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1 Departments of Urology and
2 Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With respect to the differential expression of CAR in urogential cancers, the mechanism(s) of down-regulation of the CAR gene is largely unknown. Recent studies by Kitazono et al. (11) and Hemminki et al. (12) demonstrate that a histone deacetylase (HDAC) inhibitor, FR901228 (13) , can increase CAR gene expression in several different cancer cell lines, indicating that the critical role of epigenetic control in CAR gene expression.
In this study, we cloned and characterized the promoter sequence of the human CAR gene. We have identified the core promoter sequence of CAR gene and further demonstrated that reduced CAR expression paralleled with the decreased CAR promoter activity in urogenital cancer cells. Thus, we believe that agent(s) capable of enhancing CAR promoter activity can be potential chemotherapeutic agents for urogenital cancer therapy and can also enhance the efficacy of gene therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Determination of the Transcriptional Starting Site of the CAR Gene Using 5' Rapid Amplification of cDNA Ends (RACE) Assay.
To determine the transcriptional starting site of the CAR gene, a modified 5' RACE assay, RNA ligase-mediated RACE (obtained from Ambion), was performed using three different primers, CAR6, CAR7, and CAR272 (Fig. 1A
and Table 1
). Total cellular RNA (10 µg) from RT-4 (a CAR-positive cell line) was subjected to this protocol according to the manufacturer’s instructions. PCR products were then run in a 2% NuSieve 3:1 agarose gel (Biowhittaker) to determine the length of the cDNAs.
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Clone 626 C16 was digested with HindIII to select fragments containing the 5' upstream of the untranslated region. On the basis of the predicated HindIII fragments from the gene sequence, two fragments were subcloned into pBluescript II SK vector and confirmed by DNA sequencing; one fragment was HF-740 (740 bp) and the other Clone HF4–8 (5309 bp; Fig. 2A
). To generate a variety of deletion mutants, Clone HF4–8 was further shortened using Erase-A-Base system (Promega) and several deletion fragments: 1196 (-1213 to -18), 1087 (-1213 to -127), 776 (-1213 to -438), 722 (-1213 to -492) and 382 (-1213 to -832) were generated (Fig. 2A)
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In each transfection experiment, 0.8 µg of DNA and 0.2 µg of pCMV-ß-gal DNA were added into each cell line (3 x 105 cells/well) using LipofectAMINE Plus (Invitrogen). Forty-eight hours after transfection, cells were washed twice with cold PBS and lysed with 200 µl of Reporter Lysis buffer (Promega). Cell lysate was collected after twice freeze-thawing and centrifugation; 20 µl of the supernatant were subjected to luciferase assay. All experiments were repeated at least three times in triplicate. The relative reporter gene activity was determined by normalizing the luciferase activity with the ß-gal activity. For FR9011228 treatment, the agent was added into cells 24 h after transfection and incubated for 24 h before harvesting for luciferase activity assay.
Determination of CAR mRNA Expression Using Real-Time Reverse-Transcription PCR Assay.
Cells (5 x 105) were plated in a p-100 dish 24 h before the treatment of FR901228. Cells were replaced with fresh medium containing various concentrations of FR901228. After 72 h of drug treatment, total cellular RNA was extracted with RNA-Bee reagent (Tel-Test, Houston, TX). Total cellular RNA (3.2 µg) was subjected to reverse transcription with Superscript II (Invitrogen) with 100 ng random hexamer (Invitrogen). The first strand of cDNA (2.5 µl) was further amplified by a real-time PCR using primerd CAR3 and CAR4 (Table 1)
. A 40-µl PCR reaction was carried out in a iCycler thermal cycler (Bio-Rad) using 1:100,000 of SyBr-Green (FMC Bioproducts) and Platinum Quantitative PCR SuperMix-UDG mix (Invitrogen), with a denaturing step at 95°C for 2 min, followed by 35 cycles of amplification with 92°C for 15 s, 55°C for 30 s, and 72°C for 2 min, and then an extension step at 72°C for 7 min. The glyceraldehyde-3-phosphate dehydrogenase cDNA [G3P4 (5'-AGTGAGCTTCCCGTTCAAC-3') and G3P7 (5'-GAAGGTGAAGGTCGGAGTCAACG-3')] was used as the internal control. All experiments were repeated twice in duplicate. Fold of induction of CAR mRNA was determined by normalizing the copy number of CAR cDNA with the copy number of glyceraldehyde-3-phosphate dehydrogenase cDNA of each sample.
Determination of the Association of Acetylated Histone with CAR Gene Promoter Using Chromatin Immunoprecipitation (ChIP) Assay.
A ChIP assay was performed to detect the effect of FR901228 on the association of acetylated histone with the promoter region of the CAR gene using an anti-acetyl-histone H4 antibody (Upstate Biotechnology). Cells were plated in a p-100 dish 24 h before FR901228 treatment. Seventy-two hours after treatment, an equal number of cells from either with or without FR901228 treatment were subjected to the ChIP assay as described previously (15)
. DNA fragments were further purified with phenol-chloroform and subjected to PCR reaction [98°C (3 min), 29 cycles of 98°C (30 s), 62°C (30 s), and 72°C (1 min), then 72°C (7 min)] using 3 µl of purified DNA and 1x of ThermalAce DNA polymerase (Invitrogen) with a primer set, CAR5024F and CAR5208R (Table 1)
, in a 25-µl reaction volume.
Bisulfite Genomic Sequencing.
High molecular-weight genomic DNA was obtained from the indicated cell lines and subjected to bisulfite modification (15)
. Briefly, 1–2 µg (5–10 µl) of genomic DNA were denatured by NaOH (final concentration, 0.2 M), 30 µl of 10 mM hydroquinone (Sigma), and 520 µl of 3 M sodium bisulfite (Sigma; pH 5) and incubated at 50°C for 16 h. Modified samples were purified using Wizard DNA Clean-Up System desalting columns (Promega), followed by ethanol precipitation. Bisulfite-modified DNA (100 ng) was PCR amplified with MethCAR2 and MethCAR4 primers (Table 1)
in a 25-µl reaction mixture. A hot start was performed (5 min, 95°C) by adding 0.5 units of HotStar TaqDNA polymerase (Qiagen). The PCR products were cloned into TA cloning vector pCR2.1-TOPO (Invitrogen). Five individual clones were sequenced using reverse and forward M13 primers.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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169 kb) containing the entire CAR gene was confirmed by Southern blot and DNA sequencing. We further determined the transcriptional starting site of the CAR gene by performing a 5'-RACE experiment. As shown in Fig. 1A
, three primers taken from three different locations of CAR cDNA were used, and all of the PCR products depicted the same starting point, that is, 150 bp upstream from the ATG site (Fig. 1B)
. In Clone 626C16, we estimated 30 kb of DNA sequence upstream from the ATG site. Also, the first intron in this gene is quite large and is estimated to be 34 kb in length. Using the NIH promoter search Web site,3
we were able to predict the potential promoter sequence of the CAR gene located at -470 to -719 (Fig. 1B)
upstream from the ATG site.
Determination of CAR Gene Promoter in Urogenital Cancer Cell Lines.
The promoter activity of the CAR gene was determined from the deletion mutants generated from Clone HF4–8 and HF740 (Fig. 2A)
. As shown in Fig. 2, B–D
, the reporter gene activity increased in all three cell lines when the sequence from -18 to -127 was deleted, suggesting that a putative silencer may be present. We also noticed that the reporter gene activity diminished dramatically when the deletion sequence extended beyond -492. Therefore, we believed that the promoter region of the CAR gene should be within this region. To further confirm this data, we generated two clones (pGL3–56 and pGL3–186) and demonstrated that only pGL3–186 expressed the same reporter gene activity as pGL3–776 in TCC cells (Fig. 2B)
. Noticeably, the similar pattern of reporter gene activity from each deletion mutant was observed in two other cell lines tested in this study (Fig. 2, C and D)
. We, therefore, concluded that the promoter region of the CAR gene was located between -585 and -400.
In this experiment, three cancer cell lines expressing different CAR levels were used. From our previous studies (7
, 8)
, the levels of CAR expression are as follows: 253J > PC3 > TCC. Data from the reporter gene assay clearly demonstrated that the CAR promoter activity in 253J cells was higher than in the other two cell lines tested (Fig. 2, B–D)
. We also noticed a good correlation between the percentage of CAR-positive cells (Table 2)
and the luciferase activity of pGL3–186 from each cell line (Fig. 2)
. Taken together, our data indicated that differential expression of CAR levels among urogenital cancer cell lines correlated with the promoter activity of CAR gene.
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; the total number of methylated CpG islands is very similar between CAR-positive (RT-4, 253J, SWBC, PC-3) and CAR-negative (T24 and TCC) cells. Also, we failed to detect any induction of CAR gene expression in cells treated with various concentrations of DNA hypomethylation agent (i.e., 5'-aza-2'-deoxycitidine) using real-time reverse-transcription PCR (Table 3)
. In contrast, under the same treatment condition, 5'-aza-2'-deoxycitidine was able to induce a positive control gene (i.e., hDAP1/2) expression, recently reported by us (15)
, in these cells. These data indicated that DNA methylation did not play a role in regulating the CAR gene promoter in urogenital cancer.
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demonstrated that FR901228 could induce CAR expression in PC-3, TCC, and T24 cells in a dose-dependent manner. The fold of induction ranged from 15- to 40-fold; it appeared that the higher induction seen in both T24 and PC-3 cells might be attributable to a lower basal CAR level in both cell lines. Noticeably, TCC cells were the most sensitive to FR901228 treatment among three cell lines; the peak of induction was detected at a lower concentration because of the apparent cytotoxicity induced by high concentrations of FR901228 (data not shown).
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, the highest luciferase reporter gene activity of pGL3–186 was observed in cells treated with FR901228, indicating that histone acetylation has a direct impact on the CAR gene promoter in urogenital cancer cells. To establish the correlation between the status of histone acetylation and CAR expression in cells under the routine culture condition, nuclei from the cell lines with different CAR expression were isolated and subjected to the ChIP assay. Our data (Fig. 6A)
demonstrated that the association between acetylated histone H4 and core promoter region of the CAR gene was detectable in the cell line with higher CAR expression, such as 253J (6
, 7)
, rather than in other cell lines expressing lower levels of CAR, indicating that the chromatin structure surrounding the CAR gene promoter maintains an "open" form in 253J cells. Thus, the association of acetylated histone H4 with the CAR promoter in 253J cells confirmed the constitutive expression of the CAR gene in 253J cell line. Nevertheless, the presence of FR901228 enhanced the association of acetylated histone H4 with the CAR gene promoter in TCC, T24, and PC-3 cells (Fig. 6B)
, indicating that the underlying mechanism of CAR gene regulation is via epigenetic control. Taken together, we believe that histone acetylation plays a critical role in modulating CAR gene expression in urogenital cancer cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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vß3 or vß5 (17)
. From many recent studies, CAR appears to be a high-affinity receptor for adenovirus 2 and 5 (1
, 2)
. Nevertheless, CAR also has other physiological function than as a virus receptor. For example, CAR is a typical cell-adhesion molecule with a homophilic interaction (1
, 2
, 7
, 8)
, and it colocalizes with ZO-1 in the tight junction of polarized cell (18)
, suggesting that CAR may be involved in the process of cell differentiation. In addition, recent data reported by Walter et al. (19)
indicate that the basolateral localization and cell-adhesive property of CAR protein in airway epithelium act as a barrier for preventing the spreading of newly synthesized virus into local environment. Taken together, the presence and the unique localization of CAR in each cell type could affect virus susceptibility of host cell. CAR also plays a critical physiological role in differentiated cells. From phenotypic changes in cancer cells, it is believed that cancer may undergo cell dedifferentiation. Data from our studies (6, 7, 8) further indicate that down-regulation of CAR gene expression is often seen in both prostate and bladder cancer cell lines. Thus, increased CAR levels in these cell lines can inhibit their growth both in vitro and in vivo. Using immunostaining technique, two recent studies (9 , 10) also reported that decreased CAR expression in clinical specimens of either prostate or bladder cancer. Taken together, these data support the notion that loss of CAR expression is associated with dedifferentiated phenotypes of urogenital cancers.
Obviously, the decreased CAR expression in urogenital cancers may also impose an obstacle for adenovirus based gene therapy. To circumvent this obstacle, one could change virus tropism by altering the fiber protein of virus (20) or increase endogenous CAR expression by gene transfection. Several recent findings (11 , 12 , 21 , 22) also indicated that some HDAC inhibitors could potentially turn on endogenous CAR gene expression in cells. Our studies show that increased CAR mRNA expression leads to an elevated CAR protein accumulation on the cell membrane and results in higher virus sensitivity (6, 7, 8) . These data prompted us to study the regulation of the human CAR gene in urogenital cancer cells.
Of the 17 bacterial artificial chromosomal clones, 59% are CAR pseudogenes, suggesting that CAR exists as a multigene family with many pseudogenes scattered around the human chromosome. Clone 626 C16 (169 kb) contains all of the introns, exons, and 5'-upstream regulatory sequences confirmed by PCR and 5'-RACE assay. This clone also matched GenBank sequences derived from chromosome 21 where the CAR gene has been mapped in previous study (23)
. This gene does not have a typical TATA box (Fig. 1B)
; however, we were still able to define the promoter region (186 bp) of the human CAR gene located at -400 to -585 bp from the translational initiation site. The promoter activity of this region correlated with the CAR levels in each cell line tested (Table 2)
. Moreover, there are unique trans-factor binding sites (such as E2F, Sp1) located upstream from the promoter region (Fig. 1B)
, which may be critical in modulating CAR gene expression in different cell types. For example, two groups report that mitogen-activated protein kinase kinase inhibitor (U0126) can up-regulate CAR expression in colon and pancreatic cancers (24)
; however, transforming growth factor ß and dexamethasone suppress CAR expression in HeLa and glioblastoma cell line (U87MG; Ref. 25
).
Decreased CAR is often observed in urogenital cancer cell lines and specimens (7, 8, 9, 10)
, indicating that CAR could be a potential tumor suppressor. These observations also raise a major concern for the patients who undergo adenovirus-based gene therapy. The purpose of this study was to evaluate whether or not down-regulation of CAR in urogenital cancers is attributable to epigenetic control. We showed that the association of acetylated histone protein with CAR promoter DNA correlated with the basal levels of CAR expression (Fig. 6)
. Up-regulation of the CAR gene can be observed in cells treated HDAC inhibitor (Figs. 4
and 5
) that blocks the deacetylation process of histone proteins. Although, the promoter region of CAR contains many CpG islands, DNA methylation does not seem to play any role in controlling CAR gene expression (Fig. 3
and Table 3
). Thus, these data clearly indicate that changes in chromatin structure of CAR promoter enhance the expression of CAR in urogenital cancer cells.
It is known that histone acetylation transferase unfolds nucleosome structure and leads to gene activation (26) ; histone acetylation transferase has also been identified as a transcriptional activator (27) . In contrast, HADC plays a negative feedback role in gene activation by deacetylating histone. Our recent data (16) show that HDAC inhibitors such as FR901228 and trichostatin A (TSA) can enhance not only CAR mRNA levels but also virus-mediated gene delivery in PC-3 and TCC cells. FR901228 appears to be more potent in inducing CAR gene expression than TSA. FR901228, a bicyclical depsipeptide isolated from Chromobacterium violaceum, can induce morphological reversion of H-ras-transformed NIH3T3 (28) . Currently, this agent has been evaluated in lung cancer patients in a Phase II trial.4 Using this agent, we were able to demonstrate that the status of the histone acetylation associated with the CAR gene significantly impacts on its gene transcription in cells expressing low levels of CAR. These data indicate that epigenetic control of the CAR gene modulates CAR expression in human urogenital cancer cells.
In summary, we identified the core promoter sequence from the human CAR gene and showed that this promoter activity could be enhanced by HDAC inhibitors but not by DNA hypomethylation agents in urogenital cancer cells. Thus, the human CAR gene is highly inducible in cancer cells and the promoter sequence identified in this study can be used as a screening system for searching many other potential inducers. It is likely that the increased CAR expression in target cells could reduce virus dosage and further enhance the therapeutic efficacy of gene therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Notes: Dr. Okegawa is currently in the Department of Urology, Kyroin University School of Medicine, Shinkawa, Mitaka, Tokyo, Japan.
Requests for reprints: Jer-Tsong Hsieh, Department of Urology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9110. E-mail: JT.Hsieh{at}UTSouthwestern.edu
3 bimas.dcrt.nih.gov:80/molbio/proscan. 
Received 4/ 7/03. Revised 9/15/03. Accepted 9/23/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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V and coxsackie adenovirus receptor expression in clinical bladder cancer. Urology, 60: 531-536, 2002.[Medline]
vß3 and vß5 promote adenovirus internalization but not virus attachment. Cell, 73: 309-319, 1993.[Medline]
, and TGFß. Gene Ther., 10: 198-205, 2003.[Medline]
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, unmethylated CpG; 
, methylated CpG.
