Serrated route to colorectal cancer: back street or super highway?

Morphological and molecular studies are beginning to distinguish separate evolutionary pathways for colorectal cancer, The serrated pathway encompassing hyperplastic aberrant crypt foci, hyperplastic polyps. mixed polyps, and serrated adenoma is increasingly being linked with genetic alterations, including DNA methylation, DNA microsatellite instability, Ii-ras mutation, and loss of chromosome Ip, The importance of the serrated pathway has been underestimated in terms of its frequency and potential for rapid progression, Copyright (C) 2001 John Wiley & Sons, Ltd.

Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings

High-level microsatellite instability (AISI-H) is demonstrated in 10 to 15% of sporadic colorectal cancers and in most cancers presenting In the inherited condition hereditary nonpolyposis colorectal cancer (HNPCC). Distinction between these categories of MSI-H cancer is of clinical importance and the aim of this study was to assess clinical, pathological, and molecular features that might he discriminatory. One hundred and twelve MSI-H colorectal cancers from families fulfilling the Bethesda criteria were compared with 57 sporadic MSI-H colorectal cancers. HNPCC cancers presented at a lower age (P < 0.001) with no sporadic MSI-H cancer being diagnosed before the age of 57 years. MSI was less extensive in HNPCC cancers with 72% microsatellite markers showing band shifts compared with 87% in sporadic tumors (P < 0.001). Absent immunostaining for hMSH2 was only found in HNPCC tumors. Methylation of bMLH1 was observed in 87% of sporadic cancers but also in 55% of HNPCC tumors that showed loss of expression of hMLH1 (P = 0.02). HNPCC cancers were more frequently characterized by aberrant beta -catenin immunostaining as evidenced by nuclear positivity (P < 0.001). Aberrant p53 immunostaining was infrequent in both groups. There were no differences with respect to 5q loss of heterozygosity or codon 12 K-ras mutation, which were infrequent in both groups. Sporadic MSI-H cancers were more frequently heterogeneous (P < 0.001), poorly differentiated (P = 0.02), mucinous (P = 0.02), and proximally located (P = 0.04) than RNPCC tumors. In sporadic MSI-H cancers, contiguous adenomas were likely to be serrated whereas traditional adenomas were dominant in HNPCC. Lymphocytic infiltration was more pronounced in HNPCC but the results did not reach statistical significance. Overall, HNPCC cancers were more like common colorectal cancer in terms of morphology and expression of beta -catenin whereas sporadic MSI-H cancers displayed features consistent with a different morphogenesis. No individual feature was discriminatory for all RN-PCC cancers. However, a model based on four features was able to classify 94.5% of tumors as sporadic or HNPCC. The finding of multiple differences between sporadic and familial MSI-H colorectal cancer with respect to both genotype and phenotype is consistent with tumorigenesis through parallel evolutionary pathways and emphasizes the importance of studying the two groups separately.

Caveolin and Ras function

Experimental protocols that allow confident assignment of signaling proteins to specific subdomains of the plasma membrane are essential for a full understanding of the complexities of signal transduction. This is especially relevant for Ras proteins, where the different membrane anchors of the Ras isoforms target them to functionally distinct microdomains that in turn allow quantitatively different signal outputs from otherwise highly homologous proteins. The methods outlined in this chapter, in addition to being invaluable in addressing Ras function, should also have wide utility in the study of many mammalian signal transduction pathways.

Control of the cystic fibrosis transmembrane conductance regulator by alpha G(i) and RGS proteins

The cystic fibrosis transmembrane conductance regulator (CFTR) has been shown previously to be regulated by inhibitory G proteins. In the present study, we demonstrate inhibition of CFTR by alphaG(i2) and alphaG(i1), but not alphaG(0), in Xenopus oocytes. We further examined whether regulators of G protein signaling (RGS) proteins interfere with alphaG(i)-dependent inhibition of CFTR. Activation of CFTR by IBMX and forskolin was attenuated in the presence of alphaG(i2), indicating inhibition of CFTR by alphaG(i2) in Xenopus oocytes. Coexpression of the proteins RGS3 and RGS7 together with CFTR and alphaG(i2) partially recovered activation by IBMX/forskolin. 14-3-3, a protein that is known to interfere with RGS proteins, counteracted the effects of RGS3. These data demonstrate the regulation of CFTR by alphaG(i) in Xenopus oocytes. Because RGS proteins interfere with the G protein-dependent regulation of CFTR, this may offer new potential pathways for pharmacological intervention in cystic fibrosis. (C) 2001 Academic Press.

Towards a molecular epidemiology of alcohol dependence analysing the interplay of genetic and environmental risk factors

Background Progress in identifying genetic factors protective against alcohol dependence (AlcD) requires a paradigm shift in psychiatric epidemiology. Aims To integrate analysis of research into the genetics of alcoholism. Method Data from prospective questionnaire and interview surveys of the Australian twin panel, and from a subsample who underwent alcohol challenge, were analysed. Results In men, effects of alcohol dehydrogenase ADH2*1/*2 genotype or high alcohol sensitivity (risk-decreasing), and of history of childhood conduct disorder, or having monozygotic co-twin or twin sister with AlcD (risk-increasing) were significant and comparable in magnitude. Religious affiliation (Anglican versus other) was associated with the ADH2 genotype, but did not explain the associations with AlcD symptoms. No protective effect of the ADH2*1/*2 genotype was observed in women. Conclusions The early onset and strong familial aggregation of AlcD, and opportunity for within-family tests of genetic association to avoid confounding effects, make epidemiological family studies of adolescents and young adults and their families a priority.

Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane: Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma ), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.

Interpretation of the reversible inhibition of adenosine deaminase by small cosolutes in terms of molecular crowding

Published results on the inhibitory effects of small cosolutes on adenosine deamination by adenosine deaminase [Kurz. L. C.. Weitkamp, E., and Frieden, C. (1987) Biochemistry 26, 3027-3032; Dzingeleski, G., and Wolfenden, R. (1993) Biochemistry 32, 9143 -9147] have been reexamined. Results for sucrose, dioxane, methanol, and ethanol are shown to be qualitatively consistent with thermodynamic interpretation in terms of molecular crowding effects arising from the occurrence of a minor increase in enzyme volume and/or asymmetry during the kinetic reaction-a conformational transition that could be either preexisting or ligand induced. Direct evidence for the existence of the putative isomeric transition is provided by active enzyme gel chromatography on Sephadex G-100, which demonstrates a negative dependence of enzyme elution volume upon substrate concentration and is therefore consistent with substrate-mediated conformational changes that favor a larger (or more asymmetric) isomeric state of the enzyme. There are thus experimental grounds for adopting the present description of the inhibitory effects of unrelated cosolutes on the kinetics of adenosine deamination by adenosine deaminase in terms of thermodynamic nonideality.

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b

The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as we:ll as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding try trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T reversible arrow R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.

The response of molecular isoforms of growth hormone to acute exercise in trained adult males

Circulating GH consists of multiple molecular isoforms, all derived from the one gene in nonpregnant humans. To assess the effect of a potent stimulus to pituitary secretion on GH isoforms, we studied 17 aerobically trained males (age, 26.9 +/- 1.5 yr) in a randomized, repeat measures study of rest vs. exercise. Exercise consisted of continuous cycle ergometry at approximately 80% of predetermined maximal oxygen uptake for 20 min. Serum was assayed for total, pituitary, 22-kDa, recombinant, non-22-kDa, 20-kDa, and immunofunctional GH. All isoforms increased during, peaked at the end, and declined after exercise. At peak exercise, 22-kDa GH was the predominant isoform. After exercise, the ratios of non-22 kDa/total GH and 20-kDa GH/total GH increased and those of recombinant/pituitary GH decreased. The disappearance half-times for pituitary GH and 20-kDa GH were significantly longer than those for all other isoforms. We conclude that 1) all molecular isoforms of GH measured increased with and peaked at the end of acute exercise, with 22-kBa GH constituting the major isoform in serum during exercise; and 2) the proportion of non-22-kDa isoforms increased after exercise due in part to slower disappearance rates of 20-kDa and perhaps other non-22-kDa GH isoforms. It remains to be determined whether the various biological actions of different GH isoforms impact on postexercise homeostasis.

A molecular comparison of T lymphocyte populations infiltrating the liver and circulating in the blood of patients with chronic hepatitis B: Evidence for antigen-driven selection of a public complementarity-determining region 3 (CDR3) motif

Despite a large number of T cells infiltrating the liver of patients with chronic hepatitis B, little is known about their complexity or specificity. To characterize the composition of these T cells involved with the pathogenesis of chronic hepatitis B (CHB), we have studied the clonality of V beta T cell receptor (TCR)-bearing populations in liver tissue by size spectratyping the complementarity-determining region (CDR3) lengths of TCR transcripts. We have also compared the CDR3 profiles of the lymphocytes infiltrating the liver with those circulating in the blood to see whether identical clonotypes may be detected that would indicate a virus-induced expansion in both compartments. Our studies show that in most of the patients examined, the T cell composition of liver infiltrating lymphocytes is highly restricted, with evidence of clonotypic expansions in 4 to 9 TCR V beta subfamilies. In contrast, the blood compartment contains an average of 1 to 3 expansions. This pattern is seen irrespective of the patient's viral load or degree of liver pathology. Although the TCR repertoire profiles between the 2 compartments are generally distinct, there is evidence of some T cell subsets being equally distributed between the blood and the liver. Finally, we provide evidence for a putative public binding motif within the CDR3 region with the sequence G-X-S, which may be involved with hepatitis B virus recognition.

Molecular weight changes and scission and crosslinking in poly(dimethyl siloxane) on gamma radiolysis

The molecular weight changes which occur on the gamma -radiolysis of poly(dimethyl siloxane) under vacuum between 77 and 373 K and in air at 303 K have been investigated using triple detection GPC to obtain the complete molecular weight distributions for the irradiated samples and to determine the number and weight average molecular weights. The results have been interpreted in terms of the relative yields of scission and crosslinking. The total yields for crosslinking predominate over those for scission at all the temperatures investigated for radiolysis under vacuum. Based on a solid-state Si-29 NMR analysis of PDMS irradiated under vacuum at 303 K, which yielded a value of G(Y) of 1.70, the values of G(S) = 1.15 +/-0.2 and G(H) = 1.45 +/-0.2 were obtained for radiolysis under vacuum at 303 K. For radiolysis in air at 303 K, crosslinking was also predominant, but the nett yield of crosslinking was much less than that observed for radiolysis under vacuum. Under the conditions of the radiolysis in air at 303 K, because of the low solubility of oxygen in PDMS, it is likely that the radiation chemistry is limited by oxygen diffusion. (C) 2001 Elsevier Science Ltd. All rights reserved.

Suppression of keratinocyte growth and differentiation by transforming growth factor beta 1 involves multiple signaling pathways

Transforming growth factor beta1 treatment of keratinocytes results in a suppression of differentiation, an induction of extracellular matrix production, and a suppression of growth. In this study we utilized markers specific for each of these functions to explore the signaling pathways involved in mediating these transforming-growth-factor-beta1-induced activities. In the first instance, we found that the induction of extracellular matrix production (characterized by 3TP-Lux reporter activity) was induced in both keratinocytes and a keratinocyte-derived carcinoma cell line, SCC25, in a dose-dependent manner. Furthermore, transforming growth factor beta1 also suppressed the differentiation-specific marker gene, transglutaminase type 1, in both keratinocytes and SCC25 cells. In contrast, transforming growth factor beta1 inhibited proliferation of keratinocytes but did not cause growth inhibition in the SCC25 cells. Transforming-growth-factor-beta1-induced growth inhibition of keratinocytes was characterized by decreases in DNA synthesis, accumulation of hypophosphorylated Rb, and the inhibition of the E2F:Rb-responsive promoter, cdc2, and an induction of the p21 promoter. When the negative regulator of transforming growth factor beta1 signaling, SMAD7, was overexpressed in keratinocytes it could prevent transforming-growth-factor-beta1-induced activation of the 3TP-Lux and the p21 promoter. SMAD7 could also prevent the suppression of the transglutaminase type 1 by transforming growth factor beta1 but it could not inhibit the repression of the cdc2 promoter. These data indicate that the induction of 3TP-Lux and p21 and the suppression of transglutaminase type 1 are mediated by a different proximate signaling pathway to that regulating the suppression of the cdc2 gene. Combined, these data indicate that the regulation of transforming growth factor beta1 actions are complex and involve multiple signaling pathways.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts, This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones, This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types, This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.