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Inhibition of Homologue of Slimb (HOS) Function Sensitizes Human Melanoma Cells for Apoptosis¹

Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University Medical Center, Washington, D.C. 20007 [V. A. S., A. D.], and The Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029 [Z. R., S. Y. F.]

	ABSTRACT

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Homologue of Slimb (HOS)/-transducin repeats containing proteins up-regulate nuclear factor B activity by targeting its inhibitor (IB) for ubiquitination and subsequent degradation. We investigated whether inhibition of HOS function may modulate apoptosis in human melanoma cells. Forced expression of the dominant negative HOS^F construct inhibited IB degradation and led to sensitization of melanoma cells to apoptosis induced by tumor necrosis factor with cycloheximide, as well as by cisplatin and ionizing and UV irradiation. These data indicate that HOS plays an important role in controlling the IB-dependent apoptotic pathways in human melanoma.

	Introduction

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Resistance of human malignant melanomas to radiation and commonly used chemotherapeutic agents is considered to be among the major clinical problems and reasons for poor prognosis of the metastatic disease (reviewed in Ref. 1 ). Because most anticancer agents kill susceptible cells via the induction of programmed cell death (apoptosis), the resistance of melanoma cells to apoptosis is a focus of studies aimed at clarifying the mechanisms underlying melanoma chemo- and radioresistance (1) . Recent studies have implicated NF-B³ as a critical regulator of apoptosis. NF-B is required for the regulation of survival genes (2, 3, 4) whose products can block cell death. NF-B is a ubiquitous heterodimeric complex composed of p50 and p65/RelA subunits. This complex is normally sequestered in an inactive form in the cytoplasm through interaction with members of a family of inhibitory proteins, the IBs. These proteins, when associated with NF-B, mask the nuclear localization signal of p65 of the NF-B complex and impair its ability to translocate to the nucleus and bind DNA (reviewed in Ref. 5 ). Sequential phosphorylation, ubiquitination, and proteasome-dependent degradation of IB lead to the activation of transcription factor NF-B in response to a variety of extracellular signals. IB turnover seems to play a critical role in the regulation of NF-B activities. Phosphorylation of IB at Ser³², Ser³⁶ by IB kinase marks IB for ubiquitination and proteasome-dependent degradation, thus allowing NF-B nuclear translocation and target gene expression (reviewed in Ref. 6 ). Some melanoma cells exhibit elevated basal IB kinase activity that results in an increased constitutive IB phosphorylation and degradation and NF-B activation (7) . Inhibition of NF-B activities by nondegradable IB constructs promotes apoptosis in many cancer cells (including melanoma; Refs. 8, 9, 10 ). We have recently identified an F-box/WD40 repeats-containing protein termed HOS that mediates IB degradation by recognizing the phosphorylated IB and recruiting SCF^HOS-Roc1 E3 ligase (11 , 12) . The function of HOS and of a similar relative protein, -TRCP, is required for NF-B activation (reviewed in Ref. 13 ). In this study, we have investigated the role of HOS in the regulation of melanoma cell sensitivity to apoptosis and report for the first time that inhibition of HOS function can sensitize tumor cells to cytokine- or DNA damage-induced apoptosis.

	Materials and Methods

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Plasmids.
To create a HOS^F dominant negative expression vector, we amplified the cDNA encoding amino acids 180–597 of HOS with primers entailed with SalI and NotI recognition sequences. PCR products were digested with SalI and NotI restriction enzymes and subcloned as 5'-SalI-NotI-3' fragments in pEBB vector (a gift of E. Spanopoulou). This vector is based on eukaryotic expression vector pEF-BOS (14) and provides the hemagglutinin tag at the NH₂ terminus of the fusion proteins. The marker plasmid encoding GFP, pEGFP, was purchased from Clontech. The pCI-neo-based construct encoding stable mutant -catenin^S33Y (15) was kindly provided by B. Vogelstein (Johns Hopkins University, Baltimore, MD).

Cells and Transfections.
Human melanoma Lu1205 cells (kindly provided by M. Herlyn, Wistar Institute, Philadelphia, PA) were maintained in MCDB153/L15 medium (4:1) supplemented with 5% fetal bovine serum, L-glutamine, and antibiotics at 37°C with 5% CO₂. Transfections were performed with LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer’s recommendations. The amount of plasmids in transfection mixtures was kept constant by the addition of pCDNA3. Treatment with human recombinant TNF- (20 ng/ml; R&D Systems), cycloheximide (10 µg/ml; Sigma), cisplatin (1 µM; Sigma), UV radiation (60 J/m²), or ionizing radiation (2 Gy) was performed 30 h after transfection.

DNA Fragmentation Analysis.
Cells were harvested 16 h after treatment, fixed with 70% ethanol, washed with PBS, and resuspended in 0.5 ml of PBS X-100 containing propidium iodide (40 µg/ml) and DNase-free RNase A (1 mg/ml). Cells were incubated at 25°C for 30 min and analyzed on a FACSCalibur flow cytometer (Beckton Dickinson) for propidium iodide and GFP fluorescence. The percentage of cells to the left of the diploid G₀/G₁ peak, which is diagnostic of hypodiploid cells that have lost DNA, was taken as the percentage of apoptotic cells.

Fluorescence Microscopy and Imaging Analysis for Apoptosis.
Lu1205 cells were grown on glass coverslips (Fisher Scientific) placed in 35-mm dishes. After treatment with drugs or irradiations, medium was removed, and cells were processed as described previously (16) . Briefly, cells were washed with PBS before fixation with 3.7% paraformaldehyde for 10 min. After rehydration in PBS, cells were stained with 0.5 µg/ml 4',6-diamidino-2-phenylindole nuclear dye (Sigma) for 30 min in the dark. Coverslips were then washed with PBS, blotted dry, and mounted onto glass slides using Prolong Antifade mounting medium (Molecular Probes, Inc). Epifluorescence microscopy was performed using an Olympus Vanox AH-2 microscope (Olympus, Melville, NY) equipped with differential wavelength filters and a Zeiss Plan 40X/0.65 NA objective. Images were captured using an integrating Toshiba 3 chip color charge-coupled device camera (Toshiba, New York, NY) with a RGB color framegrabber (Flashpoint, I-Cube, Crofton, MD) and analyzed using Optimas 5.2 software (Optimas Corp., Bothell, WA). A total of 400–500 cells were examined in five to seven randomly selected fields, and apoptotic cells exhibiting condensed and fragmented nuclei were scored.

Immunoblotting Analysis.
Total cell extracts (100 µg) prepared as described previously (11) were resolved on 10% SDS-PAGE, transferred to nitrocellulose, and processed by standard methods (11) . The monoclonal anti-IB antibody (Santa Cruz Biotechnology; sc-1643) and anti-p53 antibody (Calbiochem, PAb 421) were used at a 1:1,000 dilution. The secondary antibody was goat antimouse IgG conjugated to horseradish peroxidase (dilution, 1:10,000). Signals were detected using the enhanced chemiluminescence system (Amersham Pharmacia Biotech).

	Results and Discussion

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The HOS protein contains two important domains that are required for its function as a receptor for SCF-Roc1 E3 ubiquitin ligase (11) . Seven WD40 repeats serve to recognize the DS(PO3)GXS(PO3) motif on phosphorylated substrates and mediate HOS binding to those substrates (11) . The F-box located at the NH₂ terminus of HOS is a docking site for association with Skp1-Cullin1-Roc1 ubiquitin ligase complex (11 , 12) . Forced expression of HOS that lacks the F-box is expected to prevent the recruitment of ubiquitin ligase to a substrate not only by HOS but also by its relative, -TRCP protein. Indeed, transfection of human embryo kidney 293T cells as well as HeLa cells with truncated HOS mutant void of its NH₂ terminus (HOS^F) resulted in the stabilization and accumulation of two SCF^HOS-Roc1 substrates, IB and -catenin (11) . We investigated whether HOS^F expression affects the protein stability of IB in human Lu1205 melanoma cells. As evident from Fig. 1A , the TNF--induced degradation of IB is impaired in the melanoma cells transfected with the dominant negative HOS construct. These data suggest that ubiquitination and degradation of IB in human melanoma cells is regulated by HOS/-TRCP.

View larger version (35K): [in this window] [in a new window]   Fig. 1. A, level of IB in Lu1205 cells transfected with either HOSF construct (2 µg) or empty vector (2 µg; pCDNA3) and treated with TNF- (2 ng/ml) was analyzed by immunoblotting. B, immunoblotting analysis of p53 level in Lu1205 cells. Cells were harvested 30 h after transfection with HOSF (2 µg; Lane 2) and -cateninS33Y (2 µg; Lane 3) expression vectors. Lane 1, control mock-transfected (pCDNA3; 2 µg) cells.

F

F

To test whether HOS^F-mediated stabilization of IB or/and accumulation of p53 via stabilized -catenin may affect the rate of apoptosis in Lu1205 cells, we used fluorescence-activated cell sorting-based DNA fragmentation assays. Because the overall transfection efficiency was about 20%, we cotransfected cDNA encoding GFP, the expression of which allows the analysis of cells that incorporated a plasmid (GFP-positive cells). Treatment of Lu1205 cells with TNF- and cycloheximide led to a moderate increase in the rate of apoptosis of the overall cell population (Fig. 2) . Transfection of Lu1205 with cDNAs encoding GFP alone or in combination with stable -catenin^S33Y mutant did not promote TNF-/cycloheximide-induced cell death in the total cell population. Moreover, those transfections slightly inhibited apoptosis in the GFP-positive cells (Fig. 2) . Conversely, transfection of HOS^F led to a 6-fold increase in the rate of apoptosis in GFP-positive cells (Fig. 2) . These data indicate that inhibition of HOS function results in sensitization of Lu1205 melanoma cells to the programmed cell death induced by TNF- with cycloheximide.

View larger version (21K): [in this window] [in a new window]   Fig. 2. DNA fragmentation analysis in Lu1205 cells transfected with cDNAs encoding GFP (0.4 µg) and HOSF (1.6 µg). Thirty h after transfection, cells were treated with TNF- (20 ng/ml) and cycloheximide (10 µg/ml) for 16 h. Left panel, hypodiploid cells (n < 2) in the absence (top) or presence of TNF- and cycloheximide (bottom) in total cell population. Right panel, percentage of apoptotic cells in the total cell population () or in GFP-positive cells (). The representative of two experiments is shown.

F

F

To confirm that inhibition of HOS function increases the susceptibility of human melanoma cells to apoptosis, we cotransfected Lu1205 cells with HOS^F and GFP constructs and treated them with TNF-/cycloheximide as well as ionizing or UV radiation or the anticancer drug cisplatin. Apoptotic cells exhibiting the characteristic morphology (pyknosis, membrane blebbing, and cell body condensation; Fig. 3A ) were scored among GFP-negative cells (which did not uptake GFP- and HOS-expressing plasmids) and GFP-positive cells. Expression of the dominant negative mutant of HOS resulted in an increase in the rate of apoptotis after treatment with TNF- and cycloheximide (Fig. 3) , thus confirming our DNA fragmentation analysis data (Fig. 2) . Moreover, transfected cells exhibited a considerably higher level of programmed cell death in response to treatment with radiation or cisplatin (Fig. 3) . Due to the loss of dying/dead cells during cell culture and sample processing (washes), the ratio of apoptosis in transfected/untransfected cells is probably underestimated. Nevertheless, these data suggest that inhibition of HOS function results in sensitization of human melanoma cells to apoptosis induced by DNA-damaging agents.

View larger version (59K): [in this window] [in a new window]   Fig. 3. A, apoptotic morphology of Lu1205 cells transfected with pEGFP (0.2 µg) and HOSF (0.8 µg). Thirty h after transfection, cells were treated as indicated for 16 h, fixed, and stained with 4',6-diamidino-2-phenylindole, and the apoptotic cells were scored among GFP-negative and GFP-positive cells. UT, untreated control. B, the percentage of the apoptotic cells shown in A was calculated (minus the values for untreated control) and depicted.

F

At present, we cannot rule out that mechanisms other than down-regulation of NF-B activity may participate in the HOS-dependent sensitization of melanoma cells to apoptosis. Identifying the mechanisms of HOS function and regulation may lead to the design of agents capable of inhibiting HOS function that may serve as a potential target for putative antitumor agents.

	ACKNOWLEDGMENTS

 
We thank M. Herlyn for Lu1205 melanoma cells and Bert Vogelstein for pCI-neo--catenin^S33Y plasmid. Fluorescent microscopy was performed using the Microscopy/Imaging Shared Resources of the Lombardi Cancer Center, Georgetown University, Washington, D.C. (USPHS Grant 2P30-CA-51008).

	FOOTNOTES

 
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.

¹ Supported by NIH Grants CA 59908 (to Z. R.) and CA 74175 (to A. D.).

² To whom requests for reprints should be addressed, at The Ruttenberg Cancer Center, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York, NY 10029. Phone: (212) 659-5508; Fax: (212) 849-2446; E-mail: sergey_fuks{at}smtplink.mssm.edu

³ The abbreviations used are: NF-B, nuclear factor B; TNF, tumor necrosis factor; GFP, green fluorescent protein; HOS, homologue of Slimb; -TRCP, -transducin repeats-containing protein; SCF, Skp1/Cdc53/F-box protein complex; IB, inhibitor of NF-B.

⁴ V. Ivanov and Z. Ronai, unpublished observations.

Received 7/12/99. Accepted 9/ 3/99.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 

1. Serrone L., Hersey P. The chemoresistance of human malignant melanoma: an update. Melanoma Res., 9: 51-58, 1999.[Medline]
2. Wang C. Y., Mayo M. W., Korneluk R. G., Goeddel D. V., Baldwin A. S., Jr. NF-B antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science (Washington DC), 281: 1680-1683, 1998.[Abstract/Free Full Text]
3. LaCasse E. C., Baird S., Korneluk R. G., MacKenzie A. E. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene, 17: 3247-3259, 1998.[Medline]
4. Zong W. X., Edelstein L. C., Chen C., Bash J., Gelinas C. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-B that blocks TNF-induced apoptosis. Genes Dev., 13: 382-387, 1999.[Abstract/Free Full Text]
5. Stancovski I., Baltimore D. NF-B activation: the IB kinase revealed?. Cell, 91: 299-302, 1997.[Medline]
6. Verma I. M., Stevenson J. IB kinase: beginning, not the end. Proc. Natl. Acad. Sci. USA, 94: 11758-11760, 1997.[Free Full Text]
7. Devalaraja M. N., Wang D. Z., Ballard D. W., Richmond A. Elevated constitutive IB kinase activity and IB phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO transcription. Cancer Res., 59: 1372-1377, 1999.[Abstract/Free Full Text]
8. Bakker T. R., Reed D., Renno T., Jongeneel C. V. Efficient adenoviral transfer of NF-B inhibitor sensitizes melanoma to tumor necrosis factor-mediated apoptosis. Int. J. Cancer, 80: 320-323, 1999.[Medline]
9. Wang C. Y., Cusack J. C., Jr., Liu R., Baldwin A. S., Jr. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-B. Nat. Med., 5: 412-417, 1999.[Medline]
10. Paillard F. Induction of apoptosis with IB, the inhibitor of NF-B. Hum. Gene Ther., 10: 1-3, 1999.[Medline]
11. Fuchs S. Y., Chen A., Xiong Y., Pan Z. Q., Ronai Z. HOS, a human homologue of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IB and -catenin. Oncogene, 18: 2039-2046, 1999.[Medline]
12. Tan P., Fuchs S. Y., Wu K., Chen A., Gomez C., Ronai Z., Pan Z-Q. Recruitment of a ROC1: cullin1 heterodimeric ubiquitin ligase by Skp1 and F box protein HOS to catalyze the phosphorylation-dependent ubiquitination of IB. Mol. Cell, 3: 527-534, 1999.[Medline]
13. Laney J. D., Hochstrasser M. Substrate targeting in the ubiquitin system. Cell, 97: 427-430, 1999.[Medline]
14. Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res., 18: 5322 1990.[Free Full Text]
15. Morin P. J., Sparks A. B., Korinek V., Barker N., Clevers H., Vogelstein B., Kinzler K. W. Activation of -catenin-Tcf signaling in colon cancer by mutations in -catenin or APC. Science (Washington DC), 275: 1787-1790, 1997.[Abstract/Free Full Text]
16. Soldatenkov V. A., Dritschilo A. Apoptosis of Ewing’s sarcoma cells is accompanied by accumulation of ubiquitinated proteins. Cancer Res., 57: 3881-3885, 1997.[Abstract/Free Full Text]
17. Damalas A., Ben-Ze’ev A., Simcha I., Shtutman M., Leal J. F., Zhurinsky J., Geiger B., Oren M. Excess -catenin promotes accumulation of transcriptionally active p53. EMBO J., 18: 3054-3063, 1999.[Medline]
18. Brockman J. A., Scherer D. C., McKinsey T. A., Hall S. M., Qi X., Lee W. Y., Ballard D. W. Coupling of a signal response domain in IB to multiple pathways for NF-B activation. Mol. Cell Biol., 15: 2809-2818, 1995.[Abstract]
19. Xu Y., Bialik S., Jones B. E., Iimuro Y., Kitsis R. N., Srinivasan A., Brenner D. A., Czaja M. J. NF-B inactivation converts a hepatocyte cell line TNF response from proliferation to apoptosis. Am. J. Physiol., 275: C1058-C1066, 1998.[Abstract/Free Full Text]
20. Li G., Tang L., Zhou X., Tron V., Ho V. Chemotherapy-induced apoptosis in melanoma cells is p53 dependent. Melanoma Res., 8: 17-23, 1998.[Medline]

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