scholarly journals Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein.

1995 ◽  
Vol 15 (2) ◽  
pp. 1060-1070 ◽  
Author(s):  
P Sabbatini ◽  
S K Chiou ◽  
L Rao ◽  
E White
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Growth Arrest ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Dna Virus ◽  
Adenovirus E1a ◽  
Reporter Assays ◽  
Adenovirus E1b ◽  
Transforming Proteins

BRK cell lines that stably express adenovirus E1A and a murine temperature-sensitive p53 undergo apoptosis when p53 assumes the wild-type conformation. Expression of the E1B 19,000-molecular-weight (19K) protein rescues cells from this p53-mediated apoptosis and diverts cells to a growth-arrested state. As p53 likely functions as a tumor suppressor by regulating transcription, the ability of the E1B 19K protein to regulate p53-mediated transactivation and transcriptional repression was investigated. In promoter-reporter assays the E1B 19K did not block p53-mediated transactivation but did alleviate p53-mediated transcriptional repression. E1B 19K expression permitted efficient transcriptional activation of the p21/WAF-1/cip-1 mRNA by p53, consistent with maintenance of the growth arrest function of p53. The E1B 19K protein is thereby unique among DNA virus-transforming proteins that target p53 for inactivation in that it selectively modulates the transcriptional properties of p53. The E1B 19K protein also rescued cells from apoptosis induced by inhibitors of transcription and protein synthesis. This suggests that cell death may result from the inhibition of expression of survival factors which function to maintain cell viability. p53 may induce apoptosis through generalized transcriptional repression. In turn, the E1B 19K protein may prevent p53-mediated apoptosis by alleviating p53-mediated transcriptional repression.

scholarly journals ARF Function Does Not Require p53 Stabilization or Mdm2 Relocalization

2002 ◽  
Vol 22 (1) ◽  
pp. 196-206 ◽  
Author(s):  
Chandrashekhar Korgaonkar ◽  
Lili Zhao ◽  
Modestos Modestou ◽  
Dawn E. Quelle
Keyword(s):  
Transcriptional Activation ◽  
Growth Arrest ◽  
Nucleolar Localization ◽  
Wild Type ◽  
Growth Inhibitory ◽  
Nucleolar Localization Signal ◽  
Localization Signal ◽  
Reporter Assays ◽  
Dependent Growth ◽  
Block Growth

ABSTRACT It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal 31RRPR34 did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.

Bcl-2 blocks p53-dependent apoptosis

1994 ◽  
Vol 14 (4) ◽  
pp. 2556-2563
Author(s):  
S K Chiou ◽  
L Rao ◽  
E White
Keyword(s):  
P53 Protein ◽  
Growth Arrest ◽  
Suppressor Gene ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Adenovirus E1a ◽  
Induction Of Apoptosis ◽  
Adenovirus E1b ◽  
State Growth

Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity.

scholarly journals Bcl-2 blocks p53-dependent apoptosis.

1994 ◽  
Vol 14 (4) ◽  
pp. 2556-2563 ◽  
Author(s):  
S K Chiou ◽  
L Rao ◽  
E White
Keyword(s):  
P53 Protein ◽  
Growth Arrest ◽  
Suppressor Gene ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Adenovirus E1a ◽  
Induction Of Apoptosis ◽  
Adenovirus E1b ◽  
State Growth

Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity.

scholarly journals Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases.

1996 ◽  
Vol 16 (8) ◽  
pp. 4445-4455 ◽  
Author(s):  
K M Latham ◽  
S W Eastman ◽  
A Wong ◽  
P W Hinds
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Growth Arrest ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Aberrant Expression ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Rat Fibroblasts ◽  
Wild Type P53

Rat fibroblasts transformed by a temperature-sensitive mutant of murine p53 undergo a reversible growth arrest in G1 at 32.5 degrees C, the temperature at which p53 adopts a wild-type conformation. The arrested cells contain inactive cyclin-dependent kinase 2 (cdk2) despite the presence of high levels of cyclin E and cdk-activating kinase activity. This is due in part to p53-dependent expression of the p2l cdk inhibitor. Upon shift to 39 degrees C, wild-type p53 is lost and cdk2 activation and pRb phosphorylation occur concomitantly with loss of p2l. This p53-mediated growth arrest can be abrogated by overexpression of cdk4 and cdk6 but not cdk2 or cyclins, leading to continuous proliferation of transfected cells in the presence of wild-type p53 and p2l. Kinase-inactive counterparts of cdk4 and cdk6 also rescue these cells from growth arrest, implicating a noncatalytic role for cdk4 and cdk6 in this resistance to p53-mediated growth arrest. Aberrant expression of these cell cycle kinases may thus result in an oncogenic interference with inhibitors of cell cycle progression.

Fusion of adenovirus E1A to the glucocorticoid receptor by high-resolution deletion cloning creates a hormonally inducible viral transactivator

1989 ◽  
Vol 9 (9) ◽  
pp. 3878-3887
Author(s):  
D M Becker ◽  
S M Hollenberg ◽  
R P Ricciardi
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Adenovirus Type ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Adenovirus E1a ◽  
Novel Strategy

The 289-amino-acid E1A protein of adenovirus type 2 stimulates transcription from early viral and certain cellular promoters. Its mechanism is not known, and there exist no temperature-sensitive mutants of E1A that could help to elucidate the details of E1A transcriptional activation. To create for E1A such a conditional phenotype, we fused portions of E1A to the human glucocorticoid receptor (GR) to make transactivation by E1A dependent on the presence of dexamethasone. Nested subsets of the E1A coding region, centered around the 46-amino-acid transactivating domain, were substituted for the DNA-binding domain of the GR. One of the resulting chimeric proteins (GR/E1A-99), which included the entire E1A transactivating domain, stimulated expression from a viral early promoter (E3) exclusively in the presence of hormone. GR/E1A-99 did not transactivate a GR-responsive promoter. It therefore exhibited the promoter specificity of E1A while possessing the hormone inducibility of the GR. Two smaller chimeras that contained only portions of the E1A transactivating domain failed to transactivate E3. These three chimeras were constructed by a novel strategy, high-resolution deletion cloning. In this procedure, series of unidirectional deletions were made with exonuclease III on each side of the E1A coding region at a resolution of 1 to 2 nucleotides. The large number of in-frame fragments present in the collection of deleted clones facilitated the construction of the GR/E1A chimeras and can be used to create many additional fusions.

scholarly journals Human SWI-SNF Component BRG1 Represses Transcription of the c-fos Gene

1999 ◽  
Vol 19 (4) ◽  
pp. 2724-2733 ◽  
Author(s):  
Daniel J. Murphy ◽  
Stephen Hardy ◽  
Daniel A. Engel
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Protein A ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Dependent Atpase ◽  
Adenocarcinoma Cells ◽  
Cyclic Amp Response Element ◽  
Function Expression ◽  
Transcription Factor E2f

ABSTRACT Yeast and mammalian SWI-SNF complexes regulate transcription through active modification of chromatin structure. Human SW-13 adenocarcinoma cells lack BRG1 protein, a component of SWI-SNF that has a DNA-dependent ATPase activity essential for SWI-SNF function. Expression of BRG1 in SW-13 cells potentiated transcriptional activation by the glucocorticoid receptor, which is known to require SWI-SNF function. BRG1 also specifically repressed transcription from a transfected c-fos promoter and correspondingly blocked transcriptional activation of the endogenous c-fos gene. Mutation of lysine residue 798 in the DNA-dependent ATPase domain of BRG1 significantly reduced its ability to repress c-fostranscription. Repression by BRG1 required the cyclic AMP response element of the c-fos promoter but not nearby binding sites for Sp1, YY1, or TFII-I. Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb protein, transcriptional repression by BRG1 was weak unless wild-type Rb was also supplied. Interestingly, Rb-dependent repression by BRG1 was found to take place through a pathway that is independent of transcription factor E2F.

scholarly journals Concerted Regulation of Wild-Type p53 Nuclear Accumulation and Activation by S100B and Calcium-Dependent Protein Kinase C

1999 ◽  
Vol 19 (10) ◽  
pp. 7168-7180 ◽  
Author(s):  
Christian Scotto ◽  
Christian Delphin ◽  
Jean Christophe Deloulme ◽  
Jacques Baudier
Keyword(s):  
Nuclear Translocation ◽  
Growth Arrest ◽  
Nuclear Accumulation ◽  
Temperature Sensitive ◽  
Checkpoint Control ◽  
Wild Type ◽  
Calcium Dependent ◽  
Mouse Embryo Fibroblasts ◽  
Wild Type P53 ◽  
Embryo Fibroblasts

ABSTRACT The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G1 checkpoint control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272–4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G1 phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G1 arrest at the nonpermissive temperature (37.5°C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32°C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G1 phase and activation of a p53-dependent G1checkpoint control.

scholarly journals Fusion of adenovirus E1A to the glucocorticoid receptor by high-resolution deletion cloning creates a hormonally inducible viral transactivator.

1989 ◽  
Vol 9 (9) ◽  
pp. 3878-3887 ◽  
Author(s):  
D M Becker ◽  
S M Hollenberg ◽  
R P Ricciardi
Keyword(s):  
Amino Acid ◽  
High Resolution ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Adenovirus Type ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Adenovirus E1a ◽  
Novel Strategy

The 289-amino-acid E1A protein of adenovirus type 2 stimulates transcription from early viral and certain cellular promoters. Its mechanism is not known, and there exist no temperature-sensitive mutants of E1A that could help to elucidate the details of E1A transcriptional activation. To create for E1A such a conditional phenotype, we fused portions of E1A to the human glucocorticoid receptor (GR) to make transactivation by E1A dependent on the presence of dexamethasone. Nested subsets of the E1A coding region, centered around the 46-amino-acid transactivating domain, were substituted for the DNA-binding domain of the GR. One of the resulting chimeric proteins (GR/E1A-99), which included the entire E1A transactivating domain, stimulated expression from a viral early promoter (E3) exclusively in the presence of hormone. GR/E1A-99 did not transactivate a GR-responsive promoter. It therefore exhibited the promoter specificity of E1A while possessing the hormone inducibility of the GR. Two smaller chimeras that contained only portions of the E1A transactivating domain failed to transactivate E3. These three chimeras were constructed by a novel strategy, high-resolution deletion cloning. In this procedure, series of unidirectional deletions were made with exonuclease III on each side of the E1A coding region at a resolution of 1 to 2 nucleotides. The large number of in-frame fragments present in the collection of deleted clones facilitated the construction of the GR/E1A chimeras and can be used to create many additional fusions.

scholarly journals Modulation of transcriptional activation of the proliferating cell nuclear antigen promoter by the adenovirus E1A 243-residue oncoprotein depends on proximal activators.

1994 ◽  
Vol 14 (1) ◽  
pp. 543-553 ◽  
Author(s):  
G F Morris ◽  
C Labrie ◽  
M B Mathews
Keyword(s):  
Transcriptional Activation ◽  
Proliferating Cell Nuclear Antigen ◽  
Transcription Initiation ◽  
Adenovirus Type ◽  
Nuclear Antigen ◽  
Similar Response ◽  
Transcriptional Activators ◽  
Adenovirus E1a ◽  
Adenovirus E1b ◽  
Proliferating Cell

Previous analyses defined a proliferating cell nuclear antigen (PCNA) E1A-responsive element (PERE) in the PCNA promoter that is essential for transactivation by the 243-residue product of the adenovirus type 2 E1A 12S mRNA (E1A 243R). In this report, we show that the PERE activates a heterologous basal promoter and confers susceptibility to transactivation by E1A 243R, indicating that the PERE is both necessary and sufficient for the response of the PCNA promoter to this oncoprotein. Insertion of linker sequences between the PERE and the site of transcription initiation in the PCNA promoter severely impairs the promoter's response to E1A 243R transactivation. GAL4 sites can replace the function of the PERE in the E1A 243R response of the PCNA basal promoter if transcriptional activators of suitable strength are supplied as GAL4 fusion proteins. Weak transcriptional activators render the PCNA basal promoter subject to transactivation by E1A 243R but do not endow the adenovirus E1B basal promoter with a similar response. Strong transcriptional activators do not support transactivation by E1A 243R, however; instead, E1A reduces the ability of the strong activators to activate both the PCNA and E1B basal promoters. Although other mechanistic differences might determine the response, the data imply a relationship between the activation strength of promoter-proximal effectors and the response of the PCNA basal promoter to E1A 243R. These experiments indicate that the PERE can function autonomously in mediating transactivation by E1A 243R and that the PCNA basal promoter is configured in a manner that permits modulation by E1A 243R of transcriptional activation by promoter-proximal effectors.

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