Effect of low dose rate ionizing radiation on the division potential of cells in vitro. VIII. Cytogenetic analysis of human fibroblasts.

Human embryonic and adult cells were irradiated with fractionated doses of low dose rate ionizing radiation starting early during their lifespan. Adult cells were found to be more sensitive than fetal cells to ionizing radiation in terms of the number of cells produced during the lifespan of the control and the irradiated cultures. Phase-III adult control cells had fewer chromosomal aberrations than phase-III embryonic control cells. After irradiation there was an increase in chromosomal aberrations in adult cells but no increase in embryonic cells beyond those found in the control cultures. It is suggested that cells that have a higher potential for chromosomal rearrangements survive better after low dose rate ionizing radiation.

Characterization of chromosome changes in two human prostatic carcinoma cell lines (PC-3 and DU145) using chromosome painting and comparative genomic hybridization.

Using chromosome painting, a study of chromosomal abnormalities has been performed in two prostatic carcinoma cell lines, PC-3 and DU145. In PC-3, this analysis revealed a highly rearranged hypotriploid karyotype with 54 to 61 chromosomes and numerous rearrangements of chromosomes 1, 3, 5, 8, 10, and 14. At passage 73, DU145 had a hypotriploid karyotype with few rearrangements of chromosomes 1, 3, 5, 12, 13, and 20, whereas at passage 153, this cell line showed a near-tetraploid karyotype with a great number of rearrangements involving chromosomes 3, 6, 8, 10, 12, and 17. A single rearrangement was shared by the 2 cell lines, an i(5)(p10). A comparative genomic hybridization study demonstrated a noticeable amplification of bands 10q22.3-q23 and 14q22-q24 in the PC-3 cell line. No amplification signal was detected for DU145.

DNA hypomethylation in breast cancer: an independent parameter of tumor progression?

The global DNA methylation status was investigated on a series of 59 breast cancers by Southern blotting, using methylation sensitive restriction enzymes. By comparison to control DNA, almost all tumor DNAs were found globally hypomethylated. However, the demethylation was variable from tumor to tumor. Compared to other biological parameters, the methylation did not correlate with chromosome alterations, steroid hormone receptor status, or histopathological grading. Tumors which appeared to be the most evolved for other parameters were only mildly hypomethylated, whereas tumors with strongly hypomethylated DNA corresponded to those with slight alterations of the other parameters. Thus, DNA hypomethylation is a consistent characteristic of breast cancer, but its variations may not correlate with tumor progression of most breast cancers.

[Changes in methylation of tumor cells: a new in situ quantitative approach on interphase nuclei and chromosomes]. / Altérations de la méthylation dans les cellules tumorales: une nouvelle approche quantitative in situ sur les noyaux interphasiques et sur les chromosomes.

DNA methylation is known to be related to the regulation of gene expression. DNA methylation patterns are modified in tumors compared to normal tissue, and in some cases, these variations are linked to cancer progression. Molecular biology techniques are generally used to evaluate DNA methylation status. We describe here a simple and fast immunofluorescent method to quantitate in situ DNA methylation using an image analyser and a CCD camera. Quantification of relative methylation levels in interphase nuclei and metaphase chromosomes was performed on digital images of two types of cells with known methylation levels. The results from image cytometry corresponded to those obtained by molecular studies. Advantages of this approach are that all the DNA of a cell may be examined rather than a limited restriction sequence, and that it may be done on a cell by cell basis rather than on a heterogenous population. In addition, with this method, changes in methylation patterns during tumor progression could be followed and eventually used as a marker for prognosis.

[Characterization of chromosomal rearrangements by in situ hybridization in glioblastoma]. / Caractérisation par hybridation in situ des remaniements chromosomiques dans un glioblastome.

In a case of glioblastoma, the following karyotype was determined: 47, X, - Y, + der(1) t(1;9)(p21;p23), t(1;9)(p21;p23), + 3, + 7, der(9) t(Y;9)(q11;p21), - 13, t(13;16)(p13,p11), del(14)(q11q22). Classical satellite DNAs are mainly located in chromosomes 1, 9, 15, 16 and Y. Because, most of these chromosomes were implicated in the rearrangements, a detailed cytogenetic study was undertaken. This study included in situ hybridization of the satellite and alphoid DNAs of chromosomes 1, 9, 16 and Y combined with various chromosome banding methods (DA-DAPI, quinacrine mustard and R-banding). The data obtained, demonstrated that the breakpoints were always located outside the areas containing the satellite and alphoid DNAs. The situation observed here differs from that reported in breast cancers for which a high proportion of the breakpoints occur within these areas. These findings suggest that in glioblastoma, chromosome rearrangements result from different mechanisms than those implicated in breast cancers. Thus, in cancers, chromosomal instabilities may result from several mechanisms.

The nuclear skeleton and the spatial arrangement of chromosomes in the interphase nucleus of vertebrate somatic cells.

The topologic distribution of interphase chromosomes established by using various cytologic methods and data concerning the DNA-nuclear skeleton interactions in isolated nuclear fractions were reviewed and discussed. Comparison of these different data clearly showed that the position of chromosomes observed in situ is in agreement with the results obtained from isolated nuclear fractions, indicating that all DNA molecules are bound to the peripheral nuclear skeleton. Moreover, the in situ position of the rDNA near the nuclear envelope can be correlated with the existence of a nucleolar skeleton connected to the peripheral nuclear skeleton. Taking into account the discrepant results regarding the actual existence of an internal nuclear skeleton, we attempted to analyze how the various nuclear skeletal structures described in the literature can be involved in both the distribution of chromosomes and in their chromatin organization. As many questions are still unanswered, we considered the modes of investigation that seem to be the most promising.

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line.

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.

Fanconi anemia leading to acute myelomonocytic leukemia: cytogenetic studies.

A 6-year-old boy with Fanconi anemia, developed acute myelomonocytic leukemia with marker chromosomes from an interchange in the malignant clone in the bone marrow. The possible aetiological role of therapy, the morphological characteristics and chromosome aberrations of the bone marrow cell line are discussed in relation to previous reported cases of Fanconi anemia in which acute leukemia has supervened.

Evidence showing that the nucleolus-envelope region is located on the nor-bearing chromosomes in Aotus trivirgatus.

We investigated whether the existence of the Nucleolus-Envelope Region observed in dividing animal cells is related to the Nucleolus Organizer Region or to other chromosome segments of the NOR-bearing chromosomes, since NORs are usually adjacent to the chromosome segments attached to the nuclear envelope such as the centromere, telomere or heterochromatin region. We used Aotus Trivirgatus fibroblasts whose karyotype is characterized by a single pair of NORs located on the long arm of the third pair of chromosomes, far from the centromere, the telomere and any obvious heterochromatin region. All the nucleoli were seen to be clearly associated with the nuclear envelope but separated from it by the outermost layer of chromatin. These results further support the hypothesis that the NOR is a site of attachment of the chromatin to the nuclear envelope by means of the Nucleolus-Envelope Region. They show that genetically active chromatin is also attached to the nuclear envelope. A model of the arrangement of chromatin during interphase is proposed which provides a functional interpretation of the currently available data.

Analysis of the spatial organization of the cell: a statistical method for revealing the non-random location of an organelle.

A method is proposed for testing the randomness of the location of an organelle within a given area of a cell section. The approach chosen is to analyse the distance between this organelle and a specific point considered as a point of reference. The method consists of converting this distance into the ratio of one given area to another and comparing the statistical distribution of the converted values to the uniform distribution. This method has the advantage of being valid in the absence of any restrictive assumptions concerning the heterogeneity in size and/or shape of the collection of sections sampled. Detailed examples are given to illustrate the practical use of the method, and its possible extensions are discussed.

Rotation of the cell nucleus in living cells: a quantitative analysis.

Nuclear rotation is observed in a variety of cell types. However, few quantitative analyses are reported and the significance of this phenomenon is still unclear. To investigate this type of nuclear movement, we performed a quantitative analysis in mouse L-929 fibroblasts, a cell line chosen since it displays a high nuclear rotational activity. Analyses were performed using time-lapse microcinematography. The relationship between nuclear rotation and other cellular phenomena such as the cell cycle and locomotion were studied. Then, we investigated the rotation in a population of sister cells to study whether it is genetically determined. Finally, we performed a qualitative analysis of nuclear rotation in different cultured cell lines. Results show that nuclear rotations preferentially occur during the phases of the cell cycle which surround mitosis.

Growth and differentiation of rabbit tracheal outgrowths in primary cultures: influence of substrata.

We show that in rabbit tracheal outgrowths, ciliated cells can express an affinity for the isolectin BSI-B4, known to preferentially bind to basal cells. In multi-layered outgrowths grown on a thick collagen gel, a low percentage of ciliated cells are labelled by BSI-B4. This percentage increases with the aging of cultures. In monolayers developed on thin coatings of collagen or matrigel, a high percentage of ciliated cells show affinity for BSI-B4, even within young cultures.

Detection of sugar-binding sites in the fibrillar and the granular components of the nucleolus: an experimental study in cultured mammalian cells.

The intranucleolar distribution of sugar-binding sites (i.e., lectin-like molecules) was analyzed in segregated nucleoli of actinomycin D-treated HeLa cells. The detection of sugar-binding sites was performed by incubation either of permeabilized nuclei in the presence of fluorescein-labeled neoglycoproteins or of ultrathin sections cut through in situ-fixed nuclei in the presence of gold-labeled neoglycoproteins. In the former case, the fluorescent nucleolar components were identified by comparison with the nucleolar components of similarly treated cells observed in electron microscopy. For the first time, this study reveals the presence of sugar-binding sites in both the fibrillar and the granular components of the nucleolus. In view of the data already reported on the biochemical composition of the nucleolus, some of our results led us to conclude that the nucleolar sugar-binding sites are lectin-like proteins. These proteins could be associated with preribosomes since the nucleolus is the site of both synthesis and stockage of ribosomal precursors. Some results from this study, however, show that the possibility of a relationship between some lectins and a structural component cannot be excluded.

Evidence for the existence of a nucleolar skeleton attached to the pore complex-lamina in human fibroblasts.

This work deals with the types of nuclear skeletal structures obtained from human fibroblast nuclei isolated by different procedures. It is confirmed that, in somatic vertebrate cells, the pore complex-lamina is always observed, whereas the presence of internal nucleolar and extranucleolar residual structures depends upon the method of nuclear isolation used. Furthermore, the results reported here argue for the existence of a nucleolar skeleton different from the nucleolar matrix often observed in different cell types by other investigators. The conditions of nuclear isolation which allow us to visualize this nucleolar skeleton without any other internal residual structures are described. The attachment of the nucleolar skeleton to the lamina suggested by the present data is considered in relation to the in situ position of nucleoli near the nuclear envelope.

C32 human melanoma cell endogenous lectins: characterization and implication in vesicle-mediated melanin transfer to keratinocytes.

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.

Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures.

EM investigation of Ag-AS-NOR staining after short glutaraldehyde prefixation followed by Carnoy fixation maintained good ultrastructural preservation and reactive selectivity. This enables exact localization of silver deposits both in the fibrillar centers of typical or segregated nucleoli during interphase, and in chromosome NORs during mitosis. These results argue in favour of the possibility that fibrillar centers are the interphasic counterpart of chromosome NORs. Special structures such as nucleolar blobs and remnants usually considered to be of nucleolar origin, were also stained. - These findings seem to indicate a relationship between the distribution of the silver-stained proteins, the arrangement of the nucleolar structures and the degree of nucleolar activity resulting from the experimental conditions. These results are of interest at the time when the concept of the nucleolar matrix is gradually emerging.

Fluorescence-based analysis of DNA ploidy and cell proliferation within fine-needle samplings of breast tumors: a new approach using automated image cytometry.

BACKGROUND: Automated image cytometry can allow concurrent quantification of several parameters in each individual cell within a population, opening new possibilities for diagnosis and prognosis. In this study, the authors investigated the capacity of this method for performing a bivariate analysis of DNA ploidy and synthesis in fine-needle samplings obtained without aspiration from breast tumors. METHODS: Samplings from 25 unselected cases of ductal infiltrative breast adenocarcinoma and 2 cases of fibroadenoma were analyzed. For each case, 3-5 slides (containing approximately 1000 cells each) were quantified to assess experimental precision. Ploidy was determined by fluorescent staining of DNA using 4,6-diamidino-2-phenylindole (DAPI). Contaminating lymphocytes were taken as internal controls to calculate DNA indices. DNA synthesis was analyzed by immunofluorescent detection of 5-bromodeoxyuridine (BrdU) incorporation. Measurements were compared with flow cytometric data obtained from the same patients. RESULTS: Relative error in determination of DNA indices was generally below 5%. Determination of proliferation indices were more variable, with a mean relative error of 25%. Two different populations of BrdU positive cells were detected systematically, one in the diploid and another in the aneuploid fraction. For both cytometric methods, DNA indices were similar in all 27 cases, whereas BrdU labeling indices showed no significant correlation in 13 cases. The remaining cases were not comparable due to lack of flow cytometric data. Labeling indices obtained by image cytometry did not reveal any significant correlation with Scarff-Bloom-Richardson grading or clinical staging. CONCLUSIONS: Automated image cytometry allows concurrent measurement of ploidy and cell proliferation within individual breast carcinoma cells. Statistical reliability can be reached with a relative small number of cells (1000), which is crucial for samples in which the cell number is too low for flow cytometry analysis. Visual control for artifact elimination and better characterization of cell populations makes this a powerful tool for tumor cell investigation. Automated image cytometry allows the obtainment of valuable prognostic parameters of traditional flow cytometry with the relatively small number of cells obtained in aspiration procedures.

Bloom's syndrome: a probable new case with cytogenetic findings.

A 19-year-old Jordanian girl, born to first-cousin parents, has most features of Bloom's syndrome but is tall and has secondary amenorrhoea. Blood and skin cultures revealed a normal diploid female complement but about one-quarter of the cells show chromosome or chromatid gaps, breaks and rearrangements. These abnormalities were localized after trypsin banding and having been found non-randomly distributed along the chromosomes.