Pak BJ and Pang SC, J Mol Cell Cardiol 1999 Sep;31(9):1717-24.
The process of translation initiation has been postulated to play an important role in the regulation of cellular growth and proliferation. Here, we report the identification and differential expression of a fundamental translational repressor NAT1, during early
postnatal cardiac development. Differential display analysis of RNA obtained from 3-day and 4-week-old rat hearts resulted in the cloning and identification of a 396 bp cDNA fragment (DRCF-6) which corresponded to the 3' terminal portion of NAT1. Northern blot analysis revealed that the mRNA expression of NAT1 was markedly elevated during the first 2 weeks of postnatal life, with an apparent peak level of expression occurring at 1 week. NAT1 mRNA levels then steadily decreased to 4 weeks of age. The NAT1 transcript has previously been shown to be extensively edited by the enzyme APOBEC-1, which deaminates specific cytidine bases to uridine; cytidine deamination at a glutamine codon (CAA) results in the formation of a stop codon (UAA) and consequently, premature termination of translation. Accordingly, Western blot analysis detected the presence of several smaller proteins in addition to the full length NAT1 protein (97 kDa), each exhibiting a distinct pattern of expression during cardiac development. APOBEC-1 editing of NAT1 during cardiac development was further supported by primer extension analysis of cytidine 1699, which was found to be predominantly edited to uridine. Immunohistochemical staining showed that NAT1 is expressed predominantly in atrial and ventricular myocytes, although staining was also detected in vascular smooth muscle cells and in the endocardium. These results suggest that NAT1 may play a role in the postnatal development of the heart and demonstrate that APOBEC-1 editing may possibly be a novel mechanism by which translation is regulated during cardiac development.
The development of hypertension-induced cardiac hypertrophy is a complex process involving a number of biochemical pathways. In particular, the translation initiation pathway has been postulated to play an important role in controlling cellular growth and proliferation in the cardiovascular system.
Recently, a fundamental translational repressor, NAT1 (novel APOBEC target 1), has been identified. We have previously shown that NATI is developmentally-regulated in the heart of neonatal rats and its expression correlates with periods of rapid cardiac growth. The present investigation was designed to determine whether the expression of NAT1 is modified in the left ventricle of spontaneously hypertensive rats and 2-kidney-1-clip (2K1C) hypertensive rats. Northern blot analysis revealed an increase in NAT1 mRNA expression which correlates with the onset of cardiac hypertrophy. Unlike its pattern of mRNA expression, however, NAT1 protein level did not differ significantly from their respective controls throughout the time course. Interestingly, several protein species ranging in size from approximately 40-70 kDa were detected by Western blotting, in addition to the full length 97 kDa NAT1. Since the NAT1 transcript is a known substrate for the enzyme APOBEC-1 and possibly APOBEC-2, we speculate that these proteins may represent truncated fragments of NAT1 resulting from the formation of premature translation termination codons along the NAT1 transcript by APOBEC editing. Together, these results show that the ventricular expression of NAT1 is regulated at the transcriptional level during the early stages of genetic and 2K1C-induced hypertension and may be involved in the onset of left ventricular hypertrophy.
Our aim was to examine the role of NAT1 and NAT2 polymorphisms in human larynx cancer susceptibility. Genotype tests for NAT1 alleles *4, *10 and *11, and NAT2 alleles *4, *5, *6A and *7A, using PCR-RFLP analysis, were perfo
rmed in 172 healthy Portuguese individuals and 88 patients with squamous cell carcinoma of the larynx. NAT1 and NAT2 genotype frequencies were correlated between patients and control groups, using the chi-square test. Odds ratios and 95% confidence intervals were calculated from 2 x 2 tables with the Fisher's exact model. The statistical analysis of NAT1 and NAT2 genotype frequencies revealed a significant difference of NAT1*10/*11 (p = 0.038) and NAT2*5/*7 (p = 0.003) genotype distribution between cases and controls. We also observed differences concerning tumor location, since NAT1*10/*11 genotype frequency was significantly different when comparing normal control individuals with the glottic subgroup of patients. The present data suggest that NAT1 and NAT2 polymorphisms may be correlated with an increased risk of larynx cancer.
Hein DW, etal., Environ Mol Mutagen. 2002;40(3):161-7.
N-acetyltransferase-1 (NAT1) and N-acetyltransferase-2 (NAT2) are important in the metabolism of aromatic and heterocyclic amine carcinogens that induce prostate tumors in the rat. We investigated the association of genetic polymorphisms in NAT1
ght:700;'>NAT1 and NAT2, alone and in combination, with human prostate cancer. Incident prostate cancer cases and controls in a hospital-based case-control study were frequency-matched for age, race, and referral pattern. The frequency of slow acetylator NAT1 genotypes (NAT1*14, *15, *17) was 5.8% in controls but absent in cases. In contrast, in comparison with all other NAT1 genotypes the putative rapid acetylator NAT1 genotype (NAT1*10) was significantly higher in prostate cancer cases than controls (OR, 2.17; 95% CI, 1.08-4.33; P = 0.03). Combinations of NAT1*10 with NAT2 slow acetylator genotypes (OR, 5.08; 95% CI, 1.56-16.5; P = 0.008) or with NAT2 very slow (homozygous NAT2*5) acetylator genotypes (OR, 7.50; 95% CI, 1.55-15.4; P = 0.016) further increased prostate cancer risk. The results of this small pilot study suggest increased susceptibility to prostate cancer for subjects with combinations of NAT1*10 and slow (particularly very slow) NAT2 acetylator genotypes. This finding should be investigated further in larger cohorts and in other ethnic populations.
Abdel-Rahman SZ, etal., Mutat Res. 1998 Feb 26;398(1-2):43-54.
The NAT1 gene exhibits polymorphisms in the non-coding polyadenylation region with a number of alleles. Of these alleles, NAT1*10 is responsible for increased NAT1 enzyme levels and is r
eported to be associated with increased risk for colorectal and bladder cancers. In view of the possible role of the NAT1 gene product in the metabolism of a number of cigarette smoke carcinogens, we tested the possibility that genetic variation in the NAT1 gene might also be associated with increased risk for lung cancer. Allelic variances of the NAT1 gene were analyzed in 45 lung cancer patients and 47 controls who were matched with respect to age, race and gender using restriction fragment length polymorphism (RFLP) analysis and allele-specific (AS)-PCR. Our results indicate that individuals who inherited the NAT1*10 allele had a 3.7-fold increased relative risk for lung cancer (95% CL = 1.2-16.0, p < 0.02). There was a 6.8-fold increase in relative risk for lung cancer associated with the inheritance of the NAT1*10 allele in younger individuals (< 60 years of age) compared to 2.2-fold increase in older individuals (> 60 years old) (OR = 6.8; 95% CL = 1.1-40.7, p < 0.01 and OR = 2.2; 95% CL = 0.5-11.1, p = 0.2, respectively). We have also applied the sensitive fluorescence in situ hybridization (FISH) tandem probe assay to elucidate the frequency of chromosome breakage among a subgroup of the studied individuals harboring the NAT1*10 allele (17 lung cancer patients, 17 smoking controls and 7 non-smoking controls). Our results indicate a significant increase (p < 0.001) in the frequency of chromosome breaks in lung cancer patients (mean +/- SE per 100 cells = 1.45 +/- 0.11) and in smoking controls (1.30 +/- 0.13) compared to non-smoking controls (0.47 +/- 0.07). Regression analysis indicated a highly significant positive correlation between the duration of smoking in years and the frequency of chromosome breaks in lung cancer patients (r = 0.62, p = 0.008), but not in smoking controls (r = 0.02; p = 0.91). These findings suggest that NAT1 polymorphism may be an important genetic determinant of lung cancer risk. In addition, these data provide a mechanistic link between the inheritance of the NAT1*10 allele and smoking-induced lung cancer. Given that the NAT1 enzyme can mediate activation and detoxication pathways for numerous carcinogens and given that this polymorphism is prevalent in the general population (20-50% frequency), it may play a significant role in influencing the outcome of a variety of environmental cancers.
Human N-acetyltransferase 1 (NAT1) and 2 (NAT2) are important phase II enzymes involved in the biotransformation of xenobiotics. In toxicity and carcinogenicity studies, functional polymorphism of rat N-acetyltransferase is considered a model for similar human v
ariability. To accurately quantitate expression of the three rat N-acetyltransferases, we developed sensitive, specific assays for Nat1, Nat2, and Nat3 mRNAs. In male F344 rats, tissue-specific expression varied over a limited range for both Nat1 (approximately 19-fold) and Nat2 (approximately 30-fold), with the highest expression of both genes in colon. Expression of Nat3 mRNA was at least 2 to 3 orders of magnitude less than that of Nat1 or Nat2. Comparison of Nat1 and Nat2 mRNA expression in bladder, colon, liver, and lung of male and female F344 rats detected no significant gender-specific difference. In Sprague-Dawley and F344 rats ranging in age from neonate to mature adult, colon showed a >10-fold increase in Nat2 during the first postnatal month that did not correlate with changes in Nat1. In contrast, Nat2 showed no developmental change in Sprague-Dawley or F344 liver as Nat1 increased modestly. These measures of rat Nat expression confirm that Nat3 expression is negligible and that Nat1 and Nat2 are the primary determinants of arylamine acetylation activity in all tissues tested. The findings demonstrate differential tissue-specific and developmental regulation of the rat Nat1 and Nat2 genes and contribute to more complete understanding of tissue-, gender-, and development-specific expression patterns of the cognate N-acetyltransferase genes of humans and other species.
The highly polymorphic N-acetyltransferases (NAT1 and NAT2) are involved in both activation and inactivation reactions of numerous carcinogens, such as tobacco derived aromatic amines. The potential effect of the NAT genotypes in individual susceptibility to lun
g cancer was examined in a hospital based case-control study consisting of 392 Caucasian lung cancer patients [152 adenocarcinomas, 173 squamous cell carcinomas (SCC) and 67 other primary lung tumours] and 351 controls. In addition to the wild-type allele NAT1*4, seven variant NAT1 alleles (NAT1*3, *10, *11, *14, *15, *17 and *22) were analysed. A new method based on the LightCycler (Roche Diagnostics Inc.) technology was applied for the detection of the polymorphic NAT1 sites at nt 1088 and nt 1095. The NAT2 polymorphic sites at nt 481, 590, 803 and 857 were detected by polymerase chain reaction-restriction fragment length polymorphism or LightCycler. Multivariate logistic regression analyses were performed taking into account levels of smoking, age, gender and occupational exposure. An increased risk for adenocarcinoma among the NAT1 putative fast acetylators [odds ratio (OR) 1.92 (1.16-3.16)] was found but could not be detected for SCC or the total case group. NAT2 genotypes alone appeared not to modify individual lung cancer risk, however, individuals with combined NAT1 fast and NAT2 slow genotype had significantly elevated adenocarcinoma risk [OR 2.22 (1.03-4.81)] compared to persons with other genotype combinations. These data clearly show the importance of separating different histological lung tumour subtypes in studies on genetic susceptibility factors and implicate the NAT1*10 allele as a risk factor for adenocarcinoma.
Liu X, etal., EMBO Rep. 2016 Mar;17(3):349-66. doi: 10.15252/embr.201540505. Epub 2016 Feb 5.
As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2-p53 interaction upon cellular stress, while other mechanisms by which n
ucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53-mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.
Mughal AA, etal., Mol Cancer. 2015 Aug 21;14:160. doi: 10.1186/s12943-015-0432-z.
BACKGROUND: Glioblastoma (GBM) is the most common primary brain malignancy and confers a dismal prognosis. GBMs harbor glioblastoma-initiating cells (GICs) that drive tumorigenesis and contribute to therapeutic resistance and tumor recurrence. Consequently, there is a strong rationale to target this
cell population in order to develop new molecular therapies against GBM. Accumulating evidence indicates that Nalpha-terminal acetyltransferases (NATs), that are dysregulated in numerous human cancers, can serve as therapeutic targets. METHODS: Microarrays were used to study the expression of several NATs including NAT12/NAA30 in clinical samples and stem cell cultures. The expression of NAT12/NAA30 was analyzed using qPCR, immunolabeling and western blot. We conducted shRNA-mediated knockdown of NAT12/NAA30 gene in GICs and studied the effects on cell viability, sphere-formation and hypoxia sensitivity. Intracranial transplantation to SCID mice enabled us to investigate the effects of NAT12/NAA30 depletion in vivo. Using microarrays we identified genes and biochemical pathways whose expression was altered upon NAT12/NAA30 down-regulation. RESULTS: While decreased expression of the distal 3'UTR of NAT12/NAA30 was generally observed in GICs and GBMs, this gene was strongly up-regulated at the protein level in GBM and GICs. The increased protein levels were not caused by increased levels of the steady state mRNA but rather by other mechanisms. Also, shorter 3'UTR of NAT12/NAA30 correlated with poor survival in glioma patients. As well, we observed previously not described nuclear localization of this typically cytoplasmic protein. When compared to non-silencing controls, cells featuring NAT12/NAA30 knockdown exhibited reduced cell viability, sphere-forming ability, and mitochondrial hypoxia tolerance. Intracranial transplantation showed that knockdown of NAT12/NAA30 resulted in prolonged animal survival. Microarray analysis of the knockdown cultures showed reduced levels of HIF1alpha and altered expression of several other genes involved in the hypoxia response. Furthermore, NAT12/NAA30 knockdown correlated with expressional dysregulation of genes involved in the p53 pathway, ribosomal assembly and cell proliferation. Western blot analysis revealed reduction of HIF1alpha, phospho-MTOR(Ser2448) and higher levels of p53 and GFAP in these cultures. CONCLUSION: NAT12/NAA30 plays an important role in growth and survival of GICs possibly by regulating hypoxia response (HIF1alpha), levels of p-MTOR (Ser2448) and the p53 pathway.
