Enhanced skin carcinogenesis and lack of thymus hyperplasia in transgenic mice expressing human cyclin D1b (CCND1b)

Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties.Fil: Rojas, Paola Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. University of Texas; Estados UnidosFil: Benavides, Fernando. University of Texas; Estados UnidosFil: Blando, Jorge. University of Texas; Estados UnidosFil: Pérez, Carlos. University of Texas; Estados UnidosFil: Cardenas, Kim. University of Texas; Estados UnidosFil: Richie, Ellen. University of Texas; Estados UnidosFil: Knudsen, Erik S.. Thomas Jefferson University; Estados UnidosFil: Johnson, David G.. University of Texas; Estados UnidosFil: Senderowicz, Adrian M.. Department of Health and Human Services. Food and Drug Administration. Center for Drug Evaluation and Research; Estados UnidosFil: Rodriguez Puebla, Marcelo L.. University of North Carolina; Estados UnidosFil: Conti, Claudio. University of Texas; Estados Unido

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+– K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane

Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development

We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1−/− compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1−/− compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis

Cdk2 Deficiency Decreases ras/CDK4-Dependent Malignant Progression, but Not myc-Induced Tumorigenesis

We have previously shown that forced expression of CDK4 in mouse skin (K5CDK4 mice) results in increased susceptibility to squamous cell carcinomas (SCC) development in a chemical carcinogenesis protocol. This protocol induces skin papilloma development causing a selection of cells bearing activating Ha-ras mutations. We have also demonstrated that myc-induced epidermal proliferation and oral tumorigenesis (K5Myc mice) depends on CDK4 expression. Biochemical analysis of K5CDK4 and K5Myc epidermis as well as skin tumors showed that keratinocyte proliferation is mediated by CDK4 sequestration of p27Kip1 and p21Cip1, and activation of CDK2. Here, we studied the role of CDK2 in epithelial tumorigenesis. In normal skin loss of CDK2 rescues CDK4-induced, but not myc-induce epidermal hyperproliferation. Ablation of CDK2 in K5CDK4 mice results in decrease incidences and multiplicity of skin tumors as well as malignant progression to SCC. Histopathological analysis showed that K5CDK4 tumors are drastically more aggressive than K5CDK4/CDK2−/− tumors. On the other hand, we show that CDK2 is dispensable for myc-induced tumorigenesis. In contrast to our previous report K5Myc/CDK4−/− mice, K5Myc/CDK2−/− mice developed oral tumors with the same frequency as K5Myc mice. Overall we have established that ras-induced tumors are more susceptible to CDK2 ablation than myc-induced tumors, suggesting that the efficacy of targeting CDK2 in tumor development and malignant progression is dependent on the oncogenic pathway involved

GW150914: First search for the electromagnetic counterpart of a gravitational-wave event by the TOROS collaboration

We present the results of the optical follow-up conducted by the TOROS collaboration of the first gravitational-wave event GW150914. We conducted unfiltered CCD observations (0.35-1 micron) with the 1.5-m telescope at Bosque Alegre starting ~2.5 days after the alarm. Given our limited field of view (~100 square arcmin), we targeted 14 nearby galaxies that were observable from the site and were located within the area of higher localization probability. We analyzed the observations using two independent implementations of difference-imaging algorithms, followed by a Random-Forest-based algorithm to discriminate between real and bogus transients. We did not find any bona fide transient event in the surveyed area down to a 5-sigma limiting magnitude of r=21.7 mag (AB). Our result is consistent with the LIGO detection of a binary black hole merger, for which no electromagnetic counterparts are expected, and with the expected rates of other astrophysical transients.Comment: ApJ Letters, in pres

The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission

1Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs− parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs− parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito

Construct validity of a figure rating scale for Brazilian adolescents

<p>Abstract</p> <p>Background</p> <p>Figure rating scales were developed as a tool to determine body dissatisfaction in women, men, and children. However, it lacks in the literature the validation of the scale for body silhouettes previously adapted. We aimed to obtain evidence for construct validity of a figure rating scale for Brazilian adolescents.</p> <p>Methods</p> <p>The study was carried out with adolescent students attending three public schools in an urban region of the municipality of Florianopolis in the State of Santa Catarina (SC). The sample comprised 232 10-19-year-old students, 106 of whom are boys and 126 girls, from the 5th "series" (i.e. year) of Primary School to the 3rd year of Secondary School. Data-gathering involved the application of an instrument containing 8 body figure drawings representing a range of children's and adolescents' body shapes, ranging from very slim (contour 1) to obese (contour 8). Weights and heights were also collected, and body mass index (BMI) was calculated later. BMI was analyzed as a continuous variable, using z-scores, and as a dichotomous categorical variable, representing a diagnosis of nutritional status (normal and overweight including obesity).</p> <p>Results</p> <p>Results showed that both males and females with larger BMI z-scores chose larger body contours. Girls with higher BMI z-scores also show higher values of body image dissatisfaction.</p> <p>Conclusion</p> <p>We provided the first evidence of validity for a figure rating scale for Brazilian adolescents.</p