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The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.

Srougi MC, Burridge K - PLoS ONE (2011)

Bottom Line: The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2.Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death.Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America. melissa_srougi@med.unc.edu

ABSTRACT
Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

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Model of RhoB-induced cell death after IR.IR-induced DNA damage activates members of the DNA damage-sensing machinery, which may directly or indirectly activate the nuclear GEFs Ect2 and Net1 to stimulate guanine nucleotide exchange upon RhoB. Activated RhoB leads to the downstream phosphorylation of JNK that triggers the induction of the pro-apoptotic protein Bim leading to cell death. Inhibition of either RhoB or Ect2 and Net1 activation through RNAi mutes this signaling pathway and results in cellular resistance to IR.
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pone-0017108-g006: Model of RhoB-induced cell death after IR.IR-induced DNA damage activates members of the DNA damage-sensing machinery, which may directly or indirectly activate the nuclear GEFs Ect2 and Net1 to stimulate guanine nucleotide exchange upon RhoB. Activated RhoB leads to the downstream phosphorylation of JNK that triggers the induction of the pro-apoptotic protein Bim leading to cell death. Inhibition of either RhoB or Ect2 and Net1 activation through RNAi mutes this signaling pathway and results in cellular resistance to IR.

Mentions: The exposure of eukaryotic cells to genotoxic agents results in a variety of cellular responses, which can either promote or prevent survival. Deciphering the signaling pathways that modulate these signals is of important clinical relevance as a prognostic indicator of tumor response to DNA damaging agents. It has been well established that the protein expression of the small GTPase RhoB is upregulated in response to cellular stress [9] and that RhoB is required to induce apoptosis after exposure to several DNA damaging agents in Ras-transformed cell lines [22]. In the present study, we further demonstrate that RhoB activity is increased upon exposure to DNA damaging agents, and this activity is necessary for IR-induced cell death. However, it is yet unclear what the upstream regulatory processes are that control RhoB activity after DNA damage. Therefore, we sought to elucidate these upstream modulators with specific focus on Rho GEFs. We show that the nuclear GEFs Ect2 and Net1 specifically activate RhoB, which causes the downstream phosphorylation of JNK and the induction of the pro-apoptotic protein Bim, leading to cell death (Fig. 6).


The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.

Srougi MC, Burridge K - PLoS ONE (2011)

Model of RhoB-induced cell death after IR.IR-induced DNA damage activates members of the DNA damage-sensing machinery, which may directly or indirectly activate the nuclear GEFs Ect2 and Net1 to stimulate guanine nucleotide exchange upon RhoB. Activated RhoB leads to the downstream phosphorylation of JNK that triggers the induction of the pro-apoptotic protein Bim leading to cell death. Inhibition of either RhoB or Ect2 and Net1 activation through RNAi mutes this signaling pathway and results in cellular resistance to IR.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044157&req=5

pone-0017108-g006: Model of RhoB-induced cell death after IR.IR-induced DNA damage activates members of the DNA damage-sensing machinery, which may directly or indirectly activate the nuclear GEFs Ect2 and Net1 to stimulate guanine nucleotide exchange upon RhoB. Activated RhoB leads to the downstream phosphorylation of JNK that triggers the induction of the pro-apoptotic protein Bim leading to cell death. Inhibition of either RhoB or Ect2 and Net1 activation through RNAi mutes this signaling pathway and results in cellular resistance to IR.
Mentions: The exposure of eukaryotic cells to genotoxic agents results in a variety of cellular responses, which can either promote or prevent survival. Deciphering the signaling pathways that modulate these signals is of important clinical relevance as a prognostic indicator of tumor response to DNA damaging agents. It has been well established that the protein expression of the small GTPase RhoB is upregulated in response to cellular stress [9] and that RhoB is required to induce apoptosis after exposure to several DNA damaging agents in Ras-transformed cell lines [22]. In the present study, we further demonstrate that RhoB activity is increased upon exposure to DNA damaging agents, and this activity is necessary for IR-induced cell death. However, it is yet unclear what the upstream regulatory processes are that control RhoB activity after DNA damage. Therefore, we sought to elucidate these upstream modulators with specific focus on Rho GEFs. We show that the nuclear GEFs Ect2 and Net1 specifically activate RhoB, which causes the downstream phosphorylation of JNK and the induction of the pro-apoptotic protein Bim, leading to cell death (Fig. 6).

Bottom Line: The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2.Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death.Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America. melissa_srougi@med.unc.edu

ABSTRACT
Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

Show MeSH
Related in: MedlinePlus