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Carcinogenesis Advance Access originally published online on May 17, 2007
Carcinogenesis 2007 28(8):1752-1758; doi:10.1093/carcin/bgm120
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

ARA54 is involved in transcriptional regulation of the cyclin D1 gene in human cancer cells

Hirotoshi Kikuchi1,2, Chiharu Uchida2, Takayuki Hattori2, Tomoyasu Isobe1, Yoshihiro Hiramatsu1,2, Kyoko Kitagawa2, Toshiaki Oda2, Hiroyuki Konno1 and Masatoshi Kitagawa2,*

1 Second Department of Surgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan
2 Department of Biochemistry 1, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan

* To whom correspondence should be addressed. Tel: +81 53 435 2322; Fax: +81 53 435 2322;Email: kitamasa{at}hama-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Cyclin D1 is one of the major enhancers of cell cycle progression and its expression is regulated in several growth stimulatory signaling pathways. ARA54 is an androgen receptor (AR) co-activator that enhances AR-dependent transcriptional activation. Although expression of ARA54 mRNA is observed in a variety of human tissues at low levels, the AR- or androgen-independent function of ARA54 in those tissues remains unclear. In this study, we identified a novel role for ARA54 in the regulation of cyclin D1 expression in the absence of AR stimulation in human cancer cells. Depletion of endogenous ARA54 by small interfering RNA decreased both the protein and mRNA levels of cyclin D1. These changes did not result from a reduction in the half-life of either the protein or the mRNA, but from suppression of cyclin D1 gene transcription. In T98G cells, depletion of ARA54 increased the population of cells in G1 phase, but reduced the population of cells in S phase, leading to a significant increase in the G1/S ratio and impaired cell growth. Furthermore, the amount of ARA54 mRNA appeared to positively correlate with cyclin D1 mRNA levels in specimens of clinical colon carcinomas, indicating that ARA54 is not only involved in the regulation of cyclin D1 expression in cultured cell lines but also in clinical cancer specimens. These results suggest that ARA54 might participate in enhancing cell cycle progression and cell proliferation via induction of cyclin D1.

Abbreviations: AR, androgen receptor; ARA, AR-associated; BrdU, 5-bromo-2'-deoxy-uridine; RT-PCR, reverse transcriptional polymerase chain reaction; siRNA, small interfering RNA; CDK, cyclin dependent kinase; CREB, cAMP-responsive element binding protein; NF-kB, nuclear factor kappa B; TCF/LEF, T cell factor 1/lymphoid enhancer-binding factor 1; AP-1, activating protein-1; SP1, specificity protein 1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell cycle progression is controlled by cyclin–CDK complexes (1). In the G1 phase, the cyclin D–CDK4 complex is activated resulting in the phosphorylation of pRB that induces E2F-dependent expression of S phase proteins such as cyclin E, thereby promoting the transition from G1 to the S phase of the cell cycle (1). During the S phase, cyclin D1 is phosphorylated by glycogen synthase kinase 3ß, which promotes nuclear export and ubiquitination of cyclin D1 by the SCFFBX4-{alpha}B crystallin complex leading to proteasome-dependent degradation of cyclin D1 (2,3). Induction of cyclin D1 in the G1 phase is growth factor dependent and tightly regulated at the level of transcriptional activation (4,5). It has been also reported that transcription of the cyclin D1 gene can be activated by several hormones including estrogen (17ß-estradiol: E2) and angiotensin II (6,7). Several transcriptional factors such as CREB, NF-{kappa}B, TCF/LEF, AP-1 and SP1 have been found to transactivate the cyclin D1 promoter (8–13) and some transcriptional suppressors such as Tob1 and Jumonji have been reported to down-regulate cyclin D1 gene promoter activity (14,15). Over-expression of cyclin D1 enhances cell cycle progression from G1 to S phase and increases cell proliferation (5). Many studies have shown that cyclin D1 is frequently over-expressed in human cancers: the result of gene amplification, oncogene-induced signaling or a mutation that disrupts degradation (5,16,17). High expression of cyclin D is associated with a poor prognosis in many types of human cancer, such as breast, esophageal, lung and prostate cancers (18–21).

The androgen receptor (AR), a member of steroid hormone receptor superfamily, is a ligand-dependent transcriptional cofactor that mediates the biological effects of androgens (22,23). Several co-regulators have been identified as AR-associated (ARA) proteins that enhance AR-dependent transcriptional activation (24). ARA54 is an ARA protein that can interact with AR in a ligand-dependent manner and enhance transactivation by AR (25). ARA54 mRNA is expressed at the highest levels in testis but is lowly expressed in a variety of other human tissues (25,26). The physiological roles of several AR co-regulators, including ARA54, in prostate cancer progression have been well studied (27–29). However, the AR- or androgen-independent functions of AR co-regulators in normal tissues and in hormone-independent human cancer cells are still unknown.

We investigated the effect of small interfering RNA (siRNA) knock-down of ARA54 in a variety of human malignant tumor cells to identify whether endogenous ARA54 has a function in the absence of AR stimulation. Here we provide evidence to show that ARA54 is involved in regulating transcription of the cyclin D1 gene.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell culture
T98G, HCT116, HeLa and U2OS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

Antibodies
The antibodies used in this study were: anti-cyclin D1 polyclonal antibody 553 (MBL, Nagoya, Japan) and anti-ß-actin antibody AC-15 (Sigma, St Louis, MO).

RNA interference
Cells were grown to 30–50% confluence and incubated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. siRNA directed to ARA54 or non-specific control siRNA was transfected by Oligofectamine (Invitrogen, Carlsbad, CA) or Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Nucleotide sequences of siRNAs were as follows: for ARA54-1, 5'-r(GGAAGAGACCCUAGCAUAC)d(TT)-3'; for ARA54-2, 5'-r(GGAUGCAAUUUCUUAAGGA)d(TT)-3', and for ARA54-3, 5'-r(CCAAAGCUCUGAAUAGUUA)d(TT)-3'.

Quantitative reverse transcriptional polymerase chain reaction analysis
Total RNA was isolated from cultured cells using an Isogen kit (Wako, Osaka, Japan) and subject to reverse transcription with random hexanucleotide primers and SuperScript Reverse Transcriptase II (Invitrogen). The resulting cDNA was subjected to real-time polymerase chain reaction using the Rotor-Gene 3000 System (Corbett Research, Mortlake, Australia) and a QuantiTect SYBR Green PCR kit (Qiagen, Valencia, CA). Primer sequences were 5'-AGAGATGGAAAGTAAGGAGTG-3' and 5'-GACGGAAGATTAGGGAAAAAC-3' for ARA54 and 5'-GCTCCTGTGCTGCGAAGT-3' and 5'-TGTTCCTCtCAGACCTCCAG-3' for cyclin D1. Transcripts of interest were normalized to 18S rRNA or glyceraldehyde-3-phosphate dehydrogenase mRNA.

In vivo protein degradation assay
T98G cells were transfected with siRNAs and treated with cycloheximide (20 µg/ml; Sigma) for the appropriate time (0, 15, 30, 45 and 60 min). Cells were lysed and protein levels were analyzed by western blotting. For the protein of interest, band densities were quantified using the image analysis software Image Gauge 4.21 (Fujifilm, Tokyo, Japan) and normalized to ß-actin.

Transcription shut-off assays for measuring mRNA half-life
T98G cells were transfected with siRNAs and treated with actinomycin D (2.5 µg/ml: Wako) for the appropriate time (0, 2, 4, 6 and 8 h). Total RNA was isolated and analyzed by quantitative RT-PCR.

Nuclear run-on analysis
Nuclear run-on analysis was performed as described previously (30) with several modifications. Briefly, T98G cells were washed with phosphate-buffered saline (–) and suspended in cell lysis buffer [10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid, 0.15 M sucrose, 0.5% NP-40, 3 mM MgOAc and 1 mM dithiothreitol]. Intact nuclei were pelleted by centrifugation at 500g for 5 min, washed and re-suspended in storage buffer [50 mM Tris–HCl (pH 8.0), 0.1 mM ethylenediaminetetraacetic acid, 40% glycerol, 5 mM MgOAc and 1 mM dithiothreitol]. Approximately 1 x 107 nuclei were added in a 200 µl of labeling reaction mixture containing 25 mM Tris-HCl (pH8.0),120 mM KCl, 0.2 U creatine phosphokinase, 10 mM creatine phosphate, 1 mM unlabeled ATP, 0.5 mM unlabeled CTP and GTP (Roche, Indianapolis, IN) and 100 µCi of 32P-UTP (PerkinElmer, Yokohama, Japan). The reaction mixture was incubated for 30 min at 30°C, and 32P-labeled RNA was purified with an ISOGEN kit (Wako). The purified 32P-labeled RNA was re-suspended in RNase-free water and incubated at 85°C for 10 min. Target plasmid DNAs were blotted onto a Hybond-N membrane (GE Healthcare Bioscience, Tokyo, Japan) as described previously (30). For each hybridization, an equal amount of radioactive 32P-RNA was added to 1 ml of hybridization buffer (5x SSPE, 50% formamide, 5x Denhardt's solution, 0.5% sodium dodecyl sulfate and 100 µg/ml salmon testis DNA) and membranes were incubated at 50°C for 48 h. Membranes were washed at 50°C for 15 min six times: twice with 5x SSPE, twice with 1x SSPE containing 0.1% sodium dodecyl sulfate and twice with 0.1x SSPE containing 0.1% sodium dodecyl sulphate. 1 x SSPE: 180 mM NaCl, 10 mM Sodium phosphate buffer pH7.4, 1 mM EDTA. Autoradiography was performed using FLA3000 (Fujifilm).

Cell cycle analysis
T98G cells were treated with siRNAs specific to ARA54 or with a non-specific control siRNA for 48 h and then incubated with 10 ng/ml of 5-bromo-2'-deoxy-uridine (BrdU) for 20 min. Cells were harvested after trypsin treatment and fixed with 70% ethanol in phosphate-buffered saline (–) at -20°C overnight. Cells were incubated with a mouse anti-BrdU antibody (BD Bioscience, Franklin Lakes, NJ) followed by incubation in an AlexaFluor 488-labeled anti-mouse IgG antibody (Molecular Probe, Eugene, OR), and then treated with RNase and 5 µg/ml of propidium iodide. The DNA content and BrdU incorporation were analyzed by flow cytometry (Epics XL, Beckman Coulter, Fullerton, CA).

Cell growth assay
Following transfection with siRNAs, T98G cells (50,000 cells per well) were seeded into six-well plates and the number of cells was counted every 12 h until cells reached subconfluence.

Patient characteristics and tissue specimens
Specimens of colon carcinomas were obtained from 26 Japanese patients (17 men and 9 women) who underwent colectomy at the Second Department of Surgery, Hamamatsu University School of Medicine. Samples of normal mucosal control tissues were also extracted carefully to prevent the contamination of stroma. Patients did not receive therapy before this surgery and had previously given informed consent.

Statistical analysis
Data are presented as mean ± SD. Data were analyzed by Student's t-test or Fisher's exact test, where P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Depletion of ARA54 decreases cyclin D1 expression in human cancer cells
To clarify the role of ARA54 in cell cycle regulation, we investigated the effect of ARA54-depletion on the expression of various cell cycle regulators. Human glioblastoma T98G cells were transfected with one of three different siRNAs directed to ARA54 and the level of knock-down was determined by quantitative RT-PCR due to a lack of specific antibodies for endogenous ARA54 protein. Cell lysates were analyzed by western blotting and interestingly, we found that protein levels of cyclin D1 were dramatically decreased in cells that had been transfected with each of the three ARA54 siRNAs (Figure 1A, upper panel). We went on to analyze mRNA levels of cyclin D1 by quantitative RT-PCR and found them to be significantly decreased in cells depleted of ARA54 (Figure 1A, lower panel). Furthermore, we examined the effect of ARA54 siRNA knock-down in several other human cancer cell lines to determine whether this effect was specific to T98G cells. Protein and mRNA levels of cyclin D1 were also decreased by siRNA knock-down of ARA54 in HCT116, HeLa and U2OS cells (Figure 1B), suggesting that ARA54 is involved in the regulation of cyclin D1 expression in a diverse range of human cancer cells. We also analyzed protein levels of CDK4, which is known to form a complex with cyclin D1 protein resulting in cell cycle progression. The protein level of CDK4 was not changed by siRNA knock-down of ARA54 in T98G, HCT116, HeLa and U2OS cells (Supplementary Figure S1, available at Carcinogenesis Online), suggesting specific effects on cyclin D1 expression by ARA54 knock-down.


Figure 1
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  		Fig. 1. Effect of ARA54 depletion on cyclin D1 expression. (A) T98G cells were treated with one of three different siRNAs specific to ARA54, or with a non-specific control siRNA, for 48 h. Protein levels of cyclin D1 and ß-actin were analyzed by western blot using the antibodies indicated (upper panel). The level of ARA54 siRNA knock-down and the relative levels of cyclin D1 mRNA were determined by quantitative RT-PCR (lower panel) and data were normalized to the levels of 18S rRNA. All reactions were performed in triplicate and data represent the mean ± SD for n = 3 different cDNA samples. (B) HCT116, HeLa and U2OS cells were treated with an siRNA specific for ARA54 or with a non-specific control siRNA for 48 h. Protein levels of cyclin D1 and ß-actin were analyzed by western blot using the antibodies indicated (upper panel). Relative mRNA levels of ARA54 and cyclin D1 were measured by quantitative RT-PCR and normalized to 18S rRNA (lower panel). All reactions were performed in triplicate and data represent the mean ± SD for n = 3 different cDNA samples.

 
ARA54 does not affect cyclin D1 protein stability
We investigated the protein stability of cyclin D1 to determine whether the decrease in cyclin D1 protein levels induced by depletion of ARA54 was due to enhanced degradation of the cyclin D1 protein. T98G cells were transfected with control or ARA54 siRNAs (1 and 2) and 48 h after knock-down cycloheximide was added to the medium to inhibit translation. Cells were harvested at the appropriate time point (0, 15, 30, 45 and 60 min) and lysates were analyzed by western blotting. As shown in Figure 2, the half-life of cyclin D1 was not altered by depletion of ARA54, suggesting that the reduction in cyclin D1 protein levels following depletion of ARA54 is the result of decreased cyclin D1 mRNA.


Figure 2
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  		Fig. 2. Effect of ARA54 depletion on cyclin D1 protein stability. T98G cells were treated with one of two different siRNAs specific to ARA54 or with a non-specific control siRNA for 48 h, and then incubated with cycloheximide (20 µg/ml) for the time points indicated. Protein levels of cyclin D1 and ß-actin were analyzed by western blot analysis using the antibodies indicated (upper panel). Band densities were quantified using Image Gauge 4.21 image analysis software, and the relative ratio of cyclin D1 protein levels at each time point is shown in the lower panel.

 
Depletion of ARA54 inhibits transcription of the cyclin D1 gene
We next attempted to address whether ARA54 affects the production or degradation of cyclin D1 mRNA. To determine the mechanism involved in down-regulation of cyclin D1 expression, we first analyzed the stability of cyclin D1 mRNA in T98G cells. Forty-eight hours after siRNA treatment, actinomycin D was added to the culture medium to stop transcription. RNA was extracted at different time points and quantitative RT-PCR analysis was performed. We confirmed that ARA54 siRNA reduced the stability of ARA54 mRNA (Figure 3A, left panel); however, the turnover of cyclin D1 mRNA remained unchanged following inhibition of transcription (Figure 3A, right panel). As cyclin D expression is reduced following siRNA knock-down of ARA54 but remains unaltered when transfected cells are treated with actinomycin D, it appears that ARA54 does not function to decrease the stability of cyclin D1 mRNA.


Figure 3
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  		Fig. 3. Effect of ARA54 depletion on the stability of cyclin D1 mRNA and the transcription level of the cyclin D1 gene. (A) mRNA stability. T98G cells were treated with one of two siRNAs specific to ARA54 or with a non-specific control siRNA for 48 h, followed by incubation with actinomycin D (2.5 µg/ml) for the time points indicated. mRNA levels of ARA54, cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase were analyzed by quantitative RT-PCR. Graph shows the mRNA expression of ARA54 (left panel) and cyclin D1 (right panel) relative to time 0. (B) Nuclear run-on experiments. T98G cells were treated with an siRNA specific for ARA54 or a non-specific control for 48 h. Intact nuclei were then isolated and an in vitro transcription was performed to label RNA with 32P. 32P-RNA was hybridized to a Hybond-N membrane on which denatured cyclin D1 and ß-actin cDNA had been immobilized. Relative transcription rates of the cyclin D1 gene normalized to ß-actin were shown in the right panel. Data represent mean ± SD of two independent experiments. *P < 0.001, Student's t-test.

 
We went on to perform nuclear run-on analysis to evaluate the effect of ARA54 depletion on the rate of cyclin D1 gene transcription. Depletion of ARA54 had no effect on ß-actin transcription, but led to a significant decrease in cyclin D1 transcription to levels ~40% of control (Figure 3B). These data suggest that the decrease in cyclin D1 expression by depletion of ARA54 results from a reduction in the level of the cyclin D1 gene transcription.

Depletion of ARA54 decreased cell cycle progression and growth rate of T98G cells
Cyclin D1 is well known as one of major proteins that enhance cell cycle progression from G1 to S phase. To further evaluate the effect of ARA54 siRNA knock-down on cell cycle progression in T98G cells, we analyzed the percentage of cells in S phase that could incorporate BrdU. Following treatment with control or ARA54 siRNA, cells were incubated with BrdU. BrdU incorporation and DNA content were analyzed by staining with an anti-BrdU antibody or with propidium iodide, respectively, followed by flow cytometry. As shown in Figure 4, depletion of ARA54 increased the population of cells in G1 phase, but reduced the population of cells in S phase, leading to a significant increase in the G1/S ratio.


Figure 4
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  		Fig. 4. Effect of ARA54 depletion on cell cycle progression in T98G cells. T98G cells were treated with siRNAs specific to ARA54 or with a non-specific control siRNA for 48 h and then incubated with 10 ng/ml of BrdU for 20 min. Cells were harvested and stained with an anti-BrdU antibody and Alexa Fluor 488 anti-mouse IgG antibody and propidium iodide for cell cycle analysis by flow cytometry. (A) Representative flow cytometry. Incorporated BrdU-specific fluorescence is plotted against DNA content. The limits of each subpopulation of cells are indicated. (B) Ratio at each cell cycle phase. Data represent mean ± SD of three independent experiments. *P < 0.001, Student's t-test.

 
We also performed a cell growth assay. T98G cells were treated with control or ARA54 siRNA for 6 h and then seeded into six-well plates at a concentration of 50,000 cells per well. The number of cells was counted every 12 h until cells reached subconfluence (72 h) and, as expected, cell growth was inhibited in ARA54-depleted cells (Figure 5).


Figure 5
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  		Fig. 5. Effect of ARA54 depletion on growth of T98G cells. T98G cells were treated with one of two different siRNAs specific to ARA54 or with a non-specific control siRNA for 6 h and then seeded onto six-well plates (50,000 cells per well). The number of cells was counted every 12 h until cells reached subconfluence (72 h). Data represent mean ± SD of three independent experiments for each time point.

 
These results suggest that endogenous ARA54 is involved in cell cycle progression and cell growth of T98G cells. Our findings also indicate that these defects by ARA54 depletion on the cell cycle and cell growth are due to inhibition of cyclin D1 but not CDK4 expression, since CDK4 levels were not altered by ARA54 siRNA, as shown in Supplemental Figure S1 (available at Carcinogenesis Online).

mRNA levels of ARA54 correlate with cyclin D1 expression in human colon carcinomas
Finally, we investigated the relationship between ARA54 mRNA and cyclin D1 mRNA in clinical specimens of colon carcinoma and normal colon mucosa obtained from 26 patients. ARA54 mRNA was detected in 5 of the 26 specimens of normal mucosa (19.2%) at low levels (data not shown), but ARA54 mRNA expression was found to vary in 13 of the 26 specimens of carcinoma (50%). We went on to classify the cancer specimens as follows—‘low’ (n = 13): ARA54 expression was not detectable; ‘high’ (n = 7): samples where ARA54 expression levels were >10% of control (using the control sample with the highest level of expression among 26 carcinomas) and the remaining samples were classified to have ‘moderate’ expression of ARA54 mRNA (n = 6). As shown in Figure 6, expression to the levels of ARA54 mRNA appeared to positively correlate with cyclin D1 mRNA levels. This observation supports the idea that ARA54 has a physiological role in the regulation of cyclin D1 mRNA expression in cancer cell lines.


Figure 6
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  		Fig. 6. Correlation between ARA54 mRNA and cyclin D1 mRNA expression in human colon carcinomas. Total RNA was extracted from 26 clinical specimens of colon carcinomas. The mRNA levels of ARA54, cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase were quantified by quantitative RT–PCR analysis. The specimens were classified into three groups according to the expression levels of ARA54 mRNA and the correlation between ARA54 mRNA and cyclin D1 mRNA expression was analyzed. *P < 0.005, **P < 0.05, Fisher's test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
ARA54 was first identified as an AR-binding protein. Expression of ARA54 mRNA is observed in a variety of human tissues (25,26), indicating that ARA54 may function independently of androgen-mediated signaling. However, the androgen-independent function of ARA54 in human cells remained unclear. Here, we report for the first time, the function of endogenous ARA54 in human cancer cells without androgen stimulation. Although ARA54 has an impact on the cell growth of prostate cancer cells in an AR- and androgen-dependent manner (27,28), no androgen responsive element (ARE) is found in the promoter region of cyclin D1. This implies that ARA54 could participate in cyclin D1 expression without interaction of AR and ARE. Furthermore, AR expression is much less in T98G cells and it has been reported that testosterone slightly affects the growth of T98G cells (31). AR mRNA levels are also very low in HCT116 and HeLa cells (RefExA data base, http://www.lsbm.org/). Therefore, it is unlikely that the ARA54–AR complex stimulates cyclin D1 expression via unknown regulatory sites in the promoter. Taken together, these findings provide supports for our proposal that ARA54 is involved in cyclin D1 expression independently of androgen-mediated signaling in these cells.

In this study, we have identified a novel function for ARA54 in transcriptional regulation of cyclin D1 that plays an important role to enhance cell cycle progression to the S phase (1). However, the mechanisms by which ARA54 regulates transcriptional activation of the cyclin D1 gene remain unclear. The promoter region of the cyclin D1 gene contains multiple cis-elements, including binding sites for AP-1 (one site), NF-{kappa}B (three sites), STAT (two sites), Sp1 (four sites), beta-catenin/LEF (one site) and ATF/CREB (one site) in order of the distal promoter region, which are important for transcriptional activation of the cyclin D1 gene (8–13). Nagata et al. (13) reported that DNA binding of Sp1 and Sp1-meditated, but not AP-1- or CREB-mediated, cyclin D1 promoter activity is increased in the early- to mid-G1 phase and that the increase is mediated by the Ras–ERK-dependent pathway. On the other hand, transcriptional repressors such as Tob1 and Jumonji have been reported to decrease cyclin D1 gene promoter activity (14,15). Furthermore, recent studies have demonstrated the importance of histone acetyltransferase or deacetylase in control of the cyclin D1 promoter (32,33). These observations suggest that there may be several plausible mechanisms by which ARA54 modulates cyclin D1 gene expression. Firstly, ARA54 may interact with the promoter of the cyclin D1 gene as a direct activator or a co-activator that is recruited by another activator including the proteins mentioned above. Secondly, ARA54 may enhance the expression of a positive regulator of cyclin D1 gene transcription. Thirdly, ARA54 might ubiquitinate transcriptional repressors such as Tob1 and Jumonji for proteasomal degradation; it has been suggested that ARA54 is an E3 ligase due to the presence of a RING (really interesting new gene) finger domain and an auto-ubiquitination activity, although a substrate for ARA54 has not yet been identified (26,34). Recently, we reported that Skp2 interacts with Tob1 to promote its ubiquitin-dependent degradation, which leads to an increase in cyclin D1 expression (35). As with Skp2, it is plausible that ARA54 may induce cyclin D1 expression by promoting the ubiquitin-dependent degradation of a transcriptional repressor of cyclin D1 in the G1 phase. And finally, ARA54 may associate with a histone acetyltransferase, deacetylase, methylase or a binding protein to regulate histone remodeling and transcription of the cyclin D1 gene.

To date, little is known about the effect of ARA54 over-expression without androgen stimuli. We therefore examined the effect of ARA54 over-expression in a transient reporter assay using cyclin D1–luciferase reporter plasmids in several cell lines in the absence of androgen. However, we observed only a slight induction by ARA54 in some conditions (data not shown). This result may support one of our hypotheses that ARA54 functions as a co-activator or modulates acetylation/methylation, which is involved in epigenetic regulation of cyclin D1 expression. In this context, induction of cyclin D1 reporter gene expression by ARA54 over-expression might require other activators and/or functional chromatin. Further studies are needed to elucidate the precise mechanisms of transcriptional control of the cyclin D1 promoter via ARA54.

The expression of ARA54 mRNA in clinical colon cancer samples correlated with cyclin D1 expression (Figure 6). This observation suggests that in addition to its role in cyclin D1 transcription in cultured cells, over-expression of ARA54 is biologically important in human cancers. However, advanced analyses such as in-situ hybridization or immunohistochemistry are necessary to exclude the effect of stromal or inflammatory cells in our study using whole epithelial specimens because colon cancer is usually associated with inflammation not only at the carcinoma tissue itself but also in their peripheral non-neoplastic tissues. Further studies are needed to verify whether such a correlation exists between ARA54 expression and clinical outcome in cancers such as esophageal, breast and lung cancer, where over-expression of cyclin D1 has been reported to affect clinical course (18–20). Since our data indicate that siRNA knock-down of endogenous ARA54 results in suppression of growth of T98G cells (Figure 5), ARA54 may be useful as a therapeutic target molecule in cancers where cyclin D1 is over-expressed.

In conclusion, ARA54 is involved in transcriptional regulation of the cyclin D1 gene in several human cancer cells and mRNA expression of ARA54 correlates with that of cyclin D1 in human colon carcinomas. In addition, ARA54 might play an important role in cell cycle progression from the G1 to S phase.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Figure S1 can be found at http://carcin.oxfordjournals.org/.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
For Science Research from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (16021220 to M.K., 17590056 to K.K. and 17790910 to H.K.) and Center of Excellence Program of Hamamatsu University School of Medicine funded by the Ministry of Education, Science, Sports, Culture, and Technology (to M.K.).


   Acknowledgments
 
We would like to thank Sayuri Suzuki and Tomomi Abe for their technical assistance. We thank Takashi Takeuchi, Hayato Ihara, Toshiyuki Sakai and our laboratory staff for their helpful discussions.

Conflict of Interest Statement: None declared.


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

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Received February 21, 2007; revised April 25, 2007; accepted May 12, 2007.


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