Morphological aspects of human lymphoid cells grown in vitro

Summary An established immunoglobulin-producing human cell line exhibits a variety of morphological entities described as reticular, lymphoid, and plasmacytoid cells. Transitional forms are frequent, and time lapse photomicrography experiments demonstrate morphological interconversion. The round forms and their morphological precursors synthesize immunoglobulin and constitute about 44% of the cell population. This cell line displays a variety of morphological and functional properties assigned to in vivo lymphoid cell series. It is proposed as an in vitro model for the study of lymphoid cells Supported by Grant CA06939-05 and by Special Research Career Development Fellowship Award 1F03 CA42078 from the United States Public Health Service.     

Differentiation-inducing ability of sophorolipids of oleic and linoleic acids using a glioma cell line

Sophorolipids are biosurfactants produced by non-pathogenic yeasts. They show structural similarity with the membrane components of mammalian cells, i.e., glycosphingolipids and gangliosides, which are involved in processes such as signaling, oncogenesis, and differentiation. Sophorolipids have been reported to induce differentiation in several leukemic cell lines, cell death via apoptosis in a human liver cancer cell line, and necrosis in a pancreatic adenocarcinoma cell line. Here we report, for the first time, the effects of precursor fatty acids and sophorolipids of oleic and linoleic acids in pure acidic and crude forms on LN-229, a glioma cell line. In response to different sophorolipid forms, various morphological changes were observed, such as formation of long thread-like extensions arising from the ends of the cells, cell alignment, cell elongation and bundle formation in a dose-dependent manner. In this study we present the morphological evidence of the potential of sophorolipids as differentiation inducers.     

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.     

Isolation and characterization of a serially cultivated,neoplastic, epithelial cell line from theN-nitrosomethylurea induced rat mammary adenocarcinoma

Summary A new in vitro model for human breast cancer is described. Derived from anN-nitrosomethylurea (NMU) induced rat mammary adenocarcinoma, this serially cultivated cell line has been demonstrated, by a variety of criteria, to be an authentic neoplastic, rat mammary epithelial cell line. The criteria used include morphological and growth characteristics; the presence of specific cell surface antigens; steroid hormone receptors; hormone responsiveness; casein production; karyotype and isoenzyme profile analysis; anchorage independent growth and oncogenicity. Inasmuch as the NMU cell line possesses high concentrations of glucocorticoid and androgen receptors, it may provide a useful model for study of the action of these hormones in human breast cancer. In addition, the NMU line may serve as a valuable in vitro model in which to assess the effects of a variety of endogenous and exogenous agents known to influence mammary tumor growth in vivo, including drugs, nutrients, and growth factors. This work was supported by Grants CA29602 and RR05775-05 from the National Cancer Institute, Bethesda, Maryland.     

Retroviral expression of alternatively spliced forms of rat fibronectin

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.     

Characterization of a human colonic adenocarcinoma cell line,LS123

Summary An epithelial cell line, LS123, was established in 1974 from the second in a series of three primary colonic tumors resected from a Caucasian female. The cell line is aneuploid, releases low concentrations of carcinoembryonic antigen (CEA), fails to grow progressively in nude mice, and forms colonies only in enriched semisolid medium developed for tumor stem cells. LS123 cells grow on confluent cell monolayers and in either low serum or serum-free medium. In the chick embryonic skin assay, LS123 cells grew as a well-differentiated abnormal colonic epithelium with little mitotic activity but with some indication of invasion. On floating collagen gels LS123 cells formed a one to three-cell-layer-thick undifferentiated epithelial sheet. The apparent low invasiveness of the cells of this line is supported by the patient's history of three primary colon tumors without systemic metastases during the past 30 yr. Therefore, although LS123 cells possess several properties associated with neoplasia, they have little invasive potential. Thus, LS123 cells may represent an important model for the study of human colon cancer. Presented in part at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, CA, June 6–10, 1982. The work has been partially supported by U.S. Public Health Service Grants CA23871 (L. P. R.), CA24024 and RCDAK04-CA00579 (B. H. T.), and CA27124 and CA22370 (B. D. K.); the latter was awarded through the National Large Bowel Cancer Project.     

Establishment and characterization of a human colonic cancer cell line expressing carbohydrate antigen 19-9 and carcinoembryonic antigen]

Colonic cancer cell strain KE43 was established from a human colonic cancer diagnosed histologically as a predominantly well differentiated adenocarcinoma with minute foci of poorly differentiated adenocarcinoma. The well differentiated adenocarcinoma cell line was identified as the major morphological picture in xenografts of KE43 and 58 in nude mice, but this changed to poorly differentiated adenocarcinoma in passage 105. Doubling time of this cancer cell line was 22.5 hours in passage 105. The modal numbers of chromosomes were 41 and 76. Cancer cells could be heterotransplanted in 100% of the nude mice. The tumor cells produced and secreted CA19-9, CEA and Laminin into the spent medium. This cell line appears to provide a useful system for studying colonic cancer in vivo and in vitro.     

Predominance of IL-10 and TGF-β production from the mouse macrophage cell line, RAW264.7, in response to crude antigens from Clonorchis sinensis

Parasitic helminths are well-known to have the ability to modulate host immune responses. In this study, we investigated the fundamental immunoregulatory mechanism of the liver fluke Clonorchis sinensis (C. sinensis) using a murine macrophage RAW 264.7 (RAW) cell line and mouse bone marrow derived macrophages (BMDMs). We found that C. sinensis crude antigen (CA) is able to differentiate macrophage RAW cells into dendritic-like cells that can be detected by morphological observations. In addition, CA induces prominent secretion of anti-inflammatory cytokines such as IL-10 and TGF-β; however, we did not observe changes in cell surface markers that are involved in antigen recognition, antigen presentation, and T cell activation. Additionally, CA treatment induced ERK and JNK phosphorylation, and unexpectedly, elevated levels of IL-10 and TGF-β were inhibited by the addition of an ERK-specific inhibitor. Taken together, these data demonstrate that CA from C. sinensis may modulate host immune responses by upregulating anti-inflammatory cytokines via the regulation of ERK.     

Cytostatic and cytotoxic effects of (E)-2'-deoxy-2'-(fluoromethylene)-cytidine on a solid tumor and a leukemia cell line

(E)-2'-deoxy-2'-(fluoromethylene)-cytidine (FMdC), a deoxycytidine analog displaying a very high toxicity toward a variety of solid tumor cell lines and xenografts, is activated intracellularly by deoxycytidine kinase (dCK). We have compared cytotoxicity of FMdC towards a human promyeolocytic leukemia line HL-60 and a human colorectal carcinoma line COLO-205. Despite dCK activity being by far the highest in cells of lymphoid origin, the effects of FMdC were detectable at the lowest drug concentration only in a solid tumor cell line, and at higher concentrations they were qualitatively similar in the two tumor lines (increased cell protein content, cell cycle block and apoptosis). Apparently, low dCK activity in solid tumor cells sufficiently activates FMdC to yield cytotoxic effects, while high dCK activity in leukemia cells does not increase its cytotoxicity.     

Cell-to-cell contacts between lymphoreticular cells in rheumatoid synovial membrane

Nodular type and diffuse perivascular lymphoid cell infiltrates in rheumatoid synovial membranes from 72 patients were examined under the electron microscope. Attempts were made to describe the ultrastructural relations of the cells examined and to classify the different types of cell-to-cell contracts between them. Members of a complex series, composed of gradually differentiating forms of cell contacts beginning from simple membrane adherences and interdigitations and gap junctions; minute filamentous intercellular bridges: subsurface confronting cisternae and desmosome-like structures, are described and suggested as function-dependent morphological representatives of sites of cellular interactions.     

Carbonic anhydrase IX

Cell migration can be principally viewed as a chain of well-orchestrated morphological events that lead to dynamic reshaping of the cell body. However, behind the scene of such a “morphological theater” there are very complex, interrelated molecular and physiological processes that drive the cell movement. Among them, ion transport and pH regulation play a key role, with carbonic anhydrase IX (CA IX) emerging as one of the important “molecular actors.” CA IX is a highly active cell surface enzyme expressed in a broad range of solid tumors in response to hypoxia and explored as a clinically useful biomarker of hypoxia and as a therapeutic target. Its biological role is to protect tumor cells from hypoxia and acidosis in the tumor microenvironment. The study published recently by our group showed that CA IX actively contributes to cell migration and invasion. For the first time, we demonstrated CA IX accumulation in lamellipodia of migrating cells and its direct in situ interaction with bicarbonate transporters. Our findings indicate that tumor cells need CA IX not only as a pro-survival factor in hypoxia and acidosis, but also as a pro-migratory component of the cellular apparatus driving epithelial-mesenchymal transition.     

Cytomorphological charasteristics of a novel mouse cell line G1

Growth and morphological properties of a novel mouse cell line G1 have been investigated. It has been shown that cells of this cell line possess the ability of spontaneous transformation in vitro: the cells have unlimited growth in culture, grow in medium with a low serum content and form multilayer colonies on a cell monolayer and cell colonies inside agar. Using micronucleus test it has been revealed that cells of the G1 cell line possess different forms of aberrant mitosis. The results indicate the neoplastic G1 cell transformation with aberrant mitosis.     

Combined method of PAP immunocytochemistry and autoradiography: application to cell kinetic study in plasma cell dyscrasias

The plasma cell labeling index (LI), in spite of being a reliable indicator for diagnosis and prognosis of multiple myeloma, has been measured in a limited number of laboratories because of technical difficulties. We have developed a new combined technique, using the peroxidase-antiperoxidase (PAP) method and autoradiography, which has several advantages over previously described methods. The primary advantages of our method in the determination of lymphoid-plasma cell LI% are: (a) no damage to slides during storage of more than 1 year; (b) an exact LI measurement in each morphological variety of pleomorphic immunoglobulin-containing cells; (c) no problem in differentiation of lymphoid plasma cells from early red cell precursors; and (d) a separate LI measurement for those lymphoid-plasma cells composed chiefly, if not exclusively, of monoclonal or neoplastic cells. Because of these advantages, this accurate and less difficult technique will facilitate performance of lymphoid plasma cell LI in a number of laboratories.     

Growth of human malignant lymphoid cell lines in serum-free medium

Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products. This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los Angeles, and CA 09120 (C. U.)     

Changes of Plasma Membrane Properties in a Human Pre-T Cell Line Undergoing Apoptosis

A variety of cellular functions are modulated by the physical properties of the cell membrane, and the modification of intracellular transfer, resulting from loss of membrane integrity, may contribute toward setting the cell onto the pathway of apoptosis. Apoptosis in lymphoid cells can be induced in different ways and biochemical modifications occur at an early phase of cell death, while the morphological features of apoptosis are evident later. We previously reported that DMSO is an efficient apoptosis-inducing factor in the human RPMI-8402 pre-T cell line. The aim of the present study was to verify the effect of DMSO on the plasma membrane fluidity, the intracellular calcium concentration and the phosphodiesterase activity in DMSO-induced apoptosis. Our results show a modification of membrane fluidity associated with an increase of intracellular Ca²⁺ concentration. Moreover, we demonstrate that these modifications are related to a decrease in the phosphodiesterase (PDE) activity. The correlation between the proceedings of added DMSO and the induction of apoptosis will provide significant information regarding the first part of the apoptotic process.     

Comparative Characteristics of Immunological,Clinical and Morphological Features of Human Lymphoid Tumors in Respect with Tumor Genesis and Progression

We describe three lymphoid tumors with the same immunophenotype characteristic for chronic lymphoid leukemia (CD19⁺/CD5⁺, clonality of the light immunoglobulin chains, CD23⁺ and CD10^–). However, clinical picture and morphology of neoplastic cells dictate different clinical forms of these cases: chronic lymphoid leukemia, large cell transformation of chronic lymphoid leukemia and diffuse large B-cell lymphoma. Taking into account that immunophenotype reflects the origin of tumor, while clinical outcome and morphological features of cells reflect the stage of tumor progression and/or pathway of tumor formation, we discuss the approach to natural classification of lymphoid tumors based on the process of their evolution.     

Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion

Mesothelin is a glycosylphosphatidylinositol-linked cell surface molecule expressed in the mesothelial lining of the body cavities and in many tumor cells. Based on the finding that a soluble form of mesothelin specifically binds to ovarian carcinoma cell line OVCAR-3, we isolated cDNAs encoding a mesothelin-binding protein by expression cloning. The polypeptides encoded by the two cloned cDNA fragments matched to portions of CA125, an ovarian cancer antigen and a giant mucin-like glycoprotein present at the surface of tumor cells. By flow cytometric analysis and immunoprecipitation, we demonstrate that CA125 binds to mesothelin in a specific manner. Binding of CA125 to membrane-bound mesothelin mediates heterotypic cell adhesion as anti-mesothelin antibody blocks binding of OVCAR-3 cells expressing CA125 to an endothelial-like cell line expressing mesothelin. Finally, we show that CA125 and mesothelin are co-expressed in advanced grade ovarian adenocarcinoma. Taken together, our data indicate that mesothelin is a novel CA125-binding protein and that CA125 might contribute to the metastasis of ovarian cancer to the peritoneum by initiating cell attachment to the mesothelial epithelium via binding to mesothelin.     

Phospholipase D2: a pivotal player modulating RBL-2H3 mast cell structure

The current study examined the role of PLD2 in the maintenance of mast cell structure. Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PLD has two isoforms, PLD1 and PLD2, which vary in expression and localization depending on the cell type. The mast cell line RBL-2H3 was transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2. The results of this study show that PLD2CI cells have a distinct star-shaped morphology, whereas PLD2CA and RBL-2H3 cells are spindle shaped. In PLD2CI cells, the Golgi complex was also disorganized with dilated cisternae, and more Golgi-associated vesicles were present as compared with the PLD2CA and RBL-2H3 cells. Treatment with exogenous PA led to the restoration of the wild-type Golgi complex phenotype in PLD2CI cells. Conversely, treatment of RBL-2H3 and PLD2CA cells with 1% 1-Butanol led to a disruption of the Golgi complex. The distribution of acidic compartments, including secretory granules and lysosomes, was also modified in PLD2CI cells, where they concentrated in the perinuclear region. These results suggest that the PA produced by PLD2 plays an important role in regulating cell morphology in mast cells.