Combined in utero and juvenile exposure of mice to arsenate and atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors.

The effects of arsenate (As) and atrazine (Atr) on myeloid progenitors (colony-forming unit-granulocyte/macrophage, CFU-GM) cells derived from bone marrow were studied in male and female mice after combined in utero and juvenile exposure. Female adult mice were treated with arsenate in drinking water during gestation. Then, separate groups of males and females' offspring were exposed for 4 months to atrazine, to additional arsenate or to co-exposure of atrazine and arsenate together in drinking water. In male mice, arsenate and the combined exposure did not modulate the percentage of CFU-GM progenitors, whereas atrazine significantly decreases the clonogenicity of myeloid cells. In females, the percentage of CFU-GM significantly decreased after atrazine exposure did not change with arsenate treatment, but dramatically increased after the combined exposure. The expression of estrogen receptors alpha (ERalpha) and beta (ERbeta) in bone marrow cells was investigated, and an up-regulation of receptor beta was observed in both genders. A gene expression profile was generated using nylon membranes spotted with 1185 cancer-related genes. Results from microarrays indicate that atrazine alone did not stimulate the expression of any of the genes analysed in both male and female. Arsenic induced gene expression modulation only in female. Major significant changes on the gene expression resulted following the co-exposure to arsenic and atrazine in both male and female.

Relationship between p53 and p27 expression following HER2 signaling.

HER2, frequently associated with low p27 expression in breast tumors, when activated has been found to upmodulate p53 in tumor cells. The aim of this work was to investigate the role of p53 in the connection between HER2 and p27. Fifty-two breast tumor specimens, characterized for p53 mutations, were analyzed immunohistochemically (IHC) for HER2, p53 and p27 expression. p27, inversely associated with HER2, was found in 29% of tumors with IHC-negative mutated p53 versus 93% of tumors with accumulation of p53 protein and 59% with wild-type p53 (p=0.001), indicating a direct association between p53 and p27 expression. HER2-overexpressing cell lines carrying wild-type or null p53 protein, and treated with heregulin beta1 (HRG), were analyzed for expression and subcellular localization of p53 and p27. In HER2-overexpressing cells stimulated with HRG, p27 protein expression increased in parallel with p53 with no corresponding increase in p27 transcript. No p27 increase was observed in p53-null cells. Transfection with wild-type p53 restored p27 upmodulation in HRG-stimulated cells, indicating a crucial role of p53 in determining p27 upmodulation following HER2 activation. Together, our data demonstrate the crucial role of p53 in determining p27 upmodulation following HER2 activation. This could have implications in the response to Transtuzumab therapy.

A molecular view of autophagy in Lepidoptera.

Metamorphosis represents a critical phase in the development of holometabolous insects, during which the larval body is completely reorganized: in fact, most of the larval organs undergo remodeling or completely degenerate before the final structure of the adult insect is rebuilt. In the past, increasing evidence emerged concerning the intervention of autophagy and apoptosis in the cell death processes that occur in larval organs of Lepidoptera during metamorphosis, but a molecular characterization of these pathways was undertaken only in recent years. In addition to developmentally programmed autophagy, there is growing interest in starvation-induced autophagy. Therefore we are now entering a new era of research on autophagy that foreshadows clarification of the role and regulatory mechanisms underlying this self-digesting process in Lepidoptera. Given that some of the most important lepidopteran species of high economic importance, such as the silkworm, Bombyx mori, belong to this insect order, we expect that this information on autophagy will be fully exploited not only in basic research but also for practical applications.

Variability in temperature-related mortality projections under climate change.

BACKGROUND: Most studies that have assessed impacts on mortality of future temperature increases have relied on a small number of simulations and have not addressed the variability and sources of uncertainty in their mortality projections. OBJECTIVES: We assessed the variability of temperature projections and dependent future mortality distributions, using a large panel of temperature simulations based on different climate models and emission scenarios. METHODS: We used historical data from 1990 through 2007 for Montreal, Quebec, Canada, and Poisson regression models to estimate relative risks (RR) for daily nonaccidental mortality in association with three different daily temperature metrics (mean, minimum, and maximum temperature) during June through August. To estimate future numbers of deaths attributable to ambient temperatures and the uncertainty of the estimates, we used 32 different simulations of daily temperatures for June-August 2020-2037 derived from three global climate models (GCMs) and a Canadian regional climate model with three sets of RRs (one based on the observed historical data, and two on bootstrap samples that generated the 95% CI of the attributable number (AN) of deaths). We then used analysis of covariance to evaluate the influence of the simulation, the projected year, and the sets of RRs used to derive the attributable numbers of deaths. RESULTS: We found that < 1% of the variability in the distributions of simulated temperature for June-August of 2020-2037 was explained by differences among the simulations. Estimated ANs for 2020-2037 ranged from 34 to 174 per summer (i.e., June-August). Most of the variability in mortality projections (38%) was related to the temperature-mortality RR used to estimate the ANs. CONCLUSIONS: The choice of the RR estimate for the association between temperature and mortality may be important to reduce uncertainty in mortality projections.

Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori.

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase.

Implications of gender differences for human health risk assessment and toxicology.

This paper from The Human Health working group of SGOMSEC 16 examines a broad range of issues on gender effects in toxicology. Gender differences in toxicology begin at the gamete and embryo stage, continuing through development and maturation and into old age. Sex influences exposure, toxicokinetics, and toxicodynamics. The effects of sex have often been overlooked in both epidemiology and toxicology. In addition to the obvious modifying effects of the sex hormones and conditions affecting the male and female reproductive organs and sex roles, both genetic and hormonal effects influence many aspects of life and toxic responses. All aspects of toxicology should consider gender-balanced designs so that a more comprehensive understanding of differences and similarities can be obtained. Differential gene expression is a new frontier in toxicology. Risk assessment should account for gender and life cycle differences. The biological basis for altered sex ratios observed in several populations should be sought in animal models, and expanded to other compounds that might exert sex-selective effects. Wherever possible and feasible, toxicologic and environmental epidemiological studies should be designed and have sufficient statistical power to quantify differential gender-based exposures and outcomes.

Gene expression profiling of renal cell carcinoma: a DNA macroarray analysis.

OBJECTIVE: To examine differences in gene expression levels between renal cell carcinoma (RCC) tissue and 'normal' appearing renal tissue using a commercially available DNA macroarray. MATERIALS AND METHODS: Tissue was obtained from 47 consecutive radical nephrectomies, 29 of which were eligible. DNA macroarrays were analysed on the tumour and normal-appearing control tissue to measure the expression of 1185 cancer-related genes. The group of samples was also stratified according to the presence or absence of granular cells and according to tumour grade. Quantitative real-time polymerase-chain reaction (PCR) was also performed on seven key genes present on the macroarray. RESULTS: In all, 444 genes were over-expressed and 33 genes were under-expressed. Using selection criteria reduced the list to nine that were significantly over-expressed and 23 that were under-expressed. These significant genes belonged to the families of oncogenes, growth factors, interleukins, receptors, immune system components, cytoskeleton, matrix proteins and intracellular modulators, or they coded for proteins involved in DNA transcription and RNA translation, DNA repair, protein turnover, and metabolism of carbohydrates and lipids. There were differences in gene expression according to the presence or absence of granular cells and according to tumour grade. Using quantitative real-time PCR there was over-expression of epidermal growth factor receptor, c-myc, transforming growth factor-alpha, vascular endothelial growth factor and vimentin, and under-expression of TYRO3 protein tyrosine kinase. The von Hippel-Lindau gene was under-expressed but not significantly. CONCLUSIONS: A procedure for collecting and storing fresh renal tissue and subsequent gene expression profiling of RCC and normal renal tissue was established. A commercially available DNA macroarray coupled with the significance analysis of macroarrays allowed the identification of sets of differentially expressed cancer-related genes that were characteristic of RCC, compared with apparently normal renal tissue, and which distinguished among subgroups divided according to tumour grade and histological subtype. Quantitative PCR is important to validate the results of macroarray experiments.

Sensitivity of human cord blood cells to tetrachloroethylene: cellular and molecular endpoints.

The International Agency for Research on Cancer (IARC) currently lists tetrachloroethylene [perchloroethylene (PCE)] as being carcinogenic in animals. PCE is listed as possibly carcinogenic to humans upon occupational exposure. Human exposure to PCE can produce oesophageal cancer, cervical cancer, non-Hodgkin's lymphoma, urinary bladder cancer and leukemia. This work shows that PCE modulates the expression of some genes implicated in cancer induction, cell differentiation, cell-cycle progression, and the survival and clonogenic potential of human cord blood cells. After exposure to the compound, the modulated genes were involved in inflammatory responses as with the mitogen-activated protein kinase 14 (MPK 14), or in tumor and metastasis progression as with the matrix metalloproteinase 17 (MMP 17), in cell proliferation as with c-jun and c-fos, and moreover in the apoptotic process as with interferon alpha-inducible protein (IFI), BAX and BCL-2. Analysis of cord blood cells via flow cytometry showed that PCE treatment induced a statistically significant increase in necrosis after 24 h, while the clonogenicity of Human Colony-Forming Unit-Granulocyte/Macrophage (CFU-GM) and Burst-Forming Unit-Erythrocyte (BFU-E) progenitors did not change. In conclusion, our data showed that PCE affected various pathways involved in cancer induction, but its action on cell proliferation and differentiation is not yet clearly understood.

HER2 signaling enhances 5'UTR-mediated translation of c-Myc mRNA.

The increased levels of c-Myc protein observed previously in an ovarian carcinoma cell line stably transfected to express HER2 has suggested a role for the HER2 pathway in c-Myc expression. Analysis of HER2-transfected cells stimulated with heregulin beta1 (HRG) revealed increased c-Myc protein levels but not a corresponding increase in c-Myc mRNA expression or any change in c-Myc protein half-life. Transfection of HER2-overexpressing cells with a construct containing the 5' untranslated region (5'UTR) of c-Myc mRNA originated from the P2 promoter and placed upstream of the Renilla luciferase gene, enhanced reporter expression upon stimulation with HRG. The HRG-mediated increase in reporter activity correlated with the HRG-mediated induction observed for c-Myc protein, identifying the P2-derived leader (P2L) of c-Myc mRNA as the cis-element involved in c-Myc translational induction. Both the increase in c-Myc protein levels and P2L-enhanced translational activity were inhibited by the PI3K inhibitor wortmannin. Together, these results demonstrate that HRG stimulation of HER2 overexpressing cells leads to enhanced c-Myc protein synthesis through activation of the PI3K/Akt/mTOR pathway and that the P2L of c-Myc mRNA is the element responsible for induction of c-Myc translation.

Genetic progression in sporadic endometrial and gastrointestinal cancers with high microsatellite instability.

This study selected a series of 136 MSI-H (microsatellite instability at high frequency) gastric, colorectal, and endometrial carcinomas combining immunohistochemical analysis for hMLH1 or hMSH2 gene products and microsatellite study. The clinico-pathological profile of all tumours was correlated with the overall instability rates at coding and non-coding repeats, in order to clarify the role and the mutation timing of seven target genes (TGFbetaRII, IGFIIR, BAX, hMSH3, hMSH6, CHK1, and BRCA2) in the progression of an MSI-H neoplasm. Regardless of the primary site, the results confirm a model of oncogenesis in which inactivation of hMLH1, or less frequently hMSH2, may initiate a pathway culminating in a progressive accumulation of frameshifts in coding region (CDR) microsatellites. Comparing gastrointestinal and endometrial tumours, significantly lower levels of microsatellite instability at both coding and non-coding repeats were observed. Among gastric and colorectal tumours, the detection of small shortening within Bat-26 and Bat-25 markers defines a subgroup of MSI-H gastrointestinal tumours invariably characterized by early stage at diagnosis. In these tumours, mutations of TGFbetaRII or BAX genes precede frameshifts in the other tested genes. The analysis of correlations between the mutational and clinico-pathological profiles of advanced gastrointestinal tumours revealed that the higher levels of microsatellite instability at both coding and non-coding repeats were not associated with a more advanced clinico-pathological stage or a less favourable outcome. A significant association was observed between a low number of CDR frameshifts and the presence of lymph-node metastasis in advanced gastrointestinal tumours. The existence of advanced MSI-H tumours with more aggressive behaviour and a 'mild mutator phenotype' could be explained by hypothesizing an overlapping of different mechanisms of tumourigenesis, including both the mutator and the suppressor pathways; this should be tested by further studies.