Introduction
DNA damage by ionizing irradiation (IR) triggers rapid activation of DNA-damage checkpoint response, resulting in either cell-cycle arrest that allows DNA repair or induction of apoptosis, which eliminates seriously damaged or deregulated cells [
1]. Previous studies identified several intracellular signaling cascades, including signalings mediated by ataxia telangiectasia-mutated (ATM) and ATM- and rad3-related (ATR), in the activation of DNA-damage checkpoint response [
2].
The G
2/M cell-cycle checkpoint is tightly controlled by the Cdc2/cyclin B complex, whose activity is required for G
2/M transition of the cell cycle [
3]. Previous studies identified the Cdc2-Tyr15 as a critical site involved in G
2/M-checkpoint control in response to DNA damage. Cdc2-Tyr15 phosphorylation is induced and maintained during radiation-induced G
2/M arrest, and introduction in fission yeast of a mutant Cdc2-Y15F, which cannot be phosphorylated at the tyrosine 15 residue, completely abolished DNA-damage-induced G
2/M arrest [
4‐
6]. Cdc2-Tyr15 is phosphorylated by Wee1 kinase, which phosphorylates Cdc2 at Tyr15, and by Myt1 kinase, which phosphorylates Cdc2 at Thr14 and, to a lesser extent, at Tyr15 [
7,
8].
Dephosphorylation of Cdc2-Tyr15 involves Cdc25 dual-specific phosphatases [
9]. In response to DNA damage, ATM and ATR kinases are rapidly activated through phosphorylation, which, in turn, leads to the phosphorylation/activation of their downstream targets Chk1 and Chk2 kinases, respectively. Activation of Chk1 and Chk2 kinases results in phosphorylation of Cdc25, leading to the subcellular sequestration, degradation, and/or inhibition of the Cdc25 phosphatases that normally activate Cdc2/cyclin B at the G
2/M boundary [
10].
On cell transition from G
2 to mitotic phase, histone H3 is phosphorylated at Ser10, which is associated with chromosome condensation before cell division [
11]. Because both G
2 and mitotic cells have
4N-DNA content and are not distinguishable from each other by propidium iodide staining, phosphorylation of H3-Ser10 in
4N-DNA content cells has been commonly used as a specific marker indicative of mitotic cells [
12]. Furthermore, previous studies indicate that the initial phosphorylation of H3-Ser10 occurs in the late G
2 phase but only on the pericentromeric chromatin. As cells progress through mitosis, the phosphorylation spreads along chromosomes and is completed at the end of prophase [
13,
14]. Thus, a gradual increase in H3-Ser10 phosphorylation occurs from the beginning of mitosis to the end of mitosis. In log-phase growing cells, phosphorylation of H3-Ser10 in mitotic cells is detected in a wide range with flow-cytometry analysis [
15,
16]. In response to irradiation-induced G
2/M cell-cycle arrest, the phosphorylation of H3-Ser10 is suppressed in irradiated cells because of the blockage of the G
2/M transition of the cell cycle [
3,
15,
16].
Previous studies in a wide variety of cell types have shown that IR exposure results in rapid activation of MAPK family members, including ERK1/2, JNK, and p38 [
17,
18]. Although p38γ activation may be essential in IR-induced G
2/M arrest in HeLa and U2OS cells [
19], studies from our laboratory and others have demonstrated that IR-induced ERK1/2 activation is necessary for the activation of the G
2/M checkpoint response in MCF-7 breast cancer cells and that inhibition of ERK1/2 is associated with increased sensitivity to DNA-damaging agents [
16,
20,
21].
Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho family of small guanosine triphosphatases (GTPases), has been shown to play a critical role in the regulation of cytoskeleton reorganization, cell migration, and cell survival [
22]. Rac1 overexpression has been detected in many tumor types, including breast, lung, and colon cancer [
23‐
25]; and Rac1b, a fast-cycling splice variant of Rac1, has been observed to be highly expressed in some breast and colon cancers [
25,
26]. Through interaction with various downstream effectors, Rac1 has been shown to activate numerous signaling pathways, including those mediated by the members of the MAPK family [
27]. In response to various stimuli, previous studies showed that Rac1 can activate ERK1/2 signaling via p21-activated kinases 1 and 2, which phosphorylate Raf1 and MEK1 and facilitate the formation of the Raf/MEK/ERK complex [
28‐
30]. Other studies indicated that Rac1 is involved in the activation of JNK and p38 signaling in response to angiotensin II stimulation [
31]. Although the regulation of Rac1 on cytoskeleton reorganization and cell migration has been intensively investigated, the contribution of Rac1 to cell-cycle regulation has remained largely unknown. A previous study showed that expression of N17Rac1, a dominant-negative mutant of Rac1, in log-phase growing Rat 2 fibroblast cells, resulted in G
2/M cell-cycle arrest [
32]. Furthermore, a recent report detected the presence of Rac1 in the nucleus, and the level of nuclear Rac1 was increased when cells were in late G
2 phase [
33]. This evidence suggests a potential role for Rac1 in the regulation of cell-cycle progression in proliferating cells.
In the present study, we examined the effect of Rac1 on the IR-induced G2/M checkpoint response in human breast cancer cells. Results presented in this report indicate that IR exposure of cells induces Rac1 activation and that this is necessary for the activation of ERK1/2 signaling, subsequent G2/M checkpoint response, and cell survival after IR.
Materials and methods
Cell culture and treatment
Human breast cancer cell lines MCF-7, T47D, ZR-75-1, and MDA-MB-231 were obtained from American Type Culture Collection (Manassas, VA, USA). MCF-7, T47D, and ZR-75-1 cells were maintained in Dulbecco Modified Eagle medium containing 10% fetal bovine serum. MDA-MB-231 cells were maintained in the Leibovitz L-15 medium containing 10% fetal bovine serum. MCF-10A is a nontumorigenic human mammary epithelial cell line that was spontaneously immortalized previously [
34]. 76 N is a nontransformed line of primary human mammary epithelial cells immortalized by human telomerase (hTERT) [
35]. MCF-10A and 76 N cells are kind gifts from Dr. Vimla Band (University of Nebraska Medical Center). Both cell lines were maintained in Dana-Farber Cancer Institute 1 growth medium (DFCI-1). DFCI-1 medium consists of α-MEM/Ham nutrient mixture F-12 (1:1, vol/vol) supplemented with epidermal growth factor (12.5 ng/ml), triiodothyronine (10 n
M), Hepes (10 m
M), ascorbic acid (50 μ
M), estradiol (2 n
M), insulin (1 μg/ml), hydrocortisone (2.8 μ
M), ethanolamine (0.1 m
M), phosphoethanolamine (0.1 m
M), transferrin (10 μg/ml),
L-glutamine (2 m
M), sodium selenite (15 n
M), cholera toxin (1 ng/ml), 1% fetal bovine serum, and bovine pituitary extract (35 μg/ml) [
35].
Rac1-specific inhibitor NSC23766 [
36] was obtained from Tocris Biosciences (Ellisville, MO, USA) and dissolved in water. For experiments involving IR exposure, exponentially growing cells were treated with IR and then incubated at 37°C for the indicated time before analysis. For experiments involving treatment with both NSC23766 and IR, cells were incubated with NSC23766 for 1 hour before IR exposure.
Antibodies and recombinant proteins
All antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), unless otherwise indicated. These included mouse IgG for ATM (2C1) (Novus Biologicals, Littleton, CO, USA), Cdc2 (17), Chk1 (G-4), Chk2 (B-4), PARP (Ab-2) (EMD Biosciences, San Jose, CA, USA), phospho-ERK1/2 (E-4); rabbit IgG for ATM (Ab-3) (EMD Biosciences), Cdc2 (C-19), Chk1 (FL-476), Chk2 (Cell Signaling, Danvers, MA, USA) MEK1/2 (12-B), Rac1 (C-14); and goat IgG for Actin (I-19), ATR (N-19), phospho-Cdc2 (Tyr15), ERK1/2 (C-14-G), and phospho-MEK1/2 (Ser118/Ser122).
Recombinant PAK-1 protein for Rac1 activity assay was obtained from Addgene (Cambridge, NH, USA) as a glutathione
S-transferase (GST) fusion protein containing full-length human PAK1 protein. Recombinant p53 protein for ATM and ATR kinase assays was a glutathione
S-transferase (GST) fusion protein containing full-length human p53 (Addgene, Cambridge, MA, USA). Recombinant Cdc25C protein, the substrate for Chk1 and Chk2 kinase assay, was a GST fusion protein containing residues 200 to 256 of human Cdc25C (kindly provided by Dr. Helen Piwnica-Worms, Washington University School of Medicine). All GST fusion proteins were purified as described previously [
16]. GST was used as a control substrate in all kinase assays and was prepared according to standard procedures (GE Healthcare Bio-Sciences, Piscataway, NJ, USA).
Immunoblotting, immunoprecipitation, and kinase assay
Immunoblotting, immunoprecipitation, and kinase assays were performed as described previously [
16,
37,
38]. Specific protein signals on Western blots were visualized by chemiluminescence exposed to x-ray film, scanned by using EPSON Perfection 4490PHOTO scanner, and analyzed by using the ImageJ analytical program (NIH, Bethesda, MD, USA).
Rac1 activity assay
Rac1 activity was assayed by using a Rac1 assay kit (Upstate Biotechnology, Lake Placid, NY, USA), as described previously [
39,
40]. In brief, cells were lysed at 4°C in 25 m
M HEPES buffer (pH 7.4) containing 10 m
M MgCl
2, 150 m
M NaCl, 1% NP-40, 1 m
M EDTA, 2% glycerol, 1 m
M DTT, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 m
M phenylmethylsulfonyl fluoride, 1 m
M sodium fluoride, and 1 m
M sodium vanadate. Cell lysates were incubated with GST-PAK1 fusion protein for 1 hour to capture GTP-bound Rac1. The obtained GTP-bound Rac1 (Rac1-GTP) was resolved on a 4% to 20% SDS-PAGE and assessed with immunoblotting by using an anti-Rac1-specific antibody, as described by the manufacturer's instructions. As a positive control, MCF-7 cells were serum-starved for 24 hours in the medium containing 0.3% fetal bovine serum, treated with 1 μ
M phorbol 12-myristate 13-acetate (PMA) for 5 minutes, and analyzed for Rac1 activity [
41].
Cell-cycle analysis
Fluorescence-activated cell sorting (FACS) analysis was performed on 20,000 cells by using a FACS Calibur instrument (Beckon Dickinson, Mansfield, MA, USA), as described previously [
16].
Analysis for mitotic cells
MCF-7 cells were exposed to IR in the presence/absence Rac1-specific inhibitor NSC23766, harvested at the indicated times, fixed in 70% ethanol, and stained with propidium iodide (PI) and anti-phospho-histone H3 antibody (Upstate Biotechnology, Lake Placid, NY, USA) [
12]. Mitotic cells, which contain both
4N-DNA content and phospho-histone H3, were determined by using a FACSCalibur instrument (Beckon Dickinson) and analyzed by using CELLQUEST software. Each analysis was performed by using 20,000 cells.
The siRNAs and transfection
Short interfering RNA (siRNA) duplexes were obtained from Dharmacon Research (Chicago, IL, USA). Control nontargeting siRNA contains at least four mismatches to any human, mouse, or rat gene, as previously determined by the manufacturer. The sequence for Control-siRNA is 5'-UAAG GCUA UGAA GAGA UAC-3'. SMARTpool siRNAs targeting Rac1 consist of four siRNAs targeting multiple sites on Rac1 (Rac1-siRNAs). The sequences for Rac1-siRNAs are 5'-UAAG GAGA UUGG UGCU GUA-3', 5'-UAAA GACA CGAU CGAG AAA-3', 5'-CGGC ACCA CUGU CCCA ACA-3', and 5'-AUGA AAGU GUCA CGGG UAA-3'.
Cells were transfected with siRNAs at 100 nM by using DharmaFECT1 siRNA transfection reagent (Dharmacon Research, Chicago, IL, USA), according to the manufacturer's instruction. For experiments involving both siRNA transfection and IR exposure, transfected cells were first incubated for the indicated times and then exposed to IR.
Adenoviral vectors and adenoviral infections
Recombinant adenovirus N17Rac1 (Ad.N17Rac1) and control adenovirus dl312 (Ad.Control) were kindly provided by Dr. Toren Finkel (NIH, Bethesda, MD, USA). In Ad.N17Rac1, the Rac1 cDNA contains a Ser-to-Asp substitution at position 17 and functions as a dominant-negative mutant [
42].
Log-phase MCF-7 cells were infected at 50 PFU/cell with either Ad.N17Rac1 or Ad.Control for 24 hours before exposure to IR, as described previously [
43]. For studies involving cell-cycle analysis, the cells were incubated for additional 24 hours after IR and analyzed for DNA content with flow cytometry [
16]. For studies involving mitotic cell analysis, the irradiated cells were incubated for 2 hours and analyzed for cells containing both
4N-DNA content and histone H3-Ser10 phosphorylation [
12].
DAPI staining
Apoptosis was assessed with 4',6-diamidino-2-phenylindole (DAPI) staining, as described previously [
43]. Apoptotic cells were identified by condensation and fragmentation of nuclei [
44]. The percentage of viable cells was calculated as the ratio of live cells to total cells counted. At least 800 cells were counted per sample.
Cell-survival assay
Cell-survival assays were performed as described previously [
45]. In brief, log-phase growing cells were exposed to IR at the doses indicated, incubated for 7 days, and visualized for viable cells by staining with crystal violet (Sigma-Aldrich, St. Louis, MO, USA). For experiments involving treatment with both NSC23766 and IR, cells were preincubated for 1 hour with 100 μ
M NSC23766, exposed to IR, and incubated for an additional 3 hours after IR. The cells were washed and incubated in regular growth medium for 7 days before analysis. The obtained sample dishes were scanned on an EPSON Perfection 4490PHOTO scanner, and the amount of cells remaining on the culture dish was quantified by using the ImageJ analytic program.
Clonogenic assay
Clonogenic assay was performed as described previously [
46]. In brief, in the presence or absence of 100 μ
M NSC23766, MCF-7 cells were exposed to IR at the doses indicated and incubated for 3 hours after IR. The cells were then rinsed with DMEM, reseeded at the cell number indicated in duplicate, and incubated for 10 to 14 days until colonies formed. The colonies were visualized with crystal violet staining and quantified by using ImageJ software, as described previously [
47].
Discussion
G
2/M transition of the cell cycle is tightly controlled by the activity of the Cdc2/cyclin B complex, which is required for cell entry into mitosis. It has previously been shown that DNA damage induces phosphorylation of Cdc2-Tyr15, resulting in inhibition of Cdc2/cyclin B activity and ultimately G
2/M arrest [
3]. The results in this report indicate that IR exposure of MCF-7 cells induces Rac1 activation (see Figure
1C). Furthermore, inhibition of Rac1 by using the specific inhibitor, dominant-negative mutant Rac1 or specific Rac1 siRNA markedly attenuates IR-induced G
2/M arrest (see Figures
2 and
5). Additional studies in this report indicate that the inhibition of IR-induced Rac1 activation abolishes IR-induced activation of Chk1 and Chk2 kinases (see Figures
4 and
5) and subsequent Cdc2-Tyr15 phosphorylation (see Figure
3A). Because previous studies indicate that the transition of cells from G
2 to M phase of the cell cycle requires Cdc2/cyclin B activity, we also assessed the effect of Rac1 inhibition on the proportion of cells in mitosis. The studies presented in Figures
3B and
5A indicate that IR exposure of log-phase growing MCF-7 cells results in a marked decrease in mitotic cells within 2 hours after IR, and that this effect is significantly inhibited by the incubation of cells with NSC23766 or expression of the N17Rac1 dominant-negative mutant. Thus, Rac1 inhibition diminishes IR-induced G
2/M checkpoint activation and increases the entry of cells from G
2 into M phase of the cell cycle in MCF-7 cells exposed to IR. These studies suggest Rac1 as an upstream regulator of G
2/M checkpoint response after exposure of cells to IR.
Cellular response to IR-induced DNA damage involves activation of ATM and ATR signaling, which results in activation of the Wee1 kinase that phosphorylates Cdc2-Tyr15 and inhibition of the Cdc25 phosphatase that dephosphorylates Cdc2-Tyr15 [
50,
51]. Although it still remains unclear how exactly the ATM and ATR kinases are activated in response to genotoxic stress, evidence suggests that multiple mechanisms might be involved in the regulation of this biologic process. Supporting this speculation, a recent study by Wang
et al. [
52] reported that the p38MAPK pathway is required for the activation of ATR kinase after expression of hepatitis B virus X protein.
Another example is NBS1, a component of the MRE11/RAD50/NBS1 complex, which not only is involved in certain downstream steps of ATM- and ATR-dependent DNA damage response but also functions as an upstream mediator required for the ATM and ATR signaling activation after IR-induced DNA damage [
53]. The results from the present report suggest that Rac1 also plays an important role in the activation of ATM and ATR signaling after IR exposure of cells (see Figures
4 and
5).
A previous study demonstrated that incubation of MCF-7 cells with Rac1 specific inhibitor NSC23766 at 100 μ
M for 48 hours results in a G
1 cell-cycle arrest [
54]. However, in the present studies, we observe that incubation of MCF-7 cells with 100 μ
M NSC23766 for up to 24 hours does not result in a detectable increase in G
1-phase cells relative to control untreated cells (see Additional file
1, Figure S1). Furthermore, incubation of other cells, including MDA-MB-231, T47D, and ZR-75-1, with 100 μ
M NSC23766 for up to 24 hours, also does not result in an increase in percentage of G
1-phase cells (data not shown). Thus, the effect of NSC23766 on G
1-phase cells is probably time dependent. Additional studies are needed to understand the effect of prolonged Rac1 inhibition on cell-cycle regulation in log-phase growing cells.
Expression of N17Rac1 dominant-negative mutant for 72 hours has been previously shown to result in G
2/M cell-cycle arrest in Rat 2 fibroblast cells [
32]. In the present studies, after 24-hour expression of N17Rac1, we do not observe any noticeable effect by N17Rac1 on the proportion of G
2/M phase cells in log-phase growing MCF-7 cells (data not shown). Thus, the effect of N17Rac1 on G
2/M phase cells is probably cell-type specific and/or time dependent. In contrast, expression of N17Rac1 in MCF-7 cells abrogates the IR-induced activation of Rac1, and this, in turn, is associated with an attenuation of G
2/M arrest in irradiated cells and an increase in the amount of mitotic cells after irradiation (see Figure
5A).
Previous studies from several laboratories, including our own, have suggested an important role for ERK1/2 signaling in the activation of the G
2/M checkpoint response after DNA damage [
16,
20,
21]. These studies have demonstrated that DNA damage induces ERK1/2 activation and that this is associated with the induction of G
2/M arrest. Additional studies demonstrate that inhibition of ERK1/2 abrogates the G
2/M checkpoint response after DNA damage, resulting in increased sensitivity of cells to DNA-damaging agents [
16,
20,
21]. Results presented in this report indicate that Rac1 inhibition after incubation of cells with a specific inhibitor or transfection with Rac1-specific siRNA abrogates IR-induced phosphorylation of MEK1/2 and ERK1/2 (see Figure
6), as well as the IR-induced G
2/M checkpoint activation (see Figures
2 through
5), suggesting Rac1 as the upstream regulator of IR-induced ERK1/2 signaling.
A role for p53 in the regulation of the G
2/M checkpoint response has been suggested by previous studies, as several of the transcriptional targets of p53 can directly or indirectly inhibit Cdc2 kinase, which include p21
Waf1/Cip1, 14-3-3σ, and Gadd45 [
55]. However, the results of this report suggest that IR-induced G
2/M cell-cycle arrest as well as the regulation of Rac1 on the IR-induced G
2/M checkpoint response is apparently independent of p53, as among the four breast cancer cell lines used for the studies, MDA-MB-231 and T47D cells express mutant p53 [
56], whereas MCF-7 and ZR75-1 express wild-type p53. Consistent with our observation, results from other studies also show that p53 status has no influence on IR-induced G
2/M cycle arrest [
57].
The results in Figures
2 through
5 show that IR-induced G
2/M arrest in human breast cancer cells is markedly attenuated by the inhibition of Rac1. Furthermore, the results in Figure
7 and Additional file
1, Figure S4, provide evidence that Rac1 inhibition significantly increases the sensitivity of MCF-7 cells to irradiation, which involves apoptosis induction. These results suggest a strong correlation between the attenuation of G
2/M arrest and the increased radiation sensitivity in MCF-7 cells treated with IR in the presence of Rac1 inhibition. It is possible that the increased radiation sensitivity is simply a consequence of the attenuation of IR-induced G
2/M arrest by Rac1 inhibition. However, it could also be due to a new function of Rac1. Future studies must address this question.
In this report, we also tested the effect of Rac1 inhibition on IR-induced G
2/M arrest in normal human mammary epithelial cells (MCF-10A and 76N). The results are unexpected, as the Rac1 inhibition by NSC23766 does not block the IR-induced G
2/M arrest in these cells (see Additional file
1, Figure S5), whereas it blocks completely the IR-induced G
2/M arrest in human breast cancer cells (see Figures
2 through
5). The mechanism causing this difference is unclear. However, it should be noted that the growth medium DFCI-1 used for the normal human mammary epithelial cells contains additional growth factors that are not presented in the medium for maintaining the breast cancer cells, which include EGF, estradiol, and insulin (see Materials and methods). It might be that these additional components in DFCI-1 growth medium compensate for the effect of Rac1 inhibition on IR-induced G
2/M checkpoint activation. We will investigate this possibility in future studies.
Previous studies from our laboratory demonstrate that inhibition of ERK1/2 by MEK1/2 specific inhibitors or decreased ERK1/2 expression by transfection of cells with ERK1/2 siRNA abrogated the IR-induced ATR activation in MCF-7 cells but had little effect on ATM activation [
16]. Furthermore, additional studies demonstrate that ERK1/2 signaling is upstream of ATR, as decreased ATR expression in MCF-7 cells after transfection with ATR siRNA or incubation of cells with caffeine, which inhibits both ATR and ATM, has no effect on IR-induced ERK1/2 activation [
16]. Results presented in this study indicate that Rac1 activation not only is necessary for the activation of ERK1/2 and ATR kinases, but also is essential for the activation of ATM signaling after IR exposure (Figures
4 and
5).
A growing amount of evidence shows that IR exposure of breast cancer cells frequently results in G
2/M cell-cycle arrest [
58], and induction of cell-cycle arrest after DNA damage has been associated with DNA repair and cell survival [
50,
59,
60]. Thus, a better understanding of the mechanisms responsible for IR-induced G
2/M cell-cycle arrest would potentially allow identifying novel therapeutic targets that could be exploited to sensitize breast cancer cells to radiation treatment.
Results in this report provide evidence supporting a novel role for Rac1 in the activation of G2/M checkpoint response and promotion of cell survival after IR exposure.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
Experiments were performed by PG, PC, RK, and YY. The studies were designed, analyzed, and interpreted by YY and KC. The manuscript was drafted by YY and critically revised by KC. All authors read and approved the final manuscript.