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 
and that RhoB is required to induce apoptosis after exposure to several DNA damaging agents in Ras-transformed cell lines 
. 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 ().
Model of RhoB-induced cell death after IR.
Of the ≥70 identified GEFs, Ect2 and Net1 specifically localize to the nucleus at steady state. Whereas Ect2 plays a physiologic role in cytokinesis 
, the normal functions of Net1 are still being defined. Here, we describe a new function for these GEFs in the modulation of cell death after genotoxic stress. Ect2 and Net1, out of the panel of GEFs tested, were the only two activated after DNA damage. Studies by others have shown Net1-mediated RhoA activation after exposure to supra-lethal doses (20 Gy) of IR 
. However, under the lower doses of IR used in our studies, we did not observe increases in RhoA activity (). In contrast to Ect2 and Net1, the cytoplasmic GEFs, p115 RhoGEF and Vav2 were not activated after IR (). These results were not due to the inability of these GEFs to exchange upon RhoB, as overexpression of each GEF was sufficient to cause an increase in RhoB-GTP levels in cells (). Moreover, despite the transient nature of protein KD, it is apparent from our data that KD of Ect2 and Net1 at the time of insult was sufficient to prevent the later (~24 h) downstream activation of JNK () and to promote cell survival after IR () by muting RhoB-mediated death signals.
Our data demonstrate that IR activates both Ect2 and Net1, however the mechanisms that regulate their activity remain unknown. In many circumstances, GEF activation is linked to subcellular localization. Ect2 and Net1 are unique in this aspect as they both contain two nuclear localization signals within the N terminus, confining them to the nucleus at steady state. In addition to localization, GEF activation can be modulated by post-translational modification. For example, the phosphorylation of Ect2 during G2
/M increases its activity for Rho in vitro 
and the oncogenic form of Net1 was also shown to be regulated by its phosphorylation state 
. It is therefore likely that specific kinase(s) may be responsible for their regulation and/or possible cellular relocation after DNA damage. The phosphoinositide 3-kinase-related protein kinase (PIKK) family consists of large serine/threonine protein kinases involved in the response to cellular stress, and are candidates for Ect2 and Net1 activation after IR. Members of this family include Ataxia-telangiectasia mutated (ATM), DNA dependent protein kinase catalytic subunit (DNA-PKcs), and ATM and Rad3 related protein kinase (ATR). ATM is an attractive candidate as it has a pivotal role in the response to IR-induced DNA damage. The association of ATM with other DNA damage related proteins or protein-complexes facilitate its activation and function. A number of the ATM-protein interactions occur with proteins which contain a BRCA-1 C-terminal (BRCT) domain; the most notable is Brca1 (breast cancer gene 1). The BRCT domain is a highly conserved region found in many DNA damage-responsive cell cycle checkpoint proteins 
. Interestingly, Ect2 contains tandem BRCT domains in its N-terminus, which are required for proper cytokinesis 
. It is therefore possible that these BRCT regions within Ect2 may have additional functions, some of which are more intimately involved in the DNA damage response. Net1, on the other hand, contains a nuclear export signal in addition to nuclear import signals, thus implying that it can be triggered to leave the nucleus and activate Rho in the cytoplasm 
. To this effect, Schmidt and Hall have shown that the PH domain of Net1 is required for its nuclear export 
. It is therefore of interest if DNA damage-mediated ATM activation triggers Ect2 and/or Net1 activation and relocation to the cytoplasm where it can activate RhoB. Preliminary data suggest that there is an enrichment of Ect2 in the cytoplasmic fraction after damage, and that loss of ATM kinase activity decreases Ect2 and Net1 activation after IR (data not shown). The cross-talk between ATM and these nuclear GEFs may be a mechanism for amplifying death signals in cells were irreversible damage occurs, and further studies are underway to delineate the mechanisms whereby DNA damage regulates Ect2 and Net1 function.
There are many circumstances where altered GEF activity can lead to cellular transformation 
. In the case of Ect2 and Net1, truncation of the N-terminal portion of each protein has been shown to transform cells due to the mislocalization of these GEFs to the cytoplasm, which results in their constitutive activation 
. It would therefore be of interest to determine if the mislocalization of Ect2 and/or Net1 to the cytoplasm could be a potential resistance factor in human cancers to IR or other DNA damaging agents. Recently, Ect2 has been shown to be overexpressed and mislocalized to the cytoplasm of primary non-small cell lung carcinoma (NSCLC) tumor cells as well as NSCLC cell lines, but not primary normal lung epithelia 
. These cells also have low levels of RhoB, and re-expression of RhoB decreases proliferation and tumor growth in vivo 
. It is not surprising then, that the NSCLC cell line, A549 has been well studied for its radio-resistance 
. Similarly, Ect2 is also overexpressed and mislocalized in glioblastoma multiforme (GBM) compared to normal brain tissue 
and although GBMs respond to full course radiation, they tend to recur. It is therefore interesting to speculate that the mislocalization of Ect2 to the cytoplasm may be in part responsible for the relative radiation resistance and/or recurrence of these tumor cells.
RhoB plays a role in number of pathways which regulate tumor cell survival and proliferation. For example, it was demonstrated that RhoB controls endocytic trafficking and slows the internalization of the EGF receptor to the lysosome through PRK1 activation 
as well as the regulation of nuclear Akt trafficking 
. As important as RhoB is in modulating survival pathways little has been done to explore its role downstream of DNA damage. Some studies have shown that in response to farnesyl transferase inhibitor treatment, RhoB suppresses cyclin B1 leading to cell death 
and it may associate with caspase-2 in mouse cardiomyocyte apoptosis 
. In addition, RhoB represses NFκB activation after cell treatment with alkylating agents 
. Therefore, we explored the pathways activated downstream of RhoB. We observed that the IR resistance of RhoB KD cells was not due to general alterations in the cell death machinery, as these cells were still capable of undergoing apoptosis after treatment with other cytotoxic agents such as staurosporine (unpublished results). Furthermore, RhoB deficient MEFs have an intact p53 response and undergo cell cycle arrest after IR 
. These data suggest that RhoB may act downstream of the DNA damage response, functioning as a signal amplifier after cells have already been committed to die. Thus, we first examined the activation of JNK since it is commonly triggered after exposure to IR 
and particularly in MCF-7 cells where it is needed to initiate apoptosis after IR as its inhibition prevents the release of cytochrome c 
. Furthermore, overexpression of Rho family members A, B, and C can induce its activation 
. We found JNK to be downstream of RhoB activation and that suppression of RhoB activity, either by reducing RhoB protein levels directly or reducing the amount of stimulating GEFs, Ect2 and Net1, was sufficient to inhibit JNK phosphorylation (). Recently, JNK activation after IR was shown to be upstream of RhoB protein induction in Jurkat cells, however JNK activation downstream of RhoB was not examined 
. Based on our own findings, there may exist a positive feedback loop whereby RhoB activity stimulates sustained JNK phosphorylation, which potentiates cell death through the upregulation of RhoB.
Of the many pro-apoptotic cellular targets of JNK, Bim can be activated transcriptionally via activation of the transcription factors such as c-Jun 
and FOXO3a 
or after translation in response to cytotoxic stimuli 
. We found that exposure to IR increases BimEL
protein expression as early as 48 h, and that Bim protein levels are reduced by RhoB shRNA (). Alternatively, if RhoB activity is inhibited through suppression of Ect2 and Net1, BimEL
levels are also reduced (), suggesting that it is increased RhoB activity, specifically, that is necessary for this event. Since JNK-dependent dephosphorylation and nuclear accumulation of FOXO3a were found in response to paclitaxel treatment 
, based on our own data, it is likely that RhoB-mediated Bim induction is primarily through a JNK-dependent mechanism.
Collectively, our experiments delineate a novel pathway whereby the nuclear GEFs Ect2 and Net1 are activated after genotoxic stress. These GEFs activate RhoB, which is required for cell death after exposure to γ-IR in non-Ras transformed human breast cancer cells. Furthermore, we demonstrate that RhoB activation triggers the downstream activation of the SAPK/JNK pathway leading to an increase in Bim protein levels and cell death. From a therapeutic standpoint, understanding the mechanisms of IR-induced cell death is of particular clinical relevance. Therefore, exploring the regulation of these nuclear GEFs after DNA damage may initiate novel strategies for rendering tumors which are refractory to radio- and/or chemotherapy more sensitive to first line anticancer treatments.