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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 induces reversible G1 arrest of rat embryo fibroblasts (REF-52) at concentrations that do not block cell cleavage. (A) Increasing concentrations of DCB progressively suppress REF-52 proliferation. Cells were exposed to the indicated DCB concentrations, and cell counts were taken at the indicated times. The curve is representative of three independent experiments. (B) DCB induces cytokinetic failure at concentrations >2 μM. REF-52 cells were exposed to the indicated DCB concentrations for 25 h. Binucleate cells, indicative of cleavage failure, were counted. Bar graph incorporates data from three independent experiments (300–500 cells counted each time). (C) DCB induces 2N arrest at concentrations <4 μM, whereas at higher concentrations, it yields both 2N and 4N arrest. REF-52 cells were exposed to the indicated concentrations of DCB for 25 h and assayed by flow cytometry. Nocodazole exposure (0.5 μg/ml) confirms that cells not treated with DCB were cycling in the same time course. (D) 2N G1 arrest imposed by DCB is reversible. REF-52 cells were exposed to 2 or 10 μM DCB for 25 h. Cells were then either maintained in DCB (spotted bars) or released from DCB (black bars) and exposed for another 25 h to 10 μM BrdU to assay S-phase entry. BrdU incorporation was evaluated only in mononucleate cells. Values shown are the means of three independent counts of at least 300 cells each. (E) Binucleated cells do not recover from exposure to 10 μM DCB. REF-52 cells were exposed to 10 μM DCB for 25 h. DCB was then washed out, and cells were exposed to 10 μM BrdU for 25 h. Mononucleate and binucleate BrdU-positive cells were independently counted. Values shown are means from three independent counts of 300 cells each.
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fig1: DCB induces reversible G1 arrest of rat embryo fibroblasts (REF-52) at concentrations that do not block cell cleavage. (A) Increasing concentrations of DCB progressively suppress REF-52 proliferation. Cells were exposed to the indicated DCB concentrations, and cell counts were taken at the indicated times. The curve is representative of three independent experiments. (B) DCB induces cytokinetic failure at concentrations >2 μM. REF-52 cells were exposed to the indicated DCB concentrations for 25 h. Binucleate cells, indicative of cleavage failure, were counted. Bar graph incorporates data from three independent experiments (300–500 cells counted each time). (C) DCB induces 2N arrest at concentrations <4 μM, whereas at higher concentrations, it yields both 2N and 4N arrest. REF-52 cells were exposed to the indicated concentrations of DCB for 25 h and assayed by flow cytometry. Nocodazole exposure (0.5 μg/ml) confirms that cells not treated with DCB were cycling in the same time course. (D) 2N G1 arrest imposed by DCB is reversible. REF-52 cells were exposed to 2 or 10 μM DCB for 25 h. Cells were then either maintained in DCB (spotted bars) or released from DCB (black bars) and exposed for another 25 h to 10 μM BrdU to assay S-phase entry. BrdU incorporation was evaluated only in mononucleate cells. Values shown are the means of three independent counts of at least 300 cells each. (E) Binucleated cells do not recover from exposure to 10 μM DCB. REF-52 cells were exposed to 10 μM DCB for 25 h. DCB was then washed out, and cells were exposed to 10 μM BrdU for 25 h. Mononucleate and binucleate BrdU-positive cells were independently counted. Values shown are means from three independent counts of 300 cells each.

Mentions: Cytochalasins, at concentrations sufficient to fully disrupt actin structures and block cytokinesis, also arrest nontransformed cells in G1 (Bohmer et al., 1996; Assoian and Zhu, 1997; Reshetnikova et al., 2000). To establish the sensitivity of cell cycle progression to actin disruption, we exposed nontransformed REF-52 cells (primary rat embryo fibroblasts) in random cycle to different concentrations of DCB. We found that cell proliferation was significantly suppressed at 0.5 μM DCB and completely suppressed at concentrations at or above 2 μM DCB (Fig. 1 A).


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 induces reversible G1 arrest of rat embryo fibroblasts (REF-52) at concentrations that do not block cell cleavage. (A) Increasing concentrations of DCB progressively suppress REF-52 proliferation. Cells were exposed to the indicated DCB concentrations, and cell counts were taken at the indicated times. The curve is representative of three independent experiments. (B) DCB induces cytokinetic failure at concentrations >2 μM. REF-52 cells were exposed to the indicated DCB concentrations for 25 h. Binucleate cells, indicative of cleavage failure, were counted. Bar graph incorporates data from three independent experiments (300–500 cells counted each time). (C) DCB induces 2N arrest at concentrations <4 μM, whereas at higher concentrations, it yields both 2N and 4N arrest. REF-52 cells were exposed to the indicated concentrations of DCB for 25 h and assayed by flow cytometry. Nocodazole exposure (0.5 μg/ml) confirms that cells not treated with DCB were cycling in the same time course. (D) 2N G1 arrest imposed by DCB is reversible. REF-52 cells were exposed to 2 or 10 μM DCB for 25 h. Cells were then either maintained in DCB (spotted bars) or released from DCB (black bars) and exposed for another 25 h to 10 μM BrdU to assay S-phase entry. BrdU incorporation was evaluated only in mononucleate cells. Values shown are the means of three independent counts of at least 300 cells each. (E) Binucleated cells do not recover from exposure to 10 μM DCB. REF-52 cells were exposed to 10 μM DCB for 25 h. DCB was then washed out, and cells were exposed to 10 μM BrdU for 25 h. Mononucleate and binucleate BrdU-positive cells were independently counted. Values shown are means from three independent counts of 300 cells each.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172876&req=5

fig1: DCB induces reversible G1 arrest of rat embryo fibroblasts (REF-52) at concentrations that do not block cell cleavage. (A) Increasing concentrations of DCB progressively suppress REF-52 proliferation. Cells were exposed to the indicated DCB concentrations, and cell counts were taken at the indicated times. The curve is representative of three independent experiments. (B) DCB induces cytokinetic failure at concentrations >2 μM. REF-52 cells were exposed to the indicated DCB concentrations for 25 h. Binucleate cells, indicative of cleavage failure, were counted. Bar graph incorporates data from three independent experiments (300–500 cells counted each time). (C) DCB induces 2N arrest at concentrations <4 μM, whereas at higher concentrations, it yields both 2N and 4N arrest. REF-52 cells were exposed to the indicated concentrations of DCB for 25 h and assayed by flow cytometry. Nocodazole exposure (0.5 μg/ml) confirms that cells not treated with DCB were cycling in the same time course. (D) 2N G1 arrest imposed by DCB is reversible. REF-52 cells were exposed to 2 or 10 μM DCB for 25 h. Cells were then either maintained in DCB (spotted bars) or released from DCB (black bars) and exposed for another 25 h to 10 μM BrdU to assay S-phase entry. BrdU incorporation was evaluated only in mononucleate cells. Values shown are the means of three independent counts of at least 300 cells each. (E) Binucleated cells do not recover from exposure to 10 μM DCB. REF-52 cells were exposed to 10 μM DCB for 25 h. DCB was then washed out, and cells were exposed to 10 μM BrdU for 25 h. Mononucleate and binucleate BrdU-positive cells were independently counted. Values shown are means from three independent counts of 300 cells each.
Mentions: Cytochalasins, at concentrations sufficient to fully disrupt actin structures and block cytokinesis, also arrest nontransformed cells in G1 (Bohmer et al., 1996; Assoian and Zhu, 1997; Reshetnikova et al., 2000). To establish the sensitivity of cell cycle progression to actin disruption, we exposed nontransformed REF-52 cells (primary rat embryo fibroblasts) in random cycle to different concentrations of DCB. We found that cell proliferation was significantly suppressed at 0.5 μM DCB and completely suppressed at concentrations at or above 2 μM DCB (Fig. 1 A).

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