells with small interfering RNA increases the levels of p53 protein and renders these cells susceptible to etoposide-induced apoptosis in spite of the presence of activated Akt1. These results indicate that MDM2 is a critical substrate of activated Akt1 in the suppression of p53-dependent apoptosis in vivo.

scholarly journals Amyloid-ß promotes neurotoxicity by Cdk5-induced p53 stabilization

2018 ◽  
Author(s):  
Rebeca Lapresa ◽  
Jesús Agulla ◽  
Irene Sánchez-Morán ◽  
Juan P. Bolaños ◽  
Angeles Almeida
Keyword(s):  
Neuronal Apoptosis ◽  
Therapeutic Strategy ◽  
Neuronal Damage ◽  
P53 Protein ◽  
Protein A ◽  
Tumor Suppressor Protein ◽  
Protein Accumulation ◽  
Brain Areas ◽  
P53 Tumor Suppressor Protein

ABSTRACTThe p53 tumor suppressor protein, a key regulator of cell apoptosis, has been described to accumulate in affected brain areas from Alzheimer’s disease (AD) patients. However, whether p53 plays any role in AD pathogenesis remains unknown. Here, we found that exposure of neurons to oligomers of the amyloidogenic fragment 25-35 of the Aß peptide (Aβ25-35) activated Cdk5, which promoted p53 protein phosphorylation and stabilization. Moreover, Aβ25-35-mediated mitochondrial dysfunction and neuronal apoptosis were prevented by both genetic and pharmacological inhibition of either p53 or Cdk5 activities. To confirm this mechanism in vivo, Aβ25-35 was stereotaxically injected in the cerebral right ventricle of mice, a treatment that caused p53 protein accumulation, dendrite disruption and neuronal death. Furthermore, these effects were prevented in p53 knockout mice or by pharmacologically inhibiting p53. Thus, Aβ25-35 triggers Cdk5 activation to induce p53 phosphorylation and stabilization, which leads to neuronal damage. Inhibition of the Cdk5-p53 pathway may therefore represent a novel therapeutic strategy against Aβ-induced neurodegeneration.

High incidence of lung, bone, and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene

1989 ◽  
Vol 9 (9) ◽  
pp. 3982-3991
Author(s):  
A Lavigueur ◽  
V Maltby ◽  
D Mock ◽  
J Rossant ◽  
T Pawson ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Malignant Transformation ◽  
P53 Protein ◽  
P53 Gene ◽  
Cell Types ◽  
Molecular Events ◽  
High Incidence ◽  
Transgenic Lines ◽  
Functional Inactivation

We have investigated the role of the p53 gene in oncogenesis in vivo by generating transgenic mice carrying murine p53 genomic fragments isolated from a mouse Friend erythroleukemia cell line or BALB/c mouse liver DNA. Elevated levels of p53 mRNA were detected in several tissues of two transgenic lines tested. Increased levels of p53 protein were also detected in most of the tissues analyzed by Western blotting (immunoblotting). Because both transgenes encoded p53 proteins that were antigenically distinct from wild-type p53, it was possible to demonstrate that overexpression of the p53 protein was mostly, if not entirely, due to the expression of the transgenes. Neoplasms developed in 20% of the transgenic mice, with a high incidence of lung adenocarcinomas, osteosarcomas, and lymphomas. Tissues such as ovaries that expressed the transgene at high levels were not at higher risk of malignant transformation than tissues expressing p53 protein at much lower levels. The long latent period and low penetrance suggest that overexpression of p53 alone is not sufficient to induce malignancies and that additional events are required. These observations provide direct evidence that mutant alleles of the p53 oncogene have oncogenic potential in vivo and that different cell types show intrinsic differences in susceptibility to malignant transformation by p53. Since recent data suggest that p53 may be a recessive oncogene, it is possible that the elevated tumor incidence results from functional inactivation of endogenous p53 by overexpression of the mutant transgene. The high incidence of lung and bone tumors suggests that p53 transgenic mice may provide a useful model to investigate the molecular events that underlie these malignancies in humans.

scholarly journals Gap Junction–mediated Cell–Cell Communication Modulates Mouse Neural Crest Migration

1998 ◽  
Vol 143 (6) ◽  
pp. 1725-1734 ◽  
Author(s):  
G.Y. Huang ◽  
E.S. Cooper ◽  
K. Waldo ◽  
M.L. Kirby ◽  
N.B. Gilula ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neural Crest ◽  
Gap Junction ◽  
Heart Defects ◽  
Neural Crest Cells ◽  
Dominant Negative ◽  
Cardiac Neural Crest ◽  
Gap Junction Communication ◽  
Neural Crest Migration

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in α1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for α1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing α1 connexins, and from α1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative α1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and α1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous α1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and α1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the α1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium— an elevation in the CMV43 mice vs. a reduction in the α1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non–neural crest– derived tissues. Overall, these findings suggest that gap junction communication mediated by α1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of α1 connexin function.

scholarly journals Epitope-Tagged P0Glycoprotein Causes Charcot-Marie-Tooth–Like Neuropathy in Transgenic Mice

2000 ◽  
Vol 151 (5) ◽  
pp. 1035-1046 ◽  
Author(s):  
Stefano C. Previtali ◽  
Angelo Quattrini ◽  
Marina Fasolini ◽  
Maria Carla Panzeri ◽  
Antonello Villa ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Point Mutations ◽  
Dominant Negative ◽  
Epitope Tag ◽  
Charcot Marie Tooth ◽  
Demyelinating Neuropathies ◽  
Protein Zero ◽  
Homophilic Adhesion

In peripheral nerve myelin, the intraperiod line results from compaction of the extracellular space due to homophilic adhesion between extracellular domains (ECD) of the protein zero (P0) glycoprotein. Point mutations in this region of P0 cause human hereditary demyelinating neuropathies such as Charcot-Marie-Tooth. We describe transgenic mice expressing a full-length P0 modified in the ECD with a myc epitope tag. The presence of the myc sequence caused a dysmyelinating peripheral neuropathy similar to two distinct subtypes of Charcot-Marie-Tooth, with hypomyelination, altered intraperiod lines, and tomacula (thickened myelin). The tagged protein was incorporated into myelin and was associated with the morphological abnormalities. In vivo and in vitro experiments showed that P0myc retained partial adhesive function, and suggested that the transgene inhibits P0-mediated adhesion in a dominant-negative fashion. These mice suggest new mechanisms underlying both the pathogenesis of P0 ECD mutants and the normal interactions of P0 in the myelin sheath.

scholarly journals Nrf Transcription Factors in Keratinocytes Are Essential for Skin Tumor Prevention but Not for Wound Healing

2006 ◽  
Vol 26 (10) ◽  
pp. 3773-3784 ◽  
Author(s):  
Ulrich auf dem Keller ◽  
Marcel Huber ◽  
Tobias A. Beyer ◽  
Angelika Kümin ◽  
Christina Siemes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Wound Repair ◽  
Target Genes ◽  
Dominant Negative ◽  
Skin Tumor ◽  
Cellular Stress Response ◽  
Skin Development

ABSTRACT The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.

scholarly journals Effects of Mutant Ubiquitin on ts1 Retrovirus-Mediated Neuropathology

2003 ◽  
Vol 77 (13) ◽  
pp. 7193-7201 ◽  
Author(s):  
Mei Zhang ◽  
Sherry Thurig ◽  
Maria Tsirigotis ◽  
Paul K. Y. Wong ◽  
Kenneth R. Reuhl ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Therapeutic Potential ◽  
Leukemia Virus ◽  
Lewy Bodies ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Spongiform Encephalopathy ◽  
Temperature Sensitive ◽  
Transgenic Mouse Line

ABSTRACT ts1 is a temperature-sensitive mutant of Moloney murine leukemia virus that induces a rapid spongiform encephalopathy in mice infected as newborns. The pathological features include the formation of ubiquitinated inclusions resembling Lewy bodies. To determine how perturbation of the ubiquitin-proteasome pathway might affect ts1-mediated neurodegeneration, the virus was introduced into transgenic mice in which the assembly of ubiquitin chains was compromised by the expression of dominant-negative mutant ubiquitin. The onset of symptoms was greatly delayed in a transgenic mouse line expressing K48R mutant ubiquitin; no such delay was observed in mice expressing a wild-type ubiquitin transgene or K63R mutant ubiquitin. The extended latency was found to correlate with a delayed increase in viral titers. Pathological findings in K48R transgenic mice at 60 days were found to be similar to those in the other strains at 30 days, suggesting that while delayed, the neurodegenerative process in K48R mice was otherwise similar. These data demonstrate the sensitivity of retroviral replication to the partial disruption of ubiquitin-mediated proteolysis in vivo, a finding that may have therapeutic potential.

Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1808-1816 ◽  
Author(s):  
Erwan Le Scolan ◽  
Dimitri Pchejetski ◽  
Yoshiko Banno ◽  
Nicole Denis ◽  
Patrick Mayeux ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Recurrent Event ◽  
Dominant Negative Mutant ◽  
Apoptosis Resistance ◽  
Kinase Gene ◽  
Extracellular Signal

Abstract The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia. (Blood. 2005;106:1808-1816)

FGF suppresses apoptosis and induces differentiation of fibre cells in the mouse lens

Development ◽  
1995 ◽  
Vol 121 (12) ◽  
pp. 4383-4393 ◽  
Author(s):  
R.L. Chow ◽  
G.D. Roux ◽  
M. Roghani ◽  
M.A. Palmer ◽  
D.B. Rifkin ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transgenic Mice ◽  
Dominant Negative ◽  
Fgf Receptor ◽  
Fibre Cell ◽  
Dominant Negative Form ◽  
Lens Fibre ◽  
Mouse Lens ◽  
Negative Form

To determine whether fibroblast growth factor (FGF) has a role in lens development, we have generated transgenic mice expressing a dominant-negative form of the murine FGF receptor-1 (FGFRDN) in the lens. Using the fibre cell-specific alpha A-crystallin promoter to express the FGFRDN, we have asked whether FGF is required for fibre cell differentiation. The transgenic mice display diminished differentiation of fibre cells as indicated by their reduced elongation. In addition, transgenic lenses have an unusual refractile anomaly that morphological and biochemical data show results from the apoptosis of fibre cells in the central region of the lens. These results show that lens fibre cells are dependent on FGF for their survival and differentiation, and demonstrate that growth factor deprivation in vivo can lead to apoptosis.

scholarly journals A Mutant of Human Papillomavirus Type 16 E6 Deficient in Binding α-Helix Partners Displays Reduced Oncogenic Potential In Vivo

2002 ◽  
Vol 76 (24) ◽  
pp. 13039-13048 ◽  
Author(s):  
Marie Nguyen ◽  
Shiyu Song ◽  
Amy Liem ◽  
Elliot Androphy ◽  
Yun Liu ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
P53 Protein ◽  
Human Papillomaviruses ◽  
Skin Tumors ◽  
Cellular Proteins ◽  
Viral Gene ◽  
Gene Encoding ◽  
Tumor Viruses ◽  
Α Helix

ABSTRACT Human papillomaviruses (HPVs) are small DNA tumor viruses that are the causative agent of warts and are associated with many anogenital cancers. The viral gene encoding the E6 protein has been found to be involved in HPV oncogenesis. E6 is known to inactivate the cellular tumor suppressor, p53. In addition, E6 has been shown to bind to a variety of other cellular proteins. The focus of this study was to determine what role the interactions of E6 with a subset of cellular proteins which contain a common α-helical domain in their E6 binding region (α-helix partners) play in E6-mediated phenotypes. We generated transgenic mice expressing a mutant of E6, E6I128T, which is defective for binding at least a subset of the α-helix partners, including E6AP, the ubiquitin ligase that mediates E6-dependent degradation of the p53 protein, to determine whether binding of α-helix partners plays a role in E6-mediated activities in vivo. Unlike mice expressing the wild-type E6 (strain K14E6WT), the mice expressing E6I128T lacked the ability to alter the radiation-induced block to DNA synthesis and promote the formation of benign skin tumors in conjunction with chemical carcinogens. Additionally, they displayed reduced levels of skin hyperplasia, spontaneous skin tumors, and tumor progression activity compared to those of the K14E6WT mice. From these results, we conclude that a domain in E6 that mediates α-helix partner binding is critical for E6-induced phenotypes in transgenic mice.

Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle

2007 ◽  
Vol 292 (6) ◽  
pp. L1473-L1479 ◽  
Author(s):  
Moira Hagen ◽  
Karen Fagan ◽  
Wolfgang Steudel ◽  
Michelle Carr ◽  
Kirk Lane ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transgenic Mice ◽  
Bmp Signaling ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Second Hit ◽  
Protein Receptor ◽  
Bmp Pathway

The majority of familial pulmonary arterial hypertension (PAH) cases are caused by mutations in the type 2 bone morphogenetic protein receptor (BMPR2). However, less than one-half of BMPR2 mutation carriers develop PAH, suggesting that the most important function of BMPR2 mutation is to cause susceptibility to a “second hit.” There is substantial evidence from the literature implicating dysregulated inflammation, in particular the cytokine IL-6, in the development of PAH. We thus hypothesized that the BMP pathway regulates IL-6 in pulmonary tissues and conversely that IL-6 regulates the BMP pathway. We tested this in vivo using transgenic mice expressing an inducible dominant negative BMPR2 in smooth muscle, using mice injected with an IL-6-expressing virus, and in vitro using small interfering RNA (siRNA) to BMPR2 in human pulmonary artery smooth muscle cells (PA SMC). Consistent with our hypothesis, we found upregulation of IL-6 in both the transgenic mice and in cultured PA SMC with siRNA to BMPR2; this could be abolished with p38MAPK inhibitors. We also found that IL-6 in vivo caused a twofold increase in expression of the BMP signaling target Id1 and caused increased BMP activity in a luciferase-reporter assay in PA SMC. Thus we have shown both in vitro and in vivo a complete negative feedback loop between IL-6 and BMP, suggesting that an important consequence of BMPR2 mutations may be poor regulation of cytokines and thus vulnerability to an inflammatory second hit.

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