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.

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.

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.

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.

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.

The inhibition of N-acetyltransferase activity and gene expression in human bladder cancer cells (T24) by shikonin.

Shikonin has the potential to prevent, or be used in the treatment of, bladder transitional cell carcinoma induced by arylamines. We evaluated its effectiveness by measuring the amount of acetylated 2-aminofluorene (AF), AF-DNA adducts, changes of NAT mRNA and the amount of NAT enzyme. T24 human bladder cancer cells were incubated with 30 microM AF with different concentrations of shikonin for various times. T24 cells treated with shikonin (16 microM) were then harvested and used in 2 experiments: 1). T24 cells were incubated with 22.5 microM AF and shikonin (0, 16 microM) (co-treatment) for 6, 12, 18, 24 and 48 h. 2). T24 cells were incubated with various concentrations of AF and shikonin (0, 16 microM) for 24 h. AF and AAF were measured by HPLC. Then in the prepared human T24 cell cytosols different concentrations of AF and shikonin were added to measure the kinetic constants of NAT. Next, AF-DNA adducts in human T24 cells with or without treatment with shikonin were detected and measured. The final two steps included measuring the NAT Ag-Ab complex after treatment with and without shikonin and evaluating the effect of shikonin on the NAT genes. Higher concentrations of shikonin induced decreasing AF acetylation. We found that the longer the culture period, the greater the difference in AF acetylation in the same shikonin concentrations. It was also noted that increase in AAF was proportional to incubation time. In the presence of 16 microM of shikonin, N-acetylation of AF decreased by up to 72-84%. Shikonin decreased the amount of AAF production in human T24 cells in all examined AF doses. Both Km and Vmax values in the cytosolic NAT decreased after the addition of shikonin to the cytosol. Finally, shikonin decreased the amount of AAF production and AF-DNA adducts formation in human 724 cells in all examined AF doses. The percentage of cells stained by antibody was significantly different after treatment with shikonin, especially with the higher shikonin concentrations. The NAT1 mRNA level and the NAT1/beta-actin ratio decreased significantly with higher concentrations (16-24 microM) of shikonin. Shikonin affected NAT activity, gene expression (NAT1 mRNA), AF-DNA adducts formation and formation of NAT Ag-Ab in human bladder tumor T24 cells. Therefore, shikonin should be considered as a candidate agent for the prevention or treatment of transitional cell carcinoma.

Rat pineal arylalkylamine-N-acetyltransferase: cyclic AMP inducibility of its gene depends on prior entrained photoperiod.

The nocturnal biosynthesis of melatonin in the rat pineal depends on strongly enhanced expression of the enzyme N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87]. AA-NAT transcription is stimulated during darkness by adrenergic inputs to the pineal from the suprachiasmatic nucleus (SCN). Nocturnal activation of the AA-NAT promotor following stimulation of pinealocyte adrenoceptors involves cAMP-dependent stimulation of protein kinase A (PKA). The nocturnal rise in AA-NAT depends on the lighting conditions. As compared with light/dark (LD) 12:12, the delay between dark onset and the nocturnal rise in AA-NAT is shortened under long photoperiods and prolonged under short photoperiods. Here, we report that the rapidity of nocturnal AA-NAT induction depends on cAMP inducibility of the gene. Accordingly, cAMP produces a strong AA-NAT response in pineals obtained from rats housed under long photoperiods and a weak AA-NAT response under short photoperiods. Changes in AA-NAT inducibility are fully developed not earlier than after seven cycles. This observation suggests that long-term changes in the photoperiod are necessary to achieve full adjustment of cAMP inducibility of the gene. A direct relationship was found between cAMP-dependent AA-NAT inducibility and the pineal protein kinase A (PKA) activity. As compared to LD 12:12, PKA activity was increased under LD 20:4 and attenuated under LD 4:20. On the basis of the present findings, we suggest that the photoperiod determines the effectiveness of nocturnal AA-NAT induction by long-term modulation of the intrapineal pathway that transmits the cAMP signal to the AA-NAT gene.

Luteolin induces N-acetylation and DNA adduct of 2-aminofluorene accompanying N-acetyltransferase activity and gene expression in human bladder cancer T24 cell line.

Arylamine N-acetyltransferase (NAT) plays an important role in the first step of arylamines metabolism. Luteolin has shown antibacterial, antioxidant and antineoplastic activity. To glean insights into the mechanism of action of luteolin, we assessed the effects of luteolin on NAT activity and gene expression and DNA-2-aminofluorene (AF) adduct formation in human bladder cancer T24 cells. By using high performance liquid chromatography, the amounts of N-acetylation of AF and DNA adducts were determined and quantitated. Gene expression was performed by using polymerase chain reaction and gel electrophoresis. The results indicated: 1) luteolin displayed a dose-dependent inhibition of NAT activity and gene expression (NAT1 mRNA) in T24 cells; 2) time-course experiments showed that N-acetylation of AF measured from intact T24 cells was inhibited by luteolin for up to 48 hours; 3) using standard steady-state kinetic analysis, it was demonstrated that luteolin was a possible uncompetitive inhibitor of NAT activity in T24 cells; and 4) the DNA-AF adduct formation in T24 cells was inhibited by luteolin. This report is the first finding which has shown luteolin to affect human T24 cell NAT activity and gene expression (NAT1 mRNA) and DNA-AF adduct formation.

Cancer and phase II drug-metabolizing enzymes.

Cancer development results from the interaction between genetic factors, the environment, and dietary factors have been identified as modulators of carcinogenesis process. The formation of DNA adducts is recognized as the initial step in chemical carcinogenesis. Accordingly, blocking DNA adducts formation would be the first line of defense against cancer caused by carcinogens. Glutathione-S-transferases inactivate chemical carcinogens into less toxic or inactive metabolite through reduction of DNA adducts formation. There are many different types of glutathione S-transferase isozymes. For example, GST delta serves as a marker for hepatotoxicity in rodent system, and also plays an important role in carcinogen detoxification. Therefore, inhibition of GST activity might potentiate the deleterious effects of many environmental toxicants and carcinogens. In addition, approximately half of the population lacks GST Mu expression. Epidemiological evidence showed that persons possessing this genotype are predisposed to a number of cancers including breast, prostate, liver and colon cancers. In addition, individual risk of cancer depends on the frequency of mutational events in target oncogenes and tumor suppressor genes which could lead to loss of chromosomal materials and tumor progression. The most frequent genetic alteration in a variety of human malignant tumors is the mutation of the coding sequence of the p53 tumor suppressor gene. O(6)-alkylguanine in DNA leads to very high rates of G:C deltaA:T transitions in p53 gene. These alterations will modulate the expression of p53 gene and consequently change DNA repair, cell division, and cell death by apoptosis. Also, changes in the expression of BcI-2 gene results in extended viability of cells by over-riding programmed cell death (apoptosis) induced under various conditions. The prolonged life-span increases the risk of acquiring genetic changes resulting in malignant transformation. In addition, a huge variety of food ingredients have been shown to affect cell proliferation rates. They, therefore, may either reduce or increase the risk of cancer development and progression. For example, it has been found that a high intake of dietary fat accelerates the development of breast cancer in animal models. Certain diets have been suggested to act as tumor promoters also in other types of cancer such as colon cancer, where high intake of fat and phosphate have been linked to colonic hyper-proliferation and colon cancer development. Different factors such as oncogenes, aromatic amines, alkylating agents, and diet have a significant role in cancer induction. Determination of glutathione S-transferase isozymes in plasma or serum could be used as a biomarker for cancer in different organs and could give an early detection.

Expression of N-acetyltransferases in periodontal granulation tissue.

Smoking is a major risk of periodontal diseases. At the site of first contact, the gingiva is exposed to aromatic amines and polycyclic hydrocarbons. These are metabolized by the N-acetyltransferases (NAT), leading to local detoxification and/or activation reactions contributing to the risk of periodontal destruction in smokers. The purpose of this study was to detect the expression of N-acetyltransferase isoenzymes NAT1 and NAT2 in periodontal granulation tissue. In 24 specimens obtained from periodontitis patients or control subjects, mRNA encoding for NAT1 and NAT2 was detected by RT-PCR, and proteins were identified by immunohistochemistry. In periodontal granulation tissues, immunoreactivity for NAT1 and NAT2 was detected in infiltrating leukocytes and fibroblasts. In normal gingiva, both enzymes were found in epithelial cells, whereas NAT1 was also detected in endothelial cells. The results suggest that these enzymes may play a role in the defense against xenobiotics and the accelerated progression of periodontal disease in smokers.

Sulfhydryl G proteins and phospholipase A(2)-associated G proteins are involved in adrenergic signal transduction in the rat pineal gland.

The rat pineal gland with its circadian noradrenaline-regulated melatonin rhythm is an excellent model for studying adrenergic signal transduction with respect to cAMP and cGMP formation. The stimulatory G(s) proteins play a well-established role in this process. In contrast, the potential roles of the inhibitory G(i) proteins, the functionally unclear other G(o) proteins, and a number of G protein subtypes are not known. The present study examines the effects on beta(1)- and beta(1)-plus-alpha(1)-stimulated cAMP and cGMP formation of a number of G protein modulators in rat pinealocyte suspension cultures. The effects of the nitric oxide donor sodium nitroprusside on cGMP were also examined. The results showed that drugs that activate G proteins of the G(i)/G(o) family, i.e., pertussis toxin, mastoparan, and compound 48/80, had no effect on unstimulated, isoproterenol (beta(1))-stimulated, or combined isoproterenol/phenylephrine (beta(1)-plus()-alpha(1))-stimulated cAMP and cGMP accumulation. However, in this experimental paradigm, the inhibitors of sulfhydryl G proteins (N-ethylmaleimide) and those of phospholipase A2-related G proteins (isotetrandrine) exerted a clear inhibitory effect. Sodium-nitroprusside-stimulated cGMP accumulation was also inhibited. These results confirm a previous report that members of the G(i)/G(o) family, which are present in the rat pineal gland, do not play a major role in adrenergic signal transduction. The new finding that sulfhydryl G proteins and phospholipase A2-associated G proteins exert a clear stimulatory effect on adrenergic signal transduction suggests that they are subtypes of G(s) proteins.

Selective adrenergic/cyclic AMP-dependent switch-off of proteasomal proteolysis alone switches on neural signal transduction: an example from the pineal gland.

The molecular processes underlying neural transmission are central issues in neurobiology. Here we describe a novel mechanism through which noradrenaline (NA) activates its target cells, using the mammalian pineal organ as a model. In this neuroendocrine transducer, NA stimulates arylalkylamine N:-acetyltransferase (AANAT; EC 2.3.1. 87), the key enzyme regulating the nocturnal melatonin production. In rodents, AANAT protein accumulates as a result of enhanced transcription, but in primates and ungulates, the AANAT mRNA level fluctuates only marginally, indicating that other mechanisms regulate AANAT protein and activity. These were investigated in cultured bovine pinealocytes. AANAT mRNA was readily detectable in unstimulated pinealocytes, and levels did not change following NA treatment. In contrast, NA increased AANAT protein levels in parallel with AANAT activity, apparently through a cyclic AMP-mediated mechanism. Immunocytochemistry revealed that the changes in AANAT protein levels occurred in virtually all pinealocytes. Inhibition of AANAT degradation by proteasomal proteolysis alone was found to switch-on enzyme activity by increasing AANAT protein levels five- to 10-fold. Accordingly, under unstimulated conditions AANAT protein is continually synthesized and immediately destroyed by proteasomal proteolysis. NA appears to act via cyclic AMP to protect AANAT from proteolytic destruction, resulting in accumulation of the protein. These findings show that tightly regulated control of proteasomal proteolysis of a specific protein alone can play a pivotal role in neural regulation.