N-acetyltransferase 1 Expression Research Articles

Trimodal distribution of arylamine N-acetyltransferase 1 mRNA in breast cancer tumors: association with overall survival and drug resistance

BackgroundArylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that has been associated with cancer cell proliferation in vitro and with survival in vivo. NAT1 expression has been associated with the estrogen receptor and it has been proposed as a prognostic marker for estrogen receptor positive cancers. However, little is known about the distribution of NAT1 mRNA across an entire patient population or its effects on outcomes. To address this, gene expression data from breast cancer patient cohorts were investigated to identify sub-populations based on the level of NAT1 expression. Patient survival and drug response was examined to determine whether NAT1 mRNA levels influenced any of these parameters.ResultsNAT1 expression showed a trimodal distribution in breast cancer samples (n = 1980) but not in tumor tissue from ovarian, prostate, cervical or colorectal cancers. In breast cancer, NAT1 mRNA in each sub-population correlated with a separate set of genes suggesting different mechanisms of NAT1 gene regulation. Kaplan-Meier plots showed significantly better survival in patients with highest NAT1 mRNA compared to those with intermediate or low expression. While NAT1 expression was elevated in estrogen receptor-positive patients, it did not appear to be dependent on estrogen receptor expression. Overall survival was analyzed in patients receiving no treatment, hormone therapy or chemotherapy. NAT1 expression correlated strongly with survival in the first 5 years in those patients receiving chemotherapy but did not influence survival in the other two groups. This suggests that low NAT1 expression is associated with chemo-resistance. The sensitivity of NAT1 mRNA levels as a single parameter to identify non-responders to chemotherapy was 0.58 at a log(2) < 6.5.ConclusionsNAT1 mRNA can be used to segregate breast cancer patients into sub-populations that demonstrate different overall survival. Moreover, low NAT1 expression shows a distinct poor response to chemotherapy. Analysis of NAT1 expression may be useful for identifying specific individuals who would benefit from alternative therapy or drug combinations. However, additional information is required to increase the sensitivity of identifying non-responders.

Retrospective analysis of estrogen receptor1 and N‑acetyltransferase gene expression in normal breast tissue, primary breast tumors, and established breast cancer cell lines.

The expression levels of estrogen receptor 1 (ESR1), arylamine N-acetyltransferase 1 (NAT1), and arylamine N-acetyltransferase 2 (NAT2) are implicated in breast cancer; however, their co-expression profiles in normal breast tissue, primary breast tumors and established breast cancer cell lines are undefined. NAT1 expression is widely reported to be associated with ESR1 expression and is frequently investigated in breast cancer etiology. Furthermore, the NAT2 phenotype has been reported to modify breast cancer risk in molecular epidemiological association studies. Understanding the relationships between the expression levels of these genes is essential to understand their role in breast cancer etiology and treatment. In the present study, NAT1, NAT2 and ESR1 expression data were accessed from repositories of RNA-Seq data covering 57 breast cancer cell lines, 1,043 primary breast tumors and 99 normal breast tissues. The relationships between gene expression, and between NAT1 activity and RNA expression in breast cancer cell lines were evaluated using non-parametric statistical analyses. Differences in gene expression in each dataset, as well as gene expression differences in normal breast tissue compared to primary breast tumors, and stratification by estrogen receptor status were determined. NAT1 and NAT2 mRNA expression were detected in normal and primary breast tumor tissues; NAT1 expression was much higher than NAT2. NAT1 and ESR1 expression were strongly associated, whereas NAT2 and ESR1 expression were not. Although NAT1 and NAT2 expression were associated, the magnitude was moderate. NAT1, NAT2, and ESR1 expression were increased in primary breast tumor tissue compared with normal breast tissue; however, the magnitude and significance of the differences were lower for NAT2. Analysis of NAT1, NAT2, and ESR1 expression in normal and primary breast tissues and breast cancer cell lines suggested that NAT1 and NAT2 expression are regulated by distinctive mechanisms, whereas NAT1 and ESR1 expression may have overlapping regulation. Defining these relationships is important for future investigations into breast cancer prevention.

Genetic and small molecule inhibition of arylamine N-acetyltransferase 1 reduces anchorage-independent growth in human breast cancer cell line MDA-MB-231.

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer.

High N-Acetyltransferase 1 Expression Is Associated with Estrogen Receptor Expression in Breast Tumors, but Is not Under Direct Regulation by Estradiol, 5α-androstane-3β,17β-Diol, or Dihydrotestosterone in Breast Cancer Cells.

N-acetyltransferase 1 (NAT1) is an enzyme that metabolizes carcinogens, which suggests a potential role in breast carcinogenesis. High NAT1 expression in breast tumors is associated with estrogen receptor α (ERα+) and the luminal subtype. We report that NAT1 mRNA transcript, protein, and enzyme activity were higher in human breast tumors with high expression of ERα/ESR1 compared with normal breast tissue. There was a strong correlation between NATb promoter and NAT1 protein expression/enzyme activity. High NAT1 expression in tumors was not the result of adipocytes, as evidenced by low perilipin (PLIN) expression. ESR1, NAT1, and XBP1 expression were associated in tumor biopsies. Direct regulation of NAT1 transcription by estradiol (E2) was investigated in ERα (+) MCF-7 and T47D breast cancer cells. E2 did not increase NAT1 transcript expression but increased progesterone receptor expression in a dose-dependent manner. Likewise, NAT1 transcript levels were not increased by dihydrotestosterone (DHT) or 5α-androstane-3β, (3β-adiol) 17β-diol. Dithiothreitol increased levels of the activated, spliced XBP1 in ERα (+) MCF-7 and T47D breast cancer cells but did not affect NAT1 or ESR1 expression. We conclude that NAT1 expression is not directly regulated by E2, DHT, 3β-adiol, or dithiothreitol despite high NAT1 and ESR1 expression in luminal A breast cancer cells, suggesting that ESR1, XBP1, and NAT1 expression may share a common transcriptional network arising from the luminal epithelium associated with better survival in breast cancer. Clusters of high-expression genes, including NAT1, in breast tumors might serve as potential targets for novel therapeutic drug development.

Efficacy research of salazosulfamide in ankylosing spondylitis and NAT1 gene polymorphism.

The aim of this study was to explore the correlation of salazosulfamide efficacy on ankylosing spondylitis and N-acetyltransferase 1 (NAT1) gene polymorphism. Thirty-two patients with ankylosing spondylitis were recruited in the experimental group and 36 normal individuals were recruited to the control group. The experimental group received 8.0 mg of salazosulfamide (MTX) per week and the control group received isodose of normal saline. Twenty-six patients in the experimental group responded to the salazosulfamide treatment and 6 did not show response. Morning stiffness time of patients in the experimental group who responded to salazosulfamide was significantly lower than that of patients with no reaction to salazosulfamide, and similar to patients in the control group. The average tender joint count of patients in the experimental group that responded to salazosulfamide was lower than in patients with no response to treatment, and similar to patients in the control group. NAT1 gene sequencing determined that the patients sensitive to salazosulfamide treatment manifested as AA/AG at 263 locus, whereas patients not sensitive to salazosulfamide were GG. NAT1 expression was comparable between the different genotypes at the mRNA level. However, there was a significant difference of NAT1 protein between groups. Overall, salazosulfamide demonstrates curative activity for ankylosing spondylitis and we believe that NAT1 AA/GG genotype at 263 locus can promote salazosulfamide effectiveness on ankylosing spondylitis.

Untargeted polar metabolomics of transformed MDA-MB-231 breast cancer cells expressing varying levels of human arylamine N-acetyltransferase 1.

Human arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. Expression of NAT1 is elevated in several cancers including breast cancer. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear. This study explored polar metabolome differences between MDA-MB-231 breast cancer cells expressing varying levels of NAT1 activity using an untargeted approach. Three MDA-MB-231 breast adenocarcinoma cell lines that stably express wild-type, increased, and decreased levels of human NAT1 were investigated for differences in polar metabolic profile using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) system. Increased levels of human NAT1 in the transformed cell lines resulted in a statistically significant decreased abundance of the metabolite palmitoleic acid (q = 0.0006), when compared to normal and decreased levels of human NAT1. The fatty acid synthesis pathway utilizes acetyl coenzyme A (acetyl-CoA) in the first two reactions of the pathway and eventually leads to the synthesis of palmitoleic acid. These data suggest a link between increased levels of NAT1 activity and decreased flux of acetyl-CoA through this portion of the fatty acid synthesis pathway.

Expression of NAT2 in immune system cells and the relation of NAT2 gene polymorphisms in the anti-tuberculosis therapy in Mexican mestizo population.

Arylamine N-acetyltransferase 2 (NAT2) metabolizes isoniazid (INH) and Single Nucleotide Polymorphisms (SNP) responsible for its activity has been reported. The aim of this study in the Mexican mestizo population was to evaluate NAT2 expression at the protein level in immune cells, as well as the distribution and frequency of six NAT2 SNPs and their association with anti-TB therapy, by measuring the plasma levels of INH and Acetyl-INH (AcINH). We performed genotyping assays of NAT2 SNPs in 40 TB patients and 121 healthy volunteers by real-time PCR. A method for detecting NAT2 in immune cells using flow cytometry was developed. Plasma concentrations of INH and AcINH were obtained by HPLC in TB patients and the Metabolic Ratio (MR) was calculated. The phenotypes obtained in the healthy volunteers were as follows; 18.87 % of subjects had the rapid acetylator phenotype, 45.45 % had the intermediate phenotype and 39.66 % exhibited the slow acetylator phenotype. In the TB patient group, 35 % of patients had the rapid acetylator phenotype, 32.5 % were intermediate and 32.5 % showed the slow acetylator phenotype. A higher expression level of NAT2 in innate immune cells from TB patients compared to those from healthy volunteers was detected (P < 0.013). In TB patients the MR showed a bimodal distribution with an antimode of 0.7, which was used as a threshold value for acetylator classification. A high correspondence between the rapid and slow acetylator phenotype with MR was demonstrated. In conclusion, the 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, 857G>A SNPs of NAT2 gene provides accurate for prediction of the acetylator phenotype in Mexican mestizo population. A statistical difference was found in frequency of rapid metabolizer phenotype, which was higher in TB patients. In addition, the expression of NAT2 protein in immune cells can lead to further studies related to its functional role in the innate immune response against M. tuberculosis and other xenobiotics metabolized by this enzyme.

The Role of Arylamine N‐acetyltransferase 1 in Breast Cancer Progression

Breast cancer is a leading cause of cancer death among women in the United States. Arylamine N‐acetyltransferase 1 (NAT1) has been linked to breast cancer because of its role in the metabolic activation and detoxification of carcinogens. However, recent studies suggest it may play an important role in cancer biology. Higher NAT1 expression is associated with more aggressive cancers and poorer prognosis. Increased NAT1 levels in primary tumors are significantly associated with increased metastasis to the bone. We have observed NAT1 inhibition decreased proliferation and invasiveness of MDA‐MB‐231 breast cancer cells. To delineate the mechanisms by which NAT1 inhibition decreases cell invasion and proliferation, MDA‐MB‐231 and MCF‐7 breast cancer cell lines were treated with siRNA targeting NAT1, or a non‐targeting siRNA. Cells were collected at 24, 48, 72 and 96 hours after treatment. In vitro assays using a NAT1‐specific substrate, para‐aminobenzoic acid (PABA) were conducted to measure NAT1 activity by HPLC. NAT1 mRNA was measured using qRT‐PCR. Optimal knockdown of NAT1 occurred at 96 and 72 hours in MCF‐7 and MDA‐MB‐231 cells, with 65% (p &lt; 0.005) of NAT1 activity remaining after treatment in both cell lines. NAT1 mRNA decreased 60% (p &lt; 0.0001) and 34% (p &lt; 0.001) in MCF‐7 and MDA‐MB‐231 cells, respectively. These samples will undergo whole genome analysis using a GeneArray to identify potential gene expression signatures which may be altered as a result of NAT1 inhibition. [Partially supported by NIEHS T32‐ESO11564]

Identification and Characterization of Novel Arylamine NAcetyltransferase Small Molecule Inhibitors

Many environmental arylamines play a vital role in the etiology of cancer and require metabolic activation by acetylation. Arylamine N‐acetyltransferase 1 (NAT1), a xenobiotic metabolizing enzyme, catalyzes the N‐acetylation or O‐acetylation of arylamine carcinogens. O‐acetylation, which is preceded by cytochrome P450 N‐hydroxylation, results in nitrenium ions that form DNA adducts. If unrepaired, these adducts can lead to mutagenesis and initiate cancer. Molecular epidemiology studies suggest that NAT1 expression and genetic polymorphisms play an important role in individual risk for many cancers, including breast cancer. Studies also suggest that NAT1 may play an important role in cell growth and survival, as well as in cell invasion, which is a hallmark of metastatic cancer. The purpose of this study is to use NAT1 as a molecular target for the development of small molecule inhibitors for cancer prevention. We utilized in silico screening of 17 million compounds to identify those that interact with NAT active sites. A total of 150 lead compounds were identified, scored and ranked based on their active site association. Sixty of these compounds were tested for their ability to inhibit recombinant human NAT1. The IC50 values of the most efficacious inhibitors (2, 10, 11 and 16 ) of human NAT1 in vitro were &gt;2 mM, and 0.75, 191, and 202 μM, respectively. The most potent inhibitor (10 ) was further tested for inhibition of N‐acetylation in situ in Chinese hamster ovary cells. The IC50 values of inhibitor 10 were 118 μM and 81.6 μM towards the N‐acetylation of p‐aminobenzoic acid and the arylamine carcinogen 4‐aminobiphenyl. We report the in silico identification and verification of compound 10 as a potential new small molecule for cancer prevention. [NIH/NIEHS (T32‐ ES011564) and UofL Clinical and Translational Sciences Program]

Functional analysis of arylamine N-acetyltransferase 1 (NAT1) NAT1*10 haplotypes in a complete NATb mRNA construct

N-acetyltransferase 1 (NAT1) catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1*10 is the most common variant haplotype and is associated with increased risk for numerous cancers. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in messenger RNAs (mRNAs) with distinct 5'-untranslated regions (UTRs). To best mimic in vivo metabolism and the effect of NAT1*10 polymorphisms on polyadenylation usage, pcDNA5/Flp recombination target plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATb, the open reading frame and 888 nucleotides of the 3'-UTR. Following stable transfection of NAT1*4, NAT1*10 and an additional NAT1*10 variant (termed NAT1*10B) into nucleotide excision repair-deficient Chinese hamster ovary cells, N- and O-acetyltransferase activity (in vitro and in situ), mRNA and protein expression were higher in cells transfected with NAT1*10 and NAT1*10B than in cells transfected with NAT1*4 (P < 0.05). Consistent with NAT1 expression and activity, cytotoxicity and hypoxanthine phosphoribosyl transferase mutants following 4-aminobiphenyl exposures were higher in NAT1*10 than in NAT1*4 transfected cells. Ribonuclease protection assays showed no difference between NAT1*4 and NAT1*10. However, protection of one probe by NAT1*10B was not observed with NAT1*4 or NAT1*10, suggesting additional mechanisms that regulate NAT1*10B. The higher mutants in cells transfected with NAT1*10 and NAT1*10B are consistent with an increased cancer risk for individuals possessing NAT1*10 haplotypes.

Human N-acetyltransferase 1 *10 and *11 alleles increase protein expression through distinct mechanisms and associate with sulfamethoxazole-induced hypersensitivity

N-acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens. NAT1 alleles *10 and *11 have been proposed to alter protein level or enzyme activity compared with wild-type NAT1 *4 and to confer cancer risk, through uncertain pathways. This study characterizes regulatory polymorphisms and underlying mechanisms of NAT1 expression. We measured allelic NAT1 mRNA expression and translation, as a function of multiple transcription start sites, alternative splicing, and three 3'-polyadenylation sites in human livers (one of which was discovered in this study), B lymphocytes, and transfected cells. In a clinical study of 469 patients with HIV/AIDS treated with the NAT1/NAT2 substrate sulfamethoxazole (SMX), associations were tested between SMX-induced hypersensitivity and NAT1 *10 and *11 genotypes, together with known NAT2 polymorphisms. NAT1 *10 and *11 were determined to act as common regulatory alleles accounting for most NAT1 expression variability, both leading to increased translation into active protein. NAT1 *11 (2.4% minor allele frequency) affected 3'-polyadenylation site usage, thereby increasing formation of NAT1 mRNA with intermediate length 3'-untranslated region (major isoform) at the expense of the short isoform, resulting in more efficient protein translation. NAT1 *10 (19% minor allele frequency) increased translation efficiency without affecting 3'-untranslated region polyadenylation site usage. Livers and B-lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 immunoreactivity and NAT1 enzyme activity than the reference genotype *4/*4. Patients who carry *10/*10 and *11/*4 (fast NAT1 acetylators) were less likely to develop hypersensitivity to SMX, but this was observed only in individuals who are also carrying a slow NAT2 acetylator genotype. NAT1 *10 and *11 significantly increase NAT1 protein level/enzyme activity, enabling the classification of carriers into reference and rapid acetylators. Rapid NAT1 acetylator status seems to protect against SMX toxicity by compensating for slow NAT2 acetylator status.

NATb/NAT1*4 promotes greater arylamine N‐acetyltransferase 1 mediated DNA adducts and mutations than NATa/NAT1*4 following exposure to 4‐aminobiphenyl

N-acetyltransferase 1 (NAT1) is a phase II metabolic enzyme responsible for the biotransformation of aromatic and heterocyclic amine carcinogens such as 4-aminobiphenyl (ABP). NAT1 catalyzes N-acetylation of arylamines as well as the O-acetylation of N-hydroxylated arylamines. O-acetylation leads to the formation of electrophilic intermediates that result in DNA adducts and mutations. NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5'-untranslated regions (UTR). NATa mRNA is expressed primarily in the kidney, liver, trachea, and lung while NATb mRNA has been detected in all tissues studied. To determine if differences in 5'-UTR have functional effect upon NAT1 activity and DNA adducts or mutations following exposure to ABP, pcDNA5/FRT plasmid constructs were prepared for transfection of full-length human mRNAs including the 5'-UTR derived from NATa or NATb, the open reading frame, and 888 nucleotides of the 3'-UTR. Following stable transfection of NATb/NAT1*4 or NATa/NAT1*4 into nucleotide excision repair (NER) deficient Chinese hamster ovary cells, N-acetyltransferase activity (in vitro and in situ), mRNA, and protein expression were higher in NATb/NAT1*4 than NATa/NAT1*4 transfected cells (P < 0.05). Consistent with NAT1 expression and activity, ABP-induced DNA adducts and hypoxanthine phosphoribosyl transferase mutants were significantly higher (P < 0.05) in NATb/NAT1*4 than in NATa/NAT1*4 transfected cells following exposure to ABP. These differences observed between NATa and NATb suggest that the 5'-UTRs are differentially regulated.

Liver-Selective Expression of Human ArylamineN-Acetyltransferase NAT2 in Transgenic Mice

Human arylamine N-acetyltransferase 2 (NAT2) mediates the biotransformation of arylamine drugs and procarcinogens into either innocuous or reactive DNA-damaging metabolites and is expressed predominantly in liver. Interspecies differences and incongruous results between in vitro, in vivo, and epidemiological studies make it difficult to extrapolate animal results to human risk. We have generated human NAT2 transgenic mice on both C57BL/6 (hNAT2(tg)) and Nat1/2 null backgrounds [hNAT2(tg)Nat1/2(-/-)], in which liver-selective expression of human NAT2 is driven by the mouse albumin promoter. We detected expression of the human NAT2 transcript and protein in mouse liver by real-time PCR and Western blot analysis. NAT2 enzyme activity, measured using the human NAT2-selective substrate sulfamethazine (SMZ), was 40- to 80-fold higher in liver cytosols from hNAT2(tg)Nat1/2(-/-) mice than in wild-type mice. An unexpected gender difference was observed, with males displaying 2-fold higher activity than females. Transgenic mice also had an increased in vivo plasma clearance of SMZ and higher levels of N-acetylated SMZ than wild-type mice. Liver expression of human NAT2 did not affect the disposition of the human NAT1-selective substrate p-aminosalicylic acid (PAS), because hNAT2(tg)Nat1/2(-/-) mice displayed in vivo PAS pharmacokinetic profiles similar to those of Nat1/2(-/-) mice. The metabolism of 4-aminobiphenyl was similar between hNAT2(tg)Nat1/2(-/-) and wild-type mice with the exception of a more liver-restricted pattern in hNAT2(tg)Nat1/2(-/-) mice and lower activity in females. Overall, the hNAT2(tg)Nat1/2(-/-) mouse mimics human expression of NAT2 and may thus be of value in clarifying the role of human NAT2 in arylamine clearance, detoxification, and bioactivation.

RNAi-Mediated Knock-Down of Arylamine N-acetyltransferase-1 Expression Induces E-cadherin Up-Regulation and Cell-Cell Contact Growth Inhibition

Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.

Histone Deacetylase Inhibitors Increase Human Arylamine N-Acetyltransferase-1 Expression in Human Tumor Cells

Arylamine N-acetyltransferase-1 (NAT1) has been associated with disorders involving folate metabolism, such as spina bifida, as well as numerous human cancers. As a result, the transcriptional and post-transcriptional regulation of NAT1 activity has been extensively studied. However, little work has been reported on the epigenetic control of NAT1 expression. Here, we demonstrate that the histone deacetylase inhibitor trichostatin A (TSA) increases NAT1 activity in human cancer cells by increasing transcription from the proximal promoter NATb. A specific Sp1 binding site was identified as essential for optimal induction of NAT1 by TSA. However, TSA did not increase the expression of Sp1 in HeLa cells. Instead, TSA increased the acetylation of histones associated with the NATb promoter. This allowed recruitment of Sp1 to the promoter along with acetylated histones. We propose that NAT1 transcription is partially repressed by the local chromatin condensation in the vicinity of NATb and that histone deacetylase inhibition leads to up-regulation of NAT1 expression via a direct change in chromatin conformation.

Characterization of N-Acetyltransferase 1 Activity in Human Keratinocytes and Modulation by para-Phenylenediamine

N-acetyltransferase 1 (NAT1)-mediated N-acetylation in keratinocytes is an important detoxification pathway for the hair dye ingredient para-phenylenediamine (PPD). Because NAT1 can be regulated by various exogenous compounds, including some NAT1 substrates themselves, we investigated NAT1 expression in keratinocytes and the interactions between PPD and NAT1. NAT1 activity was found to be cell-cycle phase-dependent. Maximum NAT1 activities (mean: 49.7 nmol/mg/min) were estimated when HaCaT keratinocytes were arrested in G(0)/G(1) phase, whereas nonsynchronized cells showed the lowest activities (mean: 28.9 nmol/mg/min). It is noteworthy that we also found an accelerated progression through the cell cycle in HaCaT cells with high NAT1 activities. This evidence suggests an association between NAT1 and proliferation in keratinocytes. Regarding the interaction between NAT1 and PPD, we found that keratinocytes N-acetylate PPD; however, this N-acetylation was saturated with increasing PPD concentrations. HaCaT cultured in medium supplemented with PPD (10-200 microM) for 24 h showed a significant concentration-dependent decrease (17-50%) in NAT1 activity. PPD also induced down-regulation of NAT1 activity in human primary keratinocytes. Western blot studies using a NAT1-specific antibody in HaCaT showed that the loss of enzyme activity was associated with a decline in the amount of NAT1 protein, whereas no changes in the amounts of NAT1 P1 (NATb)-dependent mRNA were found by quantitative reverse transcription-polymerase chain reaction analysis, suggesting the involvement of a substrate-dependent mechanism of NAT1 down-regulation. In conclusion, these data show that overall N-acetylation capacity of keratinocytes and consequently detoxification capacities of human skin is modulated by the presence of NAT1 substrates and endogenously by the cell proliferation status of keratinocytes.

Genetic Polymorphisms of Drug-Metabolizing Enzymes and Anti-TB Drug-Induced Hepatitis

Although some genetic risk factors have been reported for the development of hepatitis due to anti-TB drugs, an extensive candidate gene approach evaluating drug-metabolizing enzymes has not been attempted. This study aimed to investigate the association of genetic polymorphisms in drug-metabolizing enzymes with anti-TB drug-induced hepatitis. We compared genotype distributions of tagging SNPs in promoter, exons and haplotypes in seven drug-metabolizing enzyme genes (CYP2C9, CYP2C19, CYP2D6, CYP2E1, NAT2, UGT1A1 and UGT1A3) between 67 cases and 159 controls. Among four tagging SNPs of N-acetyltransferase 2 (NAT2), -9796T>A in promoter and R197Q were significantly associated (p = 0.0016 and p = 0.0007, respectively). NAT2 haplotype 2 [A-A-A-G] carrying A allele of -9796T>A and A allele of R197Q showed significant association (p = 0.0004). However, there was no significant association between genotypes of other enzyme-metabolizing genes and anti-TB drug-induced hepatitis. The constructs containing -9796A of NAT2 showed significantly lower luciferase activity (p < 0.01), suggesting decreased expression of NAT2. The variant alleles and haplotype 2 showed significantly higher peak serum levels of isoniazid, lower acetyl isoniazid:isoniazid ratio and lower isoniazid clearance compared with wild-types. These findings suggest that genetic variants in the promoter and exons of NAT2 increase the risk of anti-TB drug-induced hepatitis by modifying acetylation phenotypes and/or gene expression of NAT2, and there is no essential role for genetic mutation of the other metabolizing enzymes in the development of this adverse reaction.

Identification ofN-Acetyltransferase 2 (NAT2) Transcription Start Sites and Quantitation ofNAT2-Specific mRNA in Human Tissues

Human N-acetyltransferase 2 (NAT2) genetic polymorphism is associated with drug toxicity and/or carcinogenesis in various tissues. Knowledge of NAT2 gene structure and expression is critical for understanding these associations. Previous findings suggest that human NAT2 expression is highest in liver and gut but expressed at functional levels in other tissues. A sensitive and specific TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assay with intron-spanning primers was developed and used, together with a second TaqMan RT-PCR assay based on amplification of a NAT2 open reading frame (ORF) exon segment, to measure NAT2 mRNA in 29 different human tissues. Cap-dependent amplification of mRNA 5' termini and review of public database information were done to more precisely define the NAT2 promoter(s) and to validate the quantitative RT-PCR assay design. The great majority (40/41) of NAT2 liver cDNAs had 5' termini between 8682 and 8752 nucleotides upstream of the NAT2 ORF exon, and 34 of 40 5' termini were at the -8711 and -8716 adenines. All 59 NAT2 cDNAs with 5' termini in this vicinity, including 40 of the liver isolates and 19 cDNAs in public databases from liver and other sources, showed direct splicing to the ORF exon, with no other noncoding exon detected. NAT2 mRNA was highest in liver, small intestine, and colon and was readily detected in most other tissues, albeit at much lower levels. NAT2 expression in diverse human tissues provides further mechanistic support underlying associations between NAT2 genetic polymorphism, drug toxicity, and/or chemical carcinogenesis.

Inflammatory cytokines suppress arylamine N -acetyltransferase 1 in cholangiocarcinoma cells

To evaluate the effect of inflammatory cytokines on arylamine N-acetyltransferase 1 (NAT1), which is a phase-II enzyme involved in the biotransformation of aromatic and heterocyclic amines found in food, drugs and the environment. Human cholangiocarcinoma KKU-100 cells were treated with a mixture of proinflammatory cytokines (interferon-gamma, interleukin-1 beta and tumor necrosis factor-alpha) for 48 h, and the effect on NAT1 activity was assessed by high performance liquid chromatography, while NAT1 expression was determined by reverse-transcription polymerase chain reaction. The oxidative stress on the cells was examined by the formation of nitric oxide, superoxide anion and glutathione (GSH) levels. The cells were also treated with S-nitroso-glutathione (GSNO), a nitric oxide donor, to see if the responses were similar to those obtained with the inflammatory cytokines. Cytokines suppressed NAT1 activity, reducing the Vmax without affecting the Km. Cytokines also had a significant impact on the induction of nitric oxide production and in reducing the redox ratios of glutathione (GSH) and GSH disulfide. Treatment with GSNO for 2-48 h reduced NAT1 activity without affecting the GSH ratio. Moreover, inflammatory cytokines and GSNO suppressed NAT1 mRNA expression. These findings indicate an association between inflammation and suppression of NAT1, which perhaps contributes to chemical-mediated toxicity and carcinogenesis.

Functional characterization of the A411T (L137F) and G364A (D122N) genetic polymorphisms in human N-acetyltransferase 2

Human N-acetyltransferase 2 (NAT2) genetic polymorphisms may modify drug efficacy and toxicity and cancer susceptibility from carcinogen exposure. Two human NAT2 alleles, NAT2*5I and NAT2*12D, were identified recently. In NAT2*5I, a new single nucleotide polymorphism AT (L137F) was found coexisting with single nucleotide polymorphisms TC (I114T), CT (silent) and AG (K268R). The other allele NAT2*12D consists of a new single nucleotide polymorphism GA (D122N) together with AG (K268R). We undertook a study to characterize these new single nucleotide polymorphisms and NAT2 alleles to further our understanding of genotype/phenotype relationships in human populations. Various human NAT2 alleles were cloned and recombinantly expressed in COS-1 cells and the effects of single nucleotide polymorphisms on NAT2 expression were determined. To further test our hypothesis that AT (L137F) and GA (D122N) accelerate protein degradation, various NAT2 alleles were cloned and expressed in Escherichia coli, which does not possess the ubiquitin-mediated degradation pathway. Both AT and GA reduced NAT2 immuno-reactive protein to an undetectable level without causing changes in mRNA level. Missense mutants displayed different effects on sulfamethazine N-acetylation activity for both L137 (wild-type: 70.2+/-5.2 nmol/min/mg; L137F: 1.34+/-0.03 nmol/min/mg; L137W: nondetectable; L137I: 34.2+/-2.0 nmol/min/mg; L137G: 0.52+/-0.04 nmol/min/mg) and D122 (wild-type: 70.2+/-5.2 nmol/min/mg; D122R: non-detectable; D122Q: non-detectable; D122E: 1.72+/-0.24 nmol/min/mg). In contrast to the expression in mammalian cells, recombinant NAT2 possessing either of these two single nucleotide polymorphisms showed no reduction in immuno-reactive NAT2 level when expressed in E. coli. These findings suggest that both AT (L137F) and GA (D122N) enhance NAT2 degradation, resulting in reduced NAT2 protein and catalytic activity for NAT2 5I and NAT2 12D.