Comparative karyotype using bidirectional chromosome painting: how and why?

Rat is widely used in biomedical and pharmaceutical research but its genome has been significantly less studied than that of the mouse. This represents a major limitation for studying cytogenetic and molecular mechanisms in the rat model. As Muridae species underwent an intense chromosome evolution it is not possible to directly transpose knowledge of the mouse genome to that of the rat. For establishing a comparative karyotype between rat and mouse, painting probes of both species were prepared by PARM-PCR (Priming Authorizing Random Mismatches PCR) from a low copy number of sorted chromosomes, the mouse and rat specific painting probes being then hybridized on rat and mouse metaphases, respectively. The availability of rodent species chromosome painting probes as well as the information obtained by the comparative karyotype and comparative gene mapping data are of great interest to improve knowledge on species evolution but also to better understand carcinogenesis process, as illustrated by our data concerning the cytogenetic characterization of radon-induced rat lung tumors. Detailed methods for obtaining painting probes by PARM-PCR from sorted mouse and rat chromosomes and for their hybridization in homologous or heterologous conditions are described. Usefulness of chromosome painting is illustrated by the characterization of chromosomal abnormalities in a radon-induced rat lung tumor. Advantages and limitations of this technique as compared to classical cytogenetics, FISH and CGH are discussed.

Clonal evolution of a radon-induced rat lung tumor.

Radon gas may represent a source of pulmonary radio-contamination either in mine or in domestic conditions. Since epidemiological studies are controversial, as long as biological markers of the exposure to such agents will not be identified, the question will remain open. We have previously shown a direct dose-dependent relationship between lung cancer occurrence and radon inhalation of rats. In this study, we report a cytogenetic study of a radon-induced rat lung tumor. Chromosome banding and chromosome specific paintings were performed on cultures of both fresh and xenografted tumors. We found by analyzing 17 sub-clones that all karyotypes presented a translocation involving rat chromosomes (RNO) 8 and 20, and a terminal deletion of RNO 15p suggesting a monoclonal origin of this tumor. RNO 15 is homologous to numerous human chromosomes (HSA), in particular to HSA 3p14.2, 3p22-p24.1 and 3p24.2-p24.3, this human chromosome being frequently lost in human lung carcinomas. Besides sharing chromosome alteration involving common features with those found in human lung cancer, this rat lung carcinoma represents a useful model to study tumor progression with respect to clonal evolution.

CGH analysis of radon-induced rat lung tumors indicates similarities with human lung cancers.

Epidemiological studies have shown that inhalation of radon, a radioactive gas, is associated with an increased risk for lung cancer. We have developed a model of radon-induced rat lung tumors to characterize cytogenetic and molecular events involved in radon-induced lung tumorigenesis. Using comparative genomic hybridization (CGH), gains and losses of genetic material were investigated in a series of 13 carcinomas and four adenomas of the lung. Frequent losses occurred at 4q12-21, 5q11-33, and 15q, which are homologous to human chromosome (HSA) bands 7q21-36, 1p31-36/9p21-31, and 13q14.1-14.3/3p14.2, respectively. These regions are frequently (30-80%) deleted in human lung cancer and contain tumor suppressor genes or proto-oncogenes such as MET, CDKN2A/p16/MTS1, CDKN2B/p15/MTS2, FHIT, and RB1 or yet to be identified genes. Frequent gains involved 6, 7q34-qter, and 19q; chromosomes 6 and 7 being homologous to human 2p21-25 and 8q21-24 where the MYCN and MYC oncogenes are located. The genetic similarities between rat and human lung cancer suggest common underlying mechanisms for tumor evolution in both species. Moreover, cytogenetic and molecular genetic analyses of radon-induced rat lung tumors could help to better understand the development and progression of radon-induced lung cancer in man.

Evidence for in vitro selection during cell culturing of breast cancer: detection by flow and image cytometry.

Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.

Comparative karyotype of rat and mouse using bidirectional chromosome painting.

A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G-banding morphology, heterologous chromosome painting results and available gene mapping data is proposed. Whole chromosome painting probes from both species were generated by PARM-PCR amplification of flow sorted chromosomes. Bidirectional chromosome painting identifies 36 segments of syntenic homology and allows us to propose a nearly complete comparative karyotype of mouse and rat (except for RNO 13 p and RNO 19 p12-13). Seven segments completely covered the RNO chromosomes 3, 5, 8, 11, 12, 15 and 18. Eight segments completely covered the MMU chromosomes 3, 4, 6, 7, 9, 12, 18 and 19. The RNO chromosomes 5, 8, 18 show complete homology with the MMU chromosomes 4, 9 and 18, respectively. Bidirectional hybridization results clearly assign 16 segments to subchromosomal regions in both species. Interpretation of the results allows subchromosomal assignment of all the remaining segments apart from seven distributed on chromosomes MMU 15, MMU 10 B2-D3 and MMU 17 E3-E5. The proposed comparative karyotype shows overall agreement with available comparative mapping data. The proposed repartition of syntenic homologous segments between the two species provides useful data for gene-mapping studies. In addition, these results will enable the reconstruction of the karyotype of the Cricetidae and Muridae common ancestor and the definition of the precise rearrangements which have occurred in both mouse and rat lineages during evolution.

Quantitative fluorescence in situ hybridization in lung cancer as a diagnostic marker.

The diagnosis of lung cancer is quite often hampered by the existence of various cell types within samples such as biopsies or pleural effusions. We have established a new marker for image cytometry of interphase tumor cells of the lung by using the most recurrent and early cytogenetic event in lung cancer, the loss of the short arm of chromosome 3. The method is based on the detection of the imbalance between the long and the short arms of chromosome 3 by performing two-color fluorescence in situ hybridization on both arms. Fourteen tumors were analyzed after short-term culture and compared with the corresponding cytogenetic data obtained from metaphase analysis. Results on interphase nuclei and control experiments on metaphases were the same, with imbalance ratios ranging from 1.0 to 2.0 (mean value 1.6, median 1.5). To assess the clinical significance of this approach, three pleural effusions were analyzed. Data showed that normal cells within the sample could have been distinguished from the tumor cells based on different imbalance values between the long and the short arms. Thus, our method allows refined detection of lung tumor cells within samples containing heterogeneous cell populations.

Quantitative FISH by image cytometry for the detection of chromosome 1 imbalances in breast cancer: a novel approach analyzing chromosome rearrangements within interphase nuclei.

Interphase cytogenetics have become a widespread tool for investigation of chromosome rearrangements in solid tumors. The most recurrent chromosome alteration within breast cancer affects chromosome 1, leading principally to gain of the long arm and/or loss of the short arm. We have developed a new method for detection of chromosome 1 arm imbalances in interphase nuclei. The method is based on quantitation of the fluorescence signals emitted by the hybridized two-color paintings of the short and long arms using image cytometry. The chromosome arm imbalance was determined by calculating the ratio of both fluorescence emissions of each arm. The ratio of the paintings of normal lymphocytes was used as a reference. Three breast cancer cell lines, 13 fresh tumor samples, and 6 fine-needle samplings of breast cancer were analyzed using an automated image cytometer. Whenever possible, classic cytogenetics and in situ hybridization on metaphases were performed as controls. Fluorescence ratios representing the imbalances of chromosome 1 arms with values between 1 and 3.2 were measured. Data between classic cytogenetics and interphase cytogenetics were well-correlated (r = 0.89). This method, which enables an easy detection of intrachromosomal imbalances without need of metaphase preparations, detects malignant cells and can be extended to other carcinomas for which chromosome 1 arm imbalances are recurrent or chromosome alterations specific of other malignancies. In comparison to other interphase fluorescence in situ hybridization techniques, it avoids every spot scoring problem encountered when using centromeric probes and the difficulties in interpreting structural rearrangements.

Pig standard bivariate flow karyotype and peak assignment for chromosomes X, Y, 3, and 7.

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.

Relocation of nucleolar proteins around chromosomes at mitosis. A study by confocal laser scanning microscopy.

The behaviour of nucleolar antigens known to associate with chromosomes at mitosis was investigated in mammalian cells (HeLa, HEp-2, PtK1, CHO) by immunofluorescence and confocal laser scanning microscopy. Serial optical sections through mitotic cells, from prophase to telophase, were used to generate three-dimensional images of the antigen distribution. Our results indicate that, at the onset of mitosis, these antigens leave the nucleoli in a highly ordered manner to form a network extending from the nucleoli towards the nuclear envelope. The migration begins at very early prophase, when the condensation of the chromosomes is not yet visible. After completion of the migration at late prophase, the labelling is found at the chromosome periphery. The antigens remain distributed as a sheath surrounding the chromosomes from prophase to telophase. Therefore, the proteins involved in the formation of this perichromosomal layer have different behaviour than those of the prenucleolar bodies. The antigens appear to interact strongly with chromosomes, since they are not lost during chromosome isolation in hypotonic buffer. Each chromosome is entirely covered from one telomere to the other, except in the centromeric region. Thus the relocation of these nucleolar proteins does not appear to be the result of a passive accumulation at the chromosome periphery, but seems rather to be due to an active targeting to specific sites. Consequently, these proteins may have a determining function in the progression of the cells through mitosis, possibly by participating in the protection and stabilization of the chromosomes.

Swine chromosomal DNA quantification by bivariate flow karyotyping and karyotype interpretation.

Human and swine chromosomes were analyzed separately and as a mix to obtain bivariate flow karyotypes. They were normalized to each other in order to use the human chromosomal DNA content as standard. Our results led to the characterization of the "DNA line" in swine identical to the human "DNA line." Estimation of the DNA content in mega-base pairs of the swine chromosomes is proposed. Chromosomal assignment to the various resolved peaks on the bivariate swine flow karyotype is suggested from the relation between DNA content quantified by flow cytometry and chromosomal size. Swine chromosomes 1, 13, 6, 5, 10, 16, 11, 18, and Y were assigned to peaks A, B, C, K, L, N, O, Q, and Y, respectively. Peaks D and E were assumed to contain chromosomes 2 and 14, but without specific assignment. Similarly, P and M peaks were expected to correspond to chromosomes 12 and 17. Of the remaining chromosomes (3, 7, X, 8, 15, 9, and 4), chromosomes 3, 7, and X, which were assigned previously to peaks F, G, and H, respectively, led us to deduce that chromosomes 15 and 8 belonged to peaks I and J, and chromosomes 9, 4, and X to peak H.

Immunofluorescent labeling of centromeres for flow cytometric analysis.

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.

Lamins A and C are not expressed at early stages of human lymphocyte differentiation.

Lamins are major proteins of the nuclear envelope that are members of the intermediate filament protein family. In vertebrates, nuclei from differentiated tissues usually contain both lamins of the A and B subtypes, while embryonic tissues contain the B-type lamin only. We have examined the composition of the nuclear lamina in human B and T lymphocytes representative of distinct stages of lymphoid differentiation. We show here that, in both cell lineages, while lamin B is constitutively expressed at all stages of differentiation, A-type lamin expression is restricted to later developmental stages.

Autoantibodies to 200 kD polypeptide(s) of the nuclear envelope: a new serologic marker of primary biliary cirrhosis.

Fifty sera which gave ring-like nuclear staining in immunofluorescence on rat liver tissue sections were characterized. Using immunoprecipitation and Western blotting we showed that 10 of the sera contained antibodies to 200 kD polypeptide(s) of nuclear envelope. Clinical and biological data were available for nine of the patients. Strikingly, all of these patients suffered from primary biliary cirrhosis with eight of them having anti-mitochondrial antibodies. As no control serum displayed such a reactivity, anti-200 kD polypeptide(s) antibodies can be considered as a new marker specific of a subset of primary biliary cirrhosis, being present even when anti-mitochondrial antibodies are absent. The exact identity of the target remains to be established, since several polypeptides of similar molecular weight have been reported to belong to the nuclear envelope.

Antinuclear autoantibodies specific for lamins. Characterization and clinical significance.

In 11 patients, sera displaying a ringlike nuclear immunofluorescent staining on sections of rat liver tissue were shown by Western blotting to contain antibodies to lamins. Sera from 8 patients contained autoantibodies reacting with lamin B, whereas sera from the other 3 patients reacted with lamins A and C. All patients (9 women and 2 men) had a chronic autoimmune disorder, which rarely fulfilled the usual criteria for a diagnosis of systemic lupus erythematosus. The disorder was characterized by acute or chronic (active or granulomatous) hepatitis; steroid-responsive blood cytopenia, often associated with a circulating anticoagulant, or anticardiolipin antibodies, or both; and cutaneous leukocytoclastic angiitis or probable brain vasculitis. Eight patients had at least two of these three conditions. Antilamin autoantibodies may thus be a marker for an unusual subset of autoimmune diseases.

Expression of nuclear lamins during mouse preimplantation development.

The expression of nuclear lamins during mouse preimplantation development was studied by immunofluorescence, immunoblotting and immunoprecipitation. Two sera were used, specific either for lamin B or lamins A and C. Both sera gave a positive staining of the nuclear periphery throughout preimplantation development (fertilized eggs to late blastocysts). Immunoblots revealed that the three lamins were present in eggs and blastocysts. However, lamin A from eggs was found to have a higher apparent Mr than lamin A from blastocysts and other mouse cells. Using immunoprecipitation, synthesis of lamin A was detected in eggs while synthesis of lamin B was detected in 8-cell embryos and blastocysts, indicating that at least some of the lamins used during early development do not come from a store in the egg. These results are discussed in relation to the possible role of lamins during cell differentiation.

Autoimmune serum containing an antibody against a 94 kDa nucleolar protein.

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.

A human T lymphoblastic cell line lacks lamins A and C.

Lamins A, B and C, the three major proteins of nuclear envelope, constitute a class of intermediate filament polypeptides. We have compared the amount of these polypeptides in two human cell lines, epithelial HeLa cells and T lymphoblasts KE 37. It was found that the three lamins were present in roughly equimolar stoichiometry in HeLa cells, while lamin B was the unique lamin component in T lymphoblasts. Moreover, 3-kb mRNA of lamin A and 2.1-kb mRNA of lamin C were detected with a human cDNA probe in HeLa cells but not in T lymphoblasts. These results suggest that (i) lamin B can build up the lamina structure in actively dividing somatic cells by itself, and (ii) lamin expression in lymphoid cells may be subject to important quantitative variations. Comparison of the lamin composition of human cloned T lymphocytes and Epstein-Barr virus-transformed human B lymphocytes confirmed this statement. The lamin B level was nearly equivalent in both cells but the content of lamins A and C varied to a large extent, being low in T cells and high in B cells.

Autoantibodies to nuclear lamin B in a patient with thrombopenia.

We report the characterization of novel nucleus specific autoantibodies in the serum of a patient with systemic lupus erythematosus. Immunofluorescent staining of cycling cells and absorption experiments localized the antigen to the nuclear envelope. Two-dimensional gel electrophoretic analysis of immunoprecipitated nuclear proteins show the antigen to be an acidic polypeptide (IP approximately 5.4) of 68 kDa molecular mass. It has been identified as lamin B, one of the three major nuclear envelope polypeptides of mammalian cells. Antibodies shown to be polyclonal immunoglobulin Gs, were directed against determinant(s) of the protein that have apparently been conserved during evolution. They do not appear to be related to other autoantibodies present in the serum (anti-DNA and anti-platelet). The nuclear specificity shown by these antibodies further demonstrates the antigenicity of proteins related to intermediate filament proteins in patients with autoimmune disorders.