Limits...
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.

Show MeSH

Related in: MedlinePlus

Failure of DCB-induced G1 blockage is lethal in RB pocket protein–suppressed cells. (A) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. Flow cytometric analysis of DNA content at the indicated times shows that TAG cells rapidly become aneuploid upon release from DCB, whereas REF-52 and p53DD REF-52 cells recover from DCB arrest and reach contact inhibition after a few days. (B) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. DCB was then washed out and cell counts were taken at the times indicated. (C) Asynchronous p53−/− and TKO MEFs were exposed to 10 μM DCB for 25 h. DCB was then washed out and cell counts taken at the indicated times. (D) Asynchronous HeLa, HCT 116, and A427 cells were exposed to either 10 μM DCB or 5 μM cytochalasin D (Cyto D) for 24 h. The drug was then washed out and cell counts taken at the times indicated. Control refers to untreated cells. In all cases, plateaus at late times indicate approach to confluency except for TKO MEFs, which do not reach high density.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172876&req=5

fig7: Failure of DCB-induced G1 blockage is lethal in RB pocket protein–suppressed cells. (A) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. Flow cytometric analysis of DNA content at the indicated times shows that TAG cells rapidly become aneuploid upon release from DCB, whereas REF-52 and p53DD REF-52 cells recover from DCB arrest and reach contact inhibition after a few days. (B) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. DCB was then washed out and cell counts were taken at the times indicated. (C) Asynchronous p53−/− and TKO MEFs were exposed to 10 μM DCB for 25 h. DCB was then washed out and cell counts taken at the indicated times. (D) Asynchronous HeLa, HCT 116, and A427 cells were exposed to either 10 μM DCB or 5 μM cytochalasin D (Cyto D) for 24 h. The drug was then washed out and cell counts taken at the times indicated. Control refers to untreated cells. In all cases, plateaus at late times indicate approach to confluency except for TKO MEFs, which do not reach high density.

Mentions: In the presence of DCB, p53 suppression does not abrogate G1 arrest, whereas pocket protein suppression does. Thus, in a randomly cycling population exposed to DCB, lethality will differ depending on whether the cells are suppressed for p53 activity or for RB pocket protein activity. RB pocket protein–suppressed cells can progress past G1 and past tetraploid G1, potentially leading to aneuploidy and death. In contrast, p53-incompetent cells arrest in euploid G1 in DCB and can thus recover normally upon drug release. In accord with this hypothesis, we have found that both REF-52 and p53DD REF-52 cells recovered from DCB arrest, seeded by those cells that had been arrested in euploid G1 (Fig. 7, A and B) . In contrast, TAG cells that additionally suppress RB pocket protein activity rapidly became aneuploid, as shown by a decrease in DNA content and loss of identifiable 2N and 4N peaks in DNA histograms, and died after release from DCB (Fig. 7, A and B). Similarly, RB pocket protein–suppressed MEFs (TKO) showed a highly reduced capacity to proliferate after transient exposure to DCB compared with control p53−/− MEFs (Fig. 7 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)

Failure of DCB-induced G1 blockage is lethal in RB pocket protein–suppressed cells. (A) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. Flow cytometric analysis of DNA content at the indicated times shows that TAG cells rapidly become aneuploid upon release from DCB, whereas REF-52 and p53DD REF-52 cells recover from DCB arrest and reach contact inhibition after a few days. (B) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. DCB was then washed out and cell counts were taken at the times indicated. (C) Asynchronous p53−/− and TKO MEFs were exposed to 10 μM DCB for 25 h. DCB was then washed out and cell counts taken at the indicated times. (D) Asynchronous HeLa, HCT 116, and A427 cells were exposed to either 10 μM DCB or 5 μM cytochalasin D (Cyto D) for 24 h. The drug was then washed out and cell counts taken at the times indicated. Control refers to untreated cells. In all cases, plateaus at late times indicate approach to confluency except for TKO MEFs, which do not reach high density.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Failure of DCB-induced G1 blockage is lethal in RB pocket protein–suppressed cells. (A) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. Flow cytometric analysis of DNA content at the indicated times shows that TAG cells rapidly become aneuploid upon release from DCB, whereas REF-52 and p53DD REF-52 cells recover from DCB arrest and reach contact inhibition after a few days. (B) Asynchronous REF-52, p53DD REF-52, and TAG cells were exposed to 5 μM DCB for 24 h. DCB was then washed out and cell counts were taken at the times indicated. (C) Asynchronous p53−/− and TKO MEFs were exposed to 10 μM DCB for 25 h. DCB was then washed out and cell counts taken at the indicated times. (D) Asynchronous HeLa, HCT 116, and A427 cells were exposed to either 10 μM DCB or 5 μM cytochalasin D (Cyto D) for 24 h. The drug was then washed out and cell counts taken at the times indicated. Control refers to untreated cells. In all cases, plateaus at late times indicate approach to confluency except for TKO MEFs, which do not reach high density.
Mentions: In the presence of DCB, p53 suppression does not abrogate G1 arrest, whereas pocket protein suppression does. Thus, in a randomly cycling population exposed to DCB, lethality will differ depending on whether the cells are suppressed for p53 activity or for RB pocket protein activity. RB pocket protein–suppressed cells can progress past G1 and past tetraploid G1, potentially leading to aneuploidy and death. In contrast, p53-incompetent cells arrest in euploid G1 in DCB and can thus recover normally upon drug release. In accord with this hypothesis, we have found that both REF-52 and p53DD REF-52 cells recovered from DCB arrest, seeded by those cells that had been arrested in euploid G1 (Fig. 7, A and B) . In contrast, TAG cells that additionally suppress RB pocket protein activity rapidly became aneuploid, as shown by a decrease in DNA content and loss of identifiable 2N and 4N peaks in DNA histograms, and died after release from DCB (Fig. 7, A and B). Similarly, RB pocket protein–suppressed MEFs (TKO) showed a highly reduced capacity to proliferate after transient exposure to DCB compared with control p53−/− MEFs (Fig. 7 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