Acute WNT signalling activation perturbs differentiation within the adult stomach and rapidly leads to tumour formation

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach

Both base excision repair and O-6-methylguanine-DNA methyltransferase protect against methylation-induced colon carcinogenesis

Methylating agents are widely distributed environmental carcinogens. Moreover, they are being used in cancer chemotherapy. The primary target of methylating agents is DNA, and therefore, DNA repair is the first-line barrier in defense against their toxic and carcinogenic effects. Methylating agents induce in the DNA O[superscript 6]-methylguanine (O[superscript 6]MeG) and methylations of the ring nitrogens of purines. The lesions are repaired by O[superscript 6]-methylguanine-DNA methyltransferase (Mgmt) and by enzymes of the base excision repair (BER) pathway, respectively. Whereas O[superscript 6]MeG is well established as a pre-carcinogenic lesion, little is known about the carcinogenic potency of base N-alkylation products such as N3-methyladenine and N3-methylguanine. To determine their role in cancer formation and the role of BER in cancer protection, we checked the response of mice with a targeted gene disruption of Mgmt or N-alkylpurine-DNA glycosylase (Aag) or both Mgmt and Aag, to azoxymethane (AOM)-induced colon carcinogenesis, using non-invasive mini-colonoscopy. We demonstrate that both Mgmt- and Aag-null mice show a higher colon cancer frequency than the wild-type. With a single low dose of AOM (3 mg/kg) Aag-null mice showed an even stronger tumor response than Mgmt-null mice. The data provide evidence that both BER initiated by Aag and O[superscript 6]MeG reversal by Mgmt are required for protection against alkylation-induced colon carcinogenesis. Further, the data indicate that non-repaired N-methylpurines are not only pre-toxic but also pre-carcinogenic DNA lesions.Deutsche Forschungsgemeinschaft (DFG) (FOR 527)Deutsche Forschungsgemeinschaft (DFG) (DFG KA 724/13-3)Deutsche Forschungsgemeinschaft (DFG) (WI 3304/1-1

Expression of transforming growth factor beta-1 in gastric cancer and in the gastric mucosa of first-degree relatives of patients with gastric cancer

Transforming growth factors beta (TGF-β) constitute a family of polypeptide growth factors that control cell growth, cell differentiation and migration, as well as the formation of the extracellular matrix. Recent analyses revealed the overexpression of TGF-β1 in human gastric cancers and demonstrated increased cell proliferation in the stomach of patients with gastric cancer and their first-degree relatives. Using human gastric tissues obtained from patients with gastric cancer (n = 19), biopsies from healthy first-degree relatives of gastric cancer patients (n = 18) and healthy individuals (n = 19), we analysed the expression of TGF-β1 using the reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Fifteen of 19 patients with gastric cancer expressed TGF-β1 in the tumour. In 11 of these 15 cases TGF-β1 mRNA was also detectable in the non-tumourous stomach. Interestingly, all but two individuals with a first-degree relative diagnosed with gastric cancer exhibited TGF-β1 expression in either the antrum or corpus biopsy or both. In contrast, only one of 19 individuals without a family history of gastric cancer expressed TGF-β1 in the stomach (P< 0.0001). TGF-β1 expression is detectable in a large proportion of gastric cancers and in the stomach of healthy first-degree relatives of gastric cancer patients. Since individuals without gastric cancers in their family express TGF-β1 only in one of 19 cases, the induction of TGF-β1 expression in first-degree relatives of patients with gastric cancer points to the presence of specific molecular alterations in a subgroup of individuals with an increased risk of developing gastric cancer that may precede the development of gastric cancers. © 2000 Cancer Research Campaig

Ejaculate Economics: Testing the Effects of Male Sexual History on the Trade-Off between Sperm and Immune Function in Australian Crickets

Trade-offs between investment into male sexual traits and immune function provide the foundation for some of the most prominent models of sexual selection. Post-copulatory sexual selection on the male ejaculate is intense, and therefore trade-offs should occur between investment into the ejaculate and the immune system. Examples of such trade-offs exist, including that between sperm quality and immunity in the Australian cricket, Teleogryllus oceanicus. Here, we explore the dynamics of this trade-off, examining the effects that increased levels of sexual interaction have on the viability of a male's sperm across time, and the concomitant effects on immune function. Males were assigned to a treatment, whereby they cohabited with females that were sexually immature, sexually mature but incapable of copulation, or sexually mature and capable of copulation. Sperm viability of each male was then assessed at two time points: six and 13 days into the treatment, and immune function at day 13. Sperm viability decreased across the time points, but only for males exposed to treatment classes involving sexually mature females. This decrease was similar in magnitude across both sexually mature classes, indicating that costs to the expression of high sperm viability are incurred largely through levels of pre-copulatory investment. Males exposed to immature females produced sperm of low viability at both time points. Although we confirmed a weak negative association between sperm viability and lytic activity (a measure of immune response to bacterial infection) at day 13, this relationship was not altered across the mating treatment. Our results highlight that sperm viability is a labile trait, costly to produce, and subject to strategic allocation in these crickets

Reduction in membranous expression of β-catenin and increased cytoplasmic E-cadherin expression predict poor survival in gastric cancer

β-catenin, a component of the E-cadherin–catenin cell adhesion complex, also plays a separate intracellular signalling role, interacting with APC protein. Intracellular accumulation of β-catenin is common in colorectal neoplasia. β-catenin abnormalities are associated with poor survival in gastric cancer, but previous studies do not differentiate between membrane-associated and intracellular β-catenin. In this study we aimed to determine which type of expression abnormalities for E-cadherin, β-catenin and α-catenin correlate with clinico-pathological features and survival in gastric cancer. Immunoperoxidase staining of paraffin-embedded sections from 40 gastric cancers was performed for E-cadherin, α- and β-catenins using microwave unmasking and an avidin–biotin technique. Clinical data were obtained from case records and cancer registry records. Reduced membranous expression of β-catenin occurred in 10/12 (83%) diffuse and 8/28 (29%) intestinal tumours (P = 0.0014), and was associated with poor differentiation (P = 0.0015) and short survival (P = 0.032), but not with age, sex, tumour size or nodal status. Nuclear expression of β-catenin was uncommon; cytoplasmic expression was observed in 13/40 cases (33%) but did not correlate with histology, tumour grade or survival. Reduced E-cadherin membrane expression was associated with lymph node metastasis (P = 0.02). Neither E-cadherin or α-catenin expression correlated with survival. Reduced membranous expression of β-catenin predicts poor prognosis in gastric cancer, whilst ectopic intracellular expression is relatively rare. The apparent differences in β-catenin expression from those found in colon cancer merit further study. © 1999 Cancer Research Campaig

Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling

<p>Abstract</p> <p>Background</p> <p>The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic <it>in situ </it>screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models.</p> <p>Results</p> <p>To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3' UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section <it>in situ </it>hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs.</p> <p>Conclusion</p> <p>The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.</p

Sperm Competition, Sperm Numbers and Sperm Quality in Muroid Rodents

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An “overall sperm quality” parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert

Sex Determination in the Squalius alburnoides Complex: An Initial Characterization of Sex Cascade Elements in the Context of a Hybrid Polyploid Genome

BACKGROUND:Sex determination processes vary widely among different vertebrate taxa, but no group offers as much diversity for the study of the evolution of sex determination as teleost fish. However, the knowledge about sex determination gene cascades is scarce in this species-rich group and further difficulties arise when considering hybrid fish taxa, in which mechanisms exhibited by parental species are often disrupted. Even though hybridisation is frequent among teleosts, gene based approaches on sex determination have seldom been conducted in hybrid fish. The hybrid polyploid complex of Squalius alburnoides was used as a model to address this question. METHODOLOGY/PRINCIPAL FINDINGS:We have initiated the isolation and characterization of regulatory elements (dmrt1, wt1, dax1 and figla) potentially involved in sex determination in S. alburnoides and in the parental species S. pyrenaicus and analysed their expression patterns by in situ hybridisation. In adults, an overall conservation in the cellular localization of the gene transcripts was observed between the hybrids and parental species. Some novel features emerged, such as dmrt1 expression in adult ovaries, and the non-dimorphic expression of figla, an ovarian marker in other species, in gonads of both sexes in S. alburnoides and S. pyrenaicus. The potential contribution of each gene to the sex determination process was assessed based on the timing and location of expression. Dmrt1 and wt1 transcripts were found at early stages of male development in S. alburnoides and are most likely implicated in the process of gonad development. CONCLUSIONS/SIGNIFICANCE:For the first time in the study of this hybrid complex, it was possible to directly compare the gene expression patterns between the bisexual parental species and the various hybrid forms, for an extended set of genes. The contribution of these genes to gonad integrity maintenance and functionality is apparently unaltered in the hybrids, suggesting that no abrupt shifts in gene expression occurred as a result of hybridisation

Influence of O6-benzylguanine on the anti-tumour activity and normal tissue toxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea and molecular combinations of 5-fluorouracil and 2-chloroethyl-1-nitrosourea in mice

Previous studies have demonstrated that novel molecular combinations of 5-fluorouracil (5FU) and 2-chloroethyl-1-nitrosourea (CNU) have good preclinical activity and may exert less myelotoxicity than the clinically used nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study examined the effect of O6-alkylguanine-DNA-alkyltransferase (ATase) depletion by the pseudosubstrate O6-benzylguanine (BG) on the anti-tumour activity and normal tissue toxicity in mice of three such molecular combinations, in comparison with BCNU. When used as single agents at their maximum tolerated dose, all three novel compounds produced a significant growth retardation of BCNU-resistant murine colon and human breast xenografts. This in vivo anti-tumour effect was potentiated by BG, but was accompanied by severe myelotoxicity as judged by spleen colony forming assays. However, while tumour resistance to BCNU was overcome using BG, this was at the expense of enhanced bone marrow, gut and liver toxicity. Therefore, although this ATase-depletion approach resulted in improved anti-tumour activity for all three 5-FU:CNU molecular combinations, the potentiated toxicities in already dose-limiting tissues indicate that these types of agents offer no therapeutic advantage over BCNU when they are used together with BG. © 1999 Cancer Research Campaig

Balancing repair and tolerance of DNA damage caused by alkylating agents

Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity