BCMA is a human gene expressed preferentially in mature B lymphocytes as a 1.2 kb mRNA, which encodes a 184 amino acid peptide (BCMAp). The study of BCMA mRNA expression, using human malignant B cell lines characteristic of different stages of B lymphocyte differentiation, demonstrated that the BCMA mRNA is absent in the pro-B lymphocyte stage. It is expressed faintly at the pre-B cell stage and its expression increases with B lymphocyte maturation. Polyclonal antibodies were used to show, by cellular fractionation and immunoprecipitation, that BCMAp is a non-glycosylated integral membrane protein. Furthermore, BCMAp inserts, in vitro, into canine microsomes, as a type I integral membrane protein. Cell surface labeling showed that BCMAp is not expressed in the plasma membrane of mature B lymphocytes. Immunofluorescence studies revealed that BCMAp lies in a cap-like structure near the nucleus, that was identified as the Golgi apparatus by co-localization of BCMAp with CTR433, a marker of the medial cisternae of the Golgi apparatus. Confocal scanning laser microscopy of U266 plasma cells labeled with markers of various Golgi apparatus subcompartments strongly suggests that BCMAp is located in the cis part of the Golgi apparatus. Thus, BCMAp is the first Golgi resident protein with a tissue specificity and whose expression is linked to the stage of differentiation of B lymphocytes. The location of BCMAp in the Golgi apparatus and its high expression in plasmocytes (secreting large amounts of Ig) suggest that BCMAp is implicated in the intracellular traffic of Ig.

Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. We hypothesized that the integration of environmental factors might induce specific cytoskeletal architecture patterns, characterized by quantitative image analysis. Human breast cancer cells MCF-7, flown in space in a photon capsule, were fixed after 1.5, 22, and 48 h in orbit. Cells subjected to weightlessness were compared with 1g in-flight and ground controls. Postflight, fluorescent labelings were performed to visualize cell proliferation (Ki-67), signal transduction (phosphotyrosine), three cytoskeleton components (microtubules, microfilaments, and intermediate filaments), and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network, and chromatin structure. In weightlessness, phosphotyrosine signal transduction was lower, more cells were cycling, and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade. Microtubules were altered in many cells. The perinuclear cytokeratin network was more loosely 'woven', and chromatin structure was modified. The prolongaion of mitosis can be explained by an alteration of microtubule self-organization in weightlessness, involving reaction-diffusion processes. The loosening of the perinuclear cytokeratin network and modification of chromatin distribution are in agreement with basic predictions of cellular tensegrity.

KEY w O R D s . Fractal dimensions, resolution, formulation, image analysis. S U M M A R Y

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 Fm deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-BOO (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.Key terms: DNA cytometry, confocal laser microscopy, histology, 3-D There is a considerable interest in DNA cytometry, notably because of the importance of searching for correlations of nuclear DNA content with cancer prognosis in individual patients (e.g., 22, 40, 62).The two methods commonly used a t present for nuclear DNA content analysis, image cytometry (ICM) and flow cytometry (FCM), are not devoid of limitations. Observing the tissue architecture may be important in identifying regions for cytometry and, therefore, the main shortcoming of ICM on smears or imprints and of FCM is obviously that they do not allow the study of DNA contents in situ. Even ICM on histological sections does not allow this, as the tissue structure is observed in projection and the resulting nuclear profiles are usually either incomplete if the section is thin or overlapping if the section is thick enough to include a sufficient proportion of complete nuclei. Finally, preparation procedures for ICM and FCM may produce a selection bias of a given cell type (9) and/or nuclear debris (27).'This paper is an expanded version of a work presented at the

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

DNA ploidy provides important information for the evaluation of the prognosis of prostate cancer. For the purpose of DNA cytometry and nuclear measurements, we developed an image processing system for the acquisition and processing of three-dimensional (3D) images based on confocal scanning laser microscopy (CSLM). The advantage of the CSLM is the preservation of the tissue architecture and the possibility of multilabeling. It is possible to determine both individual nuclear features and cellular features and the degree of the spatial heterogeneity of several markers. Special attention was paid to the development of the automatic method for the 3D segmentation of cell nuclei. Thick tissue slides (100 microm), stained for DNA with chromomycin A3, from 4 patients (with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), and well-and poorly-differentiated adenocarcinoma of the prostate), were studied in order to test the practicability of the developed methodology. DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, 2 peaks were observed, 1 in the diploid range and I in the tetraploid range. The case of poorly-differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinomas, we observed a considerable variation of the volume of nuclei.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.

SUMMARY A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1‐nm colloidal‐gold particles after RNA/RNA in situ hybridization, using digoxigenin‐labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras‐related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three‐dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.