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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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Actin inhibitors induce p53-independent 2N G1 arrest in nontransformed fibroblasts but not in transformed variants. (A) Nontransformed fibroblasts REF-52 and IMR-90, but not transformed TAG variants, arrest in the presence of DCB. REF-52, IMR-90, and TAG cells were exposed to the indicated concentrations of DCB for 25 h. Both REF-52 and IMR-90 arrest as 2N populations in the presence of DCB, whereas TAG cells do not arrest and progress to higher ploidy. Note that TAG cell cleavage is partially sensitive to 2 μM DCB. (B) p53 is not required for 2N G1 arrest in the presence of DCB. p53 was inactivated in REF-52 (p53DD) by expression of a dominant-negative mutant (Shaulian et al., 1992; Andreassen et al., 2001b). p53+/+ REF-52 and p53DD REF-52 cells were exposed to either 2 or 10 μM DCB for 25 h. Flow cytometry shows that p53DD REF-52 and control (p53+/+) REF-52 exhibit comparable 2N G1 arrest in the presence of DCB. (C) Latrunculin A imposes 2N G1 arrest in REF-52 cells but not in their transformed variants. REF-52 and TAG cells were exposed to the indicated concentrations of latrunculin A for 25 h. Flow cytometry shows that REF-52 cells exhibit 2N G1 arrest both at 0.2 and 0.5 μM latrunculin A, whereas TAG cells do not arrest but progress to higher ploidy at 0.5 μM latrunculin A.
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fig2: Actin inhibitors induce p53-independent 2N G1 arrest in nontransformed fibroblasts but not in transformed variants. (A) Nontransformed fibroblasts REF-52 and IMR-90, but not transformed TAG variants, arrest in the presence of DCB. REF-52, IMR-90, and TAG cells were exposed to the indicated concentrations of DCB for 25 h. Both REF-52 and IMR-90 arrest as 2N populations in the presence of DCB, whereas TAG cells do not arrest and progress to higher ploidy. Note that TAG cell cleavage is partially sensitive to 2 μM DCB. (B) p53 is not required for 2N G1 arrest in the presence of DCB. p53 was inactivated in REF-52 (p53DD) by expression of a dominant-negative mutant (Shaulian et al., 1992; Andreassen et al., 2001b). p53+/+ REF-52 and p53DD REF-52 cells were exposed to either 2 or 10 μM DCB for 25 h. Flow cytometry shows that p53DD REF-52 and control (p53+/+) REF-52 exhibit comparable 2N G1 arrest in the presence of DCB. (C) Latrunculin A imposes 2N G1 arrest in REF-52 cells but not in their transformed variants. REF-52 and TAG cells were exposed to the indicated concentrations of latrunculin A for 25 h. Flow cytometry shows that REF-52 cells exhibit 2N G1 arrest both at 0.2 and 0.5 μM latrunculin A, whereas TAG cells do not arrest but progress to higher ploidy at 0.5 μM latrunculin A.

Mentions: Arrest of randomly cycling cells in G1 by low concentrations of DCB is not restricted to REF-52 cells. Human nontransformed IMR-90 fibroblasts show the same concentration-dependent sensitivity to DCB and arrest in 2N G1 when exposed to 2 μM DCB, but arrest with approximately equal 2N and 4N status at higher DCB concentrations (Fig. 2 A). It is important to note that the arrest in 2N G1 can be distinguished from the arrest of tetraploid cells with 4N DNA content after DCB exposure (Andreassen et al., 2001b).


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)

Actin inhibitors induce p53-independent 2N G1 arrest in nontransformed fibroblasts but not in transformed variants. (A) Nontransformed fibroblasts REF-52 and IMR-90, but not transformed TAG variants, arrest in the presence of DCB. REF-52, IMR-90, and TAG cells were exposed to the indicated concentrations of DCB for 25 h. Both REF-52 and IMR-90 arrest as 2N populations in the presence of DCB, whereas TAG cells do not arrest and progress to higher ploidy. Note that TAG cell cleavage is partially sensitive to 2 μM DCB. (B) p53 is not required for 2N G1 arrest in the presence of DCB. p53 was inactivated in REF-52 (p53DD) by expression of a dominant-negative mutant (Shaulian et al., 1992; Andreassen et al., 2001b). p53+/+ REF-52 and p53DD REF-52 cells were exposed to either 2 or 10 μM DCB for 25 h. Flow cytometry shows that p53DD REF-52 and control (p53+/+) REF-52 exhibit comparable 2N G1 arrest in the presence of DCB. (C) Latrunculin A imposes 2N G1 arrest in REF-52 cells but not in their transformed variants. REF-52 and TAG cells were exposed to the indicated concentrations of latrunculin A for 25 h. Flow cytometry shows that REF-52 cells exhibit 2N G1 arrest both at 0.2 and 0.5 μM latrunculin A, whereas TAG cells do not arrest but progress to higher ploidy at 0.5 μM latrunculin A.
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fig2: Actin inhibitors induce p53-independent 2N G1 arrest in nontransformed fibroblasts but not in transformed variants. (A) Nontransformed fibroblasts REF-52 and IMR-90, but not transformed TAG variants, arrest in the presence of DCB. REF-52, IMR-90, and TAG cells were exposed to the indicated concentrations of DCB for 25 h. Both REF-52 and IMR-90 arrest as 2N populations in the presence of DCB, whereas TAG cells do not arrest and progress to higher ploidy. Note that TAG cell cleavage is partially sensitive to 2 μM DCB. (B) p53 is not required for 2N G1 arrest in the presence of DCB. p53 was inactivated in REF-52 (p53DD) by expression of a dominant-negative mutant (Shaulian et al., 1992; Andreassen et al., 2001b). p53+/+ REF-52 and p53DD REF-52 cells were exposed to either 2 or 10 μM DCB for 25 h. Flow cytometry shows that p53DD REF-52 and control (p53+/+) REF-52 exhibit comparable 2N G1 arrest in the presence of DCB. (C) Latrunculin A imposes 2N G1 arrest in REF-52 cells but not in their transformed variants. REF-52 and TAG cells were exposed to the indicated concentrations of latrunculin A for 25 h. Flow cytometry shows that REF-52 cells exhibit 2N G1 arrest both at 0.2 and 0.5 μM latrunculin A, whereas TAG cells do not arrest but progress to higher ploidy at 0.5 μM latrunculin A.
Mentions: Arrest of randomly cycling cells in G1 by low concentrations of DCB is not restricted to REF-52 cells. Human nontransformed IMR-90 fibroblasts show the same concentration-dependent sensitivity to DCB and arrest in 2N G1 when exposed to 2 μM DCB, but arrest with approximately equal 2N and 4N status at higher DCB concentrations (Fig. 2 A). It is important to note that the arrest in 2N G1 can be distinguished from the arrest of tetraploid cells with 4N DNA content after DCB exposure (Andreassen et al., 2001b).

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