Search

The association between ionizing radiation and the subsequent development of osteosarcoma has been well described, but little is known about the cytogenetic and molecular events, which could be involved in the formation of radiation-induced osteosarcomas. Here, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in a series of 16 rat osteosarcomas induced by injection of plutonium-238. Recurrent gains/amplifications were observed at chromosomal regions 3p12-q12, 3q41-qter, 4q41-qter, 6q12-q16, 7q22-q34, 8q11-q23, 9q11-q22, 10q32.1-qter, and 12q, whereas recurrent losses were observed at 1p, 1q, 3q23-q35, 5q21-q33, 8q24-q31, 10q22-q25, 15p, 15q, and 18q. The gained region at 7q22-q34 was homologous to human chromosome bands 12q13-q15/8q24/22q11-q13, including the loci of Mdm2, Cdk4, c-Myc and Pdgf-b genes. The lost regions at 5q21-q33, 10q22-q25 and 15q contained tumor suppressor genes such as p16INK4a/p19ARF, Tp53 and Rb1. To identify potential target gene(s) for the chromosomal aberrations, we compared the expression levels of several candidate genes, located within the regions of frequent chromosomal aberrations, between the tumors and normal osteoblasts by using quantitative RT-PCR analysis. The Cdk4, c-Myc, Pdgf-b and p57KIP2 genes were thought to be possible target genes for the frequent chromosomal gain at 7q22-34 and loss at 1q in the tumors, respectively. In addition, mutations of the Tp53 gene were found in 27% (4 of 15) osteosarcomas. Our data may contribute to further understanding of the molecular mechanisms underlying osteosarcomas induced by ionizing radiation in human.

We have previously studied genomic copy number changes and global gene expression patterns in rat osteosarcomas (OS) induced by the bone-seeking alpha emitter (238)Pu by comparative genomic hybridization (CGH) and oligonucleotide microarray analyses, respectively. Among the previously identified genes that were down-regulated in radiation-induced rat OS tumors, Cited2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2) and Akap12 (a kinase anchoring protein, also known as src-suppressed C-kinase substrate, SSeCKS) genes mapped to the most frequently lost regions on chromosome 1p. In the present study, relative copy number losses of Cited2 and Akap12 genes were observed in 8 of 15 (53%) and 10 of 15 (67%) tumors by quantitative PCR analysis. Loss of Cited2 and Akap12 in the tumors was confirmed at the levels of mRNA and protein expression by quantitative RT-PCR and immunoblot analyses, respectively. These results indicate that Cited2 and Akap12 are silenced in radiation-induced OS, and therefore are novel candidate tumor-suppressor genes of this tumor.

To better understand the molecular basis of radiation-induced osteosarcoma (OS), we performed global gene expression profiling of rat OS tumors induced by the bone-seeking alpha emitter (238)Pu, and the expression profiles were compared with those of normal osteoblasts (OB). The expressions of 72 genes were significantly differentially expressed in the tumors related to OB. These included genes involved in the cell adhesion (e.g., Podxl, Col18a1, Cd93, Emcn and Vcl), differentiation, developmental processes (e.g., Hhex, Gata2, P2ry6, P2rx5, Cited2, Osmr and Igsf10), tumor-suppressor function (e.g., Nme3, Blcap and Rrm1), Src tyrosine kinase signaling (e.g., Hck, Shf, Arhgap29, Cttn and Akap12), and Wnt/beta-catenin signaling (e.g., Fzd6, Lzic, Dkk3 and Ctnna1) pathways. Expression changes of several genes were validated by quantitative real-time RT-PCR analysis. Notably, all of the identified genes involved in the Wnt/beta-catenin signaling pathway were known or proposed to be negative regulators of this pathway and were downregulated in the tumors, suggesting the activation of beta-catenin in radiation-induced OS. By using immunohistochemical and immunoblot analyses, constitutive activation of the Wnt/beta-catenin signaling pathway in the tumors was confirmed by observing nuclear and/or cytoplasmic localization of beta-catenin and a decrease in its inactive (phosphorylated) form. Furthermore, we found a significant reduction in the levels of glycogen synthase kinase 3beta (GSK-3beta) protein in the tumors relative to OB. Taken together, these findings provide new insights into the molecular basis of radiation-induced OS.

International audience; Malignant mesothelioma (MM) is one of the worst cancers in terms of clinical outcome, urging the need to establish and characterize new preclinical tools for investigation of the tumorigenic process, improvement of early diagnosis and evaluation of new therapeutic strategies. For these purposes, we characterized a collection of 27 cell lines established from F344 rats, after 136 to 415 days of induction with crocidolite asbestos administered intraperitoneally. Four mesotheliomas were distinguished from 23 preneoplastic mesothelial cell lines (PN) according to their propensity to generate tumors after orthotopic transplantation into syngeneic rats, their growth pattern, and the expression profile of three genes. PN cell lines were further discriminated into groups / subgroups according to morphology in culture and the expression profiles of 14 additional genes. This approach was completed by analysis of positive and negative immunohistochemical MM markers in the four tumors, of karyotype alterations in the most aggressive MM cell line in comparison with a PN epithelioid cell line, and of human normal mesothelial and mesothelioma cells and a tissue array. Our results showed that both the rat and human MM cell lines shared in common a dramatic decrease in the relative expression of Cdkn2a and of epigenetic regulators, in comparison with PN and normal human mesothelial cells, respectively. In particular, we identified the involvement of the relative expression of the Ten-Eleven Translocation (TET) family of dioxygenases and Dnmt3a in relation to the 5-hydroxymethylcytosine level in malignant transformation and the acquisition of metastatic potential. INTRODUCTION Malignant mesothelioma (MM) is a rare, aggressive cancer mainly related to asbestos exposure [1], the long latency time between exposure and occurrence of clinical symptoms limiting the efficacy of therapeutic interventions. Given its chemoresistance, current therapies have a negligible impact on overall survival due to a lack of understanding of the complex biology of MM [2]. Thus, a better understanding of the different steps in the development of this disease should be of great interest for the identification of early markers of MM AU 1

A standard pig flow karyotype (2N = 38 chromosomes) was defined by standardization of several flow karyotypes obtained from stimulated peripheral blood lymphocytes of normal male and female pigs. Depending on the animals under study, the flow analysis of their chromosome suspensions gave rise to bivariate flow karyotypes comprising from 15 to 17 peaks, of which 11 to 15 represented single chromosomes. The results were used to propose a peak nomenclature. In addition, a male miniature pig lymphoblastoid cell line was characterized by flow cytogenetics. A very high-resolution flow karyotype, in which all peaks but one superimposed on those of the standard karyotype, was obtained. Peaks were assigned for chromosomes X and Y. Analysis of flow karyotypes obtained from translocated t(3,7)(p1.3;q2.1) pigs combined with polymerase chain reaction (PCR) studies of major histocompatibility complex (MHC)-linked sequences on flow-sorted chromosomes allowed identification of peaks 3 and 7 of normal pig chromosomes and of the derivative chromosomes associated with the t(3,7)(p1.3;q2.1) translocation.

International audience; Inhalation of radon is closely associated with an increased risk of lung cancers. While the involvement of Ink4a in lung tumor development has been widely described, the tumor suppressor gene has not been studied in radon-induced lung tumors. In this study, loss of heterozygosity (LOH) analysis of the Cdkn2a locus, common to the Ink4a and Arf genes, was performed on 33 radon-induced rat lung tumors and showed a DNA loss in 50% of cases. The analysis of p16(Ink4a) protein expression by immunohistochemistry revealed that 50% of the tumors were negative for this protein. Looking for the origin of this lack of expression, we observed a low frequency of homozygous deletion (6%), a lack of mutation, an absence of correlation between promoter methylation and Ink4a mRNA expression and no correlation between LOH and protein expression. However, a tendency for an inverse correlation between p16(Ink4a) and pRb protein expression was observed. The expressions of p19Arf, Mmd2 and Mdm4 were not deregulated and only 14% of the tumors were mutated for Tp53. These results indicated that Ink4a/Cdk4/Rb1 pathway deregulation, more than Arf/Mdm2/Tp53 pathway, has a major role in the development of these tumors through p16(Ink4a) deregulation. However, all known mechanisms of inactivation of the pathway do not play a recurrent role in these tumors and the actual origin of the lack of p16(Ink4a) protein expression remains to be established.

The patched (Ptc1) protein is a negative regulator of sonic hedgehog signaling, a genetic pathway whose perturbation causes developmental defects and predisposition to specific malignant tumors. Humans and mice with mutated Ptc1 are prone to medulloblastoma and basal cell carcinoma (BCC), both tumors showing dependence on radiation damage for rapid onset and high penetrance. Poly(ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that plays a multifunctional role in DNA damage signaling and repair. In healthy and fertile PARP-1-null mice, radiation exposure reveals an extreme sensitivity and a high genomic instability. To test for interactions between PARP-1 and sonic hedgehog signaling, PARP-1-null mice were crossed to Ptc1 heterozygous mice. PARP-1 deletion further accelerated medulloblastoma development in irradiated Ptc1(+/-) mice, showing that PARP-1 inactivation sensitizes cerebellar cells to radiation tumorigenic effects. In addition to increased formation and slowed down kinetics of disappearance of gamma-H2AX foci, we observed increased apoptosis in PARP-1-deficient granule cell progenitors after irradiation. Double-mutant mice were also strikingly more susceptible to BCC, with >50% of animals developing multiple, large, infiltrative tumors within 30 weeks of age. The results provide genetic evidence that PARP-1 function suppresses sonic hedgehog pathway-associated tumors arising in response to environmental stress.

A procedure to stain the centromeric region of chromosomes for dual beam flow cytometric analysis is described. Serum from a CREST (Scleroderma syndrome) patient presenting a high titer of anticentromeric antibodies was chosen on the basis of specificity of labeling of cells on slides. The high affinity of the antibodies to centromeres and low binding to chromosomal arms allowed the development of an indirect immunofluorescent labeling procedure using isolated and unfixed chromosomes stabilized by Mg++ ions. Discontinuous Ficoll gradients were used to separate chromosomes from unbound antibodies. With this procedure, chromosome clumping and degradation were minimal. The chromosomes were then stained with the DNA dyes Hoechst 33258 and chromomycin A3, before dual beam flow cytometric analysis. Flow karyotypes, with good chromosome peak resolution, were obtained for both human and hamster chromosomes subjected to the immunolabeling procedure. For quantification of FITC fluorescence due to bound antibody, chromosomes were counterstained with Hoechst only. The FITC intensity of antibody-labeled human and hamster chromosomes were 4-10 and 20 times greater than control chromosomes, respectively. These results suggest that the staining procedure may be suitable for immunolabeling of chromosomes with antibodies recognizing other nuclear proteins and their subsequent quantification by flow cytometry.

Little information exists on how various nucleolar proteins function in ribosome biogenesis. Of special interest is that group of nucleolar proteins which are not incorporated into mature ribosomes because they are candidates for a role in the regulation of ribosome construction. Non-ribosomal nucleolar proteins can be analyzed using autoimmune sera from scleroderma patients which often contain antinucleolar antibodies. One such serum, designated ScBr, is shown by indirect immunofluorescence to react specifically with nucleoli in cells of 3 different mammalian species, indicating that the antigen is at least partly conserved evolutionarily. It is not RNase-sensitive, but is completely eliminated after incubation with pronase and 2 M NaCl. Immuno-electron microscopy was carried out on Lowicryl ultrathin sections to localize the antigen. The labeling was observed over both the granular and the dense fibrillar component but not the fibrillar centers, indicating that the antigen is associated with ribosomal RNA transcription sites and ribosome assembly into precursor particles. In addition, the antibody was localized to small nucleoplasmic entities, termed dense nuclear bodies. This could indicate a relationship between nucleoli and dense nuclear bodies. By immunoprecipitation, only a single protein of 94 kDa molecular weight was revealed. By immunoblotting, the band at 94 kDa was found to be the only positive band for high ScBr dilutions. Observation of the behavior of the antigen during mitosis revealed that it became dispersed into the cytoplasm after breakdown of the nuclear envelope, lining most of the chromosomes rather than remaining associated with the NOR-chromosomes. The antigen appeared to be restored to nucleoli only in late telophase; phase-dense prenucleolar bodies of early telophase cells did not show positive staining for the antigen. During actinomycin-D RNA synthesis inhibition as well as in non-stimulated lymphocytes the positive staining is greatly decreased. These results were consistent with a role for the 94 kDa nucleolar protein in the process of preribosome assembly.