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Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(2):263-272; doi:10.1093/carcin/bgm251
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Proteolysis of CDH1 enhances susceptibility to UV radiation-induced apoptosis

Weijun Liu, Wenqi Li, Takeo Fujita, Qi Yang and Yong Wan*

Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213-1863

* To whom correspondence should be addressed: Tel: +1 412 623 3275; Fax: +1 412 623 7761; Email: yow4{at}pitt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
As a critical ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) governs cell cycle progression, signaling modulation and the pathogenesis of some human diseases. Recent studies implicate APC in maintaining genomic integrity, but the mechanism by which it plays such a role remains largely unknown. We report here that acute UV radiation triggers proteolysis of CDH1, an activator of APC, which is involved in regulation of apoptosis induced by UV radiation. Depletion of CDH1 by RNA interference enhances the cellular susceptibility to apoptosis in response to UV radiation, whereas overexpression of non-degradable CDH1 delays UV radiation-induced apoptosis. In addition, UV-induced degradation of CDH1 results in the accumulation of cyclin B1 and therefore to increased CDK1 activity, which is believed to enhance UV-induced apoptosis. The present results unveil a novel role for the APC in UV-induced cell death and demonstrate a new regulatory mechanism for APC/CDH1 through proteolysis.

Abbreviations: APC/C, anaphase-promoting complex/cyclosome; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutatedand Rad3 related; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SiRNA, Small interference RNA; FACS, flourescence-activated cell sorting


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
The function of anaphase-promoting complex/cyclosome (APC/C) has been initially characterized in orchestrating mitotic progression, where CDC20 in association with APC results in the initiation of anaphase whereas CDH1-activated APC leads to the exit of mitosis(1–3). Current studies have significantly extended our understanding of APC and have revealed that APC, a multifunctional E3 ligase, regulates several additional crucial cellular events, including G1/S transition, DNA replication and transforming growth factor beta-initiated signal transduction promoting growth inhibition, development and tumorigenesis (1,4–6). The activity of APC is regulated during cell cycle and certain other cellular processes by CDC20 and CDH1 (7). A number of APC substrates have been identified with substrate specificity thought to be conferred by substrate-specific activators, CDC20 and CDH1. Recognition of substrate by the substrate-specific activator is facilitated by several well-characterized degrons, including destruction box (RXXL), KEN box and A box present in the substrate (1–3). CDC20 or CDH1 binds to the substrate by recognizing the degron and subsequently making the substrate available for ubiquitylation by APC and then degradation by the 26S proteasome(1,3).

Recent pathological and epigenetic studies have demonstrated that dysfunction of components of APC pathway, including Apc6, Cdc16, Cdc23 and Cdh1 or Cdc20, is correlated to many different types of cancers such as colon cancer, B-lymphoma, gastric and lung cancer (8–11). In addition, results from recent studies have uncovered a role for APC in genomic integrity (12–14). The depletion of Cdh1 by gene knockout results in loss of checkpoint function in response to ionization (13). Moreover, the role of APC in facilitating checkpoint function is supported by the notion that APC mediates ionization-induced proteolysis of cyclin D and securin (12,15). Besides its role in the regulation of DNA damage checkpoint, APC is further suggested to be directly involved in DNA repair, where CDH1/APC targets ribonucleotide reductase R2, a critical enzyme that governs deoxynucleoside triphosphates biogenesis, for degradation thereby regulating deoxynucleoside triphosphate levels for DNA replication as well as DNA repair (16). Deregulation of APC affecting genomic integrity has been shown to promote tumorigenesis (17). However, the biochemical mechanism by which APC acts in DNA damage response remains unclear. To understand how APC is regulated in response to genotoxic stress and how APC is involved in DNA damage-induced cellular response, we have systematically analyzed the protein profile of components of APC pathway in response to different types of genotoxic stress. This analysis provides the notion that the activity of APC is dramatically responsive to certain types of genotoxic stress. Based on the loss and gain of function analyses, we have further identified several APC-targeted proteins in response to DNA damage signal. Our results have shown that severe UV radiation induces rapid CDH1 degradation, which further leads to accumulation of cyclin B1. Accumulation of cyclin B1 in turn elevates CDK1 activity and consequently promotes UV-induced apoptosis. Results from these works suggest that APC plays an important role in mediating DNA damage signal, where UV-induced downregulation of APC modulates cellular susceptibility to apoptosis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Antibodies and chemicals
Anti-CDH1 antibody (CC-43) was from Calbiochem, San Diego, CA. Anti-SKP2 antibody (SC-7164), anti-cyclin B1 antibody (SC-594), anti-p27 antibody (SC-528), anti-CDC2 antibody (SC-54), anti-cyclin H antibody (SC-1662), anti-CDC25c antibody (SC-327), anti-HA antibody (SC-805) and anti-MYC (SC-789) antibody were from Santa Cruz, Santa Cruz, CA. Anti-poly (ADP-ribose) polymerase (PARP) antibody (7D3-6), anti-phospho-CDC2 (phosphotyrosine 15) antibody (C0228) and anti-p21 antibody (556430) were from BD Pharmingen, San Jose, CA. Anti-tubulin antibody (T-5168) was from Sigma, St Louis, MO. Anti-phospho-histone H3 (Ser10) (06-570) antibody was from Upstate, Lake placid, NY. Anti-securin antibody (K0090-3) was from Cyclex, Nagano, Japan. Anti-SnoN antibody (ABM-3002) was obtained from Cascade BioScience, Winchester, MA. Cy2-conjugated anti-mouse (115-225-003) and Cy3-conjugated anti-rabbit (111-225-003) secondary antibodies were from Jackson ImmunoResearch Laboratories, West Grove, PA. Peroxidase-conjugated anti-mouse secondary antibody (W4021) and peroxidase-conjugated anti-rabbit secondary antibody (W4011) were from Promega, Madison, WI. Cycloheximide (239764) and proteasome inhibitors MG-132 (474790) and ALLN (208719) were from Calbiochem.

Construction of expression vectors
pCS2+ Myc-Cdh1 was described previously (5). Human Cdh1 cDNA and its deletion mutants were amplified using primers containing AscI and ClaI sites and pCS2+ Myc-Cdh1 as a template. Amplified Cdh1 cDNA was subcloned in-frame into pCS2+-HA vector and the resulting constructs were used for expression of the C-terminal HA-tagged Cdh1 and its deletion mutants in mammalian cells. The primers used for constructing these mutants are as follows—Cdh1 1–89: 5'-AAAAATCGATACCATGGA CCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCCAGGCCGTCTTTGCCGTTGTC-3'; Cdh1 1–124: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCCTTCTCAGGCGTGGAGGGCTG-3'; Cdh1 1–154: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCGCTGACGGGAGACAGGGAGTA-3'; Cdh1 1–177: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCCTTGAAGGGGATCTTGGAGAT-3'; Cdh1 1–257: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCGTCCCAGATCTGCACGAAGCC-3'; Cdh1 1–340: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCCCAGACCAGCAGCTTGTTGTC-3'; Cdh1 1–471: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCGTTCCAGAACCTCAGGGTCTC-3'; Cdh1 1–488: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCGAGGTTGAGCACAGACACAGA-3'; Cdh1 1–493: 5'-AAAAATCGATACCATGGACCAGGACTATGAGCGG-3' and 5'-TTGGCGCGCCCCGGATCCTGGTGAAGAGGTTGAG-3'; Cdh1 90–493: 5'-AAAAATCGATACCATGGCCTACTCTGACCTGCTCAAG-3' and 5'-TTGGCGCGCCCCGGATCCTGGTGAAGAGGTTGAG-3'; Cdh1 125–493: 5'-AAAAATCGATACCATGAAGGGTCTGTTCACGTATTCC-3' and 5'-TTGGCGCGCCCCGGATCCTGGTGAAGAGGTTGAG-3'; Cdh1 178–493: 5'-AAAAATCGATACCATGGTGCTGGACGCGCCCGAGCTG-3' and 5'-TTGGCGCGCCCCGGATCCTGGTGAAGAGGTTGAG-3'.

UV treatment
Cells were irradiated with Ultraviolet C (245 nm) at a dose rate of 0.5 J/m2 per second. During irradiation, the cells were covered by a thin layer of phosphate-buffered saline (PBS). Afterwards, PBS was removed and fresh medium was added and the culture dish was returned to the incubator for the indicated times. Control cells were treated with PBS but were not irradiated.

Western blotting and in vivo ubiquitination assay
Whole-cell lysate was prepared with CSH buffer (Tris 50 mM, pH 7.4, NaCl 2.5 M, ethylenediaminetetraacetic acid 5 mM, Triton X-100 0.1%) containing 1x complete protease inhibitors cocktail (Roche, Basel, Switzerland). Total protein (50 mg) was heated 5 min at 90°C in 4x sample buffer (Invitrogen, Carlsbad, CA) and separated on NuPAGE 4–12% Bis–Tris gel (Invitrogen), transferred to nitrocellulose membrane and probed with the indicated primary antibody. Immunocomplexes were detected by incubation with peroxidase-conjugated secondary antibody and enhanced chemiluminescence chemiluminescence detection (Amersham, Piscataway, NJ).

For CDH1 in vivo ubiquitination, HeLa cells were co-transfected with Myc-Cdh1 and HA-Ub. Twenty-four hours later, cells were treated with UV irradiation. The in vivo ubiquitination assay was performed under denaturing condition as described by Marti et al. (18). Immunoprecipitated complex with anti-Myc antibody was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The western blotting was performed with anti-HA antibody.

RNA isolation and reverse transcription–polymerase chain reaction
Cellular total RNA was isolated by TRIzol (Invitrogen). The cDNA was synthesized using random hexamers from 1 µg of RNA. Polymerase chain reaction (PCR) primers were as follows—human Cdh1: 5'-AGATCTCCAAGATCCCCTTCA-3' and 5'-CCTCCAACATGGACAGCTTCT-3', amplicon, 297 bp; human glyceraldehyde-3-phosphate dehydrogenase: 5'-AGTCAACGGATTTGGTCGTA-3' and 5'-AAATGAGCCCCAGCCTTCT-3', amplicon, 315 bp. To amplify the cDNA, 2 µl aliquots of cDNA were subjected to 22 cycles of PCR consisting of denaturation at 94°C for 45 s, annealing at 60°C for 45 s and extension at 72°C for 45 s.

Apoptosis assay
After treatment, cells were harvested and washed once with PBS. PARP cleavage, annexin V–fluorescein isothiocyanate staining and flow cytometric analysis were used to assess apoptosis. PARP cleavage was detected by western blotting. Annexin V-positive or sub-G1 peak cells were defined as percentage of apoptotic cells.

Immunofluorescence microscopy
Cells were grown on coverslips and fixed for 20 min with 4% paraformaldehyde in PBS and permeabilized for 10 min with 0.5% Triton X-100. Cells were blocked with 1% BSA for 1 h and incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal cyclin B1 (1:500) and mouse monoclonal CDH1 (1:500). Secondary antibodies labeled with Cy2 or Cy3 were added at 1:1000 dilution, and slides were incubated at room temperature for 2 h. Slides were counterstained with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Images were acquired at room temperature with an Olympus OX81 microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
CDH1 is significantly altered in response to UV radiation
In order to address the mechanism by which APC functions in genomic integrity, we have systematically analyzed the protein profile of components of APC pathway in response to different types of genotoxic stress. To our surprise, the levels of CDH1 dramatically decreased after exposure to UV radiation. As shown in Figure 1A, CDH1 protein was degraded in response to high dosage of UV radiation (>20 J/m2) in both HeLa S3 and MCF7 cells whereas CDH1 abundance was not significantly changed in response to low dosage of UV radiation after 24 hours of UV exposure. To measure the kinetics of the degradation of CDH1 protein after exposure to high-dosage UV radiation, we monitored CDH1 protein levels at different time points by immunoblotting. As shown in Figure 1B and C, the degradation of CDH1 induced by high-dosage UV radiation occurs rapidly with half-decline in ~60 min in several types of cells, including MCF7, HeLa S3 and HNF cells. The drop in CDH1 protein levels was maintained during the 18 h of observation following exposure to high dosage of UV radiation.


Figure 1
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  		Fig. 1. CDH1 protein levels are tightly regulated by UV irradiation. (A) CDH1 protein levels dropped significantly in response to severe UV irradiation (>20 J/m2) but not to low-dose UV radiation (<20 J/m2). Both HeLa S3 and MCF-7 cells were treated with various dosages of UV. Twenty-four hours after irradiation, cells were harvested and the CDH1 protein levels were detected by immunoblotting. (B) UV radiation (60 J/m2) induced rapid drop in CDH1 protein levels in a time-dependent manner in human cancer cells MCF-7, HeLa S3 and human normal fibroblast cells. (C) Quantification of CDH1 destruction in response to UV irradiation (60 J/m2). CDH1 protein levels were determined by densitometry and normalized with control, which was set at 100%. Results are the mean ± SD of three separate experiments.

 
Previous studies suggested that UV-induced major cellular response is principally mediated by ataxia telangiectasia mutated (ATM)/ataxia telangiectasia mutated and Rad3 related (ATR)–Chk1/Chk2 cascade(19). To examine whether the UV-induced CDH1 degradation is via by the above cascade, we measured the alteration of Cdh1 protein levels in response to UV radiation by inhibition of ATM or ATR activity using caffeine. As shown in supplemental Figure S1 (available at Carcinogenesis Online), inhibition of ATM/ATR resulted in attenuation of CDH1 degradation. Taken together, our results suggest that CDH1 protein levels are sensitive to UV radiation and this UV-induced CDH1 degradation is mediated by the ATM/ATR–Chk1/Chk2 cascade.

UV-induced degradation of CDH1 is mediated by ubiquitin–proteasome pathway
To examine whether transcriptional regulation is involved in UV-induced downregulation of CDH1, we measured the kinetics of Cdh1 mRNA levels after exposure to UV radiation in HeLa cells using reverse transcription–PCR. As shown in Figure 2A, no noticeable change in Cdh1 mRNA was observed when CDH1 protein levels significantly decreased in response to UV radiation, suggesting that the drop of CDH1 protein levels is due to protein degradation. To further confirm that the drop in CDH1 protein levels induced by UV radiation is due to proteolysis, we next estimated the half-life of CDH1 proteins under normal conditions or under UV radiation. HeLa cells were treated with cycloheximide and then the protein level of CDH1 was monitored. As indicated in Figure 2B and C, the half-life for CDH1 after exposure to UV radiation is 0.8 h, whereas the half-life of CDH1 under normal conditions is ~3 h. Our results suggest that the decline in CDH1 protein levels in response to UV irradiation is caused by proteolysis.


Figure 2
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  		Fig. 2. Drop of CDH1 protein levels in response to UV radiation is mediated by ubiquitin–proteasome pathway. (A) UV radiation did not affect mRNA expression of Cdh1. Total RNA was extracted from UV-irradiated HeLa S3 cells and analyzed for Cdh1 mRNA levels by RT–PCR. Human glyceraldehyde-3-phosphate dehydrogenase was amplified as a control. (B and C) Half-life of CDH1 became shorter under UV radiation conditions. HeLa S3 cells were treated with 100 µg/ml cycloheximide with or without UV radiation at 60 J/m2. CDH1 expression was detected using western blotting (B). CDH1 protein levels were determined by densitometry and normalized with control, which was set at 100%. Results are mean ± SD of three independent experiments (C). (D) UV-induced CDH1 decrease in the protein level was blocked by proteasome inhibitors MG132 and ALLN. HeLa S3 cells were preincubated with dimethyl sulfoxide (vehicle control), MG-132 and ALLN for 20 min and treated with 60 J/m2 UV. Cells were incubated with dimethyl sulfoxide and proteasome inhibitors for indicated time. CDH1 protein levels were then detected using western blotting. (E) CDH1 was polyubiquitinated in vivo in response to UV radiation. HeLa S3 cells were co-transfected with Myc-Cdh1 and HA-Ub and irradiated with 60 J/m2 UV. After immunoprecipitation with anti-Myc under denaturing condition, polyubiquitinated CDH1 was detected with anti-HA antibody. The expression of Myc-Cdh1 was detected in whole-cell lysate with anti-Myc antibody.

 
To verify that the UV-induced CDH1 degradation is mediated by ubiquitin–proteasome pathway, we tested the capacity of proteasome inhibitors including MG-132 and ALLN in blocking CDH1 degradation induced by UV radiation. As shown in Figure 2D, preincubation of HeLa cells with either MG-132 (20 µM) or ALLN (100 µM) significantly attenuated CDH1 degradation induced by UV radiation whereas incubation with dimethyl sulfoxide alone did not affect CDH1 degradation. To show that the drop in CDH1 protein levels induced by UV radiation is facilitated by ubiquitylation, we conducted a ubiquitylation assay by co-transfecting Myc-tagged CDH1 with HA-tagged ubiquitin in HeLa cells. Co-transfected cells were exposed to UV and harvested at 30, 45 and 60 min after exposure to UV radiation. Ubiquitylation of CDH1 was then detected by immunoprecipitation of CDH1 complex with anti-Myc antibody followed by immunoblotting with anti-HA antibody. As shown in Figure 2E, ubiquitin-conjugated CDH1 is detected at significant levels after 45 min following treatment with UV radiation, suggesting that UV-induced degradation of CDH1 is mediated by ubiquitin–proteasome pathway.

Proteolysis of CDH1 is independent from UV-induced alteration of cell cycle
CDH1 protein levels oscillate during the cell cycle with the abundance peaking at mitosis and dropping at G1/S transition (20,21). In addition to apoptosis, the cellular effect of UV has been shown to induce cell cycle arrest in various types of cells (22). To assess whether CDH1 degradation is due to cell cycle arrest induced by UV radiation, we measured the profile of cell cycle progression after exposure to UV radiation. As shown in Figure 3A, the alteration of cell cycle profile in response to UV at high dosage was not obvious, whereas a transient accumulation of G2/M population was observed. To further examine the effect of UV on mitosis, we measured the status of phosphorylated histone 3, a hallmark of mitosis (23), after exposure to UV using immunoblotting. As shown in Figure 3B and C, no obvious change in mitotic index as represented by the number of phosphorylated histone 3-positive cells was measured in 3 h after exposure to UV, whereas 3- to 4-fold increase in mitotic index was detected in 6–9 h after exposure to UV. Given that UV-induced CDH1 degradation occurs in 1 h in response to UV radiation, no correlation exists between the kinetics of CDH1 degradation and the cellular accumulation at G2/M after radiation, suggesting that UV-induced CDH1 degradation is independent from the UV-modulated cell cycle alteration.


Figure 3
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  		Fig. 3. Extensive UV radiation induces transient mitotic accumulation, where UV-induced CDH1 destruction is independent from UV-effected cell cycle alteration. (A–C) UV-induced transient mitotic accumulation was measured using FACS analysis (A), phosphorylated histone 3 expression (B) and mitotic index analysis (C). HeLa S3 cells were exposed to UV radiation and harvested or fixed at indicated time points followed by analyses. (D) UV-induced CDH1 destruction is independent from UV-effected cell cycle alteration. HeLa S3 cells were synchronized at G1, G1/S and G2/M. Cells were released and subsequently treated with UV. CDH1 protein levels were then determined by western blot analysis.

 
To confirm the above conclusion, we further monitored the protein levels of CDH1 at different stages (G1, G1/S transition and G2/M) in synchronized cells following exposure to UV. HeLa cells were synchronized at G1, G1/S transition or G2/M using the protocol described previously (5). Cells were subsequently treated with UV radiation and then released. The protein level of CDH1 was measured at different time points after exposure to UV. As shown in Figure 3D, the drop in CDH1 protein levels in response to UV radiation was detected in all cases described above. Again, this result confirms that UV-induced CDH1 degradation is unrelated to the UV-modulated cell cycle alteration.

Mapping of functional domain on CDH1 mediating UV-induced CDH1 degradation
Previous studies have shown that certain regions of CDH1 such as the N-terminal stretch, the WD 40 domains and the conserved isoleucine-arginine (IR) dipeptide motif motif on CDH1 are important in recruiting substrates and mediating its interaction with APC (24–26). Bioinformatic analyses indicate that there are several putative domains including destruction box, A-box, C-box and sequence enriched with praline, glutamic acid, serine and threonine domain located near the N-terminus of CDH1 (Figure 4A). To identify the region on CDH1 that mediates CDH1 degradation in response to UV radiation, we have engineered a set of CDH1 deletion mutants (Figure 4B). Based on cell-free-based protein degradation assay (5,27), we have systematically evaluated the protein stability for 11 CDH1 deletion mutants in extracts prepared from cells exposed to UV radiation. As shown in Figure 4C, mutants including CDH1 (1–89 aa), CDH1 (1–124 aa) and CDH1 (178–493 aa) were quite stable in the UV-treated cell extracts, whereas most of the other CDH1 mutants were degraded. Interestingly, all degradable CDH1 mutants contain region in the middle of CDH1-containing amino acid residues from 124 to 178, whereas mutants missing this stretch were stable in response to UV radiation. These results suggest that the region between residues 124 and 178 is sensitive to UV signal and mediate UV-induced CDH1 degradation.


Figure 4
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  		Fig. 4. Mapping of functional domains mediating CDH1 degradation induced by UV radiation. (A) Schematic presentation of CDH1 functional domains, including D-box, A-box, C-box, IR tail, sequence enriched with praline, glutamic acid, serine and threonine and WD40 domains. (B) S35 labeled in vitro-translated CDH1 mutants. (C) Analysis of protein stability for CDH1 mutants using a cell-free protein degradation assay. S35 labeled in vitro-translated CDH1 and its mutants were subjected to cell extracts prepared from UV-treated (UV+) or untreated (UV–) HeLa S3 cells. The density of CDH1 in UV (+) panel was quantified and normalized with time point 0, which was set at 100%. The CDH1 fragment with density <40% was defined as ‘degraded’.

 
Knockdown of CDH1 enhances cellular susceptibility to UV-induced apoptosis
Cells usually can survive via DNA repair and other protective mechanisms after exposure to UV radiation at low dosage, whereas severe damages caused by UV radiation at high dosage would induce cell death through the activation of apoptosis (28,29). To test the effect of UV radiation at high dosage on cell cycle and apoptosis, we have performed analyses using fluorescence-activated cell sorting (FACS) and examining cellular PARP cleavage. Consistent with Figure 2A, no obvious cell cycle arrest was observed after exposure of cells to high dosage of UV radiation, whereas transient accumulation of G2/M population was detected only 6 h after exposure to UV. Apoptotic population gradually accumulated after 12 h exposure to UV radiation (apoptotic cells increased from 1.89% for untreated cells to 19.01 and 32.09% after 12 and 24 h, respectively, in response to UV), whereas G2/M peak dropped after onset of apoptosis (Figure 5A). Furthermore, immunoblotting analysis indicate that PARP cleavage was detected after 9 h following exposure of cells to UV irradiation and such cleavage was maintained during the 18 h of observation after UV radiation, which is consistent with the FACS analysis (Figure 5B).


Figure 5
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  		Fig. 5. Depletion of CDH1 enhances the susceptibility of cells to UV-induced apoptosis. (A and B) UV-induced apoptosis in a time-dependent manner. HeLa S3 cells were irradiated with 60 J/m2 UV and harvested at indicated times. Apoptotic cells were identified as part of the sub-G1 peak by FACS (A) and characterized by PARP cleavage using western blotting (B). (C) Establishment of Cdh1 siRNA stable cells. (D–F) Depletion of Cdh1 increased cellular susceptibility to UV-induced apoptosis. Cdh1 siRNA cells (pSuper-Cdh1) and control cells (pSuper-control) were irradiated by UV at 10 or 60 J/m2 and sub-G1 was detected by FACS (D). Cdh1 siRNA cells were irradiated with 60 J/m2 UV and harvested at indicated times. Apoptosis was examined by characterizing cellular PARP cleavage (E). Both Cdh1 siRNA and control cells were treated with UV radiation at 10 J/m2, and percentage of apoptotic cells were determined by annexin-V/propidium iodide (PI) staining. The arbitrary unit of apoptotic cells (Annexin V+/– populations) was normalized with untreated group, which was set at 1 (F). Results are mean ± SD of three independent experiments.

 
To analyze the biological consequence of CDH1 degradation induced by UV and examine whether CDH1 degradation is involved in UV-induced apoptosis, we decided to engineer a stable Cdh1-depleted cell line by RNA interference and then evaluate the susceptibility of Cdh1-depleted cells to apoptosis in response to UV radiation. To date, we have engineered a Cdh1 small interference RNA (siRNA) clone based on a pSuper system described previously (27) (Figure 5C). FACS analysis showed that there is no obvious cell cycle effect observed from Cdh1 siRNA cells (data not shown), which is consistent with the previous study (13). However, Cdh1 siRNA cells are more sensitive to UV radiation, where exposure to UV at lower dosage (10 J/m2) is enough to induce cells to undergo apoptosis whereas UV-induced apoptosis in control cells only occurred after exposure to UV at high dosage (60 J/m2) (Figure 5D). This notion was further confirmed by an analysis of PARP cleavage, where low dosage of UV (10 J/m2) was sufficient to induce cleavage of PARP after 1-h exposure to UV whereas cleavage of PARP in the control cells was only observed after 9-h exposure to high dosage of UV (60 J/m2) (Figure 5E). In consistent with the results of PAPP cleavage, data from annexin V analysis showed that knockdown of Cdh1 significantly increases the population of apoptotic cells as indicated in Figure 5F. Taken together, these results suggest that the loss of Cdh1 enhances cellular susceptibility to UV-induced apoptosis. Therefore, CDH1 is critical to determine susceptibility of cell for activation of apoptosis induced by UV radiation.

Cyclin B1 is a mediator downstream of CDH1 facilitating UV-induced apoptosis
To examine the mechanism through which CDH1 degradation enhances cellular susceptibility to UV-induced apoptosis, we have observed the protein levels of several known CDH1/APC targets and cell cycle-related proteins after exposure to UV radiation, including securin, cyclin B1, SnoN, CDC2, p27, p21, polo-like kinase 1, cyclin H and CDC25c. As shown in Figure 6A, the protein levels of cyclin B1 dramatically increased after exposure to UV radiation, whereas no obvious change was observed for other tested proteins. Given the turnover of cyclin B1 is controlled by CDH1/APC during the cell cycle, the inverse correlation between the kinetics of UV-induced cyclin B1 accumulation with UV-induced CDH1 degradation suggests that cyclin B1 could be the mediator downstream of CDH1 that increase susceptibility to UV-induced apoptosis.


Figure 6
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  		Fig. 6. UV-induced CDH1 degradation resulted in accumulation of cyclin B1 and enhanced UV-induced apoptosis. (A) Cyclin B1 protein levels significantly increased in response to UV radiation. HeLa S3 cells were irradiated with 60 J/m2 UV and harvested at indicated times. A variety of proteins, including known APC/CDH1 substrates and cell cycle-related proteins were examined by western blotting. (B) Immunofluorescence staining verified that UV-induced decrease in the protein levels of CDH1, whereas cyclin B1 expression increased. Cdh1-depleted and control cells were treated with UV at 10 or 60 J/m2, respectively. Cells were then fixed 24 h later and immunostained. Red: cyclin B1; green: CDH1; blue: 4',6-diamidino-2-phenylindole (nucleus). Arrow indicates apoptotic cells. (C–E) Non-degradable CDH1 mutant, CDH1 178–493, interfered with UV-induced endogenous CDH1 degradation (C) resulting in the abrogation of cyclin B1 accumulation and delay in UV-induced apoptosis (D). HeLa S3 cells were transfected with non-degradable HA-tagged CDH1 1–89 and 178–493 and irradiated with 60 J/m2 UV. The endogenous CDH1 was detected with anti-CDH1 antibody, whereas the exogenous CDH1 was detected with anti-HA antibody. The effect on cyclin B1 protein levels (D) was quantified and normalized with untreated group, which was set at 1 (E). Results are mean ± SD of three independent experiments.

 
Cyclin B1 protein levels have been demonstrated to be critical to maintaining normal cellular function as well as promoting oncogenesis when it is aberrantly upregulated (30). Previous studies have shown that cyclin B1 protein levels rapidly increase after the cells were exposed to gamma irradiation. The gamma-induced elevation of cyclin B1 could trigger apoptosis for the exposed cells, especially hematopoietic cells (31). Consistent with this notion, our results suggest that elevation of cyclin B1 might contribute to enhanced cellular sensitivity to UV-induced apoptosis. To test whether increased cyclin B1 protein levels could lead to apoptosis, we evaluated the effect of cyclin B1 levels on apoptosis by transfection of pCMX-cyclin B1 in HeLa cells. As shown in supplemental Figure S2 (available at Carcinogenesis Online), a little effect of elevated cyclin B1 on apoptosis is observed, whereas significant increase of cellular susceptibility to UV-induced apoptosis was observed. To further address the hypothesis that CDH1/APC is pivotal to governing cellular sensitivity to genotoxic stress through orchestrating cyclin B1 protein levels, we next analyzed the UV-induced cyclin B1 alteration in CDH1-depleted cells as well as control cells by immunocytochemical analysis. As shown in Figure 6B, cyclin B1 expression levels significantly increased when apoptosis was induced by UV radiation at 60 J/m2 in HeLa cells, which was consistent with western blotting results (Figure 6A). In Cdh1-depleted cells, the basal levels of cyclin B1 is relatively higher than the basal levels of cyclin B1 in control cells (Figure 6B and Figure S3 available at Carcinogenesis Online). Elevation of cyclin B1 significantly enhanced the cellular sensitivity for induction of apoptosis by UV at 10 J/m2 as reflected by the collapse of nucleus, whereas apoptotic nucleus was only observed in the control cells when cells were treated with high dosage of UV (60 J/m2) (Figure 6B). Moreover, knockdown of cyclin B1 based on the Cdh1 siRNA stable cells attenuated cyclin B1 accumulation caused by the UV-induced Cdh1 degradation and partially blocked the UV-induced apoptosis (Figure S3, available at Carcinogenesis Online).

The above results suggest that cyclin B1 is a mediator for cellular susceptibility to UV-induced apoptosis, where UV initiates CDH1 degradation resulting in the accumulation of cyclin B1, which in turn leads to apoptosis. Given that protein abundance of cyclin B1 are controlled by CDH1/APC, we assumed that manipulation of CDH1 function could change the susceptibility of cells to apoptosis mediated by different cyclin B1 protein levels. To test this hypothesis and further understand the consequence of CDH1 degradation in UV-induced apoptosis, we have conducted an interference assay by the overexpression of non-degradable CDH1 (26). Analysis of mutagenesis suggests the CDH1 1–89 and CDH1 178–493 are non-degradable mutants (Figure 4C). To examine the effects of these non-degradable CDH1 mutants on UV-induced CDH1 degradation that increases cellular vulnerability to apoptosis, we transfected CDH1 1–89 and CDH1 178–493 into HeLa cells and then examined their effects on the alteration of endogenous CDH1 protein levels as well as PARP cleavage in response to UV. As shown in Figure 6C, overexpression of CDH1 178–493 significantly interfered with endogenous CDH1 degradation induced by UV radiation, whereas transfection of CDH1 1–89 did not show noticeable effect. Consistent with the result of interfering assay, overexpression of CDH1 178–493 but not CDH1 1–89 significantly blocks UV-induced cyclin B1 accumulation resulting in delayed onset of apoptosis (~3 h) as represented by PARP cleavage (Figure 6D and E). In summary, these results suggest that cyclin B1 is the cellular mediator for apoptosis facilitated by UV-induced CDH1 degradation.

UV-induced CDH1 degradation leads to hyperactivation of CDK1, which in turn enhances apoptosis
Cyclin B1 in association with CDC2 contributes to CDK1 activity. Previous studies have implicated that elevation of CDK1 activity is one of the pathways for the activation of apoptosis although the mechanism is unclear (32–35). To ask if accumulation of cyclin B1 caused by UV-induced CDH1 degradation results in the elevation of CDK1 activity, we first analyzed the effect of UV radiation on the inhibitory tyrosine-15 phosphorylation of Cdc2 (36). As shown in Figure 7A, no noticeable change in Cdc2 phosphorylation was detected in response to high-dose (60 J/m2) UV radiation, whereas increase in CDC2 phosphorylation at tyrosine-15 was observed in response to low-dose (10 J/m2) UV radiation. These results exclude the possibility that high-dose UV irradiation induce inhibition of CDK1 activity.


Figure 7
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  		Fig. 7. UV-induced CDH1 degradation leads to hyperactivation of CDK1, which in turn enhances apoptosis. (A) Low dosage but not high dosage of UV radiation induced inhibitory phosphorylation of CDC2. HeLa S3 cells treated with 10 or 60 J/m2 UV radiation were harvested at indicated times and proteins were examined using western blotting. (B and C) UV radiation at high dosage elevated CDK1 activity. HeLa S3 cells were irradiated with 60 J/m2 UV and harvested at indicated times. CDK1 kinase activity was measured using H1 histone as putative substrate (B). IP: immunoprecipitation. WCL: whole cell lysate. The density of phosphorylated H1 was quantified and plotted (C). Results are mean ± SD of three independent experiments.

 
To further access the effect of UV-induced CDH1 degradation on CDK1 activity, we carried out an H1 kinase assay. As shown in Figure 7B and C, UV radiation at dosage of 60 J/m2 significantly increases the incorporation of phosphate to H1 kinase, suggesting that exposure of cells to high dosage of UV radiation leads to hyperactivation of CDK1. Taken together, these results suggest a working model with CDH1/APC being targeted by UV radiation. Degradation of CDH1 induced by UV leads to the elevation of CDK1 activity via accumulation of cyclin B1. Increased CDK1 activity in turn promotes apoptosis.


   Discussion
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Uncovering the molecular basis by which APC pathway is involved in genomic integrity has attracted substantial interest recently (14,37). Systematic analysis of the protein profile of several components of the APC pathway in response to DNA damage signals reveals that CDH1, a key activator of APC, is tightly regulated by genomic stress. Extensive UV radiation causes irreversible damage to the genome, which renders the damaged cells prone to apoptosis and thereby preventing cellular transformation (38). Finding that CDH1 is dramatically downregulated after severe UV radiation indicates that CDH1, probably like other functional genomic guardians (e.g. BRCA1, p53), is required for genomic integrity whose otherwise downregulation will significantly increase its cellular susceptibility to apoptosis in response to DNA damage agents. Identification of cyclin B1 as a mediator of CDH1 after exposure to UV radiation reveals partially the mechanism by which CDH1/APC is involved in genomic integrity. CDH1/APC seems to govern CDK1 activity facilitating UV-induced apoptosis. Consistent with previous studies (17,39), this work has confirmed the hypothesis that CDH1/APC may act as a guardian of genomic integrity, which together with other guardian proteins maintains cellular genomic integrity.

CDH1, a critical regulator, plays a role in genomic integrity
Several lines of evidence support the notion that APC is an important regulator in response to DNA damage, notably when the activity of APC in response to DNA damage signal is altered leading to cell cycle arrest necessary for DNA repair or mitotic catastrophe necessary for apoptosis (14,40). The importance of the APC pathway as a response to genotoxic stress is evident in how several different steps/components of the APC pathway, including CDH1, CDC20, APC components and securin, are modulated after DNA damage. Previous gene-targeting experiment has shown that disruption of CDH1 circumvents G2/M arrest that would normally be induced following DNA damage (13). This observation suggests that CDH1 is required for the maintenance of genomic integrity although the substrate that is believed to be targeted for degradation by CDH1/APC to achieve the arrest remains unknown. Studies of mammalian cells and yeast have further revealed that both cellular ionization and UV radiation can alter the property of securin either through the regulation of its protein abundance or modification of its phosphorylation status, causing delay in mitotic exit or mitotic arrest (12,15,17). Recently, the importance of CDH1/APC in genomic integrity is further supported by the evidence that CDH1/APC directly regulates DNA repair process by governing the turnover of ribonucleotide reductase R2 and thymidine kinase 1; both are important enzymes involved in nucleotide excision repair response or DNA replication (16). Observation that CDH1 is degraded in response to genotoxic stress provides another line of novel evidence to the current paradigm that ubiquitin-dependent degradation of CDH1 is one of the mechanisms to ensure genomic integrity.

Consequence of CDH1 degradation in response to genotoxic stress
Severe DNA damage caused by extensive UV radiation leads to irreversible DNA lesion, accumulation of which can result in tumorigenesis. Under such circumstance, activation of the apoptotic program is critical to eliminate damaged cells, which in turn would prevent cellular transformation and tumorigenesis. Previous studies have shown that dysfunction of CDH1 disrupts APC-mediated DNA damage checkpoint function resulting in mis-segregation of duplicated genomes leading to mitotic catastrophe. Huang et al. (17) observed that ionization at the dose of 10 Gy significantly induces phosphorylation of CDH1 thereby attenuating the activity of CDH1/APC, which in turn blocks cyclin B1 degradation. Our experiment has demonstrated that UV radiation at high dosage (>20 J/m2) triggers rapid degradation of CDH1, similar to phosphorylation of CDH1, resulting in decreased APC activity. Observations based on different genotoxic stress strongly support the conclusion that CDH1 is an important target for DNA-damaging agents. Cellular exposure to UV radiation triggers proteolysis of CDH1, whereas cellular ionization causes phosphorylation of CDH1. Interestingly, both phosphorylation of CDH1 and degradation of CDH1 induced by different DNA-damaging agents result in similar effect: the abrogation of cyclin B1 degradation. Cyclin B1 is the regulatory subunit of the CDC2 serine/threonine kinase. Attenuation of cyclin B1 turnover leads to its aberrant accumulation resulting in the hyperactivation of CDK1. As suggested in several studies, the elevation of CDK1 activity may facilitate mitotic catastrophe or the activation of apoptosis (41,42).

UV-induced apoptosis is a highly complex process in which various signaling pathways are involved. Depending on the source of UV radiation and the type of cells, UV-facilitated apoptosis has been suggested to be mediated by various pathways, including DNA damage pathway, activation of cell-surface death receptors and the formation of reactive oxygen species (43). Activation of caspase cascade is the convergent node for apoptotic signaling mediated by diverse upstream pathways. Studies in the past decade have revealed that tumor necrosis factor receptor, CD95/Fas receptor, generation of reactive oxygen species, p53, mitogen-activated protein kinase kinase pathway, nuclear factor kappa B pathway and protein kinase B pathway could facilitate the effect of UV radiation leading to the activation of the caspase cascade (44). In complement with the above theories, many studies have shown that hyperactivation of CDK1 is an important mechanism for the onset of apoptosis (41,42,45). Previous study has demonstrated that aberrant activation of p34cdc2 (CDK1) during cell cycle can lead to apoptosis (35). Increased activation of CDK1 has also been reported in various apoptotic-inducing treatments including UV irradiation (46) and various addition of anticancer agents such as taxol (34) or camptothecin (47). However, the mechanism by which critical component of apoptotic pathway is regulated by the hyperactivated CDK1 is not clear (42). Several pathways have been proposed for involvement of CDK1 in apoptosis (41). CDK1 can induce cell death by triggering premature entry into mitosis and consequent mitotic catastrophe, followed by apoptosis (48). In addition, CDK1 can phosphorylate and inactivate the anti-apoptotic protein Bcl-2 (49). Alternatively, CDK1 can phosphorylate the proapoptotic protein BAD at Ser128 leading to the initiation of apoptosis (33). However, another study has shown that Ser128 phosphorylation has no effect on the proapoptotic activity of BAD in UV-induced apoptosis via c-Jun N-terminal kinase activation (50). Together with these findings, our results suggest that UV-induced CDK1 could phosphorylate unknown substrates that then promote the apoptosis process.

The mechanism by which CDH1 is degraded in response to DNA damage signal
CDH1 in association with APC has been shown to catalyze the degradation of a variety of substrates as part of cell cycle regulation and beyond. Data from this work suggest the presence of an unknown ubiquitin ligase that is activated in response to genotoxic stress and targets CDH1 for destruction. This hypothesis is supported by the previous observation that CDH1 is an unstable protein whose abundance oscillates substantially during cell cycle (20,21), which is probably caused by SKP, Cullin, F-box containing complex ligase-governed CDH1 degradation (20) or the autoubiquitination of CDH1 by the CDH1/APC E3 ligase as part of a negative feedback loop (21). To analyze the UV-induced degradation of CDH1 and collect information for predicting the E3 ligase that governs CDH1 ubiquitylation, we have performed a molecular mapping analysis of the functional domain on CDH1 that mediates its degradation. Result from the experiment indicates that the region between 124 and 178 amino acids is necessary for UV-induced CDH1 degradation. Amino acid analysis of the region between residues 124 and 178 has revealed the presence of multiple lysines that could serve as docking site for ubiquitin attachment. Although we have not evaluated the function of the individual lysine in mediating CDH1 ubiquitylation, finding possible ubiquitin docking sites in this region could partially explain why the absence of this stretch of amino acids leads to stabilization of CDH1 in the presence of UV radiation. In addition, a conserved sequence enriched with praline, glutamic acid, serine and threonine domain is located in this stretch of amino acids suggesting that a SKP, Cullin, F-box containing complex-related enzyme might be the candidate E3 ligase governing CDH1 degradation. At this point, putative D-box, A-box, C-box and IR tail did not seem to govern CDH1 stability in the presence of UV radiation. Interestingly, we found that C-terminal (178–493 aa) but not N-terminal CDH1 (1–89 aa) fragment could interfere endogenous CDH1 degradation, suggesting that the N-terminal CDH1 might not interact with the putative E3 ligase activated by UV irradiation. To identify the physiological relevant E3 ligase, we have engineered a TAP–CDH1 stable cell lines using retroviral technology. Purification of CDH1 complex in response to UV radiation will potentially result in the identification of E3 ligase catalyzing CDH1 ubiquitylation. Identification of E3 ligase regulating CDH1 proteolysis will significantly enhance our understanding of the regulation of CDH1, especially in response to genotoxic stress. Results from this work could provide novel therapeutic targets for anticancer treatment.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary material can be found at http://carcin.oxfordjournals.org/.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
National Institutes of Health (CA115943 [GenBank] and GM070681).


   Acknowledgments
 
We thank Wan laboratory members for critical discussion and reading of the manuscript. We appreciate Richard D. Wood, Daniel Finley, J. Wade Harper and Alan D'Andrea for discussions.Y.W. is a scholar of American Cancer Society and V Cancer Research Foundation.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received August 16, 2007; revised October 9, 2007; accepted November 3, 2007.


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