RASSF3 is the smallest member of the RASSF family of proteins that function as tumor suppressors. Unlike other members of this important family, the mechanisms through which RASSF3 suppresses tumor formation remain unknown. Here, we show that RASSF3 expression induces p53-dependent apoptosis and its depletion attenuates DNA damage–induced apoptosis. We found that RASSF3-induced apoptosis depended upon p53 expression. Exogenous expression of RASSF3 induced G1–S arrest, which was also p53 dependent. In contrast, loss of RASSF3 promoted cell-cycle progression, abrogated UVB- and VP-16–induced G1–S arrest, decreased p53 protein and target gene expression, and prevented DNA repair. RASSF3 was shown to directly interact with and facilitate the ubiquitination of MDM2, the E3 ligase that targets p53 for degradation, thereby increasing p53 stabilization. Together, our findings show the tumor suppressor activity of RASSF3, which occurs through p53 stabilization and regulation of apoptosis and the cell cycle. Cancer Res; 72(11); 2901–11. ©2012 AACR.

The mammalian RASSF proteins comprise 10 members (RASSF1–RASSF10; refs. 1–4). The C-terminal RASSF proteins (RASSF1 to RASSF6) harbor Ras association domains and Salvador/RASSF/Hippo (SARAH) domains in the C-terminus, whereas the N-terminal RASSF proteins (RASSF7 to RASSF10) have Ras association domains in the N-terminus and lack the SARAH domain. RASSF1A, a splicing variant of RASSF1, is a well-established tumor suppressor. Other RASSF proteins have also been reported to be downregulated in human cancers and considered as tumor suppressors. RASSF3 is the smallest member of the C-terminal RASSF (5). RASSF3 was reported to be upregulated in the mammary glands of tumor-resistant mouse mammary tumor virus (MMTV)/neu female mice, which do not develop mammary tumor by 11 months (6). Exogenous expression of RASSF3 reduced cell viability in breast cancer epithelial cells. Moreover, the transgenic expression of RASSF3 delayed tumor development in MMTV/neu mice. RASSF3 was expressed in MMTV/neu mouse mammary tumors, but it was lost in primary cultured cells established from the tumors, which corroborates the antisurvival property of RASSF3. Real-time (RT) PCR using human samples showed that RASSF3 is downregulated in human lung, uterus, and colon tumors than in normal tissues (6). The summary SAGE in Cancer Genome Anatomy Project (National Cancer Institute, Bethesda, MD) also supports that RASSF3 expression is lower in human stomach, liver, colon, and lung cancers. These data suggest that RASSF3 functions as a tumor suppressor like other RASSF proteins but it remains to be studied how RASSF3 suppresses tumor formation. In this study, we show that RASSF3 plays a role in p53-dependent apoptosis and G1–S arrest and that its depletion impairs DNA repair after DNA damage and causes polyploidy.

Construction of expression vectors

cDNA encoding human RASSF3 (BC055023) was purchased from Open Biosystem. cDNA for mouse RASSF3 and human MDM2 was obtained by RT-PCR from mouse brain cDNA (Clontech) with the primers (H1411, 5′-gaattcagcagcggctacagcagcct-3′ and H1412, 5′-gtcgactagccgggcttccaacctc-3′) and from human brain cDNA (Bio-Chain) with the primers (H3012, 5′-gaattcatggtgaggagcaggcaaatg-3′ and H3013, 5′-ctcgagctaggggaaataagttagca-3′), respectively. pCGN-HA-ubiquitin (Ubc) and pCMV5a human MDM2 were generously supplied by A. Kikuchi (Osaka University, Osaka, Japan) and K. Yoshida (Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan). pClneoMyc, pClneoFLAG-His6 (FH), pClneoHA, and pClneoGFP were described previously and used to generate expression constructs including pClneoMyc MDM2 and pClneoFLAG MDM2 (7–9). pClneoMyc CIN85, pClneoMyc mammalian Ste20-like kinase (MST)1, pClneoMyc MST2, and pClneoMyc modulator of apoptosis 1 (MOAP1) were described previously (9, 10). NheI/EcoRI fragment from pEGFP2 (Clontech) was ligated into NheI/EcoRI sites of pClneo to generate pClneoEGFPC2. PCR was carried out on pClneoEGFPC2 using the primers (H2962, 5′-gctagccatggggtgcgtctgctcatcaaatatggtgagcaagggcgagg-3′ and H543, 5′-caattgctcgaagcattaaccct-3′) to add MGCVCSSN (single letters code amino acids) to the N-terminus of GFP. The PCR product was ligated into pTAKN-2 TA cloning vector (Dyna Express), digested with NheI/NotI and ligated into XbaI/Not sites of iPSC hNanog (System Biosciences) to replace Nanog and red fluorescence protein with myristoylated GFP and generate pIPS-GFP to express a membrane-associated GFP.

Antibodies and reagents

Rabbit anti-RASSF3 antibody was raised against the synthetic peptide (MSSGYSSLEEDAEDFFFTARTC; single letters code amino acids) according to the previous report (6). Rabbit anti-MST2 antibody was previously described (9). The other antibodies and reagents were obtained from commercial sources: mouse anti-Myc (9E10; American Type Culture Collection); mouse anti-FLAG M2 (F3165), mouse anti-activated Bax (clone 6A7), mouse anti-α-tubulin (T9026), rabbit anti-FLAG (F7425), rabbit anti-endonuclease G (E5654), rabbit anti-actin (A5060), VP-16, Hoechst 33342, Bax-inhibiting peptide V5, cycloheximide, and propidium iodine (PI; Sigma-Aldrich); rabbit anti-cleaved caspase-3 (9661; Cell Signaling Technology); mouse anti-Bax (clone 3), and mouse anti-cytochrome C (clone 6H2.B4; BD Pharmingen); mouse anti-GFP (sc-9996), mouse anti-MDM2 (sc-965), goat anti-apoptosis–inducing factor (Sc-9416), and goat anti-p21 (SC-397G; Santa Cruz); mouse anti-HA (Roche); mouse anti-p53 (clone DO-1; Leica); mouse anti-phosphorylated histone 2AX (γ-H2AX; clone JBW301), donkey fluorescein isothiocyanate- and donkey rhodamine–conjugated secondary (Millipore); and peroxidase-conjugated secondary antibodies (ICN Cappel); MG132 and zVAD-FMK (Calbiochem); and Nutlin-3 (Cayman Chemical Company).

Cell cultures and transfection

HeLa, HEK293FT, H1299, U2OS, MCF-7, HCT116 p53+/+, and HCT116 p53−/− cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS, 10 mmol/L HEPES-NaOH at pH 7.4, 100 U/mL of penicillin, and 100 mg/L of streptomycin under 5% CO2 at 37°C. DNA transfection was carried out with Lipofectamine 2000 (Invitrogen). HCT116 p53+/+ and p53−/− cells were generous gifts of Dr. Bert Vogelstein.

Quantitative RT-PCR

Quantitative RT-PCR analysis was conducted using SYBR Green (Roche) and the DNA Engine Opticon system (Bio-Rad) as described previously (9). The used primers are listed (Supplementary Table S1).

RNA interferences

MST1, MST2, large tumor suppressor kinase (LATS)1, LATS2, and MOAP1 were knocked down as described previously (9). The double-stranded (ds) RNAs for human p53, RASSF3, and MDM2 are purchased from Ambion (Supplementary Table S2). The validity of the knockdown was confirmed by quantitative RT-PCR or immunoblotting.

Immunofluorescence

Immunofluorescence was carried out as described previously (7).

Immunoprecipitation

Various tagged proteins (FLAG-RASSF3, Myc-RASSF3, FLAG-MDM2, Myc-MST2, and GFP-MOAP1) were expressed in HEK293FT cells. The immunoprecipitation was carried out as described previously (9). Cells were lysed in the buffer [25 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 1% (w/v) Triton X-100, 10 mg/L leupeptin, 10 mg/L pepstatin A, and 10 mg/L 4-amidinophenylmethanesulfonyl fluoride]. The lysates were centrifuged at 100,000 x g for 15 minutes at 4°C. The supernatant was incubated with anti-FLAG M2 Affinity Gel (Sigma-Aldrich). The beads were washed 3 times with 25 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, and 1% (w/v) Triton X-100. To detect the interaction of endogenous RASSF3 with MST2 or MDM2, MCF-7 cells were treated with 10 μmol/L MG132 for 6 hours and the immunoprecipitation was carried out in the presence of 10 μmol/L MG132 and 1 mmol/L EDTA using anti-RASSF3 or anti-MDM2 antibody. The precipitates were analyzed by SDS-PAGE and immunoblotting.

Apoptosis detection assays

Cell survival and cell death were analyzed as described previously (7, 9). In brief, control GFP or GFP-RASSF3 was expressed in HeLa, HCT116 p53+/+, and HCT116 p53−/− cells. GFP-positive cells were gated and analyzed by flow cytometry (FACSCalibur; BD Biosciences). The Annexin-V binding assay was conducted by Annexin-V-Phycoerythrin Apoptosis Detection Kit I (BD Biosciences). DNA content was determined by PI staining to analyze sub-G1 population. Fluorescence-activated cell-sorting (FACS) data were analyzed with CellQuest software (BD Biosciences).

Cell-cycle analysis

HeLa, H1299, and U2OS cells were transfected with control pIPS-GFP, pClneoGFP-RASSF3, control dsRNA, or RASSF3 dsRNA. To evaluate the effect of GFP-RASSF3, 6 hours after transfection of pIPS-GFP or pClneoGFP-RASSF3, thymidine block was started by culturing cells in DMEM containing 10% FBS and 2 mmol/L thymidine. Eighteen hours later, cells were washed, incubated in fresh DMEM containing 10% FBS, and fixed at the indicated time points. GFP-positive cells were gated and analyzed. To evaluate the effect of RASSF3 knockdown, cells were replated at 48 hours after transfection with dsRNA and replated. The first thymidine block was started at 72 hours. Eighteen hours later, cells were released and cultured for 8 hours (HeLa cells) and 12 hours (U2OS cells). Then, the second incubation in the medium containing 2 mmol/L thymidine was done for 17 hours before washing and releasing the block. As RASSF3-expressing cells were difficult to maintain, a single thymidine block was used for the analysis of RASSF3 expression. For the FACS analysis, the cells were washed with PBS, fixed with 70% (v/v) ethanol in PBS overnight at −20°C, washed with PBS, and stained for 15 minutes with 10 mg/L PI in PBS containing 100 mg/L RNaseA. For each time, at least 104 GFP-positive cells were analyzed.

Cell proliferation and viability assessment

5-Bromo-2′-dexoyuridine (BrdUrd) incorporation assay was conducted by BrdUrd labeling and detection kit (Roche). Cell proliferation was assessed with MTT dye conversion.

Ubiquitination assay

Various tagged RASSF3 and MDM2 were expressed with HA-Ubc in HEK293FT cells. The cells were treated with dimethyl sulfoxide (DMSO) or 10 μmol/L MG132 for 6 hours and the immunoprecipitation was carried out as described earlier, except that 10 μmol/L MG132 and 1 mmol/L EDTA were added to lysis and washing buffers.

Neutral and alkaline comet assays

The comet assays were conducted according to the Trevigen CometAssay kit (Trevigen Inc.) protocol with slight modifications. U2OS or HeLa cells were transfected with either control dsRNA or RASSF3 dsRNA. Seventy-two hours later, the cells were treated with either DMSO or VP-16 (50 μmol/L) for 3 hours, or irradiated with UVB (10 J/m2), washed with the fresh medium, cultured for 24 hours, harvested using trypsin, and resuspended in the medium containing serum to stop trypsin. The final cell density was about 1.5 × 106 cells per mL. Five microliters of the cell suspension was then mixed with 120 μL of 1.0% 2-hydroxyethyl agarose (Sigma-Aldrich) in PBS at 37°C. One hundred and twenty microliters of the cell per agarose mixture was transferred and spreaded onto glass slides. Slides were incubated at 4°C for 15 minutes in dark and then immersed in prechilled Trevigen lysis buffer for 30 minutes, followed by electrophoresis in 1× Tris-borate EDTA (TBE) buffer at 1.0 V/cm for 30 minutes (neutral assay) or in 200 mmol/L NaOH, 1 mmol/L EDTA (pH > 13) at 1.0 V/cm for 20 minutes (alkaline assay) at room temperature. After electrophoresis, slides were rinsed with distilled water and stained with 2.5 mg/L PI for 15 minutes. Images were visualized under a fluorescence microscope and captured with a CCD camera. The comet tail moment was determined by CometScore software (TriTek Corporation). More than 50 cells were analyzed for each treatment group.

Statistical analysis

Statistical analyses were conducted with Student t test for the comparison between 2 samples and ANOVA with Dunnett test for the multiple comparison using the JMP statistical discovery software (SAS Institute). Data expressed as percentages were subjected to arcsine square root transformation before Student t test or ANOVA.

RASSF3 induces apoptosis in HeLa cells

RASSF3 was reported to reduce cell viability in human breast cancer cells (6). We started the study using HeLa cells to examine whether RASSF3 induces apoptosis. HeLa cells were transfected with pClneoGFP-RASSF3. At 24 hours, GFP-positive cells were detected, but thereafter, decreased in a time-dependent manner, whereas the cells expressing control GFP remained (Supplementary Fig. S1A). The flow cytometric analysis showed the increase of Annexin-V–positive 7-aminoactinomycin D (7-AAD)-negative cells and that of sub-G1 population in GFP-RASSF3–expressing HeLa cells (Supplementary Fig. S1B). In the immunofluorescence, Myc-RASSF3–expressing cells exhibited nuclear condensation, caspase-3 cleavage, Bax activation, cytochrome C release to the cytosol, and the release of apoptosis-inducing factor and endonuclease G from the mitochondria (Supplementary Fig. S1C). The caspase inhibitor, zVAD-FMK, partially suppressed the RASSF3-induced nuclear condensation (Supplementary Fig. S1D). RASSF3 suppression attenuated UVB-induced apoptosis (Supplementary Figs. S1E and S2A). These findings support that RASSF3 is implicated in apoptosis in HeLa cells.

RASSF3-induced apoptosis is p53 dependent

p53 is involved in the cell-cycle regulation by RASSF1A and Nore1A (11, 12). However, it is elusive whether p53 is involved in apoptosis mediated by RASSF proteins. When GFP-RASSF3 was expressed in p53-depleted HeLa cells, GFP-RASSF3–positive cells remained viable (Supplementary Figs. S2B and S3A). p53 knockdown reduced RASSF3-induced nuclear condensation and caspase-3 cleavage (Supplementary Fig. S3B). As p53 does not fully function in HeLa cells, we used isogenic HCT116 p53+/+ and HCT116 p53−/− cells to further confirm the involvement of p53 in RASSF3-induced apoptosis. Myc-RASSF3 induced nuclear condensation and GFP-RASSF3 increased sub-G1 population in HCT116 p53+/+ cells more remarkably than in HCT116 p53−/− cells (Fig. 1A and B). These findings support that RASSF3-induced apoptosis significantly, if not completely, depends on p53. Caspase inhibitor partially blocked apoptosis (Fig. 1C). RASSF3 knockdown reduced VP-16- and UVB-induced sub-G1 population in HCT116 p53+/+ cells (Fig. 1D). The effect of RASSF3 knockdown was more remarkable in HCT116 p53+/+ cells than in HeLa cells, which is also supportive for the implication of p53 (Fig. 1D and Supplementary Fig. S1E).

Figure 1.

RASSF3-induced apoptosis depends on p53. A, HCT116 p53+/+ (top) and HCT116 p53−/− (bottom) cells were transfected with control Myc-construct (Myc-Cont) or pClneoMyc-RASSF3 (Myc-RF3). Cells were fixed at 24 and 48 hours after transfection and immunostained with anti-Myc antibody. Nuclei were visualized by Hoechst 33342. Bars, 40 μm. The histogram shows the ratio of cells with nuclear condensation in Myc-positive cells. One hundred Myc-positive cells were observed for each experiment. B, HCT116 p53+/+ and HCT116 p53−/− cells were transfected with control pIPS-GFP (GFP) or pClneoGFP-RASSF3 (GFP-RF3). The cells were harvested 48 hours after transfection. GFP-positive cells were gated and analyzed. DNA content was determined by PI staining and the sub-G1 population was analyzed by FACS. C, 20 μmol/L zVAD-FMK reduced the RASSF3-induced nuclear condensation in Myc-RASSF3–expressing HCT116 p53+/+ cells at 48 hours after transfection. D, HCT116 p53+/+ cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1), and then treated with VP-16 (10 or 50 μmol/L) or irradiated with UVB (10 or 50 J/m2). Twenty-four hours later, the sub-G1 population was determined. Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01.

Figure 1.

RASSF3-induced apoptosis depends on p53. A, HCT116 p53+/+ (top) and HCT116 p53−/− (bottom) cells were transfected with control Myc-construct (Myc-Cont) or pClneoMyc-RASSF3 (Myc-RF3). Cells were fixed at 24 and 48 hours after transfection and immunostained with anti-Myc antibody. Nuclei were visualized by Hoechst 33342. Bars, 40 μm. The histogram shows the ratio of cells with nuclear condensation in Myc-positive cells. One hundred Myc-positive cells were observed for each experiment. B, HCT116 p53+/+ and HCT116 p53−/− cells were transfected with control pIPS-GFP (GFP) or pClneoGFP-RASSF3 (GFP-RF3). The cells were harvested 48 hours after transfection. GFP-positive cells were gated and analyzed. DNA content was determined by PI staining and the sub-G1 population was analyzed by FACS. C, 20 μmol/L zVAD-FMK reduced the RASSF3-induced nuclear condensation in Myc-RASSF3–expressing HCT116 p53+/+ cells at 48 hours after transfection. D, HCT116 p53+/+ cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1), and then treated with VP-16 (10 or 50 μmol/L) or irradiated with UVB (10 or 50 J/m2). Twenty-four hours later, the sub-G1 population was determined. Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01.

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RASSF3-induced apoptosis does not depend on the Hippo pathway but partially depends on MOAP1

RASSF1A, Nore1A, RASSF2, and RASSF6 interact with MST kinases, which are key components of the tumor suppressive Hippo pathway, via SARAH domains. Nore1A promotes MST1-mediated apoptosis (13). RASSF1A activates MST kinases and the downstream LATS2 and induces apoptosis (14–16). RASSF2 interacts with MST1 and MST2, stabilizes them and activates MST1 to induce apoptosis via c-jun-NH2-kinase (17, 18). RASSF6 blocks the activation of MST kinases and induces apoptosis independently of the Hippo pathway (9). RASSF3 also harbors the SARAH domain, and interacts with MST1 and MST2 in vitro (Supplementary Fig. S4A and data not shown). Because of the limitation of the sensitivity of the antibody, MG132 treatment was necessary to detect endogenous RASSF3. The interaction between endogenous RASSF3 and MST2 was also confirmed in MG132-treated MCF-7 cells (Supplementary Fig. S4B). However, the knockdown of MST1/2 or LATS1/2 did not inhibit RASSF3-induced apoptosis in HCT116 p53+/+ cells (Supplementary Fig. S4C). MOAP1, which activates Bax, is implicated in RASSF1A-induced and RASSF6-induced apoptosis (9, 19, 20). The interaction of RASSF3 and MOAP1 was detected in vitro (Supplementary Fig. S4D). MOAP1 knockdown partially inhibited RASSF3-induced apoptosis (Supplementary Fig. S4E). Bax-inhibiting peptide V5 also significantly reduced apoptosis (Supplementary Fig. S4F). These findings suggest that RASSF3-induced apoptosis is independent of the Hippo pathway and partially depends on MOAP1 and Bax.

RASSF3 induces G1–S arrest

RASSF1A was initially reported to regulate G1–S checkpoint and was later showed to control mitotic arrest (21–25). GFP-RASSF3 induced G1–S arrest in HeLa cells (Supplementary Fig. S5A). RASSF3 depletion facilitated the cell-cycle progression (Supplementary Fig. S5B, a black arrow). UVB induced G1–S arrest (Supplementary Fig. S5B, a gray arrow). RASSF3 depletion overrode this G1–S arrest and promoted the cell-cycle progression (Supplementary Fig. S5B, a white arrow). The main outcome of VP-16 treatment is the cell-cycle arrest in the late S and G2–M, but VP-16 also induces G1–S arrest (26). To further confirm a role of RASSF3 in G1–S checkpoint, we examined the effect of RASSF3 depletion on VP-16–induced G1–S arrest. To this end, we adopted the method that was used in the previous article by Hu and colleagues (27). HeLa cells were transfected with either control dsRNA or RASSF3 dsRNA and treated with either DMSO or VP-16. The DNA content analysis at 18 hours showed that VP-16 increased the population in G2–M and S-phases in the control dsRNA-transfected cells (Supplementary Fig. S5C). RASSF3 depletion did not significantly change the basal distribution pattern of cells, namely the increase of G2–M and S population, but reduced G1 population (Supplementary Fig. S5C, an arrow). As mouse RASSF3 is resistant to the knockdown by si RF3-1, we reintroduced GFP-mouse RASSF3 (Supplementary Fig. S5D). Under the expression of control GFP, RASSF3 knockdown reduced G1 population in VP-16–treated cells (Supplementary Fig. S5E, left, a white arrow), but mouse RASSF3 recovered it (Supplementary Fig. S5E, right, a black arrow). This result corroborates that RASSF3 indeed plays a role in VP-16–induced G1–S arrest. RASSF3 depletion had no effect on the nocodazole-mediated G2–M arrest and the mitotic exit after the release from the nocodazole treatment in HeLa cells (data not shown).

RASSF3-induced G1–S arrest depends on p53

As p53 is involved in RASSF3-induced apoptosis, it is the obvious question whether RASSF3 induces G1–S arrest via p53. We first examined whether and how RASSF3 is involved in the cell-cycle regulation in p53-normal U2OS cells. RASSF3 knockdown remarkably facilitated cell proliferation, BrdUrd incorporation, and the cell-cycle progression in U2OS cells (Fig. 2A and B). GFP-RASSF3 induced G1–S arrest (Fig. 2C). UVB and VP-16 arrest U2OS cells at G1–S (Fig. 2D, gray arrows). RASSF3 knockdown overrode UVB- and VP-16–induced G1–S arrests (Fig. 2D, white arrows). These findings support that RASSF3 regulates the cell cycle in U2OS cells as in HeLa cells. However, GFP-RASSF3 did not induce G1–S arrest in p53-negative H1299 cells (Fig. 3A). p53 knockdown increased cell proliferation and BrdUrd incorporation in U2OS cells but the combined knockdown of p53 and RASSF3 did not show additional effect (Fig. 3B). Moreover, RASSF3 knockdown increased cell proliferation and BrdUrd incorporation in HCT116 p53+/+ cells but not in HCT116 p53−/− cells (Fig. 3C). The findings support that RASSF3-induced G1–S arrest depends on p53.

Figure 2.

RASSF3 induces G1–S arrest. A, the cell proliferation was assessed by MTT assay (left) and BrdUrd incorporation (middle and right) in U2OS cells, which were transfected with control (si Cont) or RASSF3 dsRNA (si RF3-1). Forty-eight hours later, the cells were plated at 5 × 103 cells per well in triplicate in 96-well plates for MTT assay. The value at 24 hours was set to 1. For BrdUrd incorporation, the cells were plated at 5 × 104 cells per well in 12-well plates. Twenty-four hours later, the cells were incubated with BrdUrd for 30 minutes, fixed, and immunostained with anti-BrdUrd antibody. BrdUrd-positive cells were determined among 100 cells. Bar, 40 μm. B, U2OS cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). The double thymidine block was carried out. The knockdown of RASSF3 facilitates the cell-cycle progression (black arrow). C, U2OS cells were transfected with pIPS-GFP (GFP) or pClneoGFP-RASSF3 (GFP-RF3). The single thymidine block was carried out. GFP-positive cells were gated and analyzed. GFP-RASSF3 induced G1–S arrest. D, the effect of RASSF3 knockdown on UVB- and VP-16–induced G1 arrest in U2OS cells. The double thymidine block was carried out. For UVB, 16 hours after the second block, the cells were exposed to UVB (10 J/m2) and were released from thymidine blockage 1 hour later. For VP-16, 17 hours after the second block, the cells were released from thymidine blockage and treated with VP-16 (50 μmol/L). UVB and VP-16 induced G1–S arrest (left, gray arrows). The knockdown of RASSF3 facilitated the cell-cycle progression in control cells (black arrow) and overrode G1–S arrest in the UVB-exposed and VP-16–treated cells (white arrows). Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01.

Figure 2.

RASSF3 induces G1–S arrest. A, the cell proliferation was assessed by MTT assay (left) and BrdUrd incorporation (middle and right) in U2OS cells, which were transfected with control (si Cont) or RASSF3 dsRNA (si RF3-1). Forty-eight hours later, the cells were plated at 5 × 103 cells per well in triplicate in 96-well plates for MTT assay. The value at 24 hours was set to 1. For BrdUrd incorporation, the cells were plated at 5 × 104 cells per well in 12-well plates. Twenty-four hours later, the cells were incubated with BrdUrd for 30 minutes, fixed, and immunostained with anti-BrdUrd antibody. BrdUrd-positive cells were determined among 100 cells. Bar, 40 μm. B, U2OS cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). The double thymidine block was carried out. The knockdown of RASSF3 facilitates the cell-cycle progression (black arrow). C, U2OS cells were transfected with pIPS-GFP (GFP) or pClneoGFP-RASSF3 (GFP-RF3). The single thymidine block was carried out. GFP-positive cells were gated and analyzed. GFP-RASSF3 induced G1–S arrest. D, the effect of RASSF3 knockdown on UVB- and VP-16–induced G1 arrest in U2OS cells. The double thymidine block was carried out. For UVB, 16 hours after the second block, the cells were exposed to UVB (10 J/m2) and were released from thymidine blockage 1 hour later. For VP-16, 17 hours after the second block, the cells were released from thymidine blockage and treated with VP-16 (50 μmol/L). UVB and VP-16 induced G1–S arrest (left, gray arrows). The knockdown of RASSF3 facilitated the cell-cycle progression in control cells (black arrow) and overrode G1–S arrest in the UVB-exposed and VP-16–treated cells (white arrows). Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01.

Close modal
Figure 3.

RASSF3 regulates cell cycle via p53. A, p53-negative H1299 cells were transfected with control pIPS-GFP (GFP) or pClneoGFP-RASSF3 (GFP-RF3). The cell-cycle analysis was conducted as in Fig. 2C. B, U2OS cells were transfected with control dsRNA (si Cont), RASSF3 dsRNA (si RF3-1), p53 dsRNA (si p53), or the combination of RASSF3 and p53 dsRNAs (si RF3-1 + si p53). C, HCT116 p53+/+ and HCT116 p53−/− cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). MTT assay and BrdUrd incorporation assay were conducted. Error bars, SD of 3 independent experiments. n.s., not significant. *, P < 0.05; **, P < 0.01.

Figure 3.

RASSF3 regulates cell cycle via p53. A, p53-negative H1299 cells were transfected with control pIPS-GFP (GFP) or pClneoGFP-RASSF3 (GFP-RF3). The cell-cycle analysis was conducted as in Fig. 2C. B, U2OS cells were transfected with control dsRNA (si Cont), RASSF3 dsRNA (si RF3-1), p53 dsRNA (si p53), or the combination of RASSF3 and p53 dsRNAs (si RF3-1 + si p53). C, HCT116 p53+/+ and HCT116 p53−/− cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). MTT assay and BrdUrd incorporation assay were conducted. Error bars, SD of 3 independent experiments. n.s., not significant. *, P < 0.05; **, P < 0.01.

Close modal

The knockdown of RASSF3 downregulates the expression of p53 and the p53 target proteins

We examined whether RASSF3 affects p53 expression and p53-mediated gene transcriptions. In RASSF3-depleted U2OS cells, p53, p21, and Bax were downregulated at protein level (Fig. 4A). The message of p53 did not decrease, whereas the messages of p21 and PUMA decreased (Fig. 4B). MG132 treatment recovered p53 protein expression in RASSF3-depleted cells (Fig. 4C, black and white arrows). We treated U2OS cells with a protein synthesis inhibitor, cycloheximide, and examined endogenous p53 protein expression. p53 decreased in a time-dependent manner (Fig. 4D). RASSF3 depletion enhanced this decrease. The findings suggest that the proteosomal degradation of p53 is facilitated by RASSF3 depletion. MDM2 is a major E3 ligase for p53. MDM2 knockdown and Nutlin-3, which inhibits the interaction between MDM2 and p53, recovered p53 expression in RASSF3-depeleted cells (Fig. 4E and F and Supplementary Fig. S2C). These data suggest that MDM2-dependent degradation of p53 is enhanced in RASSF3-depleted cells.

Figure 4.

The knockdown of RASSF3 downregulates the expression of p53 and p53 target proteins. A to D, U2OS cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). A, seventy-two hours after transfection, the cells were harvested. The cell lysates (50 μg of total protein per each lane) were immunoblotted (IB) with the indicated antibodies. p53, p21, and Bax decreased in RASSF3-deficient cells. Actin was used as an internal marker. B, seventy-two hours after transfection, quantitative RT-PCR was carried out. The knockdown of RASSF3 decreased the messages of p21 and PUMA but not that of p53. Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01. C, seventy-two hours after transfection, U2OS cells were treated with either DMSO or 10 μmol/L MG132 for 6 hours. The cell lysates were immunoblotted with anti-p53 antibody. RASSF3 depletion decreased p53 (black arrow), whereas MG132 treatment recovered it (white arrow). D, seventy-two hours after transfection, the cells were treated with 50 mg/L cycloheximide. The endogenous p53 was determined. Actin was used as an internal control. RASSF3 depletion facilitated the degradation of p53. E, U2OS cells were transfected with control dsRNA (si Cont), RASSF3 dsRNA (si RF3-1), MDM2 dsRNA (si MDM2), or the combination of RASSF3 and MDM2 dsRNAs (si RF3-1 + si MDM2). Seventy-two hours later, the cell lysates were immunoblotted by anti-p53 or anti-actin antibody. F, seventy-two hours after transfection of dsRNA, U2OS cells were treated with DMSO or 8 μmol/L Nutlin-3 for 18 hours. The cell lysates were immunoblotted with anti-p53 antibody.

Figure 4.

The knockdown of RASSF3 downregulates the expression of p53 and p53 target proteins. A to D, U2OS cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). A, seventy-two hours after transfection, the cells were harvested. The cell lysates (50 μg of total protein per each lane) were immunoblotted (IB) with the indicated antibodies. p53, p21, and Bax decreased in RASSF3-deficient cells. Actin was used as an internal marker. B, seventy-two hours after transfection, quantitative RT-PCR was carried out. The knockdown of RASSF3 decreased the messages of p21 and PUMA but not that of p53. Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01. C, seventy-two hours after transfection, U2OS cells were treated with either DMSO or 10 μmol/L MG132 for 6 hours. The cell lysates were immunoblotted with anti-p53 antibody. RASSF3 depletion decreased p53 (black arrow), whereas MG132 treatment recovered it (white arrow). D, seventy-two hours after transfection, the cells were treated with 50 mg/L cycloheximide. The endogenous p53 was determined. Actin was used as an internal control. RASSF3 depletion facilitated the degradation of p53. E, U2OS cells were transfected with control dsRNA (si Cont), RASSF3 dsRNA (si RF3-1), MDM2 dsRNA (si MDM2), or the combination of RASSF3 and MDM2 dsRNAs (si RF3-1 + si MDM2). Seventy-two hours later, the cell lysates were immunoblotted by anti-p53 or anti-actin antibody. F, seventy-two hours after transfection of dsRNA, U2OS cells were treated with DMSO or 8 μmol/L Nutlin-3 for 18 hours. The cell lysates were immunoblotted with anti-p53 antibody.

Close modal

RASSF3 interacts with MDM2 and stabilizes p53

RASSF1A is reported to promote the autodegradation of MDM2 and to result in the stabilization of p53 (11). RASSF3 and MDM2 were coimmunoprecipitated from MCF-7 cells (Fig. 5A). Exogenously expressed RASSF3 and MDM2 interacted with each other in HEK293FT cells (Fig. 5B). When coexpressed with RASSF3, MDM2 became almost undetectable in U2OS cells (Fig. 5C, the second lane). RASSF3 expression also decreased by MDM2 coexpression. MG132 increased the basal expression of MDM2 and blocked the RASSF3-induced reduction (Fig. 5C, the fourth lane: an arrowhead, and the fifth lane: a star). The ubiquitination of MDM2 was enhanced by RASSF3 (Fig. 5D). The ubiquitination of RASSF3 was also slightly enhanced by MDM2 (Fig. 5E). These data support that RASSF3 binds to MDM2 and that this interaction causes ubiquitination and degradation of both proteins.

Figure 5.

RASSF3 enhances the ubiquitination of MDM2. A, coimmunoprecipitation of RASSF3 and MDM2 from MG132-treated MCF-7 cells. RASSF3 (top) or MDM2 (bottom) was immunoprecipitated from MG132-treated MCF-7 cells. The immunoprecipitate was immunoblotted with anti-MDM2 and anti-RASSF3 antibodies. B, Myc-RASSF3 was coimmunoprecipitated with FLAG-MDM2 by anti-FLAG-M2 Affinity Gel from MG132-treated HEK293FT cells. C, Myc-RASSF3 and Myc-MDM2 were expressed in U2OS cells with or without MG132 treatment. MDM2 expression remarkably decreased under the coexpression of RASSF3 (second lane, arrow). MG132 enhanced MDM2 expression (fourth lane, arrowhead) and canceled the effect of RASSF3 (fifth lane, star). RASSF3 slightly decreased under the coexpression of MDM2 (second lane, bottom). White arrow, a dimer of Myc-RASSF3. To avoid nonspecific inhibition, we added control pIPS-GFP to adjust the total DNA amount and to express GFP for lanes 1, 3, 4, and 6. D, FLAG-MDM2 and HA-Ubc were expressed with control GFP or GFP-RASSF3 in MG132-treated HEK293FT cells. The immunoprecipitation was done with anti-FLAG M2 Affinity Gel. The input and the immunoprecipitate were immunoblotted with anti-FLAG, anti-GFP, and anti-HA antibodies as indicated. RASSF3 enhanced the ubiquitination of MDM2. E, FLAG-RASSF3 and HA-Ubc were expressed with or without Myc-MDM2 in MG132-treated HEK293FT cells. The immunoprecipitation was done with anti-FLAG M2 Affinity Gel. The input and the immunoprecipitate were immunoblotted with anti-FLAG, anti-Myc, and anti-HA antibodies as indicated. MDM2 enhanced the ubiquitination of RASSF3.

Figure 5.

RASSF3 enhances the ubiquitination of MDM2. A, coimmunoprecipitation of RASSF3 and MDM2 from MG132-treated MCF-7 cells. RASSF3 (top) or MDM2 (bottom) was immunoprecipitated from MG132-treated MCF-7 cells. The immunoprecipitate was immunoblotted with anti-MDM2 and anti-RASSF3 antibodies. B, Myc-RASSF3 was coimmunoprecipitated with FLAG-MDM2 by anti-FLAG-M2 Affinity Gel from MG132-treated HEK293FT cells. C, Myc-RASSF3 and Myc-MDM2 were expressed in U2OS cells with or without MG132 treatment. MDM2 expression remarkably decreased under the coexpression of RASSF3 (second lane, arrow). MG132 enhanced MDM2 expression (fourth lane, arrowhead) and canceled the effect of RASSF3 (fifth lane, star). RASSF3 slightly decreased under the coexpression of MDM2 (second lane, bottom). White arrow, a dimer of Myc-RASSF3. To avoid nonspecific inhibition, we added control pIPS-GFP to adjust the total DNA amount and to express GFP for lanes 1, 3, 4, and 6. D, FLAG-MDM2 and HA-Ubc were expressed with control GFP or GFP-RASSF3 in MG132-treated HEK293FT cells. The immunoprecipitation was done with anti-FLAG M2 Affinity Gel. The input and the immunoprecipitate were immunoblotted with anti-FLAG, anti-GFP, and anti-HA antibodies as indicated. RASSF3 enhanced the ubiquitination of MDM2. E, FLAG-RASSF3 and HA-Ubc were expressed with or without Myc-MDM2 in MG132-treated HEK293FT cells. The immunoprecipitation was done with anti-FLAG M2 Affinity Gel. The input and the immunoprecipitate were immunoblotted with anti-FLAG, anti-Myc, and anti-HA antibodies as indicated. MDM2 enhanced the ubiquitination of RASSF3.

Close modal

RASSF3 is involved in DNA repair and the depletion of RASSF3 gives birth to the polyploid cells

We tested the idea that RASSF3 depletion impairs G1–S checkpoint and leads to the genomic instability, which is one of the major hallmarks of cancer cells (28, 29). We treated U2OS cells with VP-16 and immunostained γ-H2AX. γ-H2AX was detected immediately after the VP-16 treatment but remarkably diminished at 24 hours after VP-16 removal in the control cells (Fig. 6A). In contrast, γ-H2AX signals remained in the RASSF3-depleted cells (Fig. 6A). The comet assay also showed the delay of DNA repair in RASSF3-depeleted U2OS cells (Fig. 6B). RASSF3 depletion delayed DNA repair in UVB-exposed U2OS cells (Supplementary Fig. S6A and S6B). VP-16 treatment caused polyploidy in U2OS cells and RASSF3 depletion further increased polyploid cells in VP-16–treated U2OS cells (Fig. 6C). The similar result was obtained for UVB-exposed U2OS cells (Supplementary Fig. S6C). These data indicate that RASSF3 depletion compromises DNA repair and eventually leads to polyploidy. In the final set of the experiments, we addressed a question whether or not RASSF3 functions as a tumor suppressor even in p53 partially defective HeLa cells. RASSF3 depletion reduced p53, p21, and Bax at protein level (Supplementary Fig. S7A, left). p53 expression recovered by MG132 treatment (Supplementary Fig. S7A, middle). The additional knockdown of MDM2 also recovered p53 expression (Supplementary Fig. S7A, right). p53 degradation was more rapid in HeLa cells than in U2OS cells (Fig. 4D and Supplementary Fig. S7B). RASSF3 depletion further accelerated this degradation. VP-16 treatment increased p53 and p21, but RASSF3 knockdown reduced this increase (Supplementary Fig. S7C). We confirmed that VP-16 treatment caused DNA damage in HeLa cells and that RASSF3 knockdown impaired DNA repair using γ-H2AX staining and the comet assay (data not shown). In HeLa cells, VP-16 treatment itself caused polyploidy and RASSF3 depletion increased it (Supplementary Fig. S7D). These findings support that RASSF3 contributes to the prevention of polyploidy even in HeLa cells.

Figure 6.

RASSF3 is involved in DNA repair and the depletion of RASSF3 gives birth to the polyploid cells. A, U2OS cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). Seventy-two hours after transfection, the cells were treated with 50 μmol/L VP-16 for 3 hours and washed with fresh medium. The cells were immediately fixed or further cultured for 3 or 24 hours. γ-H2AX was immunostained. The positive cells were evaluated among 100 cells for each condition. Bars, 40 μm. B, U2OS cells were treated as in A. The neutral comet assay was conducted. Fifty cells were analyzed for each condition. Bars, 40 μm. C, U2OS cells were treated with VP-16 and cultured for 96 hours before flow cytometric profilings. Three independent experiments were carried out. Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01; n.s., not significant.

Figure 6.

RASSF3 is involved in DNA repair and the depletion of RASSF3 gives birth to the polyploid cells. A, U2OS cells were transfected with control dsRNA (si Cont) or RASSF3 dsRNA (si RF3-1). Seventy-two hours after transfection, the cells were treated with 50 μmol/L VP-16 for 3 hours and washed with fresh medium. The cells were immediately fixed or further cultured for 3 or 24 hours. γ-H2AX was immunostained. The positive cells were evaluated among 100 cells for each condition. Bars, 40 μm. B, U2OS cells were treated as in A. The neutral comet assay was conducted. Fifty cells were analyzed for each condition. Bars, 40 μm. C, U2OS cells were treated with VP-16 and cultured for 96 hours before flow cytometric profilings. Three independent experiments were carried out. Error bars, SD of 3 independent experiments. *, P < 0.05; **, P < 0.01; n.s., not significant.

Close modal

Mouse and human RASSF3 were identified from EST clones by homology search to identify similar sequences to the Ras association domain of RASSF1A (5). The message of human RASSF3 is detected ubiquitously. CpG islands are predicted in the promoter regions of RASSF3 but no promoter methylation has been reported for human cancers. RASSF3 is experimentally shown to suppress mammary tumor development in MMTV/neu transgenic mice (6). Its expression is downregulated in human lung, uterus, stomach, colon, and liver tumors. RASSF3 is similar to other C-terminal RASSF proteins in the molecular structure and reduces cell viability. Therefore, it is likely that RASSF3 functions as a tumor suppressor. However, the basal characters of RASSF3 have not been clear enough.

We showed that exogenous expression of RASSF3 induces apoptosis and that RASSF3 depletion attenuates UVB- and VP-16–induced apoptosis. The C-terminal RASSF proteins harbor the SARAH domain and interacts with MST1 and MST2, the core kinases of the proapoptotic Hippo pathway. RASSF1A and Nore1 regulate apoptosis via MST kinases (13–16). In contrast, RASSF6 induces apoptosis independently of MST1/2 (9). We confirmed the interaction between RASSF3 and MST1/2. However, the knockdown of MST1/2 or LATS1/2 had no effect on RASSF3-induced apoptosis. Therefore, RASSF3 induces apoptosis independently of the Hippo pathway. RASSF1A- and RASSF6-induced apoptosis partially depend on MOAP1, which is an activator of Bax (9, 19, 20). The knockdown of MOAP1 and Bax-inhibiting peptide V5 block RASSF3-induced apoptosis, suggesting that MOAP1 and Bax are involved in RASSF3-induced apoptosis. As apoptosis is suppressed in p53-negative cells, p53 is necessary for RASSF3-induced apoptosis.

RASSF3 expression remarkably influences the cell cycle. RASSF3 depletion facilitates proliferation in U2OS cells and HCT116 p53+/+ cells but not in HCT116 p53−/− cells. Exogenous expression of RASSF3 induces G1–S arrest in HCT116 p53+/+ cells, and in HeLa cells, in which p53 is expressed but its degradation is facilitated. However, RASSF3 does not cause G1–S arrest in p53-negative H1299 cells. These findings support that RASSF3 regulates the cell cycle via p53.

We hypothesized that RASSF3 modulates p53 expression. RASSF3 depletion slightly increases p53 mRNA but significantly decreases p53 protein in U2OS cells. As MG132 treatment recovers p53 protein expression, it is considered that RASSF3 depletion promotes the proteosomal degradation of p53. The major E3 ligase to disrupt p53 is MDM2 (30). RASSF1A promotes self-ubiquitination and subsequent degradation of MDM2 (11). The coexpression of RASSF3 with MDM2 enhances the ubiquitination of MDM2 and remarkably reduces MDM2 expression. On the other hand, RASSF3 expression is reduced by MDM2, indicating that RASSF3 is a substrate of MDM2. The findings support that RASSF3 induces the self-degradation of MDM2 to protect p53. MDM2 knockdown and Nutlin-3 fully recover p53 expression in RASSF3-depleted U2OS cells. This result suggests that RASSF3 stabilizes p53 mainly through MDM2 in U2OS cells.

p53 plays an important role in DNA damage responses (30–32). Its significance is well studied in the context of ionizing irradiation and DNA double-strand break-inducing reagents, but UVB exposure also stabilizes p53, which induces G1–S arrest and is important for DNA repair (33, 34). RASSF3 depletion blunts DNA repair in VP-16–treated and UVB-exposed U2OS cells and generated polyploid cells. RASSF3 also induces apoptosis and regulates cell cycle in HeLa cells. Moreover, RASSF3 suppresses the generation of polyploidy after DNA damage in HeLa cells. In HeLa cells, p53 is expressed but its degradation is promoted by E6AP (35). When MDM2 was knocked down in addition to RASSF3, p53 expression recovered but MG132 treatment was slightly more effective in HeLa cells. It is possible that MDM2 knockdown was not complete, but it is also possible that RASSF3 stabilizes p53 not only through MDM2 degradation but also through other mechanisms in HeLa cells. Importantly, RASSF3 prevents polyploidy in HeLa cells. This observation suggests that RASSF3 prevents oncogenesis in p53 function–compromised cells as a sort of second line of defense.

K. Nakagawa: commercial research grant, Daiichi Sankyo TaNeDS. Y. Hata: commercial research grant, Daiichi Sankyo TaNeDs. No potential conflicts of interest were disclosed by the other authors.

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Kudo

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Kudo

Writing, review, and/or revision of the manuscript: T. Kudo, Y. Hata

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Kudo, M. Ikeda, M. Nishikawa, Z. Yang, K. Ohno, K. Nakagawa

Study supervision: M. Ikeda, Y. Hata

The authors thank Kiyotsugu Yoshida (Tokyo Medical and Dental University), Hirofumi Teraoka (Tokyo Medical and Dental University), Ryo Sakasai (Kyushu University, Fukuoka, Japan), Akira Kikuchi (Osaka University), Hitoshi Nakagama (National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan), Koji Okamoto (National Cancer Center Research Institute), and Bert Vogelstein (Johns Hopkins University, Baltimore, MD) for the materials and the advice and Shintaro Ohgake and Yuko Tateishi for their contributions at the initial stage of the study.

This work was supported by research grants from the Ministry of Education, Sports, Science, and Technology (17081008) and from Japan Society for the Promotion of Science (JSPS; 22790275, 22590267). Z. Yang was supported by Japanese Government (Monbukagakusho; MEXT) scholarship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Mol Cell Biol
1993
;
13
:
775
84
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Supplementary data