Molecular profiling of male breast cancer - lost in translation?

Breast cancer is the most common cancer form in women and it has been extensively studied on the molecular level. Male breast cancer (MBC), on the other hand, is rare and has not been thoroughly investigated in terms of transcriptional profiles or genomic aberrations. Most of our understanding of MBC has therefore been extrapolated from knowledge of female breast cancer. Although differences in addition to similarities with female breast cancer have been reported, the same prognostic and predictive markers are used to determine optimal management strategies for both men and women diagnosed with breast cancer. This review is focused on prognosis for MBC patients, prognostic and predictive factors and molecular subgrouping; comparisons are made with female breast cancer. Information was collected from relevant literature on both male and female breast cancer from the MEDLINE database between 1992 and 2014. MBC is a heterogeneous disease, and on the molecular level many differences compared to female breast cancer have recently been revealed. Two distinct subgroups of MBC, luminal M1 and luminal M2, have been identified which differ from the well-established intrinsic subtypes of breast cancer in women. These novel subgroups of breast cancer therefore appear unique to MBC. Furthermore, several studies report inferior survival for men diagnosed with breast cancer compared to women. New promising prognostic biomarkers for MBC (e.g. NAT1) deserving further attention are reviewed. Further prospective studies aimed at validating the novel subgroups and recently proposed biomarkers for MBC are warranted to provide the basis for optimal patient management in this era of personalized medicine. This article is part of a Directed Issue entitled: Rare Cancers.

Liver-selective expression of human arylamine N-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.

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.

Effect of environmental substances on the activity of arylamine N-acetyltransferases.

Arylamine N-acetyltransferases (NAT) are xenobiotic-metabolizing enzymes responsible for the acetylation of many aromatic arylamine and heterocyclic amines, thereby playing an important role in both detoxification and activation of numerous drugs and carcinogens. Two closely related isoforms (NAT1 and NAT2) have been described in humans. NAT2 is mainly expressed in liver and gut, whereas NAT1 is found in a wide range of tissues. Interindividual variations in NAT genes have been shown to be a potential source of pharmacological and/or pathological susceptibility. In addition, there is now evidence that non genetic factors, such as substrate-dependent inhibition, drug interactions or cellular redox conditions may also contribute to NAT activity. The recent findings reviewed here provide possible mechanisms by which these environmental determinants may affect NAT activity. Interestingly, these data could contribute to the development of selective NAT inhibitors for the treatment of cancer and microbial diseases.

High potency of bioactivation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in mouse colon epithelial cells with Apc(Min) mutation.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a prominent heterocyclic aromatic amine (HAA) found in meat and fish cooked at moderate to high temperature. It is considered as a potent dietary factor promoting colon carcinogenesis. However, the role of intestinal cells in PhIP bioactivation has not been fully explained, particularly when cells are pre-malignant. Loss of function of the adenomatous polyposis coli (APC) gene product is an early and frequent event in human colorectal carcinogenesis. Normal (Apc(+/+)) and pre-malignant (Apc(Min/+), where Min=multiple intestinal neoplasia) colonic epithelial cells of mice can be used to study promotion of carcinogenesis, but these cells have not been characterized for bio-activation of HAA. We investigated the metabolism of (14)C-PhIP in these two murine cell lines. Cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) metabolized PhIP into 4'-OH-PhIP as the main metabolite in PhiP detoxification. Besides, 5-OH-PhIP was identified, revealing the formation of intermediary reactive metabolites, since it results from a degradation of conjugates of N-acetoxy-PhIP. Apc(Min/+) cells produce significantly higher amounts of these metabolites. Demethylated metabolites are also observed, indicating that the colon contains a significant CYP1 family dependent metabolic activity. A minor hydroxy-glucuronide-PhIP metabolite is observed in Apc(Min/+) cells, the glucuronidation being known as an important step in the detoxification pathway. Quantitative real-time reverse transcription polymerase chain reaction experiments demonstrate that induction by TCDD has prevailing effects in gene expression of CYP1A1, CYP1A2 and CYP1B1 in Apc(Min/+) cells. In these cells, N-acetyltransferase-2 is also expressed at higher levels. So, the more important potency to metabolically bio-activate PhIP, as measured in Apc(Min/+) cells, can be linked to a higher probability to generate new in situ mutations.

Promoter hypomethylation of the N-acetyltransferase 1 gene in breast cancer.

Arylamine N-acetyltransferase type 1 (NAT1) is reported to be involved in the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of hydrazine and arylamine drugs or carcinogens. Gene-specific hypomethylation frequently occurs in a range of cancers and hypomethylation of the genes often correlates well with increased transcription levels. This study was conducted in order to evaluate the methylation status and the transcriptional activity of NAT1 in breast cancer tissues (n=72), benign breast tissues (n=31) and morphologically normal breast tissues (n=30). Our findings showed that the methylation of the NAT1 gene was identified in 39 of the breast carcinomas (54.2%), 23 normal (76.7%) and 25 benign breast tissue samples (80.6%). The breast cancer tissues showed significantly lower methylation rates of the NAT1 promoters than the normal and benign tissues (P=0.012). Furthermore, cancer tissues showed lower methylation density rates than normal and benign breast tissues (P=0.012). The tissues that showed aberrant methylation of NAT1 showed significantly less mRNA expression compared with the unmethylated cases by a thousand fold (P<0.001). Twenty cancers from the methylated group showed positive staining for the estrogen receptor (ER) (51.3%), while 72.7% from the unmethylated group stained positive (P=0.063). Our results suggest that DNA hypomethylation in the NAT1 gene appears to be present in cancerous breast tissues thus indicating that this type of methylation may significantly influence the transcriptional activation of the gene. Therefore, hypomethylation of the NAT1 gene plays a significant role in breast carcinogenesis.

Arylamine N-acetyltransferase 1 expression in breast cancer cell lines: a potential marker in estrogen receptor-positive tumors.

The prognosis for patients with estrogen receptor (ER)-positive breast cancer has improved significantly with the prescription of selective ER modulators (SERMs) for ER-positive breast cancer treatment. However, only a proportion of ER-positive tumors respond to SERMs, and resistance to hormonal therapies is still a major problem. Detailed analysis of published microarray studies revealed a positive correlation between overexpression of the drug metabolizing enzyme arylamine N-acetyltransferase type 1 (NAT1) and ER positivity, and increasing evidence supports a biological role for NAT1 in breast cancer progression. We have tested a range of ER-positive and ER-negative breast cancer cell lines for NAT1 enzyme activity, and monitored promoter and polyadenylation site usage. Amongst ER-positive lines, NAT1 activities ranged from 202 +/- 28 nmol/min/mg cellular protein (ZR-75-1) to 1.8 +/- 0.4 nmol/min/mg cellular protein (MCF-7). The highest levels of NAT1 activity could not be attributed to increased NAT1 gene copy number; however, we did detect differences in NAT1 promoter and polyadenylation site usage amongst the breast tumor-derived lines. Thus, whilst all cell lines tested accumulated transcripts derived from the proximal promoter, the line expressing NAT1 most highly additionally initiated transcripts initiating at a more distal, "tissue"-specific promoter. These data pave the way for investigating NAT1 transcripts as candidate prognostic markers in ER-positive breast cancer.

Mouse arylamine N-acetyltransferase 2 (Nat2) expression during embryogenesis: a potential marker for the developing neuroendocrine system.

Arylamine N-acetyltransferase (NAT) genes in humans and in rodents encode polymorphic drug metabolizing enzymes. Human NAT1 (and the murine equivalent mouse Nat2) is found early in embryonic development and is likely to have an endogenous role. We report the detailed expression of the murine gene (Nat2) and encoded protein in mouse embryos, using a transgenic mouse model bearing a lacZ transgene inserted into the coding region of mouse Nat2. In mouse embryos, the transgene was expressed in sensory epithelia, epithelial placodes giving rise to visceral sensory neurons, the developing pituitary gland, sympathetic chain and urogenital ridge. In Nat2+/+ mice, the presence and activity of Nat2 protein was detected in these tissues and their adult counterparts. Altered expression of the human orthologue in breast tumours, in which there is endocrine signalling, suggests that human NAT1 should be considered as a potential biomarker for neuroendocrine tissues and tumours.

Arylamine N-acetyltransferases: characterization of the substrate specificities and molecular interactions of environmental arylamines with human NAT1 and NAT2.

Arylamine N-acetyltransferases (NATs) are phase II xenobiotic metabolism enzymes that catalyze the detoxification of arylamines by N-acetylation and the bioactivation of N-arylhydroxylamines by O-acetylation. Endogenous and recombinant mammalian NATs with high specific activities are difficult to obtain in substantial quantities and in a state of homogeneity. This paper describes the overexpression of human wild-type NAT2 as a dihydrofolate reductase fusion protein containing a TEV protease-sensitive linker. Treatment of the partially purified fusion protein with TEV protease, followed by chromatographic purification, afforded 2.8 mg of homogeneous NAT2 from 2 L of cell culture. The kinetic specificity constants ( k cat/ K m) for N-acetylation of arylamine environmental contaminants, some of which are associated with bladder cancer risk, were determined with NAT2 and NAT1. The NAT1/NAT2 ratio of the specificity constants varied almost 1000-fold for monosubstituted and disubstituted alkylanilines containing methyl and ethyl ring substituents. 2-Alkyl substituents depressed N-acetylation rates but were more detrimental to catalysis by NAT1 than by NAT2. 3-Alkyl groups caused substrates to be preferentially N-acetylated by NAT2, and both 4-methyl- and 4-ethylaniline were better substrates for NAT1 than NAT2. NMR-based models were used to analyze the NAT binding site interactions of the alkylanilines. The selectivity of NAT1 for acetylation of 4-alkylanilines appears to be due to binding of the substituents to V216, which is replaced by S216 in NAT2. The contribution of 3-alkyl substituents to NAT2 substrate selectivity is attributed to multiple bonding interactions with F93, whereas a single bonding interaction occurs with V93 in NAT1. Unfavorable steric clashes between 2-methyl substituents and F125 of NAT1 may account for the selective NAT2-mediated N-acetylation of 2-alkylanilines; F125 is replaced by S125 in NAT2. These results provide insight into the structural basis for the substrate specificity of two NATs that play major roles in the biotransformation of genotoxic environmental arylamines.

Induction of human arylamine N-acetyltransferase type I by androgens in human prostate cancer cells.

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 EC(50) 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.

DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation.

NAT10 (N-acetyltransferase 10) is a protein with histone acetylation activity and primarily identified to be involved in regulation of telomerase activity. The presented research shows its transcriptional activation by genotoxic agents and possible role in DNA damage. NAT10 mRNA could be markedly increased by using hydrogen peroxide (H2O2) or cisplatin in a dose- and time-dependent way, and the immunofluorescent staining revealed that the treatment of H2O2 or cisplatin induced focal accumulation of NAT10 protein in cellular nuclei. Both H2O2 and cisplatin could stimulate the transcriptional activity of the NAT10 promoter through the upstream sequences from -615 bp to +110 bp, with which some nuclear proteins interacted. Ectopic expression of NAT10 could enhance the number of survival cells in the presence of H2O2 or cisplatin. The above results suggested that NAT10 could be involved in DNA damage response and increased cellular resistance to genotoxicity.

Orofacial cleft risk is increased with maternal smoking and specific detoxification-gene variants.

Maternal smoking is a recognized risk factor for orofacial clefts. Maternal or fetal pharmacogenetic variants are plausible modulators of this risk. In this work, we studied 5,427 DNA samples, including 1,244 from subjects in Denmark and Iowa with facial clefting and 4,183 from parents, siblings, or unrelated population controls. We examined 25 single-nucleotide polymorphisms in 16 genes in pathways for detoxification of components of cigarette smoke, to look for evidence of gene-environment interactions. For genes identified as related to oral clefting, we studied gene-expression profiles in fetal development in the relevant tissues and time intervals. Maternal smoking was a significant risk factor for clefting and showed dosage effects, in both the Danish and Iowan data. Suggestive effects of variants in the fetal NAT2 and CYP1A1 genes were observed in both the Iowan and the Danish participants. In an expanded case set, NAT2 continued to show significant overtransmission of an allele to the fetus, with a final P value of .00003. There was an interaction between maternal smoking and fetal inheritance of a GSTT1-null deletion, seen in both the Danish (P=.03) and Iowan (P=.002) studies, with a Fisher's combined P value of <.001, which remained significant after correction for multiple comparisons. Gene-expression analysis demonstrated expression of GSTT1 in human embryonic craniofacial tissues during the relevant developmental interval. This study benefited from two large samples, involving independent populations, that provided substantial power and a framework for future studies that could identify a susceptible population for preventive health care.

Functional properties of an alternative, tissue-specific promoter for human arylamine N-acetyltransferase 1.

Variable expression of human arylamine N-acetyltransferase 1 (NAT1) due to genetic polymorphism, gene regulation or environmental influences is associated with individual susceptibility to various cancers. Recent studies of NAT1 transcription showed that most mRNAs originate at a promoter, P1, located 11.8 kb upstream of the single open reading frame (ORF) exon. We have now characterized an alternative NAT1 promoter lying 51.5 kb upstream of the NAT1 ORF. In the present study, analysis of human RNAs representing 27 tissue types by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR showed the upstream 51.5 kb promoter, designated P3, to be most active in specific tissues, including kidney, liver, lung, and trachea. All NAT1 P3 mRNAs included 5'-untranslated region (5'-UTR) internal exons of 61 and 175 nucleotides in addition to the 79 nucleotide 5'-UTR exon present in P1 mRNA. CAP-dependent amplification of 5'-P3 mRNA termini defined an 84 bp transcription start region in which most start sites are centrally clustered. The hepatoma-derived HepG2 cell line expressed a high level of P3 mRNA with the same spliced structure and start site pattern as found in normal tissues. A 435-bp minimal promoter was defined by transfection of HepG2 with luciferase expression constructs containing genomic fragments from the P3 start region. These findings imply a fundamental role for P3 in NAT1 regulation and define additional regions for genetic polymorphisms associated with enhanced cancer risk.

Heterologous co-expression of human cytochrome P450 1A2 and polymorphic forms of N-acetyltransferase 2 for studies on aromatic amines in V79 Chinese hamster cells.

V79 Chinese hamster cells were genetically engineered for the stable co-expression of human cytochrome P450 1A2 and the polymorphic N-acetyltransferase 2 alleles *4, *5B, *6A and *13, in order to generate an in vitro tool for studying the metabolism-dependent toxicity of aromatic amines. N-acetyltransferase 2*4-encoding cDNA was generated by the polymerase chain reaction (PCR) with defined primers from the genomic DNA of a human liver donor homozygous for *4, and served as a template to generate the *5B, *6A and *13 isoforms by site-directed mutagenesis. Human cytochrome P450 (CYP) 1A2-encoding cDNA was generated by the PCR from genomic DNA of the recombinant V79MZh1A2 cell line. All the cDNAs were inserted into a CMV promoter-containing plasmid in conjunction with the selectable marker genes, neomycin and hydromycin. The recombinant expression plasmids were transfected for stable integration into the genomic DNA of the V79 cells. Several cellular clones were obtained and checked for the genomic integration of intact cDNAs with the PCR on the genomic DNA of the recombinant cells. Stable expression was confirmed by the reverse transcriptase PCR (RT-PCR) on RNA preparations. Metabolic function was tested with ethoxyresorufin as a marker substrate for CYP1A2, and 2-aminofluorene and N-sulphametazine for N-acetyltransferase activity, and compared to data obtained from biological samples. 7-Ethoxyresorufin-O-deethylase activities ranged from 0.2 to 4 pmol resorufin/min/mg total protein. The N-acetylation of sulphametazine ranged from 0.07 to 1.7 nmol N-acetyl-sulphametazine/mg total protein/min. Selected clones showing activities in the range of physiological activities were submitted to metabolism dependent mutagenicity studies. In particular, the polymorphism-dependent N-acetylation of 2-aminofluorene and the role of CYP1A2 and N-acetyltransferase in the mutagenicity of 2-aminofluorene, were investigated. Surprisingly, the mutagenicity of 2-aminofluorene is dramatically reduced in V79 cells co-expressing CYP1A2 and N-acetyltransferase, compared to V79 cells expressing CYP1A2 only, pointing to a significant species-dependent difference in the metabolic activation of aromatic amines between rats and humans.

Effect of inhibition of aloe-emodin on N-acetyltransferase activity and gene expression in human malignant melanoma cells (A375.S2).

Arylamine carcinogens and drugs are N-acetylated by cytosolic N-acetyltransferase (NAT), which uses acetyl-coenzyme A as a cofactor. NAT plays an initial role in the metabolism of these arylamine compounds. 2-Aminofluorene is one of the arylamine carcinogens which have been demonstrated to undergo N-acetylation in laboratory animals and humans. Our previous study showed that human cancer cell lines (colon cancer, colo 205; liver cancer, Hep G2; bladder cancer, T24; leukemia, HL-60; prostate cancer, LNCaP; osteogenic sarcoma, U-2 OS; malignant melanoma, A375.S2) displayed NAT activity, which was affected by aloe-emodin in human leukemia cells. The purpose of this study was to determine whether aloe-emodin could affect the enzyme activity and gene expression of NAT at the mRNA and protein levels in malignant human melanoma A375.S2 cells. The results showed that aloe-emodin inhibited NAT1 activity (decreased N-acetylation of 2-aminofluorene) in intact cells in a dose-dependent manner. The effect of aloe-emodin on NAT1 at the protein level was determined by Western blotting and the mRNA levels were examined by polymerase chain reaction (PCR) and cDNA microarray. These results clearly indicate that aloe-emodin inhibits the mRNA expression and enzyme activity of NAT1 in A375.S2 cells.

N-acetyltransferase is involved in baicalein-induced N-acetylation of 2-aminofluorene and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells.

Many arylamine and hydrazine drugs are acetylated by cytosolic N-acetyltransferase (NAT). The human promyelocytic leukemia cell line (HL-60) has been shown to acetylate arylamine and contain NAT activity. The purpose of this study was to determine whether or not baicalein could affect N-acetylation of 2-aminofluorene (AF) in HL-60 cells. Acetylated and nonacetylated AF were determined by using high performance liquid chromatography. Baicalein displayed a dose-dependent inhibition of cytosolic and intact cells' NAT activity and reduced the number of viable cells. Time-course experiments showed that N-acetylation of AF, measured from intact HL-60 cells, was inhibited by baicalein for up to 48 h. Baicalein also decreased AF-DNA adduct formation in the examined cells. The effects of baicalein on NAT were examined by flow cytometry and NAT gene expression was examined by polymerase chain reaction. The results demonstrated that baicalein inhibited NAT1 mRNA gene expression and reduced the level of NAT in HL-60 cells. These results show that baicalein can affect the NAT activity of human leukemia cells in vitro.

Irreversible inactivation of arylamine N-acetyltransferases in the presence of N-hydroxy-4-acetylaminobiphenyl: a comparison of human and hamster enzymes.

Arylamine N-acetyltransferases (NATs) catalyze the N-acetylation of arylamines, the O-acetylation of N-arylhydroxylamines, and the conversion of N-(aryl)acetohydroxamic acids to N-acetoxyarylamines. NATs also undergo irreversible inactivation in the presence of N-(aryl)acetohydroxamic acids. We previously established that inactivation of hamster NAT1 by N-hydroxy-2-acetylaminofluorene is the result of sulfinamide adduct formation with Cys68. The purpose of this research was to determine the kinetics of inactivation of hamster NAT1, hamster NAT2, and human NAT1 by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), to identify the amino acids that are modified upon NAT-catalyzed bioactivation of N-OH-4-AABP, to characterize the adducts and to identify factors that influence the propensity of NATs to undergo inactivation by N-arylhydroxamic acids. Mass spectrometric analysis of the NATs, after incubation with N-OH-4-AABP, revealed that the principal adduct of each protein was a (4-biphenyl)sulfinamide. Proteolysis of the adducted NATs caused hydrolysis of the sulfinamides to sulfinic acids. Tandem mass spectrometric analysis of the modified peptides revealed that each NAT isozyme contained a sulfinic acid on the Cys68 side chain. Minor adducts were identified as 4-aminobiphenyl conjugates of tyrosines. Hamster NAT1 was more rapidly inactivated by N-OH-4-AABP than either hamster NAT2 or human NAT1, and it was demonstrated that 4-nitrosoobiphenyl, which forms the sulfinamide adducts, accumulates during incubation of N-OH-4-AABP with hamster NAT2 and human NAT1 but not during incubations with hamster NAT1. Steady state kinetic analysis of the hydrolysis of acetylated NATs revealed that the half-lives of acetylated hamster NAT2 and human NAT1 are 7-8-fold greater than that of acetylated hamster NAT1. These results support the proposal that the mechanism of inactivation of NATs by N-OH-4-AABP involves initial deacetylation to produce N-OH-4-aminobiphenyl, which after oxidative conversion to 4-nitrosobiphenyl reacts with Cys68 to form a sulfinamide. The relatively short half-life of the acetylated form of hamster NAT1 contributes to its greater susceptibility to inactivation by N-OH-4-AABP.

Arylalkylamine N-acetyltransferase gene expression in retina and pineal gland of rats under various photoperiods.

The present study examines how the circadian oscillators in the retina and the suprachiasmatic nucleus (SCN) respond to changes in photoperiod. Arylalkylamine N-acetyltransferase (aa-nat) gene expression studied by quantitative RT-PCR revealed that in adult Sprague-Dawley rats kept under different light-dark (LD) cycles for two weeks the temporal pattern of AA-NAT mRNA expression was identical in retina and pineal gland. In both tissues, the time span between the onset of darkness and the nocturnal rise in AA-NAT mRNA expression was 3 h under LD 20:4, 6 h under LD 12:12, and 15 h under LD 4:20. As aa-nat expression in the pineal gland is regulated by the circadian oscillator in SCN, the results suggest that the photoperiodic differences accompanying the seasons of the year are imprinted in more than one oscillator and that this may accentuate the important message regarding 'time of year.'

Embryonic and post-embryonic expression of arylalkylamine N-acetyltransferase and melatonin receptor genes in the eye and brain of chum salmon (Oncorhynchus keta).

Melatonin and arylalkylamine N-acetyltransferase (AANAT), the rate-limiting enzyme in melatonin synthesis, have taken on special importance in vertebrate circadian biology. Recent identification of genes encoding two AANAT (AANAT(1) and AANAT(2)) and two subtypes of melatonin receptor (Mel-R; Mel(1a) and Mel(1b)) in several fish species has led to rapid advances in characterizing the physiological roles of melatonin. In the present study, partial cDNAs encoding these four genes were cloned from the eye and brain of chum salmon (Oncorhynchus keta). Based on the nucleotide sequences, we developed highly sensitive real-time PCR systems for these four mRNAs. The development of daily rhythmicity in AANAT(1), AANAT(2), Mel(1a), and Mel(1b) transcript levels was examined in the eye and brain of chum salmon during embryonic and post-embryonic stages (from day -9 to day +180). In a parallel experiment, ocular and brain melatonin levels were measured by radioimmunoassay. Parallelism in developmental changes and in circadian rhythms of AANAT mRNAs and melatonin levels in the eye and the brain supports a hypothesis that the developmental increases of nocturnal melatonin levels results partly from the elevated transcription of AANAT genes. Moreover, abundant expression of AANAT and Mel-R mRNAs in the optic tectum, thalamus, hypothalamus, cerebellum, and eye indicates possible roles of melatonin in visual processing and neuroendocrine regulation, through which melatonin might be involved in migratory behavior of chum salmon.

PCR and flow cytometric analysis of paclitaxel-inhibited arylamine N-acetyltransferase activity and gene expression in human osteogenic sarcoma cells (U-2 OS).

Human epidemiological studies suggest an association between N-acetyltransferase (NAT) activity and the incidence of bladder and colorectal cancers. In this study, paclitaxel was selected to examine the inhibition of arylamine NAT activity, gene expression and 2-aminofluorene-DNA adduct formation in a human osteogenic sarcoma cell line (U-2 OS). The activity of NAT was determined by high performance liquid chromatography (HPLC) assay for the amounts of acetylated 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) and nonacetylated AF and PABA. Human osteogenic sarcoma cell cytosols and intact cells were used to examine the NAT activity, gene expression and AF-DNA adduct formation. The results demonstrated that NAT activity percent of NAT in examined cells, gene expression (NAT1 mRNA) and AF-DNA adduct formation in human osteogenic sarcoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The results also demonstrated that paclitaxel decreased the apparent values of Km and Vmax from intact human osteogenic sarcoma cells (U-2 OS). Thus, paclitaxel is an uncompetitive inhibitor of the NAT enzyme.