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Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53.

Lohez OD, Reynaud C, Borel F, Andreassen PR, Margolis RL - J. Cell Biol. (2003)

Bottom Line: We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53.Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage.Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean Ebel (Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique-Université Joseph Fourier), Grenoble cedex 1, France.

ABSTRACT
p53 and the retinoblastoma (RB) pocket proteins are central to the control of progression through the G1 phase of the cell cycle. The RB pocket protein family is downstream of p53 and controls S-phase entry. Disruption of actin assembly arrests nontransformed mammalian fibroblasts in G1. We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53. Thus, mammalian fibroblasts with normal pocket protein function reversibly arrest in G1 on exposure to actin inhibitors regardless of their p53 status. By contrast, pocket protein triple knockout mouse embryo fibroblasts and T antigen-transformed rat embryo fibroblasts lacking both p53 and RB pocket protein function do not arrest in G1. Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage. Interestingly, G1 arrest is accompanied by inhibition of surface ruffling and by induction of NF2/merlin. The combination of failure of G1 control and of tetraploid checkpoint control can cause RB pocket protein-suppressed cells to rapidly become aneuploid and die after exposure to actin inhibitors, whereas pocket protein-competent cells are spared. Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.

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DCB does not interfere with ERK kinase activation and nuclear translocation upon serum stimulation of quiescent cells. (A) Serum-starved REF-52 cells were reactivated with 10% FCS in the presence of 2 or 10 μM DCB. Flow cytometry shows that cells remain arrested in G1 25 h after release. Nocodazole exposure of cells in the absence of DCB indicates that control cells were cycling in the same time course. (B) ERK1/2 is normally activated in the presence of 2 μM DCB. At time intervals after add-back of serum to serum-starved REF-52, cells were harvested and subjected to Western blotting using either antibodies to total ERK or to activated phospho-ERK. Experiments were run either in the presence of 2 μM DCB (DCB, bottom two strips) or absence of DCB (Control, top two strips). ST designates quiescent serum-starved controls. (C) ERK1/2 is normally translocated to the nucleus in the presence of 2 μM DCB. 1 h after serum add-back to serum-starved REF-52 cells, ERK1/2 kinases were immunolocalized using anti–pan ERK antibodies. Experiments were run either in the presence of 2 μM DCB (DCB, right) or in the absence of DCB (Control, left). Nuclei were counterstained with propidium iodide (PI). Bars, 50 μm.
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fig5: DCB does not interfere with ERK kinase activation and nuclear translocation upon serum stimulation of quiescent cells. (A) Serum-starved REF-52 cells were reactivated with 10% FCS in the presence of 2 or 10 μM DCB. Flow cytometry shows that cells remain arrested in G1 25 h after release. Nocodazole exposure of cells in the absence of DCB indicates that control cells were cycling in the same time course. (B) ERK1/2 is normally activated in the presence of 2 μM DCB. At time intervals after add-back of serum to serum-starved REF-52, cells were harvested and subjected to Western blotting using either antibodies to total ERK or to activated phospho-ERK. Experiments were run either in the presence of 2 μM DCB (DCB, bottom two strips) or absence of DCB (Control, top two strips). ST designates quiescent serum-starved controls. (C) ERK1/2 is normally translocated to the nucleus in the presence of 2 μM DCB. 1 h after serum add-back to serum-starved REF-52 cells, ERK1/2 kinases were immunolocalized using anti–pan ERK antibodies. Experiments were run either in the presence of 2 μM DCB (DCB, right) or in the absence of DCB (Control, left). Nuclei were counterstained with propidium iodide (PI). Bars, 50 μm.

Mentions: Adherence to the substratum is intact in 2 μM DCB-treated cells by the criterion of the activation of extracellular signal–regulated kinase (ERK) on serum stimulation in the presence of 2 μM DCB. Fibroblast cell lines maintained in suspension fail to activate the ERK pathway in response to growth factor stimulation (Lin et al., 1997; Renshaw et al., 1997; Aplin et al., 1999). REF-52 cells arrest in G0 after serum starvation, and 2 μM or 10 μM DCB, but not nocodazole, inhibits progression past G1 upon serum add-back (Fig. 5 A). In accord with previous findings (Reshetnikova et al., 2000), we have found that REF-52 cells released from G0 serum starvation in the presence of low concentrations of DCB showed time-dependent ERK activation after serum add-back that is indistinguishable from controls (Fig. 5 B). Further, after serum stimulation, ERK migrates to the nuclei of DCB-blocked cells (Fig. 5 C).


Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53.

Lohez OD, Reynaud C, Borel F, Andreassen PR, Margolis RL - J. Cell Biol. (2003)

DCB does not interfere with ERK kinase activation and nuclear translocation upon serum stimulation of quiescent cells. (A) Serum-starved REF-52 cells were reactivated with 10% FCS in the presence of 2 or 10 μM DCB. Flow cytometry shows that cells remain arrested in G1 25 h after release. Nocodazole exposure of cells in the absence of DCB indicates that control cells were cycling in the same time course. (B) ERK1/2 is normally activated in the presence of 2 μM DCB. At time intervals after add-back of serum to serum-starved REF-52, cells were harvested and subjected to Western blotting using either antibodies to total ERK or to activated phospho-ERK. Experiments were run either in the presence of 2 μM DCB (DCB, bottom two strips) or absence of DCB (Control, top two strips). ST designates quiescent serum-starved controls. (C) ERK1/2 is normally translocated to the nucleus in the presence of 2 μM DCB. 1 h after serum add-back to serum-starved REF-52 cells, ERK1/2 kinases were immunolocalized using anti–pan ERK antibodies. Experiments were run either in the presence of 2 μM DCB (DCB, right) or in the absence of DCB (Control, left). Nuclei were counterstained with propidium iodide (PI). Bars, 50 μm.
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Related In: Results  -  Collection

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fig5: DCB does not interfere with ERK kinase activation and nuclear translocation upon serum stimulation of quiescent cells. (A) Serum-starved REF-52 cells were reactivated with 10% FCS in the presence of 2 or 10 μM DCB. Flow cytometry shows that cells remain arrested in G1 25 h after release. Nocodazole exposure of cells in the absence of DCB indicates that control cells were cycling in the same time course. (B) ERK1/2 is normally activated in the presence of 2 μM DCB. At time intervals after add-back of serum to serum-starved REF-52, cells were harvested and subjected to Western blotting using either antibodies to total ERK or to activated phospho-ERK. Experiments were run either in the presence of 2 μM DCB (DCB, bottom two strips) or absence of DCB (Control, top two strips). ST designates quiescent serum-starved controls. (C) ERK1/2 is normally translocated to the nucleus in the presence of 2 μM DCB. 1 h after serum add-back to serum-starved REF-52 cells, ERK1/2 kinases were immunolocalized using anti–pan ERK antibodies. Experiments were run either in the presence of 2 μM DCB (DCB, right) or in the absence of DCB (Control, left). Nuclei were counterstained with propidium iodide (PI). Bars, 50 μm.
Mentions: Adherence to the substratum is intact in 2 μM DCB-treated cells by the criterion of the activation of extracellular signal–regulated kinase (ERK) on serum stimulation in the presence of 2 μM DCB. Fibroblast cell lines maintained in suspension fail to activate the ERK pathway in response to growth factor stimulation (Lin et al., 1997; Renshaw et al., 1997; Aplin et al., 1999). REF-52 cells arrest in G0 after serum starvation, and 2 μM or 10 μM DCB, but not nocodazole, inhibits progression past G1 upon serum add-back (Fig. 5 A). In accord with previous findings (Reshetnikova et al., 2000), we have found that REF-52 cells released from G0 serum starvation in the presence of low concentrations of DCB showed time-dependent ERK activation after serum add-back that is indistinguishable from controls (Fig. 5 B). Further, after serum stimulation, ERK migrates to the nuclei of DCB-blocked cells (Fig. 5 C).

Bottom Line: We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53.Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage.Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Structurale Jean Ebel (Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique-Université Joseph Fourier), Grenoble cedex 1, France.

ABSTRACT
p53 and the retinoblastoma (RB) pocket proteins are central to the control of progression through the G1 phase of the cell cycle. The RB pocket protein family is downstream of p53 and controls S-phase entry. Disruption of actin assembly arrests nontransformed mammalian fibroblasts in G1. We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53. Thus, mammalian fibroblasts with normal pocket protein function reversibly arrest in G1 on exposure to actin inhibitors regardless of their p53 status. By contrast, pocket protein triple knockout mouse embryo fibroblasts and T antigen-transformed rat embryo fibroblasts lacking both p53 and RB pocket protein function do not arrest in G1. Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage. Interestingly, G1 arrest is accompanied by inhibition of surface ruffling and by induction of NF2/merlin. The combination of failure of G1 control and of tetraploid checkpoint control can cause RB pocket protein-suppressed cells to rapidly become aneuploid and die after exposure to actin inhibitors, whereas pocket protein-competent cells are spared. Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.

Show MeSH
Related in: MedlinePlus