Spontaneously immortalized mouse mesothelial cells display characteristics of malignant transformation

Abstract. Objectives: Mesotheliomas occur in occult serous cavities after chronic exposure of mesothelial cells to asbestos fibres. Molecular events that contribute to the development of this cancer are therefore not readily accessible for study. We have used in vitro culture systems to study and compare induced and spontaneous transformation events in primary mouse mesothelial cells. Materials and methods: Mouse mesothelial cells were cultivated until small populations of proliferating cells emerged from senescing cultures. Spontaneously transformed cultures of cells were characterized and compared to malignantly transformed cells. Results: Human mesothelial cells had a finite lifespan of 10–15 population doublings when cultured in vitro; mouse mesothelial cells typically exhibit this same pattern. Here, we show that mouse mesothelial cells can be cultured for extended periods and that these cells can transform spontaneously. Lines of spontaneously transformed cells generated in this study are immortal and growth factor‐independent. They display the salient characteristic features of transformation, including increased proliferation rate, lack of contact inhibition, aneuploidy and ability to grow in anchorage‐independent conditions. A subset of these cell lines developed into tumours in syngeneic mice. Comparative gene expression analysis demonstrated that spontaneously transformed cell lines were more closely related to neoplastic cells than to primary cells. Conclusion: These findings have implications for interpretation of in vitro transformation studies, demonstrating broad similarity between spontaneous and induced genetic changes.     

Alterations in cell nuclei during apoptosis

Apoptosis is a genetically programmed phenomenon that aids in maintaining homeostasis in multicellular organisms. The characteristic morphological features of apoptosis are highly conservative and are dependent on the cell type and the apoptotic inducer. The nuclear events occurring during apoptosis include changes at the molecular level (i.e. DNA cleavage, modifications of nuclear polypeptides, and proteolysis of several proteins important for cell maintenance), and, consequently, alterations at the morphological level (i.e. chromatin condensation, nuclear shrinkage, DNA fragmentation and apoptotic body formation). These events are still not fully understood. It is very probable that a progressive decrease in pH could also be an essential factor for the induction of nuclease and protease activities, and an important element of the optimal conditions for their function. This review details the current state of knowledge on apoptotic nuclear events, with particular focus on the proteins involved in the execution of apoptosis in cell nuclei, and on the differences in substrate cleavage profiles for different types of cell undergoing cell death.     

Immunomodulating activity in supernatants from EBV immortalized lymphocytes

Summary Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed.Supported by grants from the Concern Foundation and the Brigham Surgical Foundation     

Human neonatal lymphocytes immortalized after microinjection of Epstein-Barr virus DNA.

Epstein-Barr virus (EBV) is a highly efficient acute transforming agent in human cells, provided that the intact virus is used. To investigate the ability of viral DNA alone to transform cells, we introduced the EBV genome into human lymphocytes. After microinjection of EBV DNA into neonatal B lymphocytes, we established a cell line that in early passages contained multiple viral fragments. This cell line retained sequences from the short, unique (Us) region of the EBV genome and sequences from EcoRI-E. The viral sequences were not expressed; however, the cells expressed a 2.3-kilobase polyadenylated message homologous to the c-fgr oncogene, a cellular locus believed to be activated by EBV infection [M. S. C. Cheah, T. J. Ley, S. R. Tronick, and K. C. Robbins, Nature (London) 319:238-240.]. The cell line was monoclonal with rearrangement at the immunoglobulin locus and had a reciprocal translocation t(1;7)(p34;q34) and a deletion of sequences within the locus for the beta chain of the T-cell receptor. The close proximity of the translocation to the chromosomal loci for c-fgr on chromosome 1 and the T-cell receptor beta chain on chromosome 7 suggests that structural alteration of these genes was critical to this transformation event.     

Physical characteristics of mouse sperm nuclei.

The nuclei of epididymal sperm, isolated from C57BL/6J and CBA/J inbred mice by their resistance to trypsin digestion, retain the shape differences of the intact sperm head. Various physical characteristics of these nuclei were measured and compared. The measurement of the projected dimensions of nuclei showed that the CBA nuclei are 13.5% longer than C57BL/6 nuclei (8.64 +/- 0.02 mum compared with 7.61 +/- 0.02 mum), 0.8% narrower (3.51 +/- 0.01 vs. 3.54 +/-0.01 mum) with 6.8% more area (22.34 +/- 0.10 vs. 20.91 +/- 0.09 mum2). However, the volumes of the nuclei as based on reconstructing calibrated electronmicrographs of serial sections of the nuclei indicated that CBA are about 7% smaller than C57BL/6 nuclei (3.72 +/- 0.08 vs. 4.01 +/- 0.03 mum3). The buoyant density of the CBA nuclei is 1.435 +/- 0.002 g/cm3 compared with 1.433 +/- 0.002 g/cm3 for the C57BL/6 nuclei as determined on linear CsCl and Renografin-76 density gradients and confirmed by a technique utilizing physiological tonicities. Therefore, the average mass of the CBA nuclei is less than that of the C57BL/6 nuclei (5.34 +/- 0.12 vs. 5.75 +/- 0.05 pg). The sedimentation velocities at unit gravity of nuclei from 11 inbred strains differ over a range of more than 6% with CBA nuclei sedimenting about 2.0% more slowly than C57BL/6 nuclei. We show that for these nuclei the sedimentation velocity can be related to their buoyant density, volume and a sedimentation shape factor. Within the errors of our measurements of these various characteristics, it was found that C57BL/6 and CBA nuclei have similar sedimentation shape factors. Therefore, the difference in sedimentation velocity between these nuclei appears to be primarily a result of differences in volume. The possible applications of these techniques to the physical separation of sperm are evaluated in the discussion.     

Alterations in expression and glycosylation pattern of the Thy-1 glycoprotein during maturation and transformation of mouse T lymphocytes

The mouse Thy-1 glycoprotein of normal and transformed lymphoid cells was studied with regard to amount per cell, apparent m.w., and glycosylation characteristics. Thy-1 was measured by a solid-phase radioimmunoassay calibrated with pure mouse brain Thy-1. Thymocytes were shown to contain five times the amount of Thy-1 found in lymph node cells (1 X 10(6) vs 2 X 10(5) molecules per cell), whereas the T cell lymphomas studied (P52-127-166, RBL-5, YWA, Y191, Y274, YAC-1, RL male 1, and BW5147) varied in their Thy-1 content. The apparent m.w. of Thy-1, as determined by SDS-PAGE, was in all cases 25,000 to 30,000. However, the appearance of the Thy-1 bands revealed a size heterogeneity that was less pronounced with material from lymph node cells than from thymocytes. This band broadening seemed to be inversely correlated to the affinity for lentil lectin. Whereas half the Thy-1 molecules from thymocytes were bound to the lectin, lymph nodes Thy-1 showed 75% binding. All T lymphomas but one (BW5147) contained Thy-1 also heterogeneous in lentil lectin binding. The charge, previously shown to be dependent on the sialic acid content, was shifted to more acidic forms for lymph node Thy-1 compared to thymocytes. The T lymphomas possessed Thy-1 with charge properties similar to those of the thymocytes; the only exception was BW5147, which showed more basic forms. These results show that the expression and the glycosylation of Thy-1 is altered when thymocytes mature into immunocompetent cells and after malignant transformation of lymphocytes.     

Flow cytometry of DNA in mouse sperm and testis nuclei.

Mutations that occur in spermatogenic cells may be expressed as changes in DNA content, but developmentally-dependent alteration of its staining properties complicates the quantitation fo DNA in individual germ cells. These alterations have been studied with flow cytometric techniques. Nuclei from mouse testis cells and sperm were stained by the acriflavine-Feulgen method. The fluorescence intensity frequency distribution of nuclei of testis cells was characterized by 2 major and 5 minor peaks. Nuclei sorted from the various peaks with a fluorescence-activated cell sorter were identified microscopically. These data were confirmed by generation of peaks with nuclei prepared from cell suspensions enriched in specific cell types. One of the major peaks corresponded to round spermatid nuclei. The other major peak, located at 0.6 of the fluorescence intensity of the round nuclei, corresponded to elongated spermatid nuclei. Purified nuclei of epididymal and vas deferens spermatozoa displayed asymmetric fluorescence distributions. A minor peak at 0.8 the intensity of the round spermatid nuclei was tentatively assigned to elongating spermatids. 2 of the minor peaks, located at 1.7 and 2.0 times the fluorescence intensity of the round nuclei, corresponded to clumps of 2 haploid and diploid nuclei. The additional peaks, located at 3.0 and 3.7 times the fluorescence intensity of round spermatid nuclei correspond to leptotene and zygotene spermatocytes and to late pachytene spermatocytes, respectively. These peaks contained clumps of nuclei. The homogeneity of the nuclei sorted from the peaks, as well as the relative sizes of the peaks, was enhanced when the nuclei were prepared from cells enriched in specific stages of development. The relative fluorescence intensities of the various testis nuclei were characteristic and repeatedly found but were not stoichiometric with the DNA content of the nuclei.     

Quantitative fluorometry of abnormal mouse sperm nuclei.

An extensive quantitative analysis of deformed mouse spermatozoa was undertaken. Improvements over previous studies included the isolation and purification of sperm nuclei, a multifaceted analytical approach using several fluorochromes and the analysis of individual nuclei classified into shape categories. Malformed sperm nuclei in BALB/c mice could not be distinguished from normal ones in terms of total and basic proteins, sulfhydryl and disulfide group concentration, DNA concentration and chromatin organization. The shape of sperm nuclei is therefore probably determined by the manner in which the internal biochemical components are assembled.     

Physical heterogeneity of mouse thymus lymphocytes.

Mouse thymocytes have been separated by velocity sedimentation in a density gradient. The resulting fractions have been analyzed using electrophoretic light scattering. The electrophoretic distributions of the individual sedimentation fractions reveal the presence of physically distinct subpopulations. Comparison of the mean mobilities of each fraction indicates that the faster-sedimenting cells tend to have a higher electrophoretic mobility.     

Identification of human and mouse chromosomes in human-mouse hybrids by centromere fluorescence

Differences between the centromeric regions of mouse and human chromosomes have been revealed with a staining technique which is based on the quenching by 5-bromodeoxyuridine of the fluorescence of the dye 33258 Hoechst. The differences can be used to distinguish between human and mouse chromosomes in human-mouse hybrids.     

Ultrastructural demonstration of NAD-pyrophosphorylase activity in mouse liver nuclei.

Ultrastructural demonstration of NAD-pyrophosphorylase activity (E.C.2.7.7.1) in isolated mouse liver nuclei was investigated with the use of an electronhistochemical procedure based on the precipitation of pyrophosphate ions with lead ions under conditions permitting simultaneous ATPase inhibition by formaldehyde/ethanol prefixation. In isolated mouse liver nuclei activity of NAD-pyrophosphorylase was found in nucleoli, in interchromatin granules, coiled bodies and strand-like structures in nucleoplasm.     

A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells

Leukocyte-endothelial cell (EC) interactions regulate the entry of immune effectors into the tissues. This interaction occurs in lymphoid tissues and inflammatory sites at post-capillary high endothelial venules, as opposed to large capacitance vessels lined with flat EC. Transient SV40 infection of mouse EC derived from lymph node stroma has resulted in a cell line, SVEC4-10, that retains morphological and functional characteristics of normal EC. SVEC4-10 cells grow efficiently on plastic as a monolayer with a characteristic epithelioid morphology. They require as little as 2% FCS and are independent of other exogenous growth factors or matrix components. When grown on a synthetic basement membrane, SVEC4-10 forms branching tube-like networks. SVEC4-10 expresses Factor VIII related Ag as measured by indirect immunofluorescence using a rabbit antiserum to human FVIII-associated protein and incorporates acetylated low density lipoprotein. SVEC4-10 specifically binds mouse lymphocytes in vitro. IFN-gamma induces expression of MHC class II Ag in a time course identical to normal EC and the cell line is susceptible to lysis by anti SV40 H-2k CTL clones. Thus, this SV40 immortalized line retains much of the normal cellular physiology of EC.     

Dynamics of the endo-DNAase activity of cell nuclei of mouse thymus and spleen lymphocytes during the immune response

Endo-DNAse (mostly Ca/Mg-dependent endonuclease) activity was studied in extracts of lymphocyte cellular nuclei from the spleen and thymus of mice upon their immunization with sheep red blood cells. Endo-DNAses were detected by their action on super-stranded DNA pBR 322. It has been established that endo-DNAse activity considerably changes in the course of immune response. The changes start in the early (induction) phase of immune response, are characterized by certain regularities and are distinct in thymus and spleen lymphocytes. It is assumed that endo-DNAses of lymphocyte cellular nuclei are involved in antigen-dependent lymphocyte differentiation.     

Occludin and claudin-1 concentrate in the midbody of immortalized mouse hepatocytes during cell division.

It has been believed that epithelial cells maintain tight junctions at all times, including during cell division, to provide a continuous epithelial seal. However, changes in localization of integral tight junction proteins during cell division have not been examined. In this study, using SV40-immortalized mouse hepatocytes transfected with human Cx32 cDNA, in which tight junction strands and the endogenous tight junction proteins occludin, claudin-1, ZO-1, and ZO-2 were induced, we examined changes in localization of the tight junction proteins at all stages of cell division. All tight junction proteins were present between mitotic cells and neighboring cells throughout cell division. In late telophase, the integral tight junction proteins occludin and claudin-1, but not the cytoplasmic proteins ZO-1 and ZO-2, were concentrated in the midbody between the daughter cells and were observed at cell borders between the daugher and neighboring cells. These results indicate that the integral tight junction proteins are regulated in a different manner from the cytoplasmic proteins ZO-1 and ZO-2 during cytokinesis.     

Morphometric characterization of nuclei in non-Hodgkin's malignant lymphoma

In addition to the nuclear area and a form factor, four morphometric parameters of nuclear shape (ID, R1, R2 and ND), obtained by the application of the principles of mathematical morphology, were used to characterize the nuclear contours in non-Hodgkin's malignant lymphomas. The values for each parameter were determined in 58 cases of non-Hodgkin's lymphoma categorized according to the Kiel and National Cancer Institute classifications. Small-cell, mixed and large-cell lymphomas could be distinguished on the basis of the mean nuclear area. The shape parameters R1, R2 and ID were efficient discriminators of the large centrocytic (cleaved-cell) lymphomas. Neither size nor shape factors could distinguish between centroblastic and immunoblastic tumors. The good correlation between the morphometric findings and the histopathologic categories suggest that morphometry may provide a quantitative and objective method for grading lymphomas.