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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-arrested REF-52 cells have markers characteristic of G1. (A) Asynchronous cells (Rdom), contact-inhibited cells (CI), and cells exposed for 25 h to either 2 μM DCB (DCB) or 2 mM HU (HU) were harvested and subjected to Western blotting procedures using the indicated antibodies. (B) The kinase activities of Cdk2 and Cdk4 were determined after immunoprecipitation of the kinases and incubation with histone H1 (Cdk2) or RB peptide (ckd4) as substrates. Autoradiographs of 32P incorporation are shown (top strips) and compared with the kinase expression levels as determined by Western blotting (bottom strips). (C) p27Kip1 accumulation is not incidental to DCB treatment. REF-52 cells were exposed for 25 h to either 2 mM HU or 2 μM DCB. Alternatively, cells were exposed to 2 mM HU for 25 h before addition of 2 μM DCB for 25 h in the continuous presence of HU. Samples were subjected to Western blotting analysis using anti-p27Kip1 antibodies.
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fig6: DCB-arrested REF-52 cells have markers characteristic of G1. (A) Asynchronous cells (Rdom), contact-inhibited cells (CI), and cells exposed for 25 h to either 2 μM DCB (DCB) or 2 mM HU (HU) were harvested and subjected to Western blotting procedures using the indicated antibodies. (B) The kinase activities of Cdk2 and Cdk4 were determined after immunoprecipitation of the kinases and incubation with histone H1 (Cdk2) or RB peptide (ckd4) as substrates. Autoradiographs of 32P incorporation are shown (top strips) and compared with the kinase expression levels as determined by Western blotting (bottom strips). (C) p27Kip1 accumulation is not incidental to DCB treatment. REF-52 cells were exposed for 25 h to either 2 mM HU or 2 μM DCB. Alternatively, cells were exposed to 2 mM HU for 25 h before addition of 2 μM DCB for 25 h in the continuous presence of HU. Samples were subjected to Western blotting analysis using anti-p27Kip1 antibodies.

Mentions: Our results indicating up-regulation of NF2/merlin during DCB-induced arrest and the requirement for RB proteins both suggested that arrest would be in G1. Further, Bohmer et al. (1996) found, in timed experiments with synchronous cells, that arrest of fibroblasts with high concentrations of cytochalasin occurred in mid to late G1. To assess the physiological status of randomly cycling cells arrested in G1 by 25 h exposure to 2 μM DCB, we assayed for the presence and activity of different cell cycle markers, compared with the presence of these markers in contact-inhibited cells in G0/G1, in control cycling cells, or in cycling cells arrested in S phase (Fig. 6) . S-phase cells were obtained by exposure for 25 h to 2 mM hydroxyurea (HU), which blocks cells at the G1/S boundary with active Cdk2 and suppressed p27 (Borel et al., 2002a). The levels of different markers in DCB-arrested cells resembled those found in contact-inhibited cells. Cyclin A was absent in both cases, and p27Kip1, a CKI (Polyak et al., 1994a,b), was elevated. p21WAF1, a CKI transactivated by p53 el-Deiry et al., 1993, was not elevated, consistent with the insensitivity of arrest to p53 status. RB was hypophosphorylated both in contact-inhibited cells and in DCB-treated cells. This profile is consistent with suppression of Cdk2 activity in both contact-inhibited and DCB-blocked status (Fig. 6 B). In contrast to contact-inhibited cells, drug-arrested cells showed reduced Cdk4 activity (Fig. 6 B). The induction of p27Kip1 was not incidental to DCB treatment, as it did not occur in cells that were first arrested in S phase with HU and then treated with DCB (Fig. 6 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-arrested REF-52 cells have markers characteristic of G1. (A) Asynchronous cells (Rdom), contact-inhibited cells (CI), and cells exposed for 25 h to either 2 μM DCB (DCB) or 2 mM HU (HU) were harvested and subjected to Western blotting procedures using the indicated antibodies. (B) The kinase activities of Cdk2 and Cdk4 were determined after immunoprecipitation of the kinases and incubation with histone H1 (Cdk2) or RB peptide (ckd4) as substrates. Autoradiographs of 32P incorporation are shown (top strips) and compared with the kinase expression levels as determined by Western blotting (bottom strips). (C) p27Kip1 accumulation is not incidental to DCB treatment. REF-52 cells were exposed for 25 h to either 2 mM HU or 2 μM DCB. Alternatively, cells were exposed to 2 mM HU for 25 h before addition of 2 μM DCB for 25 h in the continuous presence of HU. Samples were subjected to Western blotting analysis using anti-p27Kip1 antibodies.
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Related In: Results  -  Collection

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fig6: DCB-arrested REF-52 cells have markers characteristic of G1. (A) Asynchronous cells (Rdom), contact-inhibited cells (CI), and cells exposed for 25 h to either 2 μM DCB (DCB) or 2 mM HU (HU) were harvested and subjected to Western blotting procedures using the indicated antibodies. (B) The kinase activities of Cdk2 and Cdk4 were determined after immunoprecipitation of the kinases and incubation with histone H1 (Cdk2) or RB peptide (ckd4) as substrates. Autoradiographs of 32P incorporation are shown (top strips) and compared with the kinase expression levels as determined by Western blotting (bottom strips). (C) p27Kip1 accumulation is not incidental to DCB treatment. REF-52 cells were exposed for 25 h to either 2 mM HU or 2 μM DCB. Alternatively, cells were exposed to 2 mM HU for 25 h before addition of 2 μM DCB for 25 h in the continuous presence of HU. Samples were subjected to Western blotting analysis using anti-p27Kip1 antibodies.
Mentions: Our results indicating up-regulation of NF2/merlin during DCB-induced arrest and the requirement for RB proteins both suggested that arrest would be in G1. Further, Bohmer et al. (1996) found, in timed experiments with synchronous cells, that arrest of fibroblasts with high concentrations of cytochalasin occurred in mid to late G1. To assess the physiological status of randomly cycling cells arrested in G1 by 25 h exposure to 2 μM DCB, we assayed for the presence and activity of different cell cycle markers, compared with the presence of these markers in contact-inhibited cells in G0/G1, in control cycling cells, or in cycling cells arrested in S phase (Fig. 6) . S-phase cells were obtained by exposure for 25 h to 2 mM hydroxyurea (HU), which blocks cells at the G1/S boundary with active Cdk2 and suppressed p27 (Borel et al., 2002a). The levels of different markers in DCB-arrested cells resembled those found in contact-inhibited cells. Cyclin A was absent in both cases, and p27Kip1, a CKI (Polyak et al., 1994a,b), was elevated. p21WAF1, a CKI transactivated by p53 el-Deiry et al., 1993, was not elevated, consistent with the insensitivity of arrest to p53 status. RB was hypophosphorylated both in contact-inhibited cells and in DCB-treated cells. This profile is consistent with suppression of Cdk2 activity in both contact-inhibited and DCB-blocked status (Fig. 6 B). In contrast to contact-inhibited cells, drug-arrested cells showed reduced Cdk4 activity (Fig. 6 B). The induction of p27Kip1 was not incidental to DCB treatment, as it did not occur in cells that were first arrested in S phase with HU and then treated with DCB (Fig. 6 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