ONCOLOGY REPORTS 32: 2047-2053, 2014

CUL4B promotes proliferation and inhibits of human osteosarcoma cells

Zhi Chen1*, Bao-liang Shen2*, Qing-ge Fu3*, Fei Wang1, Yi‑xing Tang1, Cang-long Hou1 and Li Chen1

1Department of Orthopedics, Changhai Hospital Affiliated to the Second Military Medical University, Shanghai 200433; 2Department of Orthopedics, Shanghai Jiading Central Hospital, Shanghai 201800; 3Department of Emergency and Trauma, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, P.R. China

Received March 4, 2014; Accepted June 10, 2014

DOI: 10.3892/or.2014.3465

Abstract. 4B (CUL4B) is a component of the Introduction Cullin4B-Ring E3 ligase complex (CRL4B) that functions in proteolysis and is implicated in tumorigenesis. Here, we report Osteosarcoma is a primary bone malignancy with high rates that CUL4B is associated with tumorigenesis by promoting of metastasis, mortality and disability, and usually occurs in proliferation and inhibiting apoptosis of human osteosarcoma children and adolescents (1). The most common treatments cells. We performed RNA interference (RNAi) with a lentiviral for osteosarcoma are surgery, chemotherapy and biotherapy. vector system to silence the CUL4B using osteosarcoma However, the prognosis of osteosarcoma is still poor because SAOS-2 cells. The negative control included the normal of the high degree of malignancy, rapid disease progression target cells infected with the negative control virus whereas and early metastasis (2). the knockdown cells included the normal target cells trans- Gene amplification can be defined as increased copy fected with the RNAi target virus. We assessed the inhibition numbers of certain regions of the genome. Gene amplifica- resulting from the decreased expression of the CUL4B gene on tion often results in the upregulation of by the proliferation rate of SAOS-2 cells, and also evaluated the increasing the number of templates available for transcription. distribution, apoptosis and clonability. Compared Gene amplification is commonly found in tumor cells and is with the negative control, the CUL4B gene expression was considered an important mechanism whereby tumor cells gain significantly inhibited in the SAOS-2 cells at the mRNA and increased levels of expression of critical . Thus, identi- levels in the knockdown group (P<0.01). Furthermore, fication and characterization of these genes should provide in the knockdown group, the cell proliferation rate and important insights into the pathobiology of cancer. clonability were also significantly inhibited (P<0.01). The Culllin 4B (CUL4B) is a component of the Cullin4B-Ring apoptosis rate increased significantly (P<0.05). A significant E3 ligase complex (CRL4B) that functions in proteolysis. It decrease in the number of cells in the G1 phase (P<0.01) and has been implicated in tumorigenesis since the core compo- significant increases in the S (P<0.01) and G2 phases (P<0.05) nents participate in a broad variety of physiologically and were observed. The silencing of CUL4B gene expression can developmentally controlled processes such as cell progres- effectively inhibit osteosarcoma cell proliferation and induce sion, replication and DNA damage response (3). In mammals, apoptosis. These findings may provide a novel biomarker for there are two Cullin 4 members, CUL4A and CUL4B, which the treatment of osteosarcoma. share 82% sequence identity. Although Cul4a-null mice do not exhibit evident developmental defects (4-6), CUL4A has been shown to target p53 and several cyclin-dependent kinase inhibitors, such as and p27 for proteolysis in cultured cells (7-9) and was found to be highly expressed in breast and hepatocellular carcinomas (10,11). Loss-of-function mutation in the X-linked CUL4B, in contrast, causes mental retarda- Correspondence to: Dr Li Chen, Department of Hematology, tion, short state, absence of speech and other phenotypes in Changhai Hospital Affiliated to the Second Military Medical University, 168 Changhai Road, Shanghai 200433, P.R. China humans (12,13), and Cul4B knockout mice are embryonically E-mail: [email protected] lethal, indicating a unique function of CUL4B that cannot be compensated by CUL4A. Recently, Yang et al (14) showed that *Contributed equally depletion of CUL4B resulted in loss of not only H2AK119 mono-ubiquitination but also H3K9 tri-methylation and DNA Key words: CUL4B, osteosarcoma cells, proliferation, apoptosis methylation, leading to depression of a collection of genes, including the tumor‑suppressor IGFBP3. Further experiments revealed that CUL4B promoted cell proliferation and invasion, 2048 CHEN et al: Function of CUL4B in Osteosarcoma which are consistent with a tumorigenic phenotype, at least linearize the pGCSIL-GFP vector. We modified the double- partially by repressing IGFBP3. Further findings revealed stranded DNA after annealing and linked it with the that the expression of CUL4B was markedly upregulated in pGCSIL-GFP vector following the double digestion. We used samples of human cervical carcinoma and was negatively calcium chloride to prepare competent cells of Escherichia correlated with the expression of IGFBP3. coli afresh and cultured it at 37˚C for 16 h. We used computer- Although experiments have shown that CULB possesses an aided high-throughput cloning of bacteria in liquid medium intrinsic transcription repressive activity, (3,15) the exact role for sequencing (Shanghai Genechem Co., Ltd.). of CULB in the process and progression of osteosarcoma is still poorly understood. Here, we established an osteosarcoma Preparation and grouping of cells. The human osteosarcoma cell model in vitro, and transfected osteosarcoma SAOS-2 cell line SAOS-2 was cultured in the RPMI-1640 culture cells using CUL4B RNAi and assessed the apoptosis of solution with fetal calf serum (volume fraction 10%) and

SAOS-2 cells. We demonstrated that CUL4B promotes cell incubated with 5% CO2 at 37˚C. The cells that remained in the proliferation and inhibits the apoptosis of osteosarcoma cells. logarithmic phase were divided into two groups: i) negative These results add to the understanding of the role of CUL4B control, in which the normal target cells were infected withthe in the proliferation and apoptosis of osteosarcoma cells and its negative control virus and ii) knockdown, in which the normal molecular mechanisms. target cells were infected with the RNAi target virus.

Materials and methods Real-time PCR and western blotting to test knockdown effi- ciency. The human osteosarcoma SAOS-2 cells grew well on Cell line and culture conditions. Human osteosarcoma cell the day prior to viral introduction was recovered. The cell line SAOS-2 was purchased from the Shanghai Institute of the suspension was incubated with 5% CO2 at 37˚C. When the Chinese Academy of Sciences (Shanghai, China) and grown degree of cell fusion reached 30%, adequate viral load was in Dulbecco's modified Eagle's medium (DMEM) with 10% introduced in the different groups, to a multiplicity of infec- (v/v) fetal calf serum, streptomycin (100 U/ml) and penicillin tion (MOI) of 100. Following incubation for three days, the (100 U/ml). DMEM, fetal bovine serum (FBS) and Dimethyl expression level of GFP was observed under a fluorescence sulfoxide (DMSO) were purchased from Gibco Biotechnology microscope. The culture was continued if the efficiency of (Gibco-BRL, MD, USA). Cultures were maintained at 37˚C in infection exceeded 50%. After incubation for five days, the an incubator with a humidified atmosphere of 5% CO2. cells were collected; the mRNA expression of the gene of interest was analyzed using real-time PCR for RNA interfer- Reverse transcription-polymerase chain reaction (RT-PCR) ence. The upstream primer sequence for the CUL4B gene was analysis of CUL4B gene expression in tumor cells. Total‑RNA 5'-GGGAAAGGAATGGTGAA-3' while the downstream was extracted under RNase-free conditions (carried out primer sequence was 5'-TGCATAGAGCCGGTTAG-3'. The according to the operation manual for TRIzol; Invitrogen cell culture solution was aspirated and the cells were washed Corp., Carlsbad, CA, USA) with glyceraldehyde-3-phosphate twice in phosphate-buffered saline (PBS). An adequate amount dehydrogenase (GAPDH) as an internal reference. Primer of pre-cooled 2X lysis buffer was added. After deplating, the design was as follows: the CUL4B upstream sequence was cells were transferred to the tube and then lysed on ice for 5'-GGGAAAGGAATGGTGAA-3'; and the downstream 10-15 min. The protein concentration was determined and sequence was 5'-TGCATAGAGCCGGTTAG-3'. The GAPDH adjusted to 2 µg/µl. Next, 2X loading buffer was added to each upstream sequence was 5'-TGACTTCAACAGCGACAC sample and subsequently boiled for 5-10 min. Forty micro- CCA-3'; and its downstream sequence was 5'-CACCCTGTT grams of total protein was loaded into each well containing GCTGTAGCCAAA-3'. The Access RT-PCR kit was used to 10% SDS-polyacrylamide gel, subjected to electrophoresis at perform single-step reverse transcription and PCR amplifica- 30 mA for 2 min, and then transferred to a polyvinylidene tion. An aliquot of 5 µl of the amplified products was subjected fluoride (PVDF) membrane at 400 mA for 2 h. The membrane to electrophoresis on 2% agarose gel. The gels were examined was blocked with 5% milk in Tris-buffered saline (TBS) for under a UV lamp. 1 h at room temperature. The primary antibody was added and incubated with the membrane for 2 h at room temperature, and Construct design: lentiviral-mediated small interfering RNA then washed three times with TBS/Tween-20. A secondary delivery system. We targeted the gene of interest by designing antibody was added to the membrane and incubated at room small interfering RNAs (siRNAs) using the design software temperature with gentle agitation. Two hours later, the developed by Ambion Corp. (Naugatuck, CT, USA) to select membrane was washed three times with TBS/Tween-20 for the best parameters for the RNA interference target. We deter- 10 min per wash. The bands were visualized using an enhanced mined the effective target sequence: PSCSI2891, chemilluminescence (ECL) kit followed by exposure to X-ray AGCAGTGGAAGCTATTCAGAA (CUL4B mRNA). We film. designed the DNA oligonucleotides of siRNAs (Shanghai Genechem Co., Ltd.): PSCSI2891-1, 5'-CcggAGCAGTGGA Cellomics to test inhibition of osteosarcoma cell proliferation AGCTATTCAGAATTCAAGAGATTCTGAATAGCTTCCA following downregulation of the CUL4B gene. After tryp- CTGCTTTTTTg-3' and PSCSI2891-2, 5'-aattcaaaaaAGCAG sinization, the cell suspension was resuspended in complete TGGAAGCTATTCAGAATCTCTTGAATTCTGAATAGCT medium and density was adjusted to 2x104/ml. We used the TCCACTGCT-3'. After annealing, the double-stranded DNA blood counting chamber to count the cells, laying them at was digested with Agel and EcoRI (New England Biolabs) to 2,000 cells/well. Each group comprised three to five compound ONCOLOGY REPORTS 32: 2047-2053, 2014 2049 perforations. Each perforation was filled with 100 µl, and the same quantity of cells. The cells were incubated with 5%

CO2 and cultured at 37˚C. Starting the next day, the plates were tested and read once a day with Cellomics for five days. After adjusting the input parameters of Cellomics ArrayScan (Thermo Fisher Scientific Inc., Waltham, MA, USA), the quan- tity of cells with green fluorescence was accurately calculated while scanning the perforations in the plates. The data were collected and analyzed to create a proliferation curve for the five days. Figure 1. CUL4B gene mRNA expression in SAOS-2 cells.

Fluorescence-activated cell sorting (FACS) to assess osteo- sarcoma cell cycle distribution following CUL4B gene in complete medium. Using a blood counting chamber, we silencing. The cell culture supernatant was aspirated when inoculated the 96-well culture plate at the rate of 500 cells per the coverage rate for the 6-cm dish cells in the experimental perforation in each experimental group. We set three compound group increased to 80%, ensuring that the cells did not enter perforations in each experimental group, transferred the cells the plateau phase. The cells were washed once with D-Hank's that were already inoculated into the incubator and they were solution and subjected to trypsinization. The complete medium cultured for three days or when the number of cells in most was removed and the cells were collected in a 5-ml centrifuge single clones exceeded five. During the process, the solution tube. We set three compound perforations in each group and was changed and cells were monitored every three days. performed timed cycle tests ensuring an adequate number of Using Cellomics ArrayScan, we scanned and photographed cells for computerized analysis, with at least 1,000,000 each the perforations, analyzed the number and size of the clones time. We used PBS (pH, 7.2-7.4) that was pre-cooled at 4˚C to within the perforations and the number of cells in each clone. wash and precipitate cells once. The cells were collected after centrifugation at 1,500 rpm for 5 min. The cells were fixed Statistical analysis. All data are expressed as mean ± standard with 70% ethanol, which was pre-cooled to 4˚C, for at least deviation (SD). We used the statistical software SPSS 12.0 to 1 h. The stationary liquid was abandoned by centrifugation perform the relevant analysis. The significance level of statis- at 1,500 rpm for 5 min. We used PBS to wash and precipitate tics was set at P<0.05. cells once. Adequate quantity of cell staining fluid (1-1.5 ml) was added for suspension, based on the volume of cells, Results ensuring that the pass rate of cells reached 200-350 cell/sec for computer analysis. We used a 300-mesh screen cloth to filter Expression of the CUL4B gene in SAOS-2 cells, preparation within the tube while streaming onto the computer. of RNA-interfering lentivirus vector and assessment of knock- down efficiency. The results of the semi-quantitative PCR, Fluorescence-activated cell sorting (FACS) analysis of apop- which set GAPDH as an internal reference, showed that the tosis following downregulation of the CUL4B gene. We used CUL4B gene was abundantly expressed in the osteosarcoma D-Hank's solution to wash cells once after collecting the culture SAOS-2 cells (Fig. 1). The length of the PCR fragment in the supernatant from each experimental group following transfer positive clone that annealed with the fragment of vshRNA into the 5-ml centrifuge tube. We used pancreatic enzymes to was 343 bp. After transfection with the siRNA lentivirus for digest the cells. The culture supernatants were then abandoned, three days, GFP expression was observed under fluorescence and cells were collected into one 5-ml centrifuge tube, with microscopy (Fig. 2). After five days, it was found that the three compound perforations in each group. The supernatants mRNA expression level of the CUL4B gene in the SAOS-2 were aspirated after centrifugation at 1,500 rpm for 5 min. We cells of the knockdown group was inhibited, which was signifi- used PBS to wash and precipitate the cells once and then cells cantly different compared with the negative control group. The were collected after centrifugation at 1,500 rpm for 5 min. A western blotting results confirmed inhibition of the protein 1X binding buffer was then used to wash and precipitate cells level by the silencing of the CUL4B gene in SAOS-2 cells. once, and centrifuged at 1,500 rpm for 5 min. The collected The RNA-interfering lentivirus of the CUL4B gene construct cells were resuspended using 1 ml 1X staining buffer; the effectively inhibited the expression of the CUL4B gene and volume of staining buffer solution was determined according several targets following gene silencing (Fig. 3). to the precipitation capacity of the cells, adjusting the final density of the cell suspension to 1x106-1x107 cells/ml. We took Cellomic analysis of inhibition of the proliferation of osteo- a 100-µl cell suspension (1x105-1x106 cells) and added 5 µl sarcoma cells following downregulation of the CUL4B gene. Annexin V-APC for dyeing. The mixture was placed in a dark After transfection with the siRNA lentivirus, the proliferation place at room temperature for 10-15 min, and then transferred rate of SAOS-2 cells in the knockdown group was inhibited to the tube for streaming onto the computer for further analysis. together with the negative control from the third day (P<0.01). The results suggested that downregulation of the CUL4B gene Inhibition of clonability of the osteosarcoma cells following inhibited the proliferation of SAOS-2 (Fig. 4). downregulation of the CUL4B gene. We digested cells that remained in the logarithmic phase in each experimental FACS analysis of cell cycle distribution following CUL4B gene group, with pancreatic enzymes. The cells were resuspended silencing. Following transfection with the siRNA lentivirus, 2050 CHEN et al: Function of CUL4B in Osteosarcoma

Figure 2. GFP expression in SAOS-2 cells as detected by fluorescence microscope. (A) Negative-control; magnification x100. (B) Negative-control; magnifica- tion x200. (C) CUL4B-siRNA; magnification x100. (D) CUL4B-siRNA; magnification x200.

Figure 3. CUL4B gene expression in SAOS-2 cells following knockdown of the CUL4B gene. (A) In the knockdown group, the mRNA expression of the CUL4B gene in the SAOS-2 cells was decreased (**P<0.01, CUL4B-siRNA vs. negative-control). (B) In the knockdown group, the protein expression of CUL4B in the SAOS-2 cells was decreased.

the percentage of SW1116 cells in the G1 phase was signifi- cantly different from the negative control group (P<0.01). The cantly decreased (P<0.01), while cells in the S (P<0.01) and results suggest that downregulation of the CUL4B gene largely G2 phases increased significantly (P<0.05) in the knockdown inhibited the clonability of the SAOS-2 cells (Fig. 8). group compared with the negative control group. This indi- cates that downregulation of the CUL4B gene was apparently Discussion related to regular distribution of the SW1116 cells (Fig. 5). In the present study, using RNA interference with a lentiviral FACS analysis of apoptosis following CUL4B gene silencing. vector containing the CUL4B gene, we knocked down the Transfection by siRNA lentivirus increased the rate of apop- expression of the CUL4B gene in osteosarcoma SAOS-2 cells. tosis significantly (P<0.05) in the knockdown group compared Our results demonstrated that the knockdown of CUL4B inhib- with the negative control group, suggesting that the CUL4B ited osteosarcoma SAOS-2 cell proliferation and clonability gene silencing stimulated the apoptosis of SAOS-2 cells and significantly induced apoptosis. Our findings suggest that (Figs. 6 and 7). silencing of CUL4B gene may be used in the treatment of human osteosarcoma. Analysis of inhibition of clonability following CUL4B gene Cancer cells commonly develop mechanisms by which silencing. We noticed a decrease in the number of SW1116 they resist cell death either through disruption of apoptotic cell colonies in the knockdown group following interference processes or activation of survival signals. Within a growing by siRNA lentivirus. Furthermore, the number of cells in the tumor mass, the sequential acquisition of a number of genetic colonies that had already formed decreased, which was signifi- and epigenetic alterations during tumor progression also enable ONCOLOGY REPORTS 32: 2047-2053, 2014 2051

Figure 4. Downregulation of the CUL4B gene inhibits proliferation of SAOS-2 cells. (A) Proliferation of SAOS-2 cells in the knockdown group was inhibited when compared with the negative-control group (**P<0.01, CUL4B-siRNA vs. negative-control). (B) Proliferation rate of SAOS-2 cells in the knockdown group was inhibited when compared with the negative-control group (**P<0.01, CUL4B-siRNA vs. negative-control).

Figure 5. Changes in the cell cycle distribution of SAOS-2 cells in the knockdown group (**P<0.01, CUL4B-siRNA vs. negative-control; *P<0.05, CUL4B- siRNA vs. negative-control).

cancer cells to gain the ability to escape apoptosis, induce Although the mechanism of CUL4B in cancer develop- angiogenesis and metastasize to distant organs. Notably, ment and progression remains unclear, studies have shown that CUL4B carries a nuclear localization signal in its N terminus CUL4B may function together with other , such as and is also localized in the nucleus (15,16), suggesting that cyclin D1 and p53 (7,18). The cyclin D1 level was low in quies- CUL4B might be involved in nuclear-based functions. cent cells, and it increased as cells progressed into the G1 phase Recently, one study indicated that CUL4B is a transcriptional and served as a control protein (19). P53 also known as cellular corepressor that regulates transcription by recruiting PRC2. tumor antigen p53 or tumor-suppressor p53 is a protein that in It was demonstrated that CUL4B functions as a transcription humans is encoded by the TP53 gene. The p53 protein is crucial corepressor and a potential oncogene, supporting the use of in multicellular organisms, where it regulates the cell cycle and, CUL4B as a target for cancer therapy (17). thus, functions as a tumor suppressor, preventing cancer (20). 2052 CHEN et al: Function of CUL4B in Osteosarcoma

Figure 6. Downregulation of the CUL4B gene stimulates apoptosis of SAOS-2 cells (Peak diagram). (*P<0.05, CUL4B-siRNA vs. negative-control).

Figure 7. Downregulation of the CUL4B gene promotes apoptosis of SAOS-2 cells (Scatter diagram). (*P<0.05, CUL4B-siRNA vs. negative-control).

Figure 8. Compared with the negative-control group, the clonability of SAOS-2 cells in the knockdown group was significantly inhibited (*P<0.01, CUL4B‑siRNA vs. negative-control).

CUL4-mediated ubiquitination, which is involved in other X- inactivation (21-23), may play an important types of histone modifications and epigenetic mechanisms, role in cancer development. CUL4B expression was found such as heterochromatin formation, parental imprinting or to be significantly higher in tumor samples compared to ONCOLOGY REPORTS 32: 2047-2053, 2014 2053 adjacent normal tissue, and the level of CUL4B expression 6. Yin Y, Lin C, Kim ST, et al: The E3 Cullin 4A regulates meiotic progression in mouse spermatogenesis. Dev was negatively correlated with the level of IGFBP3 expres- Biol 356: 51-62, 2011. sion. Moreover, IGFBP3 is induced by wild-type p53 (24) 7. Banks D, Wu M, Higa LA, et al: L2DTL/CDT2 and PCNA and enhances the p53-dependent apoptotic response of tumor interact with p53 and regulate p53 polyubiquitination and protein stability through MDM2 and CUL4A/DDB1 complexes. Cell cells to DNA damage (25). Aberrant promoter methylation of Cycle 5: 1719-1729, 2006. IGFBP3 at the p53 regulatory element causes gene silencing 8. Higa LA, Yang X, Zheng J, et al: Involvement of CUL4 ubiquitin resistant to p53 (26). Interestingly, it has been reported that E3 ligases in regulating CDK inhibitors Dacapo/p27Kip1 and cyclin E degradation. Cell Cycle 5: 71-77, 2006. CRL4A can degrade p53 to promote cell cycle progression and 9. Nishitani H, Shiomi Y, Iida H, Michishita M, Takami T and immortalization (7,27). Thus, it is reasonable to speculate that Tsurimoto T: CDK inhibitor p21 is degraded by a proliferating CRL4 negatively controls the p53-IGFBP3 axis. Collectively, in cell nuclear antigen-coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation. J Biol Chem 283: 29045-29052, addition to the hypothesis that CRL4B promotes tumorigenesis 2008. through degradation of several cyclin-dependent kinase inhibi- 10. Schindl M, Gnant M, Schoppmann SF, Horvat R and Birner P: tors (8,9) and coordinates with PRC2 in H3K27me3‑mediated Overexpression of the human homologue for Caenorhabditis elegans cul-4 gene is associated with poor outcome in node- transcriptional silencing (17), recent research has also shown negative breast cancer. Anticancer Res 27: 949-952, 2007. that CUL4B controls DNA methylation‑based transcriptional 11. Singhal S, Amin K, Kruklitis R, et al: Alterations in cell cycle repression adding a new element to the understanding of the genes in early stage lung adenocarcinoma identified by expression profiling. Cancer Biol Ther 2: 291-298, 2003. oncogenic potential of CRL4B. 12. Tarpey PS, Raymond FL, O'Meara S, et al: Mutations in CUL4B, Progression of human cancers is known to involve multiple which encodes a ubiquitin Ε3 ligase subunit, cause an Χ-linked genetic changes that lead to upregulation or downregulation mental retardation syndrome associated with aggressive outbursts, seizures, relative macrocephaly, central obesity, hypogonadism, of expression of critical genes (28). Gene amplification is a pes cavus, and tremor. Am J Hum Genet 80: 345-352, 2007. common mechanism whereby tumor cells increase expres- 13. Zou Y, Liu Q, Chen B, et al: Mutation in CUL4B, which encodes sion of genes that are critical for malignancy. However, not a member of cullin-RING ubiquitin ligase complex, causes X-linked mental retardation. Am J Hum Genet 80: 561-566, 2007. all genes present in multiple copies are necessarily relevant 14. Yang Y, Liu R, Qiu R, et al: CRL4B promotes tumorigenesis to malignancy. Amplicons may contain irrelevant genes that by coordinating with SUV39H1/HP1/DNMT3A in DNA meth- are present by virtue of their physical proximity to target ylation-based epigenetic silencing. Oncogene: Dec 2, 2013 (Epub ahead of print). doi: 1038/onc.2013.522. genes. In this study, we investigated the biological function of 15. Nakagawa T and Xiong Y: X-linked mental retardation gene the CUL4B gene in tumorigenesis and proposed the possible CUL4B targets ubiquitylation of H3K4 methyltransferase mechanism based on previous research. We provide a new component WDR5 and regulates neuronal gene expression. Mol Cell 43: 381-391, 2011. prospective for the role of CUL4B in tumorigenesis which 16. Zou Y, Mi J, Cui J, et al: Characterization of nuclear localization warrants further research. signal in the N terminus of CUL4B and its essential role in cyclin In summary, in our study, using RNA interference with E degradation and cell cycle progression. 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