Altered patterns of DNA methylation on chromosomes from leukemia cell lines: identification of 5-methylcytosines by indirect immunodetection.

An immunodetection technique has been developed to map with high resolution the methylated sites of human chromosomes. We have used this method to define the methylated areas of chromosomes from normal donors and from leukemia cell lines. The chromosomes were exposed for a short time to UV light to induce mild denaturation. The methylated sites were detected in situ by using monoclonal antibodies against 5-methylcytosine (prepared in mouse), and fluorescein-conjugated antimouse immunoglobulins. The chromosomes from normal cells exhibited a fluorescent pattern with RCT banding, although some differences from previously reported patterns could be detected. With this method we have been able to show the presence of two types of R-bands: High fluorescence R-band (HFR) and low fluorescence R-band (LFR). Chromosomes from leukemia cell lines exhibited low global staining with disrupted RCT banding of the chromosomes. The decreased level of the methylation status of the chromosomes from leukemia cells was confirmed by detection of 5-methylcytosines on total immobilized DNA. Thus, we have shown that this method can be used to determine the methylated status of chromosomes and, in turn, to map not only the structural (banding) but also the functional (methylation status) properties of the different chromosome domains in normal and pathologic human cells.

Use of confocal microscopy to localize the SHBG interaction with human breast cancer cell lines--a comparison with serum albumin interaction.

This work has allowed a comparison between the interaction of two principal plasma estradiol-binding proteins, serum albumin and Sex Hormone Binding Globulin (SHBG), with human breast cancer cells in culture (MCF-7 and MDA-MB 231), using a protocol which protects the integrity of cell structure. We showed that serum albumin was highly internalized by cells whereas SHBG interacted essentially at the plasma membrane level, and this whatever the contents of the receptor estrogen cells. If, by its high plasma concentration, serum albumin is internalized in a non-specific way and can thus fit into intracellular traffic, SHBG, by its membrane binding, seems to have a specific action toward target cells.

Internalization and biological effects of serum albumin in the breast cancer MCF-7 and MDA-MB 231 cells.

We show that albumin is internalized by human breast cancer cells (MDA-MB 231 and MCF-7) in culture by using confocal laser scanning microscopy. Moreover, albumin has an effect on the level of radioactivity incorporated when the cells are incubated with radioactive estradiol, and it is necessary to observe the mitogenic effect of estradiol towards the MCF-7 cells. This finding opens some possibilities regarding the internalization mechanisms and fate (degradation, recycling) of albumin as well as the role played by this protein in the intracellular metabolism of estradiol and in the intra-extracellular traffic of estradiol and its metabolites.

Overexpression of human cyclin A advances entry into S phase.

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S-phase control in mammalian cells. Using a genomic construction corresponding to the human cyclin A gene under the control of its own promoter, we have established stable transfectants overexpressing cyclin A protein. Experiments assisted by laser scanning image cytometry showed that this overexpression begins from late G1 phase onwards and is therefore cell cycle-regulated in this model. We demonstrated that this overexpression advances entry into S phase, leading to a contraction of the overall cell generation time. These results provide evidence that cyclin A can be a rate-limiting factor with respect to the control of the transition to S phase in mammalian cells.

Distribution of MHC class I and of MHC class II molecules in macrophages infected with Leishmania amazonensis.

Macrophages, being apparently the only cells that in vivo allow the growth of the intracellular pathogen Leishmania, are likely candidates to present antigens to Leishmania-specific CD4+ and CD8+ T lymphocytes, known to be involved in the resolution or in the development of lesions induced by these parasites, and recognizing processed antigens bound to MHC class I and MHC class II molecules, respectively. In the present study, we analysed by confocal microscopy and by immunoelectron microscopy the subcellular distribution of both MHC class I and class II molecules in mouse (Balb/c and C57BL/6 strains) bone marrow-derived macrophages infected for 12 to 48 hours with Leishmania amazonensis amastigotes and activated with gamma interferon to determine the intracellular sites where Leishmania antigens and MHC molecules meet and can possibly interact. Double labelings with anti-MHC molecule antibodies and with either propidium iodide or an anti-amastigote antibody allowed localization of MHC molecules with regard to the endocytic compartments housing Leishmania amastigotes, organelles known as the parasitophorous vacuoles (PV) and which most likely contain the highest concentration of parasite antigens in the host cell. Both uninfected and infected macrophages from Balb/c mice expressed the MHC class I molecules H-2Kd and H-2Dd on their cell surface but no significant amount of these molecules could be detected in the PV, which indicates that, if infected macrophages play a role in the induction of Leishmania-specific CD8+ T lymphocytes, PV are probably not loading compartments for MHC class I molecules. In contrast, MHC class II molecules were found to be associated with the PV membranes as shown previously with microscopic techniques at lower resolution (Antoine et al. Infect. Immun. 59, 764-775, 1991). In addition, we show here that, 48 hours after infection of Balb/c macrophages, in about 90% of PV containing MHC class II molecules, the latter were mainly or solely localized at the attachment zone of amastigotes to PV membranes. This peculiar distribution, especially well demonstrated using confocal microscopy, was confirmed by subcellular fluorescence cytometry of infected macrophages stained for the MHC class II molecules. The following data agree with the idea that PV-associated MHC class II molecules establish specific interactions with plasma membrane components of amastigotes. First, the polarized localization of class II appeared specific to these molecules, since the distribution of the lysosomal glycoproteins Igp110 and Igp120, of the macrosialin (a macrophage-specific marker of endocytic compartments) and of the GTP-binding protein rab7p, shown here as being PV membrane components, was homogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of maitotoxin on calcium entry and phosphoinositide breakdown in the rabbit ciliated tracheal epithelium.

Maitotoxin induces a concentration-dependent 45Ca uptake in primary cultures of rabbit tracheal epithelial cells. This response is insensitive to the calcium channel antagonists nifedipine, diltiazem and verapamil up to 20 microM. However, verapamil at 200 microM completely prevents 45Ca uptake. Measurements of indo-1 fluorescence show that MTX induces a very sustained (> or = 2 h) [Ca]i rise, which is completely inhibited by 200 microM of verapamil. Genistein (110 microM) (an inhibitor of tyrosine kinases) also strongly inhibits it. The inhibitory effect of 50 microM miconazole (an inhibitor of cytochrome P450) is only partial. Okadaic acid (inhibitor of protein-phosphatases) primarily delays the response to the toxin without decreasing its magnitude. MTX induces the formation of (1,4,5) inositol trisphosphate (IP3). The MTX response curve is biphasic. Stimulation is transient (< or = 10 min) and is not inhibited by chelation of intracellular Cai with BAPTA, nor by verapamil (200 microM) or U73122 (10 microM) (an inhibitor of activation of PLC beta 1 through a trimeric G protein). Results suggest that MTX independently activates a calcium transport process (which might imply phosphorylation on tyrosine residues) and a PLC not linked to a trimeric G protein.

Use of a biotinyl-estradiol derivative to demonstrate estradiol-membrane binding sites on adherent human breast cancer MCF-7 cells.

A biotinyl-derivative of 17 beta-estradiol has been used to demonstrate a site of recognition and binding of estradiol located on the plasma membrane of human breast cancer MCF-7 cells by using the biotin/avidin-FITC system. The specificity of this binding has been shown by a displacement of the fluorescent label by 17 beta-estradiol. No displacement was observed when testosterone was added. Quantification of this phenomenon has been shown by laser scanning cytometry while preserving the cells adhesiveness to their growth support as well as their membrane integrity. An analysis by confocal laser scanning microscopy suggested that the fluorescence distribution on MCF-7 cells treated with estradiol-biotin was on the cell periphery. The results obtained are in favour of the recognition and binding site of 17 beta-estradiol located on the plasma membrane of MCF-7 cells and they would indicate that the biological activity of estradiol, among others, could be initiated by an interaction with the membrane.

Image and flow cytometry: companion techniques for adherent and non-adherent cell analysis and sorting.

Flow cytometry (FMC) is an analytical and preparative technique whereas image analysis is only applied to cell analysis. Recently, image analysis has been adapted as a preparative method using a new technique: image cytometry for analysis and sorting (ICAS). FCM and ICAS are complementary. Flow cytometry allows rapid, quantitative and precise study of fluorescence and light scattering in a large number of cells in suspension, while ICAS analyses fewer cells (adherent cells or tissue) on the basis of fluorescence, morphology and size. ICAS can use these criteria to destroy unwanted cells and hence sort selected cells. ICAS can also be used for confocal microscopy and laser surgery.

Characterization of a panel of somatic cell hybrids for subregional mapping along 11p and within band 11p13. Subdivision of the WAGR complex region.

The short arm of chromosome 11 carries genes involved in malformation syndromes, including the aniridia/genitourinary abnormalities/mental retardation (WAGR) syndrome and the Beckwith-Wiedemann syndrome, both of which are associated with an increased risk of childhood malignancy. Evidence comes from constitutional chromosomal aberrations and from losses of heterozygosity, limited to tumor cells, involving regions 11p13 and 11p15. In order to map the genes involved more precisely, we have fused a mouse cell line with cell lines from patients with constitutional deletions or translocations. Characterization of somatic cell hybrids with 11p-specific DNA markers has allowed us to subdivide the short arm into 11 subregions, 7 of which belong to band 11p13. We have thus defined the smallest region of overlap for the Wilms' tumor locus bracketed by the closest proximal and distal breakpoints in two of these hybrids. The region associated with the Beckwith-Wiedemann syndrome spans the region flanked by two 11p15.5 markers, HRAS1 and HBB. These hybrids also represent useful tools for mapping new markers to this region of the human genome.

Cell subpopulations within proliferative and differentiating compartments of epidermis.

The heterogeneous population of newborn rat keratinocytes was separated into different subgroups according to their cell size. The relation between cell size, position in the cell cycle, RNA content, and proliferative potential in culture was examined. A reserve stem cell population of Go/G1 cells, low in RNA, giving rise to colonies of undifferentiated phenotype in cell culture, has been separated from more differentiated transit basal cells. In the fractions of the larger cells, several subgroups, probably corresponding to different stages of differentiation, were identified: G2M cells with low RNA content, large S-phase cells rich in RNA, and small Go/G1 cells low in RNA. The clonogenic cells from these fractions have limited growth potential and give rise to moderately or terminally differentiated colonies. The selective sorting of stem cell populations may be useful for elucidating the mechanism of carcinogenesis in epidermis and other proliferative tissues. Analysis of the relative proportions of cell subpopulations represents a novel approach leading to the refinement of the concepts of epidermal structure in physiological and pathological states. It also could, by extension, shed new light on the behavior of other proliferative tissues.

The gene for catalase is assigned between the antigen loci MIC4 and MIC11.

Genetic analysis of the cells of a WAGR patient (W, predisposition to Wilms tumor; A, aniridia; G, genitourinary abnormalities; R, mental retardation), bearing a partial deletion of band 11p13, was performed with biochemical and antigenic 11p markers by using gene dosage, somatic hybridization, molecular hybridization, and indirect immunofluorescence techniques. These studies allowed the regional assignment of the gene for catalase, which is linked to the Wilms tumor locus, between MIC4 and MIC11, two loci encoding for membrane antigens previously mapped to band 11p13.

Immunoregulatory functions of paf-acether. II. Decrease of CD2 and CD3 antigen expression.

Paf-acether (platelet-activating factor) is a phospholipid initially described as a potent platelet-aggregating compound. It is produced by numerous cell types and is now considered as an important mediator of cell-cell interactions. The effect of paf-acether on the expression of CD2 and CD3, two human T cell surface glycoproteins, was investigated by indirect immunofluorescence and flow cytometry. Paf-acether partially down-regulated, in a time- and dose-dependent manner, CD2 and CD3 but not HLA class I antigen expression on peripheral human T cells and Jurkat cells. Lysophosphatidylcholine, a phospholipid closely related to paf-acether, had no detectable modulatory effect on CD2 and CD3 expression. In addition to CD2/CD3 modulation, paf-acether markedly inhibited T cell proliferative response not only to phytohemagglutinin or concanavalin A but also to anti-CD3 or a stimulatory combination of anti-CD2 monoclonal antibodies. These data demonstrate for the first time that lipid mediators such as paf-acether might be involved in the regulation of the expression of cell surface glycoproteins that are essential in the execution of T cell function.

[Flow cytogenetics. Principles, advantages and limitations. What trends for onco-hematology?]. / Cytogénétique en flux. Principes, avantages et limites. Quelles perspectives pour l'onco-hématologie?

Human chromosomes can be observed after coloration with ADN specific fluorochromes. Measurement of fluorescence intensity may be done by flow cytometry and it allows achievement of flow karyotypes. It is possible to define a standard karyotype and-by comparison-to bring to the fore chromosomes abnormalities (translocation, deletion, polysomy). Various genomic abnormalities are observed with leukemia. Flow cytometry allows a multiparameter analysis which could be used to detect rare events, unknown in classic karyotyping. With Flow Cytometry and cell sorting, the abnormal chromosomes could be separated and secondly observed after Q banding or analysed after molecular hybridization to confirm leukemia diagnosis or prognostic. Flow cytometry, an analytical technology already used in onco-hematology, allows, which chromosome analysis associated with other technics (molecular biology,...) a new approach of particular diagnosis.

Repopulation of spleens of irradiated mice after injection of spleen cell subpopulations.

Haemopoietic stem cells present in the spleen of adult mice were analysed by grafting X-irradiated animals with polystyrene-nonadherent (NABS) and polystyrene-adherent (ABS) B-enriched splenocytes from syngeneic donors. The progeny of the haemopoietic stem cells present in NABS and ABS subsets were studied with respect to size, surface markers, and response to mitogens and antigens. Ninety-six per cent of the precursors of the myeloid cell lineage (CFU-S) were present in the NABS fraction (50-fold enrichment). The presence in NABS of progenitors of functional T and B lymphocytes was also demonstrated. Twelve days after grafting with NABS, more than 80% of the recipient splenocytes were large and nonadherent granulocyte-like cells. These cells had surface similarities with NABS from normal mice, since both populations reacted with peanut agglutinin and with a rabbit anti-NABS (RAN) serum.

Flow cytometry: technical description and some applications in France.

After reviewing basic technical considerations, we discuss some applications of flow cytometry in French laboratories. This methodology is used in several areas: oncology, cellular pharmacotoxicology, molecular biology and genetics, immunology, as well as cellular biology and physiology. We also examine the evolution of this technique in two directions: on the one hand, the appearance of increasingly sophisticated instruments; on the other, the development of less expensive and less complicated apparatuses principally directed at clinical applications.

Direct hybridization of sorted human chromosomes: localization of the Y chromosome on the flow karyotype.

A method is described for directly hybridizing a small number of sorted chromosomes with specific DNA probes. The chromosomes are analyzed by flow cytometry and sorted by deflecting the droplets containing the desired chromosomes onto a nitrocellulose filter. By using probes specific for the human Y chromosome, it has been possible to unambiguously identify the peak corresponding to the Y chromosome in the flow karyotypes of a variety of male cell lines. The position of this peak was found to vary significantly from individual to individual, correlating with the heterochromatin chromosomal polymorphism of the human Y chromosome. The sensitivity of the hybridization was such that, with a probe for a male-specific repetitive sequence, only 2,500 sorted chromosomes were enough to obtain a clear, positive signal; 10,000 were needed with a probe specific for a weakly repeated (maximum, 3-fold) sequence of Y chromosome. With this new method, chromosome sorting may be a rapid and efficient way to assign DNA sequences to chromosomes.