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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-induced G1 blockage is dependent on the RB pocket protein family. p53−/− MEFs, MEFs deleted for the three RB pocket proteins (TKO), ARF−/− MEFs, and RB−/− MEFs were exposed to 10 μM DCB or to nocodazole (0.5 μg/ml) for 25 h and analyzed for cell cycle distribution. Flow cytometric analysis shows that p53−/− MEFs exhibit 2N G1 arrest, whereas TKO MEFs do not and instead progress to higher ploidy. ARF−/− MEFs exhibit 2N G1 arrest like that of p53−/− MEFs, whereas RB−/− MEFs exhibit partial 2N G1 arrest unlike TKO MEFs, which do not arrest. In all cases, nocodazole exposure confirms that control cells, not exposed to DCB, were cycling in the same time course.
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fig3: DCB-induced G1 blockage is dependent on the RB pocket protein family. p53−/− MEFs, MEFs deleted for the three RB pocket proteins (TKO), ARF−/− MEFs, and RB−/− MEFs were exposed to 10 μM DCB or to nocodazole (0.5 μg/ml) for 25 h and analyzed for cell cycle distribution. Flow cytometric analysis shows that p53−/− MEFs exhibit 2N G1 arrest, whereas TKO MEFs do not and instead progress to higher ploidy. ARF−/− MEFs exhibit 2N G1 arrest like that of p53−/− MEFs, whereas RB−/− MEFs exhibit partial 2N G1 arrest unlike TKO MEFs, which do not arrest. In all cases, nocodazole exposure confirms that control cells, not exposed to DCB, were cycling in the same time course.

Mentions: p53-dependent controls can be suppressed by expression of a dominant-negative truncation mutant of p53 (p53DD) (Shaulian et al., 1992). We have established a REF-52 cell line (p53DD REF-52) that expresses dominant-negative p53 (Andreassen et al., 2001b). To distinguish if p53 was involved in DCB suppression of G1 progression, we exposed p53DD REF-52 cells to both 2 and 10 μM DCB. Results (Fig. 2 B) clearly demonstrate that DCB-dependent arrest in G1 with 2N DNA content is independent of p53 function as cells arrest equally in G1, regardless of p53 status. We have obtained similar results with p53−/− MEF cells (Fig. 3) . We conclude that p53 is not involved in the DCB-dependent arrest of nontransformed fibroblasts in G1.


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-induced G1 blockage is dependent on the RB pocket protein family. p53−/− MEFs, MEFs deleted for the three RB pocket proteins (TKO), ARF−/− MEFs, and RB−/− MEFs were exposed to 10 μM DCB or to nocodazole (0.5 μg/ml) for 25 h and analyzed for cell cycle distribution. Flow cytometric analysis shows that p53−/− MEFs exhibit 2N G1 arrest, whereas TKO MEFs do not and instead progress to higher ploidy. ARF−/− MEFs exhibit 2N G1 arrest like that of p53−/− MEFs, whereas RB−/− MEFs exhibit partial 2N G1 arrest unlike TKO MEFs, which do not arrest. In all cases, nocodazole exposure confirms that control cells, not exposed to DCB, were cycling in the same time course.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172876&req=5

fig3: DCB-induced G1 blockage is dependent on the RB pocket protein family. p53−/− MEFs, MEFs deleted for the three RB pocket proteins (TKO), ARF−/− MEFs, and RB−/− MEFs were exposed to 10 μM DCB or to nocodazole (0.5 μg/ml) for 25 h and analyzed for cell cycle distribution. Flow cytometric analysis shows that p53−/− MEFs exhibit 2N G1 arrest, whereas TKO MEFs do not and instead progress to higher ploidy. ARF−/− MEFs exhibit 2N G1 arrest like that of p53−/− MEFs, whereas RB−/− MEFs exhibit partial 2N G1 arrest unlike TKO MEFs, which do not arrest. In all cases, nocodazole exposure confirms that control cells, not exposed to DCB, were cycling in the same time course.
Mentions: p53-dependent controls can be suppressed by expression of a dominant-negative truncation mutant of p53 (p53DD) (Shaulian et al., 1992). We have established a REF-52 cell line (p53DD REF-52) that expresses dominant-negative p53 (Andreassen et al., 2001b). To distinguish if p53 was involved in DCB suppression of G1 progression, we exposed p53DD REF-52 cells to both 2 and 10 μM DCB. Results (Fig. 2 B) clearly demonstrate that DCB-dependent arrest in G1 with 2N DNA content is independent of p53 function as cells arrest equally in G1, regardless of p53 status. We have obtained similar results with p53−/− MEF cells (Fig. 3) . We conclude that p53 is not involved in the DCB-dependent arrest of nontransformed fibroblasts in G1.

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