Sensitive response of the Greenland Ice Sheet to surface melt drainage over a soft bed.

The dynamic response of the Greenland Ice Sheet (GrIS) depends on feedbacks between surface meltwater delivery to the subglacial environment and ice flow. Recent work has highlighted an important role of hydrological processes in regulating the ice flow, but models have so far overlooked the mechanical effect of soft basal sediment. Here we use a three-dimensional model to investigate hydrological controls on a GrIS soft-bedded region. Our results demonstrate that weakening and strengthening of subglacial sediment, associated with the seasonal delivery of surface meltwater to the bed, modulates ice flow consistent with observations. We propose that sedimentary control on ice flow is a viable alternative to existing models of evolving hydrological systems, and find a strong link between the annual flow stability, and the frequency of high meltwater discharge events. Consequently, the observed GrIS resilience to enhanced melt could be compromised if runoff variability increases further with future climate warming.

Critical roles for the COOH terminus of the Cu-ATPase ATP7B in protein stability, trans-Golgi network retention, copper sensing, and retrograde trafficking.

ATP7A and ATP7B are copper-transporting P-type ATPases that are essential to eukaryotic copper homeostasis and must traffic between intracellular compartments to carry out their functions. Previously, we identified a nine-amino acid sequence (F37-E45) in the NH(2) terminus of ATP7B that is required to retain the protein in the Golgi when copper levels are low and target it apically in polarized hepatic cells when copper levels rise. To understand further the mechanisms regulating the intracellular dynamics of ATP7B, using multiple functional assays, we characterized the protein phenotypes of 10 engineered and Wilson disease-associated mutations in the ATP7B COOH terminus in polarized hepatic cells and fibroblasts. We also examined the behavior of a chimera between ATP7B and ATP7A. Our results clearly demonstrate the importance of the COOH terminus of ATP7B in the protein's copper-responsive apical trafficking. L1373 at the end of transmembrane domain 8 is required for protein stability and Golgi retention in low copper, the trileucine motif (L1454-L1456) is required for retrograde trafficking, and the COOH terminus of ATP7B exhibits a higher sensitivity to copper than does ATP7A. Importantly, our results demonstrating that four Wilson disease-associated missense mutations behaved in a wild-type manner in all our assays, together with current information in the literature, raise the possibility that several may not be disease-causing mutations.

Alterations in the proteome of the NHERF2 knockout mouse jejunal brush border membrane vesicles.

To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.

Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles.

Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCß3, E-cadherin, p120, ß-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.

NH2-terminal signals in ATP7B Cu-ATPase mediate its Cu-dependent anterograde traffic in polarized hepatic cells.

Cu is an essential cofactor of cellular proteins but is toxic in its free state. The hepatic Cu-ATPase ATP7B has two functions in Cu homeostasis: it loads Cu+ onto newly synthesized apoceruloplasmin in the secretory pathway, thereby activating the plasma protein; and it participates in the excretion of excess Cu+ into the bile. To carry out these two functions, the membrane protein responds to changes in intracellular Cu levels by cycling between the Golgi and apical region. We used polarized hepatic WIF-B cells and high-resolution confocal microscopy to map the itinerary of endogenous and exogenous ATP7B under different Cu conditions. In Cu-depleted cells, ATP7B resided in a post-trans-Golgi network compartment that also contained syntaxin 6, whereas in Cu-loaded cells, the protein relocated to unique vesicles very near to the apical plasma membrane as well as the membrane itself. To determine the role of ATP7B's cytoplasmic NH2 terminus in regulating its intracellular movements, we generated seven mutations/deletions in this large [approximately 650 amino acid (AA)] domain and analyzed the Cu-dependent behavior of the mutant ATP7B proteins in WIF-B cells. Truncation of the ATP7B NH2 terminus up to the fifth copper-binding domain (CBD5) yielded an active ATPase that was insensitive to cellular Cu levels and constitutively trafficked to the opposite (basolateral) plasma membrane domain. Fusion of the NH2-terminal 63 AA of ATP7B to the truncated protein restored both its Cu responsiveness and correct intracellular targeting. These results indicate that important targeting information is contained in this relatively short sequence, which is absent from the related CuATPase, ATP7A.

Na(+)/H(+) exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells.

Cell biological approaches were used to examine the location and function of the brush border (BB) Na(+)/H(+) exchanger NHE3 in the opossum kidney (OK) polarized renal proximal tubule cell line. NHE3 epitope tagged with the vesicular stomatitis virus glycoprotein epitope (NHE3V) was stably expressed and called OK-E3V cells. On the basis of cell surface biotinylation studies, these cells had 10-15% of total NHE3 on the BB. Intracellular NHE3V largely colocalized with Rab11 and to a lesser extent with EEA1. The BB location of NHE3V was examined by confocal microscopy relative to the lectins wheat germ aggluttinin (WGA) and phytohemagluttin E (PHA-E), as well as the B subunit of cholera toxin (CTB). The cells were pyramidal, and NHE3 was located in microvilli in the center of the apical surface. In contrast, PHA-E, WGA, and CTB were diffusely distributed on the BB. Detergent extraction showed that total NHE3V was largely soluble in Triton X-100, whereas virtually all surface NHE3V was insoluble. Sucrose density gradient centrifugation demonstrated that total NHE3V migrated at the same size as approximately 400- and approximately 900-kDa standards, whereas surface NHE3V was enriched in the approximately 900-kDa form. Under basal conditions, NHE3 cycled between the cell surface and the recycling pathway through a phosphatidylinositol (PI) 3-kinase-dependent mechanism. Measurements of surface and intracellular pH were obtained by using FITC-WGA. Internalization of FITC-WGA occurred largely into the juxtanuclear compartment that contained Rab11 and NHE3V. pH values on the apical surface and in endosomes in the presence of the NHE3 blocker, S3226, were elevated, showing that NHE3 functioned to acidify both compartments. In conclusion, NHE3V in OK cells exists in distinct domains both in the center of the apical surface and in a juxtanuclear compartment. In the BB fraction, NHE3 is largely in the detergent-insoluble fraction in lipid rafts and/or in large heterogenous complexes ranging from approximately 400 to approximately 900 kDa.

Absence of direct delivery for single transmembrane apical proteins or their "Secretory" forms in polarized hepatic cells.

The absence of a direct route to the apical plasma membrane (PM) for single transmembrane domain (TMD) proteins in polarized hepatic cells has been inferred but never directly demonstrated. The genes encoding three pairs of apical PM proteins, whose extracellular domains are targeted exclusively to the apical milieu in Madin-Darby canine kidney cells, were packaged into recombinant adenovirus and delivered to WIF-B cells in vitro and liver hepatocytes in vivo. By immunofluorescence and pulse-chase metabolic labeling, we found that the soluble constructs were overwhelmingly secreted into the basolateral milieu, which in vivo is the blood and in vitro is the culture medium. The full-length proteins were first delivered to the basolateral surface but then concentrated in the apical PM. Our results imply that hepatic cells lack trans-Golgi network (TGN)-based machinery for directly sorting single transmembrane domain apical proteins and raise interesting questions about current models of PM protein sorting in polarized and nonpolarized cells.

Synapsin I is expressed in epithelial cells: localization to a unique trans-Golgi compartment.

Synapsin I is abundant in neural tissues. Its phosphorylation is thought to regulate synaptic vesicle exocytosis in the pre-synaptic terminal by mediating vesicle tethering to the cytoskeleton. Using anti-synapsin antibodies, we detected an 85 kDa protein in liver cells and identified it as synapsin I. Like brain synapsin I, non-neuronal synapsin I is phosphorylated in vitro by protein kinase A and yields identical (32)P-peptide maps after limited proteolysis. We also detected synapsin I mRNA in liver by northern blot analysis. These results indicate that the expression of synapsin I is more widespread than previously thought. Immunofluorescence analysis of several non-neuronal cell lines localizes synapsin I to a vesicular compartment adjacent to trans-elements of the Golgi complex, which is also labeled with antibodies against myosin II; no sub-plasma membrane synapsin I is evident. We conclude that synapsin I is present in epithelial cells and is associated with a trans-Golgi network-derived compartment; this localization suggests that it plays a role in modulating post-TGN trafficking pathways.

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells.

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.

A novel cellular phenotype for familial hypercholesterolemia due to a defect in polarized targeting of LDL receptor.

Basolateral targeting of membrane proteins in polarized epithelial cells typically requires cytoplasmic domain sorting signals. In the familial hypercholesterolemia (FH)-Turku LDL receptor allele, a mutation of glycine 823 residue affects the signal required for basolateral targeting in MDCK cells. We show that the mutant receptor is mistargeted to the apical surface in both MDCK and hepatic epithelial cells, resulting in reduced endocytosis of LDL from the basolateral/sinusoidal surface. Consequently, virally encoded mutant receptor fails to mediate cholesterol clearance in LDL receptor-deficient mice, suggesting that a defect in polarized LDL receptor expression in hepatocytes underlies the hypercholesterolemia in patients harboring this allele. This evidence directly links the pathogenesis of a human disease to defects in basolateral targeting signals, providing a genetic confirmation of these signals in maintaining epithelial cell polarity.

Isolation of rat hepatocyte plasma membrane sheets and plasma membrane domains.

This unit describes a method for isolation of plasma membrane sheets from rat liver. It also includes protocols for preparation of plasma membrane domains isolated from plasma membrane sheets and indirect immunofluorescence localization of marker proteins associated with plasma membrane sheets. The unit has been updated with assays for the marker enzymes alkaline phosphodiesterase I, 5' nucleotidase, and K+-stimulated.

Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat.

The regulation of intracellular vesicular trafficking is mediated by specific families of proteins that are involved in vesicular budding, translocation, and fusion with target membranes. We purified a vesicle-associated protein from hepatic microsomes using sequential column chromatography and partially sequenced it. Oliogonucleotides based on these sequences were used to clone the protein from a rat liver cDNA library. The clone encoded a novel protein with a predicted mass of 137 kDa (p137). The protein had an N terminus WD repeat motif with significant homology to Sec31p, a member of the yeast COPII coat that complexes with Sec13p. We found that p137 interacted with mammalian Sec13p using several approaches: co-elution through sequential column chromatography, co-immunoprecipitation from intact cells, and yeast two-hybrid analysis. Morphologically, the p137 protein was localized to small punctate structures in the cytoplasm of multiple cultured cell lines. When Sec13p was transfected into these cells, it demonstrated considerable overlap with p137. This overlap was maintained through several pharmacological manipulations. The p137 compartment also demonstrated partial overlap with ts045-VSVG protein when infected cells were incubated at 15 degrees C. These findings suggest that p137 is the mammalian orthologue of Sec31p.

Microsomal epoxide hydrolase gene polymorphism and susceptibility to colon cancer.

We examined polymorphisms in exons 3 and 4 of microsomal epoxide hydrolase in 101 patients with colon cancer and compared the results with 203 control samples. The frequency of the exon 3 T to C mutation was higher in cancer patients than in controls (odds ratio 3.8; 95% confidence intervals 1.8-8.0). This sequence alteration changes tyrosine residue 113 to histidine and is associated with lower enzyme activity when expressed in vitro. This suggests that putative slow epoxide hydrolase activity may be a risk factor for colon cancer. This appears to be true for both right- and left-sided tumours, but was more apparent for tumours arising distally (odds ratio 4.1; 95% confidence limits 1.9-9.2). By contrast, there was no difference in prevalence of exon 4 A to G transition mutation in cancer vs controls. This mutation changes histidine residue 139 to arginine and produces increased enzyme activity. There was no association between epoxide hydrolase genotype and abnormalities of p53 or Ki-Ras.

Evidence for apical endocytosis in polarized hepatic cells: phosphoinositide 3-kinase inhibitors lead to the lysosomal accumulation of resident apical plasma membrane proteins.

The architectural complexity of the hepatocyte canalicular surface has prevented examination of apical membrane dynamics with methods used for other epithelial cells. By adopting a pharmacological approach, we have documented for the first time the internalization of membrane proteins from the hepatic apical surface. Treatment of hepatocytes or WIF-B cells with phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, led to accumulation of the apical plasma membrane proteins, 5'-nucleotidase and aminopeptidase N in lysosomal vacuoles. By monitoring the trafficking of antibody-labeled molecules, we determined that the apical proteins in vacuoles came from the apical plasma membrane. Neither newly synthesized nor transcytosing apical proteins accumulated in vacuoles. In wortmannin-treated cells, transcytosing apical proteins traversed the subapical compartment (SAC), suggesting that this intermediate in the basolateral-to-apical transcytotic pathway remained functional. Ultrastructural analysis confirmed these results. However, apically internalized proteins did not travel through SAC en route to lysosomal vacuoles, indicating that SAC is not an intermediate in the apical endocytic pathway. Basolateral membrane protein distributions did not change in treated cells, uncovering another difference in endocytosis from the two domains. Similar effects were observed in polarized MDCK cells, suggesting conserved patterns of phosphoinositide 3-kinase regulation among epithelial cells. These results confirm a long-held but unproven assumption that lysosomes are the final destination of apical membrane proteins in hepatocytes. Significantly, they also confirm our hypothesis that SAC is not an apical endosome.

Direct detection of eae-positive bacteria in human and veterinary colorectal specimens by PCR.

A PCR test based on the amplification of an eae-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenic Escherichia coli and Citrobacter spp. (amplification of eae open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions. Positive PCR results were observed with eae-positive reference strains of E. coli and Citrobacter rodentium (Citrobacter freundii biotype 4280). Known eae-negative reference strains of E. coli and other laboratory strains of enteric bacteria were negative by the amplification test. The sensitivity of the PCR for detection of eae-positive E. coli and C. rodentium was between 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template. Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing) E. coli were PCR positive for bacterial eae genome. Sections from control animals were negative. Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial eae genome. The PCR test was a simple and quick method of detecting bacterial eae genome in human and veterinary clinical specimens. This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions. The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.

Apical plasma membrane proteins and endolyn-78 travel through a subapical compartment in polarized WIF-B hepatocytes.

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37 degreesC by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5'nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti-endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.

N-acetyl transferase 1: two polymorphisms in coding sequence identified in colorectal cancer patients.

Increased cancer risk has been associated with functional polymorphisms that occur within the genes coding for the N-acetyltransferase enzymes NAT1 and NAT2. We detected two NAT1 polymorphisms in colorectal cancer patients by heteroduplex analysis. DNA sequencing revealed the wild-type sequence (NAT1*4) and two single base substitutions at adjacent positions 999 bp (C to T, NAT1*14) and 1000 bp (G to A, NAT1*15) of the gene, changing Arg187 to a stop codon and Arg187 to Gln respectively. NAT1 alleles NAT1*4 (0.98) and NAT1*15 (0.02) were present at a similar frequency in patients with colorectal cancer (n=260) and in a Scottish control group (n=323). The third allele, NAT1*14, was present only in the colorectal cancer group at a frequency of 0.006. NAT1 genotype NAT1*4/ NAT1*15 was significantly less frequent in individuals that had a slow NAT2 genotype. This was observed in both cancer and control groups and suggests that this association was unrelated to cancer risk. We conclude that polymorphisms within the coding region of the NAT1 gene are infrequent and do not appear to have an independent association with colorectal cancer risk. However, the relationship between NAT1 and NAT2 polymorphisms appears non-random, suggesting a linkage between these enzymes.

Endogenous syntaxins 2, 3 and 4 exhibit distinct but overlapping patterns of expression at the hepatocyte plasma membrane.

To investigate the mechanisms regulating polarized vesicle delivery to the cell surface in hepatocytes, we have characterized the endogenous plasma membrane (PM)-associated syntaxins. These integral membrane proteins are components of the membrane docking/fusion apparatus and are thought to function as vesicle receptors at the PM. In hepatocytes, the PM is divided into two domains, the apical and basolateral. If syntaxins are mediating the specific recognition of vesicles delivered to either membrane surface, the simple prediction is that each domain expresses one syntaxin isoform. However, we report that rat hepatocytes express three endogenous PM-associated syntaxin isoforms, syntaxins 2, 3 and 4. By biochemical subfractionation, we determined that the syntaxins exhibit distinct, but overlapping patterns of expression among the PM domains. Syntaxin 4 is primarily expressed at the basolateral surface while syntaxins 2 and 3 are enriched at the apical PM. The immunolocalization of syntaxins 2 and 4 in rat hepatocytes and PM sheets revealed similarly complex patterns of PM expression with enhanced apical staining for both. A significant proportion of syntaxin 3 (25%) was detected in subcellular fractions containing transport vesicles. We have used quantitative immunoblotting to determine that the syntaxins are relatively abundant PM molecules (11-260 nM) in rat liver, spleen and kidney. Also, we determined that the syntaxin binding protein, Munc-18, is present at concentrations from 1.5-20 nM in the same tissues. Although this fundamental quantitative and morphological information is lacking in other systems, it is critical not only for defining syntaxin function, but also for predicting the specific mechanisms that regulate vesicle targeting in hepatocytes and other tissues.

N-acetyltransferase 2 genotype in colorectal cancer and selective gene retention in cancers with chromosome 8p deletions.

BACKGROUND: Genetic polymorphisms in N-acetyltransferase (NAT2) can change the normally fast acetylation of substrates to slow acetylation, and have been associated with the development of some cancers. The NAT2 locus may also suffer dysregulation during cancer progression, as the gene resides on chromosome 8p22, a region which is frequently deleted in colorectal cancer. SUBJECTS AND METHODS: A polymerase chain reaction based method was used to determine NAT2 genotype in 275 patients with colon cancer and 343 normal control DNAs. Within the cancer group, 65 cases known to contain deletions in chromosome 8p were examined for loss of heterozygosity at the NAT2 locus. RESULTS: Overall, there was no statistical difference in frequency or distribution of NAT2 alleles and genotype between colon cancer and control groups. There was a significant association between the slow acetylation genotype and early age of onset. NAT2 genotype did not vary with other clinical features of colon cancer, which included Dukes's stage, site of tumour, and sex. Of 48 informative cases, only three (6%) showed loss of heterozygosity, indicating that the NAT2 locus is not commonly deleted in colorectal cancer. This suggests that NAT2 is retained during the process of allele loss possibly because of its proximity to a gene necessary for cell viability. CONCLUSIONS: NAT2 does not play a major role in colorectal cancer risk, but may influence risk in some age groups. The nature of the loss of heterozygosity at the chromosome 8p site is complex and is worthy of further study.

Screening status in relation to biological and chronological characteristics of breast cancers: a cross sectional survey.

OBJECTIVE: To determine the pathological and biological characteristics of breast cancers diagnosed by screening and examined at the Edinburgh University pathology department. METHODS: These cancers were classified by screening status: never screened (n = 111), prevalence screen detected (n = 105), and previously screened (n = 74). The last category arose in women who had been regularly screened during the trial; the cancers were diagnosed as interval cases before the first invitation to service screening (n = 33) or were incidence screen detected at that time (n = 41). RESULTS: Association (for operable invasive cancers, n = 250) of cancer characteristics with screening status reflects influences of biology (aggressiveness) or chronology (time of diagnosis), or both. The prognostic indicators tumour grade, histological type, and oestrogen receptor status were found in a smaller percentage of the patients with poor prognosis among the prevalence screen detected cases (9%, 77%, 18%) than among those previously screened (29%, 84%, 35%). The chronological factors size and node status were found in a smaller percentage of patients with poor prognosis among women previously screened (31%, 24%) than among those never screened (62%, 39%). Apart from these two, no other factors improved the diagnosis in the previously screened group compared with the never screened group. CONCLUSIONS: These results suggest that favourable characteristics of screen detected cases are often due to the effects of length bias on "biological factors" and fail to show that current local screening practice has succeeded in advancing the diagnosis of breast cancers to a less aggressive phase.