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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.

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.

Clonal heterogeneity is a major difficulty in the analysis of chromosome rearrangements within tumor tissue. Using in situ hybridization, a cell-to-cell analysis can be performed and should allow a better understanding of the genetic process. In addition, detection of pre-neoplastic lesions with only a few cells involved may improve the diagnosis of such lesions and their precocious treatment.Automated analysis was performed on tissue sections with our previously described two-color fluorescence in situ hybridization-based method for quantitative determination of chromosome arm imbalance. The imbalance between the long and short arms of chromosome 3 was determined in 24 cases of non-small-cell and small-cell lung cancers in which only small snap-frozen sections were used, allowing other simultaneous molecular analyses, such as TP53 gene mutation detection.Specifically developed software allowed localization of each nucleus within the section with regard to its chromosome imbalance and to reconstitute a multi-clonal panel within an apparently homogeneous sample. In some cases, discrepancies in the imbalance values were observed between the biopsy and the tumor obtained after surgery from the same patient.The discrepancies observed between biopsies and tumors, likely linked to the samples' heterogeneity, demonstrate the necessity to analyze tissue sections collected at various locations. The fully automated approach developed in this study rendered such investigations possible.

Objectives Presently, conventional cytogenetic analysis of metaphase chromosomes remains the reference approach in prenatal diagnosis. However, this method is labor-intensive and time-consuming. The first step toward the rapid identification of aneuploidies is achieved by interphase fluorescence in situ hybridization (FISH) with centromeric or locus-specific probes. Spot counting using this type of probes is a reliable approach, but is very time-consuming with some technical and biological limitations. In this study, we present a new FISH method using image cytometry for the detection of trisomy 21 within interphase nuclei. Methods The method is based on a comparative quantitation of the fluorescence signals emitted by whole chromosome 21 and 22 painting probes cohybridized on interphase nuclei. The chromosomal imbalance was determined with an automated image cytometer by detecting an abnormal ratio of both fluorescence emissions when compared with the ratio obtained in normal cells. Results Ten blood samples and twenty amniotic fluids were analyzed. Results from FISH and standard cytogenetics were compared and 100% correlation was achieved. Conclusions This method, which enables an easy detection of chromosomal imbalances without a need for metaphase preparations, can be applied to the diagnosis of trisomy 21 and extended to other disorders with chromosomal imbalances. Compared to other interphase FISH techniques, it avoids spot-scoring difficulties. Copyright © 2003 John Wiley & Sons, Ltd.

The detection of DNA aneuploid cells using flow cytometry is an indication for the presence of tumor cells, but when DNA diploid cells are found in 25-33% of the cases, the diagnostic and prognostic significance of DNA ploidy is more limited. We analyzed interphase nuclei after in situ hybridization and using image cytometry on 50 breast tumors with diploid DNA content to investigate whether early chromosome rearrangements were detectable and if their occurrence was clinically significant. Imbalances between the two arms of chromosome 1 were found in 55% of the cases and values ranged from 1.5-3.0. Comparison with histological data showed that Grade I tumors mainly have imbalances (67%) and that Grade III tumors were mainly without the imbalance (67%), whereas Grade II tumors were intermediate (50% imbalance). These data suggest that the diagnosis of DNA diploid cases may be improved by using interphase FISH. In addition, the data also indicates that early breast tumors may have different genetic origins, which is important in the comprehension of tumor malignancy in early stages, especially for preinvasive lesions.

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.