Cell transformation assays for prediction of carcinogenic potential: State of the science and future research needs

Copyright @ 2011 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting

Tuning transcription factor availability through acetylation-mediated genomic redistribution

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability

Cloning and expression of SAG: A novel marker of cellular senescence

Unlike immortalized cell lines, normal human fibroblasts in culture undergo replicative senescence in which the number of population doublings is limited. While fibroblasts display a variety of changes as they senesce in vitro, little is known about how gene expression varies as a function of population doubling level. We have used differential hybridization screening to identify human genes that are preferentially expressed in senescent cells. While we found several isolates that were up-regulated in late-passage cells, all appeared to be variants of the same cDNA, which we named senes-cence-associated gene (SAG). Our data show that SAG expression is threefold higher in senescent fibroblasts and closely parallels the progressive slowdown in growth potential, but is not cell-cycle regulated. Thus, SAG serves as an accurate marker for fibroblast growth potential during replicative senescence. Further studies demonstrated that SAG is a novel gene active in nearly all tissue types tested and that it is conserved through evolution. DNA sequencing data indicate that SAG contains a potential DNA-binding domain, suggesting that SAG may function as a regulatory protein.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30134/1/0000511.pd

Cancer Genomics Identifies Regulatory Gene Networks Associated with the Transition from Dysplasia to Advanced Lung Adenocarcinomas Induced by c-Raf-1

Background: Lung cancer is a leading cause of cancer morbidity. To improve an understanding of molecular causes of disease a transgenic mouse model was investigated where targeted expression of the serine threonine kinase c-Raf to respiratory epithelium induced initialy dysplasia and subsequently adenocarcinomas. This enables dissection of genetic events associated with precancerous and cancerous lesions. Methodology/Principal Findings: By laser microdissection cancer cell populations were harvested and subjected to whole genome expression analyses. Overall 473 and 541 genes were significantly regulated, when cancer versus transgenic and non-transgenic cells were compared, giving rise to three distinct and one common regulatory gene network. At advanced stages of tumor growth predominately repression of gene expression was observed, but genes previously shown to be upregulated in dysplasia were also up-regulated in solid tumors. Regulation of developmental programs as well as epithelial mesenchymal and mesenchymal endothelial transition was a hall mark of adenocarcinomas. Additionaly, genes coding for cell adhesion, i.e. the integrins and the tight and gap junction proteins were repressed, whereas ligands for receptor tyrosine kinase such as epi- and amphiregulin were up-regulated. Notably, Vegfr- 2 and its ligand Vegfd, as well as Notch and Wnt signalling cascades were regulated as were glycosylases that influence cellular recognition. Other regulated signalling molecules included guanine exchange factors that play a role in an activation of the MAP kinases while several tumor suppressors i.e. Mcc, Hey1, Fat3, Armcx1 and Reck were significantly repressed. Finally, probable molecular switches forcing dysplastic cells into malignantly transformed cells could be identified. Conclusions/Significance: This study provides insight into molecular pertubations allowing dysplasia to progress further to adenocarcinoma induced by exaggerted c-Raf kinase activity

Mouse Chromosome 11

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46996/1/335_2004_Article_BF00648429.pd

Effects Of Huntite/hydromagnesite On Capacity Of Flame Retardancy, Mechanical And Physical Properties Of Polypropylene

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2012Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2012Bu çalışmada, polipropilenin aleve dayanımını arttırmak için, huntit/hidromanyezit (H/HM) minerallerinden oluşan alev geciktirici sistem üzerine çalışılmıştır. H/HM yüzdeleri farklı 4 ticari karışım (U1250, U1253, LH15, H2090) kullanılmıştır. İdeal formül için, H/HM karışımları %30-40-50-60 yükleme seviyelerinde polipropilene çift vidalı ekstruderde eklenmiştir. H/HM karışımlarının kullanılan oranlarının polipropilen üzerindeki alev geciktirici performansı ve mekanik özelliklere etkisi incelendi. Çekme deneyi sonuçlarına göre, kompozit içerisindeki katkı maddesi oranının artmasıyla birlikte malzemenin kırılganlığı artmaktadır. Polipropilen içerisindeki H/HM oranının arttırılmasıyla birlikte, polipropilenin alev geciktirici performansının arttığı görülmektedir. UL 94-yatay yanmazlık testi, kızgın tel testi ve limit oksijen indeks ölçümü test sonuçlarına göre, her yükleme seviyesi için yüksek oranda (%50-60) hidromanyezit içeren karışım (LH15), yüksek oranda (%60-80) huntit içeren karışımlara (U1250, U1253, H2090) göre daha aktif bir performans göstermiştir. Polipropilenin alev geciktirici özelliklerini arttırmak amacıyla yapılan çalışmada ise %50 LH15 ve %10 çinko borat beraber kullanılmıştır. Test sonuçlarına göre LH15 ve çinko boratı birlikte içeren kompozit, mekanik ve yanmazlık özellikler açısından %60 oranında LH15 içeren kompozitten daha iyi sonuçlar göstermiştir.In this study, in order to increase the flame retardancy of polypropylene, usage of a flame retardant system comprising huntite and hydromagnesite was suggested. Four different commercial grades (U1250, U1253, LH15, H2090) of H/HM mixtures were used. In order to determine the ideal formulation, mixtures of H/HM were added to twin-screw extruder at 30-40-50-60% loading levels. The ratios of used H/HM mixtures on flame retardancy performances and mechanical properties of polypropylene were evaluated. The brittleness of the composite materials increased and tensile strengths of the test samples decreased with increasing the additive percentage in the composite. The addition of H/HM in polypropylene matrix increased flammability resistance of polypropylene. UL94-horizontal flammability, glow wire test and limiting oxygen index results showed that polypropylene composites containing higher hydromagnesite content (50-60%) (LH15) had more effective performance than that of the mixtures comprising higher huntite content (60-80%) (U1250, U1253, H2090) for each loading level. In order to improve flame retardancy properties of polypropylene, 10%zinc borate and 50%LH 15 were used together. According to the test results; the addition of zinc borate with LH 15 in the polypropylene matrix showed that a better flame retardancy performances and mechanical properties than that of 60% LH15 containing polypropylene composite.Yüksek LisansM.Sc