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Inhibition of F-Box Protein p45^SKP2 Expression and Stabilization of Cyclin-Dependent Kinase Inhibitor p27^KIP1 in Vitamin D Analog-Treated Cancer Cells

Departments of Physiology (R.L., T.T.W., J.H.W.) and Medicine (R.L., T.T.W., J.H.W.), McGill University, Montréal, Québec H3G 1Y6, Canada; and Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital (W.H.M.), Montréal Centre for Experimental Therapeutics in Cancer (W.H.M., J.H.W.), Montréal, Québec H3T 1E2, Canada

Address all correspondence and requests for reprints to: John H. White, Department of Physiology, McGill University, McIntyre Building, Room 1128, 3655 Drummond Street, Montréal, Québec H3G 1Y6, Canada.

	Abstract

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Treatment of cancer cells with 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] or its analogs induces growth arrest and expression of the cyclin-dependent kinase inhibitor p27^KIP1. Although 1,25(OH)₂D₃ transiently enhances p27^kip1 gene transcription in some cells, its effects on p27^KIP1 protein levels are generally more gradual and sustained. This suggests that 1,25(OH)₂D₃ treatment may be stabilizing p27^KIP1 protein, which is sensitive to modification by the SCF^SKP2 protein ubiquitin ligase and proteosomal degradation. Here, we show that treatment of AT-84 head and neck squamous carcinoma cells with the 1,25(OH)₂D₃ analog EB1089 increases p27^KIP1 protein levels without significantly affecting expression of its mRNA. EB1089 treatment repressed expression of mRNAs encoding the F-box protein p45^SKP2, a marker of poor head and neck cancer prognosis, and the cyclin kinase subunit CKS1, which is essential for targeting p45^SKP2 to p27^KIP1. This coincided with a reduction of total p45^SKP2 protein, and p45^SKP2 associated with p27^KIP1. Consistent with these findings, turnover of p27^KIP1 protein was strongly inhibited in the presence of EB1089. A similar reduction in p45^SKP2 expression and stabilization of p27^KIP1 protein was observed in 1,25(OH)₂D₃-sensitive UF-1 promyelocytic leukemia cells, which also respond by transiently increasing p27^kip1 gene transcription. Our results reveal that 1,25(OH)₂D₃ analogs increase levels of p27^KIP1 in different cell types by inhibiting expression of SCF^SKP2 subunits and reducing turnover of p27^KIP1 protein.

	Introduction

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SIGNAL TRANSDUCTION BY the active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], primarily controls calcium transport in the intestinal epithelia and modulates bone resorption (1). However, 1,25(OH)₂D₃ and its analogs also stimulate cell differentiation and inhibit proliferation in a number of cellular and animal models, and its analogs are of interest because of their broad potential as anticancer agents (1). We are studying the antiproliferative and potential chemopreventive actions of 1,25(OH)₂D₃ analogs in head and neck squamous carcinoma (HNSCC; Refs. 2, 3, 4), where development of second primary carcinomas after treatment of an initial malignancy is a major clinical problem (5).

1,25(OH)₂D₃ and its analogs regulate the expression cyclin-dependent kinase inhibitors p21^waf1/cip1 and p27^kip1. 1,25(OH)₂D₃ affects p21^waf1/cip1 expression strongly in a cell specific matter (e.g. Refs. 2, 6, 7, 8), whereas its induction of p27^kip1 is generally more consistent. For example, p27^kip1 was induced in HNSCC lines under conditions where p21^waf1/cip1 was repressed (2, 8). In addition, the rapid and transient induction of p27^kip1 transcripts in monocytic cells was accompanied by a delayed and more sustained increase in p27^KIP1 protein (7, 9). p27^KIP1 protein is a substrate for protein ubiquitin ligase (E3) SCF^SKP2 (10, 11, 12). The p45^SKP2 subunit of SCF^SKP2 binds directly to the cyclin kinase subunit CKS1 (10, 11, 12, 13), which directs p45^SKP2 to p27^KIP1. Significantly, CKS^-/- mice express elevated levels of p27^KIP1 (13).

We were interested in determining whether p27^KIP1 protein turnover was affected in cells treated with 1,25(OH)₂D₃ analogs. Here, we show that p27^kip1 mRNA is unchanged in EB1089-treated AT-84 cells. Rather, treatment represses expression of p45^skp2 and cks1 mRNAs, leading to reduced association of p45^SKP2 protein with p27^KIP1 and strongly reduced p27^KIP1 protein turnover. A similar reduction in p45^SKP2 expression and stabilization of p27^KIP1 protein was observed in promyelocytic UF-1 cells. Taken together, the results indicate that 1,25(OH)₂D₃ analogs enhance p27^KIP1 expression in diverse cell types by reducing its turnover.

	Materials and Methods

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Tissue culture
Mouse AT-84 HNSCC cells and human promyelocytic UF-1 cells were cultured as described (2, 9). Effects of EB1089 were analyzed by seeding cells in 100-mm dishes at 60% confluence in 10 ml of medium containing charcoal-stripped serum. Media were changed after 24 h to charcoal-stripped medium containing 0.1 µM EB1089. Media were changed every 48 h, and fresh ligand was added.

RT-PCR
RT-PCR analysis was performed essential as described (4). For analysis of p45^skp2, p27^kip1, and cks1 mRNA expression, 1 µl of reverse transcription reactions was amplified by PCR as follows: 30-sec denaturation at 94 C, 45-sec elongation at 72 C, and 30-sec annealing starting at 60 C, down 1 C per cycle to 55 C, and continuing 20 cycles amplification (94 C for 30 sec, 57.5 C for 30 sec, 72 C for 45 sec). cDNAs for p45^skp2, p27^kip1, and cks1 were amplified using 5' primer 5'-CCTAAGCAGCTGTCCCAGAC-3', 3' primer 5'-GTGTCAGTCGGCATTTGATG-3' for p45^skp2, 5' primer 5'-GGATGGACGCCAGACAAG-3', 3' primer 5'-GGGGAACCGTCTGAAACATT-3' for p27^kip1, and 5' primer 5'-TTGGACAAATACGACGACGA-3', 3' primer 5'-CTTTGTTTTCTCGGGTAGTGG-3' for cks1. For amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1 µl of reverse transcription reaction was subjected to 18 cycles amplification (95 C for 30 sec, 56 C for 1 min, 72 C for 25 sec) using 5' primer 5'-GGTGAAGGTCGGTGTCAACG-3', and 3' primer 5'-CAAAGTTGTCATGGATGACC-3'. All of the above reactions were performed in 25 µl of 1.5 mM MgCl₂, 50 mM KCl, and 10 mM Tris-HCl (pH 9.0) using 2.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Baie d’Urfé, Québec, Canada). PCRs were loaded on 2% agarose gel, transferred for Southern blotting to a nylon membrane (Hybond-N⁺, Amersham Pharmacia Biotech), and fixed by UV cross-linker. The membrane was soaked in 3x standard saline citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at 50 C, and prehybridized at 42 C in 50 mM-phosphate buffer (pH 6.5), 5x SSC, 10% Denhardt’s solution containing 250 µg/ml sheared, and denatured salmon sperm DNA. Hybridization was carried out in the same solution by the addition of ³²P-end-labeled oligonucleotides 5'-GAGCTGAACCTCTCCTGGTG-3' for p45^skp2, 5'-CAAATGCCTGACTCGTCAGA-3' for p27^kip1, 5'-TCACATCTTGCTGTTCCGG-3' for cks1 and 5'-TCTTCACCACCATGGAGAAG-3' for GAPDH. Following hybridization, the membrane was washed four times in 2x SSC and 0.2% SDS for 5 min, three times in 0.1x SSC. and 0.2% SDS for 30 min at 50 C, dried, and autoradiographed. All experiments were repeated at least three times.

Immunoprecipitation and Western blot analysis
Cells were rinsed with PBS, harvested by scraping in 1 ml of PBS, and centrifuged (10,000 rpm, 5 min) at 4 C. Pellets were resuspended in 100 µl of ice-cold lysis buffer (10 mM Tris-HCl, pH 8.0; 60 mM KCl; 1 mM EDTA; 1 mM dithiothreitol; 0.5% Nonidet P-40) containing protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany) and incubated on ice for 15 min. Lysates were centrifuged at 4 C (10,000 rpm, 10 min), and supernatants were recovered. Total protein was quantitated using the Bio-Rad Laboratories, Inc. (Richmond, CA) protein assay reagent. Lysates containing 100 µg of total proteins normalized to a 1-ml volume in lysis buffer were precleared by incubation with 30 µl of 50% slurry protein A-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 45 min on a rotator at 4 C. Protein complexes were immunoprecipitated from the precleared lysates by addition of either 1 µg of rabbit anti-p27^kip1 (Santa Cruz Biotechnology, Inc.) or anti-p45^skp2 (Zymed Laboratories, Inc., South San Francisco, CA) for 1 h at 4 C with rotation, followed by addition of 30 µl of 50% slurry protein A-agarose for 1 h on a rotator at 4 C. After three washes with lysis buffer, pelleted beads were boiled for 3 min in 2x SDS-PAGE loading buffer. Immunoprecipitates and lysates (30 µg) were resolved on 20% SDS-PAGE and analyzed by Western blotting with rabbit anti-p45^skp2 (0.5 µg/ml) (Zymed Laboratories, Inc.) and rabbit anti-p27 (0.4µg/ml), and secondary antibody goat antirabbit IgG horseradish peroxidase conjugate (0.04 µg/ml) (Santa Cruz Biotechnology, Inc.). The reaction was developed by the chemiluminescence method (NEN Life Science Products, Beverly, MA).

Metabolic labeling experiments
Cells treated with 10^-7 M EB1089 or dimethylsulfoxide (DMSO) vehicle for 48 h were rinsed with methionine-free RPMI 1640, labeled with 5 ml of 50 µCi/ml [³⁵S]methionine for 3 h, washed with medium containing charcoal-stripped serum and incubated in medium containing DMSO or EB1089. Cells were collected at 0, 1, 2, 3, 6, and 8 h and lysed with lysis buffer. One hundred micrograms of total protein normalized to a 1-ml volume in lysis buffer were precleared by incubation with 30 µl of 50% slurry protein A-agarose (Santa Cruz Biotechnology, Inc.) for 45 min on a rotator at 4 C. Proteins were immunoprecipitated from the precleared lysates by addition of 1 µg of rabbit anti-p27 (Santa Cruz Biotechnology, Inc.) for 1 h at 4 C with rotation, followed by 30 µl of 50% slurry protein A-agarose for 1 h on a rotator at 4 C. After three washes with lysis buffer, pelleted beads were boiled for 3 min in 2x SDS-PAGE loading buffer, resolved in 20% SDS-polyacrylamide gels and autoradiographed. Signals were quantified using Kodak (Rochester, NY) digital science 1D Image Analysis software.

	Results

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Treatment of AT-84 cells with 1,25(OH)₂D₃ or EB1089 induces G0/G1 arrest and is accompanied by up-regulation of p27^KIP1 protein (2). We probed further this regulation by RT-PCR analysis of p27^kip1 transcripts in EB1089-treated AT-84 cells. Strikingly, EB1089 treatment did not substantially alter p27^kip1 transcript levels over a 48-h period (Fig. 1A), in contrast to results obtained in other cell lines where a strong but transient induction was observed (6, 7, 9). To investigate the possibility that mechanisms regulating the turnover of p27^KIP1 protein may be affected in EB1089-treated AT-84 cells, we analyzed the expression of transcripts encoding the F-box protein p45^SKP2 and the cyclin kinase subunit CKS1. RT-PCR analyses showed that expression of both transcripts was strongly and rapidly (<24 h) repressed in treated cells (Fig. 1, B and C). These results were consistent with those obtained by Northern blotting of RNA from EB1089-treated AT-84 cells (data not shown). In agreement with these and our previous studies (2), treatment with EB1089 for 72 h led to a gradual accumulation of p27^KIP1 protein in AT-84 cells (Fig. 2A). Significant changes in p27^KIP1 protein were observed after 24 h and levels continued to increase through 72 h. No change in p27^KIP1 levels was observed in control experiments with vehicle-treated cells (data not shown). The accumulation of p27^KIP1 over 72 h coincided with a progressive reduction in p45^SKP2 protein over the same period as assessed by direct Western blotting (Fig. 2B) or immunoprecipitation of p45^SKP2 following by Western blotting (Fig. 2C).

View larger version (43K): [in this window] [in a new window]   FIG. 1. Effects of EB1089 treatment on expression of p27kip1, p45skp2, and cks1 mRNA levels. AT-84 cells in culture were treated with EB1089 for the times indicated, and expression of transcripts encoding p27KIP1 (A), p45SKP2 (B), and CKS1 (C) was analyzed by RT-PCR followed by Southern blotting with radiolabeled internal oligonucleotides. Southern blots of typical experiments are shown and the results of three experiments are presented in histograms. A control for GAPDH expression is shown at the bottom.

View larger version (37K): [in this window] [in a new window]   FIG. 2. Effects of EB1089 treatment on p27KIP1 protein and its association with p45SKP2. A and B, Extracts of EB1089-treated AT-84 cells were immunoblotted for expression of p27KIP1or p45SKP2. C, Cell extracts were immunoprecipitated with an anti-p45SKP2 antibody and immunoprecipitates were probed for p45SKP2. D, Cell extracts were immunoprecipitated with an anti-p27KIP1 antibody and immunoprecipitates were probed for p27KIP1 (inset) or p45SKP2. Note that in A–D, Western blots show duplicate lanes corresponding to each time point. Histograms show the results of triplicate experiments.

The reduced association of p45^SKP2 with p27^KIP1 suggested that p27^KIP1 was being turned over less rapidly in EB1089-treated cells. The effects of EB1089 on p27^KIP1 turnover in AT-84 cells were analyzed in metabolic labeling experiments (Fig. 3). Cells were treated with EB1089 or vehicle for 48 h, labeled with ³⁵S-methionine, and chased with serum for the times indicated (Fig. 3). Although radiolabeled p27^KIP1 immunoprecipitated from control cells diminished during the 8-h chase, no p27^KIP1 turnover was observed in immunoprecipitates of EB1089-treated cells. Taken together, our results indicate that EB1089 up-regulates p27^KIP1 by inhibiting of expression of the p45^SKP2 subunit of the SCF^SKP ubiquitin ligase and reducing turnover of p27^KIP1 protein.

View larger version (30K): [in this window] [in a new window]   FIG. 3. EB1089 treatment stabilizes p27KIP1 protein. Metabolic labeling experiments performed in vehicle- (A) and EB1089-treated AT-84 cells (B) are presented. Cells were labeled for 3 h with 35S-methionine after 48 h of treatment with either vehicle or EB1089, and chased as indicated. Extracts were immunoprecipitated with an anti-p27KIP1 antibody and immunoprecipitates were analyzed by gel electrophoresis. Histograms present the results of triplicate experiments. C, Western analysis showing that EB1089 treatment enhances p27KIP1 protein expression and down-regulates expression of p45SKP2 in human promyelocytic UF-1 cells. Metabolic labeling experiments performed as above in vehicle- (D) and EB1089-treated UF-1 cells (E) reveal that EB1089 treatment reduces p27KIP1 turnover in UF-1 cells (see Materials and Methods for details).

	Discussion

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P27^KIP1 is a key regulator of the G1/S checkpoint (14), and several studies have shown that its up-regulation underlies the antiproliferative effects of 1,25(OH)₂D₃ and its analogs (2, 6, 7, 8, 9). Our results indicate that 1,25(OH)₂D₃ analogs can act by two cell type-dependent mechanisms to enhance expression of p27^KIP1; induction of expression of its gene, and inhibition of p27^KIP1 protein turnover. In contrast to results obtained in other cell lines (6, 7, 9), EB1089 had little effect on p27^kip1 mRNA levels in AT-84 cells over a 48-h period. The varying effects of 1,25(OH)₂D₃ analogs on p27^kip1 gene transcription may due in part to the fact that induction of expression is dependent on an Sp1 element and a CCAAT box in the p27^kip1 promoter and not a vitamin D response element (15), suggesting that these sites function cell specifically.

In spite of the lack of response at the mRNA level, p27^KIP1 protein increased steadily over 72 h in EB1089-treated AT-84 cells, consistent with studies in other cell lines (6, 7, 9). Metabolic labeling experiments indicated that the sustained increase was due to inhibition of p27^KIP1 degradation, which was consistent with the observed reduction in expression of p45^skp2 transcripts and protein. The inhibition of p45^skp2 gene expression by EB1089 was rapid, occurring within 24 h, whereas the drop in p45^SKP2 protein levels continued over a 72-h period. This reduced the total amount of p45^SKP2 associated with p27^KIP1 by coimmunoprecipitation even though p27^KIP1 protein levels were increasing. Taken together, these results indicate that the increase in levels of p27^KIP1 protein in EB1089-treated AT-84 calls arose from protein stabilization, and not from increased gene expression. A similar stabilization of p27^KIP1 protein was also observed in UF-1 promyelocytic leukemia cells.

The effect of EB1089 observed on p27^KIP1 protein expression in AT-84 cells (2-fold) is not dramatic. However, it is important to stress that progression through G1 into S phase is regulated by threshold levels of key regulators (14), and p27^KIP1 levels control the activity of the CDK2/cyclin E complex, whose function is critical for entry into S phase. Subtle changes in p27^KIP1 expression can have profound effects on cell function. Indeed, p27^KIP1 heterozygous mice are predisposed to tumors in a number of tissues after irradiation or exposure to carcinogens (16). Therefore, it is likely that the 2-fold increase observed in p27^KIP1 protein levels in treated AT-84 cells is critical for the antiproliferative effects of EB1089.

Just as reduced levels of p27^KIP1 are associated with cancer development, overexpression of p45^SKP2 is oncogenic. For example, elevated expression of p45^SKP2 was found in 49% of oral squamous cell carcinomas and was correlated with a poor prognosis of affected patients (17). Another study found an inverse correlation between p45^SKP2 and of p27^KIP1 levels in oral cancers (18) and also showed that of p45^SKP2 could cooperate with H-Ras^G12V to transform primary fibroblasts. Similarly, p45^SKP2 cooperated with N-Ras in induction of T cell lymphomas in a mouse transgenic model, leading to significantly reduced survival times (19). Taken together, these studies strongly suggest that the down-regulation of p45^SKP2 and consequent sustained up-regulation of p27^KIP1 are key elements in the antiproliferative and anticancer actions of vitamin D analogs.

	Acknowledgments

 
We are grateful to Dr. Lise Binderup (Leo Laboratories, Ballerup, Denmark) for the generous gift of EB1089 and to Dr. Masahiro Kizaki, Keio University School of Medicine (Tokyo, Japan) for the generous gift of UF-1 cells.

	Footnotes

 
This work was supported by Operating Grant MOP57763 (to J.H.W.) from the Canadian Institutes of Health Research.

¹ R.L. and T.T.W. contributed equally to this work.

Abbreviations: CKS, Cyclin kinase subunit; DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HNSCC, head and neck squamous carcinoma; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; SDS, sodium dodecyl sulfate; SSC, standard saline citrate.

Received November 12, 2002.

Accepted for publication December 20, 2002.

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