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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 stress fibers and substratum adherence are not perturbed but NF2/merlin is up-regulated after exposure of REF-52 cells to 2 μM DCB. REF-52 cells were exposed to 2 μM DCB for 25 h. Cells were then analyzed for actin distribution with TRITC-phalloidin (A and B, left), and vinculin (A, right) and phosphotyrosine (B, right) distribution were determined by immunolabeling. For each pair (untreated/DCB) in both A and B, the intensity settings used for confocal microscopy were kept constant. Bars, 40 μm. (C) Higher magnification images show that ruffling was suppressed in cells exposed to 2 μM DCB for 25 h (DCB), but reappeared rapidly (arrowheads) in cells released from DCB (30 min) and persisted at 25 h of release. Cells were imaged for actin with TRITC-phalloidin. Bar, 40 μm. (D) NF2/merlin is up-regulated and dephosphorylated in DCB-arrested cells. REF-52 cells, either random cycling (Rdom), contact inhibited (CI), or random cycling while exposed to 2 μM DCB for 25 h (DCB), were harvested and subjected to Western blotting procedures using anti-NF2/merlin antibodies. An equivalent amount of protein was loaded for each sample.
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fig4: Actin stress fibers and substratum adherence are not perturbed but NF2/merlin is up-regulated after exposure of REF-52 cells to 2 μM DCB. REF-52 cells were exposed to 2 μM DCB for 25 h. Cells were then analyzed for actin distribution with TRITC-phalloidin (A and B, left), and vinculin (A, right) and phosphotyrosine (B, right) distribution were determined by immunolabeling. For each pair (untreated/DCB) in both A and B, the intensity settings used for confocal microscopy were kept constant. Bars, 40 μm. (C) Higher magnification images show that ruffling was suppressed in cells exposed to 2 μM DCB for 25 h (DCB), but reappeared rapidly (arrowheads) in cells released from DCB (30 min) and persisted at 25 h of release. Cells were imaged for actin with TRITC-phalloidin. Bar, 40 μm. (D) NF2/merlin is up-regulated and dephosphorylated in DCB-arrested cells. REF-52 cells, either random cycling (Rdom), contact inhibited (CI), or random cycling while exposed to 2 μM DCB for 25 h (DCB), were harvested and subjected to Western blotting procedures using anti-NF2/merlin antibodies. An equivalent amount of protein was loaded for each sample.

Mentions: There is substantial evidence that the capacity of nontransformed fibroblasts to progress in the cell cycle is coupled to substratum adherence (Stoker et al., 1968; Assoian, 1997; Assoian and Schwartz, 2001). However, even at 10 μM DCB, we have found that cells that are no longer capable of undergoing cleavage remain adherent (unpublished data). At 2 μM DCB, there is little overt evidence of disturbance of the actin cytoskeleton in randomly cycling interphase cells and no evidence of disturbance of adhesion (Fig. 4) . Compared with controls, cells retain abundant actin cables, and focal adhesion plaques appear intact, as assayed by both vinculin antibody (Fig. 4 A) and antiphosphotyrosine antibody (Fig. 4 B). One notable change during arrest is that, in comparison to controls, cells exposed to 2 μM DCB have smooth margins and do not appear to undergo ruffling (Fig. 4 C). The absence of ruffling is rapidly reversible on release from DCB, and ruffling is equally evident at 30 min and a full day after DCB release (Fig. 4 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)

Actin stress fibers and substratum adherence are not perturbed but NF2/merlin is up-regulated after exposure of REF-52 cells to 2 μM DCB. REF-52 cells were exposed to 2 μM DCB for 25 h. Cells were then analyzed for actin distribution with TRITC-phalloidin (A and B, left), and vinculin (A, right) and phosphotyrosine (B, right) distribution were determined by immunolabeling. For each pair (untreated/DCB) in both A and B, the intensity settings used for confocal microscopy were kept constant. Bars, 40 μm. (C) Higher magnification images show that ruffling was suppressed in cells exposed to 2 μM DCB for 25 h (DCB), but reappeared rapidly (arrowheads) in cells released from DCB (30 min) and persisted at 25 h of release. Cells were imaged for actin with TRITC-phalloidin. Bar, 40 μm. (D) NF2/merlin is up-regulated and dephosphorylated in DCB-arrested cells. REF-52 cells, either random cycling (Rdom), contact inhibited (CI), or random cycling while exposed to 2 μM DCB for 25 h (DCB), were harvested and subjected to Western blotting procedures using anti-NF2/merlin antibodies. An equivalent amount of protein was loaded for each sample.
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fig4: Actin stress fibers and substratum adherence are not perturbed but NF2/merlin is up-regulated after exposure of REF-52 cells to 2 μM DCB. REF-52 cells were exposed to 2 μM DCB for 25 h. Cells were then analyzed for actin distribution with TRITC-phalloidin (A and B, left), and vinculin (A, right) and phosphotyrosine (B, right) distribution were determined by immunolabeling. For each pair (untreated/DCB) in both A and B, the intensity settings used for confocal microscopy were kept constant. Bars, 40 μm. (C) Higher magnification images show that ruffling was suppressed in cells exposed to 2 μM DCB for 25 h (DCB), but reappeared rapidly (arrowheads) in cells released from DCB (30 min) and persisted at 25 h of release. Cells were imaged for actin with TRITC-phalloidin. Bar, 40 μm. (D) NF2/merlin is up-regulated and dephosphorylated in DCB-arrested cells. REF-52 cells, either random cycling (Rdom), contact inhibited (CI), or random cycling while exposed to 2 μM DCB for 25 h (DCB), were harvested and subjected to Western blotting procedures using anti-NF2/merlin antibodies. An equivalent amount of protein was loaded for each sample.
Mentions: There is substantial evidence that the capacity of nontransformed fibroblasts to progress in the cell cycle is coupled to substratum adherence (Stoker et al., 1968; Assoian, 1997; Assoian and Schwartz, 2001). However, even at 10 μM DCB, we have found that cells that are no longer capable of undergoing cleavage remain adherent (unpublished data). At 2 μM DCB, there is little overt evidence of disturbance of the actin cytoskeleton in randomly cycling interphase cells and no evidence of disturbance of adhesion (Fig. 4) . Compared with controls, cells retain abundant actin cables, and focal adhesion plaques appear intact, as assayed by both vinculin antibody (Fig. 4 A) and antiphosphotyrosine antibody (Fig. 4 B). One notable change during arrest is that, in comparison to controls, cells exposed to 2 μM DCB have smooth margins and do not appear to undergo ruffling (Fig. 4 C). The absence of ruffling is rapidly reversible on release from DCB, and ruffling is equally evident at 30 min and a full day after DCB release (Fig. 4 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