Potent inhibitors of cyclin-dependent kinase 2 induce nuclear accumulation of wild-type p53 and nucleolar fragmentation in human untransformed and tumor-derived cells

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.

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Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6565-6577.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6565-6577

Author(s):

G Shaulsky ◽

N Goldfinger ◽

A Ben-Ze'ev ◽

V Rotter

Keyword(s):

Nuclear Localization ◽

Cell Nucleus ◽

P53 Protein ◽

Simian Virus 40 ◽

T Antigen ◽

Nuclear Accumulation ◽

Mutant P53 ◽

Wild Type ◽

Nuclear Localization Signals ◽

Wild Type P53

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.

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Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6565 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6565-6577 ◽

Cited By ~ 200

Author(s):

G Shaulsky ◽

N Goldfinger ◽

A Ben-Ze'ev ◽

V Rotter

Keyword(s):

Nuclear Localization ◽

Cell Nucleus ◽

P53 Protein ◽

Simian Virus 40 ◽

T Antigen ◽

Nuclear Accumulation ◽

Mutant P53 ◽

Wild Type ◽

Nuclear Localization Signals ◽

Wild Type P53

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.

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Autologous Dendritic Cells Transduced with Wild-type p53 Adenovirus Vaccine

10.32388/g85lo1 ◽

2020 ◽

Author(s):

Keyword(s):

Dendritic Cells ◽

Wild Type ◽

Adenovirus Vaccine ◽

Wild Type P53

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Faculty Opinions recommendation of Wild-Type p53 Promotes Cancer Metabolic Switch by Inducing PUMA-Dependent Suppression of Oxidative Phosphorylation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.734958049.793558103 ◽

2019 ◽

Author(s):

Christoph Borner

Keyword(s):

Oxidative Phosphorylation ◽

Wild Type ◽

Metabolic Switch ◽

Wild Type P53

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Characterization of CD8 + cytotoxic T lymphocyte/tumor cell interactions reflecting recognition of an endogenously expressed murine wild-type p53 determinant

Cancer Immunology Immunotherapy ◽

10.1007/s002620000156 ◽

2001 ◽

Vol 49 (11) ◽

pp. 603-612 ◽

Cited By ~ 27

Author(s):

Mary Hilburger Ryan ◽

Scott I. Abrams

Keyword(s):

Tumor Cell ◽

T Lymphocyte ◽

Cytotoxic T Lymphocyte ◽

Cell Interactions ◽

Wild Type ◽

Wild Type P53

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An MDM2 inhibitor achieves synergistic cytotoxic effects with adenoviruses lacking E1B55kDa gene on mesothelioma with the wild-type p53 through augmenting NFI expression

Cell Death and Disease ◽

10.1038/s41419-021-03934-y ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Thao Thi Thanh Nguyen ◽

Masato Shingyoji ◽

Michiko Hanazono ◽

Boya Zhong ◽

Takao Morinaga ◽

...

Keyword(s):

Wild Type ◽

Inhibitory Effects ◽

Cytotoxic Effects ◽

P53 Expression ◽

Nuclear Factor I ◽

Infected Cells ◽

Wild Type P53 ◽

Mdm2 Inhibitor ◽

P16 Expression

AbstractA majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

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