Germline BRCA1 alterations in a population-based series of ovarian cancer cases.

The objective of this study was to provide more accurate frequency estimates of breast cancer susceptibility gene 1 ( BRCA1 ) germline alterations in the ovarian cancer population. To achieve this, we determined the prevalence of BRCA1 alterations in a population-based series of consecutive ovarian cancer cases. This is the first population-based ovarian cancer study reporting BRCA1 alterations derived from a comprehensive screen of the entire coding region. One hundred and seven ovarian cancer cases were analyzed for BRCA1 alterations using the RNase mismatch cleavage assay followed by direct sequencing. Two truncating mutations, 962del4 and 3600del11, were identified. Both patients had a family history of breast or ovarian cancer. Several novel as well as previously reported uncharacterized variants were also identified, some of which were associated with a family history of cancer. The frequency distribution of common polymorphisms was determined in the 91 Caucasian cancer cases in this series and 24 sister controls using allele-specific amplification. The rare form of the Q356R polymorphism was significantly ( P = 0.03) associated with a family history of ovarian cancer, suggesting that this polymorphism may influence ovarian cancer risk. In summary, our data suggest a role for some uncharacterized variants and rare forms of polymorphisms in determining ovarian cancer risk, and highlight the necessity to screen for missense alterations as well as truncating mutations in this population.

Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p-aminosalicylate as an in-vivo and in-vitro probe.

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT1-selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to N-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n = 144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n = 19) of this sample. Urinary and plasma ratios correlated moderately (r = 0.74, p < 0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent K(m) for PAS and a 90-fold decreased Vmax for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1 x 14 and NAT1 x 15, in case 244, as well as one variant, NAT1 x 11, which has been observed in previous investigations. NAT1 x 14 contained a missense mutation (G560-->A) that is predicted to change a single amino acid (Arg187-->Gln), as well as two 3' non-coding region mutations (T1088-->A and C1095-->A) that have previously been observed in the NAT1 x 10 allelic variant. NAT1 x 15 had a single nonsense mutation (C559-->T; Arg187-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1 x 11, NAT1 x 14 and NAT1 x 15 were 0.021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in our predominantly Caucasian sample.

Human acetyltransferase polymorphisms.

Conjugation of primary amino and hydroxylamino groups with acetate, catalyzed by acetyl CoA-dependent arylamine acetyltransferase (NAT) enzymes, may play an important role in the intricate series of metabolic pathways that produce or prevent toxicity following exposure to homo- and heterocyclic arylamine and hydrazine xenobiotics. Two independently regulated and kinetically distinct human acetyltransferases are now known to exist, namely NAT1 and NAT2. Interindividual variation in NAT2 function is associated with the classical isoniazid acetylation polymorphism which was discovered over forty years ago. At last count, fifteen variant alleles at the NAT2 gene locus have been linked to the isoniazid 'acetylator phenotype', and each of these can be identified in population studies using specific PCR-based genotyping tests. On the other hand, NAT1 shows kinetic selectivity for compounds whose disposition is unrelated to the classical isoniazid acetylation polymorphism. NAT1 expression is also phenotypically variable in human populations, at least in part due to allelic differences at the NAT1 gene locus. Nine NAT1 variant alleles have been described to date, of which NAT1* 14 and NAT1* 15 clearly produce defective NAT1 proteins and lead to functional impairment in the metabolism of NAT1-selective substrates both in vivo and in vitro. On the other hand, it has been reported that the NAT1* 10 variant associates with elevated NAT1 activity and increased risk for cancers of the bladder and colon. Because of the important toxicologic consequences of allelic variation in NAT1 and NAT2 function for the metabolic activation of arylamine and heterocyclic amine procarcinogens, further studies are needed to improve our understanding of the extent of NAT allelic variation, to determine the functional capacity of each variant gene product, and to develop accurate methods of detecting them in population and epidemiological studies.

Structure-function studies of human arylamine N-acetyltransferases NAT1 and NAT2. Functional analysis of recombinant NAT1/NAT2 chimeras expressed in Escherichia coli.

The human arylamine N-acetyltransferases NAT1 and NAT2 catalyze the biotransformation of primary aromatic amine or hydrazine drugs and xenobiotics. These enzymes share 81% amino acid sequence identity, yet differ markedly with respect to their acceptor substrate selectivities and intrinsic in vitro stabilities. To define the contribution of large regions of NAT1 and NAT2 polypeptide structure to enzyme integrity and catalytic specificity, we used selected restriction endonuclease digestions and fragment religation into the tac promoter-based phagemid pKEN2 to construct a panel of 18 NAT1/NAT2 hybrid gene vectors for heterologous expression in Escherichia coli. Induction of hybrid gene expression in recombinant transformants of E. coli strain XA90 led to the production of soluble, catalytically active acetylating enzymes in all cases. Chimeric proteins produced in this fashion were then compared to wild-type NAT1 and NAT2 with respect to their enzyme kinetic constants (apparent Km, Vmax, and Vmax/Km) for the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid and sulfamethazine, respectively, and for their in vitro stabilities at 37 degrees C. The ratio of the Vmax/Km for sulfamethazine to that for p-aminosalicylic acid allowed for the unambiguous classification of each enzyme as either NAT1 or NAT2 type, except for one novel chimera possessing a low Michaelis constant and a high maximal velocity for the acetylation of both substrates. A central region (amino acids 112-210) within the 290-residue polypeptide appeared to play a role in determining NAT1- or NAT2-type behavior. On the other hand, the region (residues 47-111) encompassing the putative active site cysteine (Cys68) was important in contributing to a low apparent Km for p-aminosalicylic acid but not for sulfamethazine, while amino acids 211-250 affected Km for sulfamethazine and 251-290 influenced Km for both substrates. Maximal velocities were highest for both substrates when the central 112-210 amino acid region was derived from NAT1. Finally, the region from amino acids 211-250 in NAT2 was important in determining its greater intrinsic enzyme stability than that exhibited by NAT1.

Dose-dependent effects of dietary phytosterol on epithelial cell proliferation of the murine colon.

Phytosterols are a group of compounds in plants that have been found to inhibit tumour development and decrease enhanced colonic epithelial cell proliferation in carcinogen-treated rats. The mechanism by which phytosterols may inhibit tumour development and alter cell proliferation is unknown. However, studies have shown that dietary phytosterol intake may alter levels of certain promoters in the colonic lumen, leading to altered levels of colonic epithelial cell proliferation. In this study, the effect of dietary phytosterol on the proliferative status of the intestinal epithelium was investigated in mice. Inbred C57B1/6J mice were fed semi-synthetic diet (control); 0.1% cholic acid, 0% phytosterol (cholic acid control); and cholic acid plus 0.3, 1.0 or 2.0% phytosterol. Dietary cholic acid significantly increased colonic epithelial cell proliferation and the highest labelled cell position by 92 and 35%, respectively. Phytosterol significantly reduced the enhanced labelling index and the position of the highest labelled cell in a dose-dependent manner. Mitotic index was also reduced significantly by phytosterol but not in a dose-dependent manner. The results of this study indicate that phytosterol influences the colonic epithelial cell morphometrics, that are important preneoplastic events in colon carcinogenesis, and may thus contribute to a reduced risk of cancer.

Dietary cholesterol enhances the induction and development of colonic preneoplastic lesions in C57BL/6J and BALB/cJ mice treated with azoxymethane.

The effect of dietary cholesterol on the induction and development of colonic precursor lesions was determined in two mouse strains, C57BL/6J and BALB/cJ, which differ in their metabolism of cholesterol. Mice were randomized into four groups and fed a cholesterol-free or a 1.25% cholesterol diet during and/or subsequent to four weekly injections of azoxymethane (5 mg/kg body wt.). Dietary cholesterol significantly increased the number of aberrant crypt foci (P less than 0.0001), enhanced cell proliferation (P less than 0.0001) and induced alterations in the proliferative pattern and crypt morphometrics in the colonic epithelium of both mouse strains. While C57BL/6J mice developed a greater number of aberrant crypt foci than BALB/cJ mice (p less than 0.0001), a significant diet-strain interaction effect was not observed. The present results indicate that dietary cholesterol enhances the induction and development of chemically-induced colonic precancerous lesions but this process is not affected by genetic differences in cholesterol metabolism, as represented by the two strains of mice studied.

Dietary cholesterol enhances preneoplastic aberrant crypt formation and alters cell proliferation in the murine colon treated with azoxymethane.

The effect of dietary cholesterol on the development of colonic preneoplastic aberrant crypts, as well as its influence on the proliferative status of the intestinal epithelium, was investigated in mice exposed to the chemical carcinogen azoxymethane. Two strains of mice, C57BL/6J and BALB/cJ, were fed a semisynthetic diet containing 0% (control), 1.25%, or 5.00% cholesterol for eight weeks. During the first four weeks of the experiment, mice were given weekly injections of azoxymethane. Cholesterol supplementation significantly increased the formation of aberrant crypts (p less than 0.0001), enhanced the rate of cell proliferation (p less than 0.0001), altered the cell proliferative pattern, and increased crypt height (p less than 0.05) and the total number of cells per crypt (p less than 0.01) in the colonic epithelium of both mouse strains. C57BL/6J mice developed a greater number of aberrant crypts (p less than 0.0001). However, a diet-strain interaction was not observed. The results of this study indicate that dietary cholesterol enhances colon carcinogenesis in the murine colon and therefore may be an important factor in the etiology of large bowel cancer in humans.

The role of dietary phytosterols in colon carcinogenesis.

Epidemiological and experimental studies have shown that increased intake of plant foods and decreased meat consumption are correlated with a decreased risk for colon cancer. Many components of plant foods are suggested to mitigate colon carcinogenesis, including vitamins, minerals, and dietary fiber. Phytosterols are a common component of plant foods consumed in relatively large quantities by vegetarians, who are at lower risk for colon cancer development than individuals on a Western diet low in phytosterols. In addition, phytosterols have been shown experimentally to inhibit colon cancer development. Dietary cholesterol, although structurally similar to the phytosterols, is correlated etiologically to the incidence of colon cancer, with changes in serum cholesterol levels and fecal bile acid profiles suggested to increase susceptibility to colon tumorigenesis. The objective of this paper is to discuss the effect of dietary phytosterols on cholesterol and bile acid metabolism and how these effects may lead to a decreased risk for colon cancer development.

Tridimensional ultrastructure of perfusion fixed gastrointestinal epithelial cells by high resolution scanning electron microscopy.

Improvements in the design of modern scanning electron microscopes (SEM) and new methods of specimen preparation incorporating chemical removal of the cytosol and cytoskeleton, now make it possible to view cells and their organelles in three dimensions (3D) at high magnification. In this experiment, high resolution SEM (HRSEM) utilizing new methods of tissue preparation was used to study the intracellular structures of the mouse ileum. In addition, in vivo intestinal perfusion was used to further enhance cellular preservation. Using these modifications it was possible to visualize, in 3D, the fine structure of intestinal epithelial cells and intracellular organelles such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi complex, as well as microvilli and cell membrane. Whole mitochondria appeared as irregularly shaped organelles which contained tubular cristae. Plate-like cristae were not observed. The brush border was found to be closely packed array of cylindrical projections. The extensive folding and structural intricacy of lateral cell membranes between absorptive cells could only be appreciated by viewing this tissue with 3D HRSEM. The use of HRSEM to study 3D ultrastructure of cells and their organelles will improve our understanding of the structure-function relationships in both the healthy and diseased gastrointestinal tract.