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Cancer Research 67, 85, January 1, 2007. doi: 10.1158/0008-5472.CAN-06-2635
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Induction of Human Arylamine N-Acetyltransferase Type I by Androgens in Human Prostate Cancer Cells

Neville J. Butcher, Natasha L. Tetlow, Catherine Cheung, Gysell M. Broadhurst and Rodney F. Minchin

School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland, Australia

Requests for reprints: Neville J. Butcher, School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia. Phone: 617-3365-2684; Fax: 617-3365-1766; E-mail: n.butcher{at}uq.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human arylamine N-acetyltransferases (NAT) bioactivate arylamine and heterocyclic amine carcinogens present in red meat and tobacco products. As a result, factors that regulate expression of NATs have the potential to modulate cancer risk in individuals exposed to these classes of carcinogens. Because epidemiologic studies have implicated well-done meat consumption as a risk factor for prostate cancer, we have investigated the effects of androgens on the expression of arylamine N-acetyltransferase type I (NAT1). We show that NAT1 activity is induced by R1881 in androgen receptor (AR)–positive prostate lines 22Rv1 and LNCaP, but not in the AR-negative PC-3, HK-293, or HeLa cells. The effect of R1881 was dose dependent, with an EC50 for R1881 of 1.6 nmol/L. Androgen up-regulation of NAT1 was prevented by the AR antagonist flutamide. Real-time PCR showed a significant increase in NAT1 mRNA levels for R1881-treated cells (6.60 ± 0.80) compared with vehicle-treated controls (1.53 ± 0.17), which was not due to a change in mRNA stability. The increase in NAT1 mRNA was attenuated by concurrent cycloheximide treatment, suggesting that the effect of R1881 may not be by direct transcriptional activation of NAT1. The dominant NAT1 transcript present following androgen treatment was type IIA, indicating transcriptional activation from the major NAT1 promoter P1. A series of luciferase reporter deletions mapped the androgen responsive motifs to a 157-bp region of P1 located 745 bases upstream of the first exon. These results show that human NAT1 is induced by androgens, which may have implications for cancer risk in individuals. [Cancer Res 2007;67(1):85–92]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is one of the most commonly diagnosed cancers in men from Western countries (1, 2). Whereas the etiology of prostate cancer is not completely understood, there is compelling epidemiologic evidence implicating intake of dietary components such as animal fat, meat, and dairy products as a significant risk factor for the disease (3–5). Studies into the genetic susceptibility for prostate cancer have identified a number of genes that either increase or decrease risk. These include genes that encode proteins involved in androgen metabolism and signaling (6, 7) and the metabolic activation/detoxification of carcinogens (7).

Of particular interest are the arylamine N-acetyltransferases (NAT; EC 2.3.1.5), which catalyze the bioactivation and detoxification of many arylamine and heterocyclic amine carcinogens. Human exposure to these carcinogens occurs during consumption of meat and through the use of tobacco products. Both classes of carcinogens induce prostate tumors in animal models (8, 9). Recently, it has been reported that human prostate tissue, as well as prostate cells in culture, contains measurable levels of the enzymes required for the activation of arylamine carcinogens (10–14).

There are two functional NATs in humans (NAT1 and NAT2), and both exhibit genetic polymorphisms that affect enzyme activity (15). One allele of the NAT1 gene, NAT1*10, has been associated with an increased risk for a number of different cancers, including cancer of the prostate (16–18). In some studies, this allele leads to an elevation in enzyme activity, in particular tissues such as the colon and bladder (19). However, the NATs are influenced by environmental factors that can both induce and inhibit cellular activity (20, 21). An understanding of how these genes are regulated by the environment is therefore important for deciphering their role in diseases.

Several studies have shown that mouse NAT2 (which is homologous to human NAT1) expression in the kidney exhibits a sexual dimorphism (22, 23). Smolen et al. (22) showed that the developmental difference in mouse kidney NAT2 activity was due to an increase in testosterone. Androgenic modulation of NAT2 was caused by an increase in mRNA levels and subsequent elevation in NAT2 protein content.

An androgen responsive element has been identified in an intronic sequence of the mouse NAT2 gene (24). However, the human homologue (NAT1) seems to have a different gene structure and promoter elements (25, 26) than that described for the mouse NAT2 gene (27). We were therefore interested in whether the human NAT1 gene is regulated by androgens. Using human prostate cancer cells that were androgen receptor (AR) positive or AR negative, we found in the present study that NAT1 activity was induced by AR activation as a result of increased NAT1 gene expression. However, activated AR did not seem to directly regulate NAT1. We mapped a 157-bp region of NAT1 promoter 1 (P1) that contains the motif responsible for androgen up-regulation of NAT1. In addition, we show that NAT1 expression in human prostate tissue is greatest in epithelial cells, the cell type where AR is predominantly expressed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Human prostate cancer cell lines LNCaP, 22Rv1, and PC-3 were obtained from the American Type Culture Collection (Manassas, VA), as were the cervical cancer cell line HeLa and the human embryonic kidney cell line 293. Cells were cultured in DMEM supplemented with 5% fetal bovine serum (FBS) and penicillin/streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2 in air. For androgen-free conditions, cells were cultured in phenol red–free DMEM supplemented with 5% dextran-coated charcoal-stripped FBS. Cells were treated with up to 100 nmol/L R1881 (Perkin-Elmer, Melbourne, Victoria, Australia) or vehicle (DMSO) for up to 48 h under androgen-free conditions. AR antagonists bicalutamide (Toronto Research Chemicals, North York, ON, Canada) and flutamide (Sigma, Castle Hill, New South Wales, Australia) were used at 10 µmol/L. Actinomycin D (Sigma) was used at 5 µg/mL and cycloheximide (Sigma) at 20 µg/mL.

Assay of NAT1 activity. Cells were washed twice with PBS, resuspended in buffer [20 mmol/L Tris (pH 7.4), 1 mmol/L EDTA, and 1 mmol/L DTT], and lysed on ice by sonication. Cell lysates were then centrifuged at 4°C and the supernatant assayed for NAT1 activity as previously described (21). Protein concentrations were determined by the method of Bradford (28).

Western blot analysis. Cell lysates containing equal amounts of protein were electrophoresed on 12% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with a polyclonal NAT1-specific antibody (29) as previously described (21).

DNA cloning of NAT1 P1. A 4,228-bp fragment of NAT1 P1 was amplified from 22Rv1 genomic DNA by PCR using primers and conditions previously described (25). This fragment was digested with HindIII and cloned into HindIII-digested, shrimp alkaline phosphatase–treated pGL3-enhancer (Promega, Annandale, New South Wales, Australia), yielding pGL3-ex4-3657, where the number indicates base pairs upstream of exon 4. NAT1 P1 construct pGL3-ex4-257 was made by PCR using pGL3-ex4-3657 as template and forward primer FP4 (see Supplementary Table S1), which contains an artificial KpnI restriction site, together with the reverse primer used to PCR the 4,228-bp fragment. This PCR fragment was digested with KpnI and HindIII and cloned into the same sites of pGL3-enhancer. A series of deletion constructs was made by PCR using forward primers FP1, FP2, FP3, FP5, FP6, and FP7 (see Supplementary Table S1) together with reverse primer RP4, yielding pGL3-ex4-2972, pGL3-ex4-1865, pGL3-ex4-902, pGL3-ex4-745, pGL3-ex4-587, and pGL3-ex4-438, respectively. All primers contained KpnI sites and PCR fragments were cloned into KpnI-digested, shrimp alkaline phosphatase–treated pGL3-ex4-257. All clones were verified by DNA sequencing.

Transient transfection. 22Rv1 cells were seeded into 24-well plates at a density of 2.5 x 105 per well and allowed to adhere overnight. Cells were then transfected with 200 ng of the androgen-responsive probasin promoter luciferase construct pARR3-TK-Luc (ref. 30; courtesy of Prof. Peter Leedman, Western Australian Institute for Medical Research, Perth, Western Australia, Australia) using LipofectAMINE 2000 (Invitrogen, Melbourne, Victoria, Australia) according to the manufacturer's instructions, and incubated for 24 h under androgen-free conditions. Cells were then treated with R1881 and/or AR antagonists under androgen-free conditions for up to 24 h, washed twice with PBS, and prepared for luciferase assay. PC-3 cells were transfected with the AR expression vector pCMV-AR3.1 (ref. 31; courtesy of Wayne Tilley, Hanson Institute, Adelaide, New South Wales, Australia) and pARR3-TK-Luc by electroporation using a Bio-Rad GenePulser Xcell (280 V, 200 µF, 50 {Omega}; Bio-Rad, Hercules, CA). Briefly, 15-µg pCMV-AR3.1 and 5-µg pARR3-TK-Luc were added to 0.4-mL cells (1 x 107/mL) in PBS and electroporated in a 2-mm cuvette. Electroporated cells were then diluted in DMEM (5% FBS) and seeded at a density of 2.5 x 105/mL in 24-well plates. The cells were allowed to adhere overnight and then were treated with either 100 nmol/L R1881 or vehicle under androgen-free conditions for 24 h. Cells were washed twice with PBS and cell lysates prepared for either NAT1 assay or luciferase assay. For promoter studies, cells were seeded at a density of 2.5 x 105/mL in 24-well plates and allowed to adhere overnight. Cells were transfected with 1 µg DNA using LipofectAMINE 2000 and incubated for 24 h, after which they were treated with vehicle or 100 nmol/L R1881 under androgen-free conditions for a further 24 h. Cells were then washed with PBS and lysates prepared for luciferase assay. The transfection efficiency of electroporated cells was determined by transfection with 20-µg pEGFP (Scientifix, Cheltenham, Victoria, Australia). Transfected cells were determined by flow cytometry 48 h after transfection.

Luciferase assay. Cells were lysed in passive lysis buffer and firefly luciferase activity was measured using a luciferase assay system (Promega) as outlined in the manufacturer's instructions. Luciferase activities were normalized to total protein.

Extraction of total RNA and reverse transcription. Monolayer cultures were washed with PBS and total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. RNA was resuspended in RNase-free water and reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen) and oligo(dT)15 primer (Promega) as outlined in the manufacturer's instructions. For each RNA sample, a reaction lacking reverse transcriptase was done and used in subsequent PCR to ensure no DNA contamination.

Identification of NAT1 transcripts by reverse transcription-PCR. First-strand cDNA was amplified by PCR using forward primers designed to detect each of the exonic sequences known to exist in the 5'-untranslated region (5'-UTR) of NAT1, in combination with a common reverse primer located in the NAT1 coding sequence. The primers and PCR conditions have previously been reported (26).

Quantitative real-time PCR. Expression levels of NAT1 mRNA were determined using the iCycler IQ Real-time PCR Detection System (Bio-Rad). First-strand cDNA was amplified using specific primers for NAT1 or ß-actin (see Supplementary Table S1). Reactions contained iQ Supermix (Bio-Rad), 6 pmol of each primer, and 2 µL of cDNA mix in a total volume of 25 µL. Samples were amplified using the following conditions: initial denaturation at 95°C for 90 s, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing/extension at 51°C for 60 s. A melting curve was obtained to verify specificity of the PCR. Samples were analyzed using the standard curve method. Standard curves were generated for NAT1 and ß-actin using 10-fold serial dilutions of cDNA from controls. Expression values for samples were then determined from the standard curves and NAT1 values were normalized to ß-actin.

Immunohistochemistry. Formalin-fixed, paraffin-embedded normal human prostate tissue was prepared using conventional histologic methods. Serial tissue sections (6 µm) were deparaffinized in xylene, rehydrated through graded alcohol, and rinsed in distilled water. Antigen retrieval was done in 10 mmol/L citrate buffer (pH 3.0) in a microwave oven (AR staining) or by proteinase K treatment (NAT1 staining). Endogenous peroxidase activity was blocked with 3% H2O2 in water for 5 min. Slides were blocked with rabbit immunoglobulin G (IgG; 50 µg/mL) for AR staining or with rabbit preimmune serum (1:100) for NAT1 staining. Rabbit IgG and preimmune serum epitopes were blocked by treatment with Alexa Fluor 488 goat anti-rabbit IgG (20 µg/mL; BioScientific, Gymea, New South Wales, Australia). Slides were then treated with affinity-purified polyclonal rabbit anti-human AR antibody (50 µg/mL; AB561P, Chemicon, Boronia, Victoria, Australia) or human NAT1 rabbit antiserum (1:100; produced as described in ref. 29) at 4°C overnight. After washing, slides were incubated with Dako EnVision Plus HRP (Dako, Botany, New South Wales, Australia) for 30 min. Diaminobenzidine Plus was used as the chromogen. Nuclei were lightly counterstained with hematoxylin and sections were dehydrated and permanently mounted.

Data analysis and statistics. Data are expressed as mean ± SE. Statistical comparisons between different treatments were assessed by Student's t tests or one-way ANOVA assuming significance at P ≤ 0.05. EC50 values for R1881 were estimated by fitting a standard sigmoidal dose-response model to the data using Prism 4 (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
R1881 induced NAT1 activity in AR-positive prostate cancer cells. Initially, the effect of the androgen R1881 on NAT1 activity was investigated in the AR-positive prostate cell lines 22Rv1 and LNCaP as well as the AR-negative cell lines PC-3 (prostate), HK-293 (kidney), and HeLa (cervical). Both AR-positive lines showed a higher basal level of NAT1 activity (46–51 nmol/min/mg protein) compared with the AR-negative lines (<15 nmol/min/mg protein). Following treatment with 100 nmol/L R1881 for 24 h, there was a significant increase in NAT1 activity in both 22Rv1 (2.5-fold) and LNCaP (1.5-fold) cells but not in PC-3, HK-293, or HeLa cells (Fig. 1A ). The effect of androgen was dose dependent (Fig. 1B, closed symbols). In 22Rv1 cells, a maximum NAT1 activity of 140 nmol/min/mg protein was seen at R1881 concentrations >10 nmol/L. The estimated EC50 for R1881 was 1.6 nmol/L (95% confidence interval, 1.3–2.0 nmol/L). When 22Rv1 cells were transfected with the AR reporter construct pARR3-TK-Luc, the EC50 for R1881 induction of luciferase was significantly less than that for induction of NAT1 (54 pmol/L; Fig. 1B, open symbols), but similar to the reporter EC50 for this drug in other AR-dependent expression systems (32).


Figure 1
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  		Figure 1. Androgen induces NAT1 activity in AR-positive cell lines. A, AR-positive 22Rv1 and LNCaP cells and AR-negative PC-3, HK-293, and HeLa cells were treated with 100 nmol/L R1881 (closed columns) or vehicle (open columns) for 24 h under androgen-free conditions. Cells were then lysed and assayed for NAT1 activity. *, P < 0.05, versus untreated controls. B, 22Rv1 cells were transfected with the androgen-responsive reporter pARR3-TK-Luc and, 24 h later, treated with various concentrations of R1881 for 24 h under androgen-free conditions. Cells were then lysed and assayed for NAT1 (bullet) and luciferase ({circ}) activities. C, induction of NAT1 activity was prevented by AR antagonists. 22Rv1 cells were transfected with pARR3-TK-Luc and, 24 h later, treated with 0.3 nmol/L R1881 for 24 h under androgen-free conditions in the absence or presence of either 10 µmol/L flutamide or 10 µmol/L bicalutamide. Cell lysates were prepared and assayed for NAT1 (closed columns) and luciferase (open columns) activities. D, 22Rv1 cells were incubated under androgen-free conditions in the absence or presence of 10 µmol/L bicalutamide for up to 48 h (open columns). Some cells were treated with 0.3 nmol/L R1881 for 24 h in the absence or presence of 10 µmol/L bicalutamide (closed columns). Cell lysates were prepared and assayed for NAT1 activity.

 
To determine whether the induction of NAT1 by R1881 involved the AR, 22Rv1 cells were treated with the drug (0.3 nmol/L) in the absence or presence of the AR competitive antagonists flutamide or bicalutamide. Both antagonists completely inhibited the effects of R1881 on NAT1 activity, confirming their inhibitory action on AR in these studies (Fig. 1C, closed bars). Moreover, in studies under identical conditions with the AR reporter construct, both antagonists also reversed the effects of R1881 (Fig. 1C, open bars). These results indicate that the induction of NAT1 by R1881 involved activation of the AR.

Because the basal level of NAT1 activity was significantly higher in the AR-positive cells (see above), an experiment was conducted to determine if this could be explained by the endogenous androgens found in bovine serum used to culture the cells. The AR-positive line 22Rv1 was grown for up to 48 h in medium supplemented with charcoal-stripped serum and devoid of phenol red to reduce endogenous androgenic activity (33). In addition, bicalutamide was added to further ensure inhibition of any endogenous androgen. Figure 1D shows that, under these conditions, the AR antagonist could completely inhibit the effect of R1881 (closed bars, positive control). However, the activity of NAT1 in cells not treated with R1881 did not change over 48 h in the presence of the antagonist. Because we have previously shown that the half-life of NAT1 is ~22 h in human cells (34), we interpreted these results to suggest that the high basal level of NAT1 activity in the AR-positive lines was not due to endogenous androgen activation of NAT1 expression.

Androgens increase NAT1 transcription. Western blot analysis of 22Rv1 cells treated with 100 nmol/L R1881 showed an increase in cellular NAT1 protein compared with vehicle-treated controls (Fig. 2A ). Quantification by densitometry (Quantity One software, Bio-Rad) indicated a 2.2-fold increase in NAT1 protein content, which was similar to the observed increase in NAT1 activity (Fig. 1A). To determine if the increase in NAT1 protein was a result of elevated NAT1 mRNA levels, NAT1 mRNA from 22Rv1 cells treated with 100 nmol/L R1881 was quantified by real-time PCR. A significant increase in NAT1 mRNA for R1881-treated cells (6.60 ± 0.80) compared with vehicle-treated controls (1.53 ± 0.17) was seen (Fig. 2B).


Figure 2
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  		Figure 2. NAT1 mRNA and protein expression are up-regulated by androgen. A, Western blot with a NAT1-specific antibody on lysates from 22Rv1 cells treated with 100 nmol/L R1881, compared with vehicle-treated control cells. Arrow, NAT1 at 33 kDa. B, real-time PCR using RNA extracted from 22Rv1 cells treated with 100 nmol/L R1881 or vehicle for 24 h under androgen-free conditions. RNA was reverse transcribed and amplified with primers for NAT1 and ß-actin. Results were normalized to ß-actin (n = 8). C, effect of R1881 on NAT1 mRNA stability. 22Rv1 cells were treated with 100 nmol/L R1881 (bullet) or vehicle ({circ}) for 24 h under androgen-free conditions. Cells were then treated with 5 µg/mL actinomycin D for up to 6 h. RNA was extracted and reverse transcribed and NAT1 mRNA levels were quantified by real-time PCR as outlined in Materials and Methods. NAT1 mRNA levels were normalized to ß-actin. Half-lives were calculated using a single exponential decay curve fit (Prism 4, GraphPad Software). D, ethidium bromide–stained agarose gel showing exons present in NAT1 transcripts following treatment of 22Rv1 cells with vehicle (left) or 100 nmol/L R1881 (right). Reverse-transcribed RNA was amplified by PCR using forward primers designed to detect the eight identified exons in the 5'-UTR of NAT1 and a common reverse primer located in the coding region. Amplification gave a series of products depending on the combination of exons. The different transcripts present can be deduced by the product sizes.

 
Because androgens have been shown to regulate gene expression in part by stabilizing mRNA (35), we determined the decay rate of NAT1 mRNA in the absence and presence of R1881 by real-time PCR of 22Rv1 cells when transcription was blocked with actinomycin D (Fig. 2C). In the absence of the drug, the half-life of NAT1 message was 3.49 ± 0.18 h, which was not significantly different from that in the presence of the drug (3.62 ± 0.14 h). Taken together, the above results suggest that R1881 increases NAT1 activity, at least in part, by an increase in gene transcription without affecting message stability.

NAT1 mRNA has previously been shown to consist of several splice variants that differ in the sequence of the 5'-UTR (25, 26). In addition, at least two independent promoters (P1 and P3 according to the nomenclature in ref. 25) have been identified that use markedly different transcriptional start sites. Amplification of the different splice variants as previously described (26) showed that the major splice variant in untreated 22Rv1 cells was type IIA (Fig. 2D, left), which consists of exons 4 and 8 in the 5'-UTR, together with exon 9, which contains the protein coding region. There also was little or no evidence of the minor transcripts type IIB (exons 4, 7, 8, and 9), type IID (exons 4, 6, 8, and 9), and type IIE (exons 4, 5, 6, 8, and 9) in untreated cells. In addition, there was no evidence for transcripts originating from P3. Following treatment with 100 nmol/L R1881, minor transcripts originating from P1 became evident but type IIA remained the most prominent. Again, there was no indication of transcripts originating from P3 (Fig. 2D, right). These results suggest that the increase in NAT1 gene transcription induced by R1881 is due to activation of the P1 promoter.

Androgen induction of NAT1 is an indirect effect. The marked difference in the EC50 for R1881 activation of NAT1 activity and activation of luciferase activity from the AR reporter construct suggested that the mode of activity of the drug may not be the same for each gene. To investigate this possibility further, the time course of R1881-dependent induction for NAT1 and the AR reporter construct pARR3-TK-Luc was determined in 22Rv1 cells. A significant increase in NAT1 activity was seen after 8-h treatment and induction continued up to 48 h (Fig. 3A, closed circles ). By contrast, luciferase activity increased after only 2 h of treatment and was maximal by 16 h (Fig. 3A, open circles). This difference in time course supported the notion that the activation of the two genes may not proceed by a common mechanism.


Figure 3
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  		Figure 3. Induction of NAT1 by androgen is an indirect effect. A, time course of R1881-induction of NAT1 and luciferase activities. 22Rv1 cells were transfected with pARR3-TK-Luc for 24 h and then treated with either vehicle or 100 nmol/L R1881 for various times up to 48 h. Cell lysates were prepared and NAT1 activity (bullet) or luciferase activity ({circ}) was measured. B, 22Rv1 cells were treated with vehicle, 10 µg/mL cycloheximide, 100 nmol/L R1881, or 100 nmol/L R1881 plus 10 µg/mL cycloheximide for 24 h. RNA was extracted and reverse transcribed, and then quantified by real-time PCR. Results were normalized to ß-actin.

 
If NAT1 transcription is directly up-regulated by androgen, then the lack of effect of R1881 in AR-negative cells should be overcome by transfection of these cells with an AR expression vector. However, when PC-3 cells were transiently transfected with pCMV-AR3.1, NAT1 activity was not affected by treatment with 100 nmol/L R1881 (Table 1 ), despite a >50% transfection efficiency. To show that the expression vector was functional under these conditions, PC-3 cells were cotransfected with pCMV-AR3.1 and the AR reporter construct pARR3-TK-Luc. Following treatment with R1881 (100 nmol/L), a significant increase in luciferase expression was observed (Table 1), indicating functional AR in these cells, which is similar to several other reports (36, 37).


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  		Table 1. Effect of AR expression in PC-3 cells on endogenous NAT1 activity

 
The lack of induction of NAT1 in AR-transfected PC-3 cells, along with the differences in dose and time kinetics compared with the AR reporter construct, suggested that the effect of R1881 may not be by direct transcriptional activation of the NAT1 gene. To test this possibility, cycloheximide inhibition of translation was used to determine whether protein synthesis was a necessary step in NAT1 mRNA induction. Figure 3B shows that R1881 increased NAT1 mRNA by ~6-fold compared with untreated cells, and that this induction was attenuated by concurrent treatment with cycloheximide. Therefore, NAT1 transcriptional activation by R1881 requires prior protein synthesis and suggests that the AR may induce a secondary transcription factor that functions at the NAT1 promoter.

Identification of a region of P1 that responds to androgen. Initially, the luciferase reporter construct pGL3-ex4-3657 (where the number indicates bp upstream of exon 4 and is the same as pGL3-4228 used in ref. 25) was transfected into 22Rv1 cells and then treated with either vehicle or 100 nmol/L R1881. The luciferase activity from androgen-treated cells was 2.6-fold higher than that of vehicle-treated cells (Fig. 4A ). Because the motif responsible for androgen up-regulation of NAT1 seemed to be in this region of P1, a series of deletion constructs was made (pGL3-ex4-2972, pGL3-ex4-1865, pGL3-ex4-902, and pGL3-ex4-257) to map this region further. These reporter constructs were transfected into 22Rv1 cells, which were then treated with either vehicle or R1881. Cells transfected with pGL3-ex4-2972, pGL3-ex4-1865, or pGL3-ex4-902 showed 2.6-, 2.6-, and 2.4-fold increases in luciferase activity, respectively (Fig. 4A). Cells transfected with pGL3-ex4-257 showed no evidence of R1881 induction. Therefore, the region responding to androgen was located between –902 and –257 bp, relative to the start of exon 4. To map the androgen-responsive site further, pGL3-ex4-745, pGL3-ex4-587, and pGL3-ex4-439 reporter constructs were made. Transfection into 22Rv1 cells and treatment with androgen revealed that none of the new deletion constructs responded to R1881 treatment (Fig. 4B). It was concluded that the 157-bp region between –902 and –745 bp upstream of exon 4 contains elements responsible for androgen up-regulation of NAT1. This region of the NAT1 gene is shown in Fig. 4C. Putative transcription factor binding sites are indicated on the sequence.


Figure 4
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  		Figure 4. Identification of a region of NAT1 P1 that is responsive to androgen. A, various lengths of P1 were inserted into the pGL3-enhancer vector and were transiently transfected into 22Rv1 cells. Cells were treated 24 h later with either 100 nmol/L R1881 (closed columns) or vehicle (open columns) for a further 24 h, and then lysed and luciferase activity was measured. B, the R1881-responsive region of NAT1 P1 identified above was fine mapped by creating further deletion constructs. C, sequence of the 157-bp NAT1 P1 region that contains the promoter elements responsible for androgen up-regulation of NAT1 activity. The sequence was examined using MatInspector (39) to identify putative transcription factor binding sites (underlined).

 
AR and NAT1 proteins are colocalized in human prostate. Immunohistochemistry was done on sequential human prostate tissue slices to determine if the expression of NAT1 protein was higher in those cell types that expressed AR. AR exhibited a typical luminal epithelial cell distribution, similar to that reported elsewhere (38). The majority of the intracellular AR protein was localized to the nuclei, although faint staining was evident in the extranuclear space as well (Fig. 5, top ). NAT1 was strongly localized to AR-positive epithelium and dispersed throughout the cell (Fig. 5, bottom). Some staining of nuclei as well as cytoplasmic bodies was also seen. Control IgG staining is shown in Fig. 5 (left).


Figure 5
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  		Figure 5. Colocalization of NAT1 and AR in human prostate tissue. AR was restricted to nuclei of the epithelial cells (top right). NAT1 expression was strongest in the epithelial cells and seemed to be localized both in the cytoplasm and nucleus (bottom right). Inset, staining of NAT1 in cytoplasmic bodies (arrowhead) and nuclei (arrows). Left, control IgG staining.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A sexual dimorphism has been described for mouse kidney NAT2 activity, with males having about a 2-fold higher activity than females (22). In the same study, castration reduced male kidney NAT2 activity to female levels, whereas testosterone replacement restored original levels of activity. This led the authors to suggest that androgen modulation of NAT2 acetylating capacity may be responsible for the observed higher susceptibility of male mice to arylamine-induced carcinogenesis. Characterization of the mouse NAT2 promoter identified a hormone response element that may be responsible for the androgenic regulation of NAT2 in mouse kidney (23). However, the hormone response element resides in an intronic sequence and was not shown to be functional in the context of the promoter. The above observations prompted us to investigate the possibility that human NAT1 expression was regulated by androgens.

The synthetic androgen R1881 induced NAT1 activity in the AR-positive prostate cell lines 22Rv1 and LNCaP in a time- and concentration-dependent manner. The induction of NAT1 was prevented by AR antagonists, indicating that AR activation was required. This is also supported by immunohistochemical data showing NAT1 protein staining to be greatest in the epithelial cells where AR is almost exclusively expressed in the prostate. The localization of NAT1 in human prostate tissue to the epithelium is similar to that reported elsewhere (11). The mechanism by which androgen increased NAT1 protein levels was primarily by increased transcription. There was a >4-fold increase in NAT1 transcript in the presence of androgen, and this was not a result of message stabilization as the mRNA decay rates in the absence and presence of androgen were not significantly different.

The human NAT1 gene gives rise to mRNA consisting of several different splice variants that differ only in the 5' noncoding sequence, and at least two independent promoter regions have been identified (25, 26). Amplification of the different mRNA splice variants from 22Rv1 cells treated with androgen showed that all detectable transcripts contained exon 4 and therefore originated from the P1 promoter. Using a reporter construct containing a 3,657-bp fragment upstream of exon 4, we were able to show that the motif responsible for androgen responsiveness was localized in this region. A series of deletion constructs then allowed the androgen responsive sequence to be mapped to a 157-bp sequence located between –745 and –902 bp, relative to the start of exon 4. Analysis of this sequence using MatInspector (39) identified 25 putative transcription factor binding sites. Of particular interest was the presence of a putative X-box motif, which binds the basic region leucine zipper transcription factor X-box binding protein-1 (XBP-1). XBP-1 is present in the prostate and has been shown to be up-regulated in prostate cancer (40). Close examination of the androgen-responsive sequence did not identify any AR binding elements.

When the AR-negative PC-3 cells were transiently transfected with AR, NAT1 expression was not affected by androgen treatment. This suggested that the androgen signaling pathway responsible for NAT1 up-regulation in LNCaP and 22Rv1 cells was not intact in these cells. In addition, the finding that cycloheximide abrogates androgen-mediated increases in NAT1 mRNA indicated that increased transcription of the NAT1 gene requires prior protein synthesis, possibly of an androgen-regulated transcription factor. Together, these data indicate that the activated AR does not directly bind to the NAT1 promoter to increase transcription but modulates the expression of a secondary transcription factor that regulates NAT1. The indirect nature of androgen up-regulation of NAT1 also is supported by the absence of an androgen response element in the region of P1 identified as androgen responsive. Interestingly, both AR-positive prostate cell lines that exhibited androgen-mediated up-regulation of NAT1 have unusually high basal expression levels, which is not due to androgen present in the serum used to culture the cells. This may be due to high basal expression of the transcription factor that directly activates NAT1 gene expression and is subject to androgen regulation.

The potential role of the arylamine NATs in the development of cancers through the bioactivation of procarcinogens is well established (41, 42). As such, tissue-specific factors that up-regulate expression of the NATs have the potential to enhance mutagenesis and promote cancer. The up-regulation of NAT1 by androgens may have functional significance in the prostate, as well as in the many other tissues that express AR.

In conclusion, the present study has shown androgen-dependent regulation of NAT1, which is mediated by increased transcriptional activity of the NAT1 gene. Identification of the precise mechanism that leads to this change in transcription is under investigation. Because the arylamine NATs are able to bioactivate a number of important human carcinogens (41), androgenic modulation may have implications for cancer risk in individuals.


   Acknowledgments
 
Grant support: Department of Veteran Affairs, Australia (DVA 303155) and National Health and Medical Research Foundation, Australia (NHMRC 401563).

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.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 7/17/06. Revised 9/27/06. Accepted 11/ 3/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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