In vitro characterization of estrogen induced syrian hamster renal tumors: Comparison with an immortalized cell line derived from diethylstilbestrol-treated adult hamster kidney

Summary Primary diethylstilbestrol-induced kidney tumors from Syrian hamsters were grown in vitro and maintained in culture for 6 mo. Combined immunohistochemical studies using antibodies to intermediate filaments and ultrastructural studies of tumor cells in culture exhibited characteristics similar to tumor cells in vivo. Furthermore, the cells manifested transformed properties in culture; they grew both as multilayered colonies attached to the tissue culture substrate and as floating multicellular colonies (spheroids). When cultured cells were injected into diethylstilbestrol-treated recipient hamsters, tumors developed at the injection sites. In contrast, renal tubules or whole kidney cortex from control hamsters cultured in the same medium underwent only short-term growth, with senescence developing after approximately 1 mo. However, cell cultures of kidney cortex from animals treated in vivo for 5 mo. with diethylstilbestrol formed a cell line. This diethylstilbestrol-induced cell line has been maintained in culture for 1.5 yr and has the following characteristics: a) it is anchorage-dependent, b) it is negative in in vivo tumorigenicity tests, and c) cultured cells are histochemically and ultrastructurally similar to cultured tumor cells. This culture system should prove to be of use in studying hormonal carcinogenesis in vitro. This study was supported by the Medical Research Service, Department of Veterans Affairs, Washington, DC, and by grant CA-22008 from the National Cancer Institute, NIH, DHHS, Bethesda, MD.     

Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23187 and sodium periodate) whose mechanism and site of action differ. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to impair [³H]thymidine incorporation by prestimulated blast cells at the higher drug concentrations tested. The results presented in this communication show that the Vinca alkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Preliminary studies on the cultivation and characterization of mini-pig prostate epithelial cells

Mini-pig prostate epithelial cells exhibited the unique metabolic characteristics associated with the specialized function of production and secretion of high levels of citric acid. Epithelial cell suspensions from mini-pig prostate were successfully grown in primary and secondary cultures. The cultured epithelial cells exhibited rapid proliferation reaching confluency in approximately 6 days. Growth and proliferation of fibroblasts were markedly restricted by the dominance of epithelial cell growth. Confluent cultures could be maintained for approximately 6 weeks. The epithelial cells retained their polymorphic appearance in primary and secondary cultures and exhibited the characteristic formalin-resistant acid phosphatase reaction. Testosterone stimulated mitochondrial aspartate aminotransferase (mAAT) activity and citrate production by confluent epithelial cell cultures. These initial results indicate that cultured epithelial cells derived from mini-pig prostate might be an excellent model related to human for studies of prostate biology and hormonal regulation.     

Characterization of a monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus (L.) G. Don

Conditions have been established for the optimization of the specific activity of a membrane-bound monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus. In time course studies, the hydroxylase and NADPH-cytochrome c reductase exhibited maximal activities 18–20 days after inoculation, i.e., during early stationary phase. By late stationary phase, enzyme activity had declined. In contrast an enzyme of primary metabolism achieved optimal specific activity by the 12th day and remained constant through day 26, synchronous with general growth. Effects of nutritional and hormonal factors on the specific activity of the hydroxylase and cell growth were evaluated. Inhibitors of hydroxylase activity were also assessed in vitro. A soluble form of the monoterpene hydroxylase has been detected in cultured cells possibly affording a useful source of this enzyme for further purification.     

Multidrug resistance (MDR) genes in haematological malignancies

The emergence of drug resistant cells is one of the main obstacles for successful chemotherapeutic treatment of haematological malignancies. Most patients initially respond to chemotherapy at the time of first clinical admission, but often relapse and become refractory to further treatment not only to the drugs used in the first treatment but also to a variety of other drugs. Laboratory investigations have now provided a cellular basis for this clinical observation of multidrug resistance (MDR). Expression of a glycoprotein (referred to as P-glycoprotein) in the membrane of cells made resistantin vitro to naturally occurring anticancer agents like anthracyclines, Vinca alkaloids and epipodophyllotoxins, has been shown to be responsible for the so-called classical MDR phenotype. P-glycoprotein functions as an ATP-dependent, unidirectional drug efflux pump with a broad substrate specificity, that effectively maintains the intracellular cytotoxic drug concentrations under a non-cytotoxic threshold value. Extensive clinical studies have shown that P-glycoprotein is expressed on virtually all types of haematological malignancies, including acute and chronic leukaemias, multiple myelomas and malignant lymphomas. Since in model systems for P-glycoprotein-mediated MDR, drug resistance may be circumvented by the addition of non-cytotoxic agents that can inhibit the outward drug pump, clinical trials have been initiated to determine if such an approach will be feasible in a clinical situation. Preliminary results suggest that some haematological malignancies, among which are acute myelocytic leukaemia, multiple myeloma and non-Hodgkin's lymphoma, might benefit from the simultaneous administration of cytotoxic drugs and P-glycoprotein inhibitors. However, randomised clinical trials are needed to evaluate the use of such resistance modifiers in the clinic.Abbreviations ALL acute lymphocytic leukaemia - AML acute myelocytic leukaemia - BM bone marrow - CAT chloramphenicol acetyltransferase - CLL chronic lymphocytic leukaemia - CML chronic myelocytic leukaemia - CR complete remission - HCL hairy cell leukaemia - MDR multidrug resistance - MDS myelodysplastic syndrome - MM multiple myeloma - MoAb monoclonal antibody - NHL non-Hodgkin's lymphoma - PB peripheral blood - PCR polymerase chain reaction - PLL prolymphocytic leukaemia - RMA resistance modifying agent - VAD vincristine, doxorubicin, dexamethasone     

Accessory cell requirements for lymphoma growth in vitro and in irradiated mice.

Growth of the transplantable B-cell lymphoma, PU-5, is markedly diminished in γ-irradiated as compared to normal BALB/c mice. Transfer of bone marrow, but not of lymph node or peritoneal exudate cells, partially restored the ability of irradiated mice to support lymphoma growth. In vitro growth of PU-5 cells is promoted by silica-sensitive, adherent cells, bearing surface Ia antigen and present in peritoneal exudates, spleen and lymph node, but not in bone marrow. Their action on PU-5 growth can be shown only in rocking cultures; the cells do not have to be histocompatible, they act synergistically with 2-mercaptoethanol (2-ME) in the medium. The growth-promoting action in vitro is decreased 24 hr after γ-irradiation of the adherent cells in vitro. Growth of transplantable reticulum cell sarcoma in SJL/J mice has previously also been shown to be inhibited by prior irradiation of the host and to be restored by transfer of lymphoid cells including a phagocytic component, but in the present studies no consistent growth-promoting effect of accessory cells on reticulum cell sarcomas has been shown in vitro. Both lymphomas are stimulated by the presence of 2-ME in stationary cultures. The relationship between the in vivo and in vitro lymphoma growth-promoting activities of macrophage-like cells is discussed.     

Anticarcinogenic Alkaloids in the Cultured Cells of Cephalotaxus fortunei

Calli were induced from the leaves and stems of Cephalotaxus fortunei Hook. f. on MS medium supplemented with 0. 1 mg/L KT and 3 mg/L NAA, and from which the suspension culture cell line of this plant was established for the first time. Factors such as light, pH value of the medium, concentration of plant hormone, carbon resources and addition of substances to the medium, which affect the growth of suspension cells were investigated. The results showed that suspension cells grew appropriately at pH 5.8 with a low concentration of sucrose or glucose, and a low level of NAA. No difference effect on cell growth was seen between sucrose and glucose. Phenylalanine and protein hydrolysate were not suitable for cell growth in suspension cultures, and light inhibited cell growth. A sensitive and rapid high-performance liquid chromatographic method has been developed for detecting the alkaloids in cultured cells. The results revealed the following contents of cephalotaxine and its anticancer esters in cultured cells: harringtonine, isoharringtonine and homoharringtonine. The total alkaloid production in cell suspension cultures was doubled as that in solid cultures. The relative amounts of cephalotaxine, drupacine, harringtonine, homoharringtonine and isoharringtonine in suspension cells was 22%, 6%, 8%, 23% and 41% respectively. In addition, other alkaloid as deoxyharringtonine and some steroids, including ergdst-5-en-3-ol. stigmasta-5, 22-dien-3-ol, β-sitosterin and 2-naphthalenamine have also been detected in cell cultures using GC/MS combined technique.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Cytokeratin provides a specific marker for human arachnoid cells grown in vitro

Cells from cranial and spinal arachnoid membranes of humans were grown in culture. Their growth characteristics, morphology and details of their cytoskeletal composition are described. Arachnoid membranes, obtained at autopsy, were finely minced and incubated in tissue culture medium. Monolayers of cells of homogeneous morphology grew from these tissue fragments. The cells were flat and polygonal. They divided slowly to form non-overlapping monolayers of low cell density. Electron microscopic examination of cultured arachnoid cells revealed numerous desmosome-like tight junctions and abundant intermediate filaments (tonofilaments). Both morphological features are characteristic of arachnoid cells in situ, but not of cells in the fibroblast-rich dura mater. Immunofluorescence microscopy with monoclonal antibodies demonstrated cytokeratin in the cytoplasm of primary cultures of arachnoid cells. Thus we demonstrated that these cultured cells retained certain of the specific differentiated properties of arachnoid cells in situ and that they are not fibroblasts (which lack tight junctions and cytokeratins). To our knowledge, there have been no previous reports of in vitro growth of arachnoid cells. This in vitro model should be useful in studying the response of arachnoid cells to a variety of substances thought to be involved in the chronic inflammatory condition of the meninges known as arachnoiditis.     

Morphological and functional characteristics of human temporal-bone cell cultures

Morphology and calcium metabolism have been studied on five different cell cultures from human normal adult temporal-bone biopsies obtained during five stapedectomies. Control cell cultures were obtained from normal human skin. Four different cell types were observed in the bone biopsies: 1) osteoblast-like cells; 2) osteoclast-like cells; 3) fibroblast-like cells; 4) intermediate cells. However, morphology by itself is inadequate for clear differentiation of the four cell types. Hormonal stimulation with calcitonin and dibutyryl-cAMP in presence of 45Ca++ showed a clear-cut difference in 45Ca++ uptake between cultured cells deriving from bone and skin. Functional responses to hormonal stimulation are therefore more specific than cell shape and morphology in differentiating fibroblasts from bone cells. Since responses to hormonal stimulation confirm that temporal-bone cell cultures actually contain bone cells, such cultures seem to be a good experimental model for the study of bone morphology and physiology.     

Modelling of malignant lymphoma in rabbits using primate oncogenic viruses. Preliminary report

Inoculation of rabbits with permanent B-cell line cultures obtained from Stumptailed Macaques M. arctoides (MAL) which contain lymphotropic herpesvirus and C-type particles has led to the development of generalized lymphomas. The lymphoma cells had rabbit karyotype and did not contain surface an cytoplasmic immunoglobulins. Permanent suspension of lymphoid cell line independent of growth factors was obtained from rabbit lymphoma. The serum of a rabbit with lymphoma transmitted from another rabbit with MAL-induced lymphoma did not react with virus-negative human Raji cells when tested in immunofluorescence. But this serum reacted positively with cytoplasmic antigens of simian 594S-F9 cells (producing lymphotropic herpesvirus and two types of retroviruses, namely, endogenous C-type and HTLV-I-like) and human C91-PL cells (producing HTLV-I). The results obtained demonstrated high oncogenicity of the viruses produced by simian permanent cellular MAL lines for rabbits.     

Insulin-like growth factor-I supports proliferation of autocrine thymic lymphoma cells with a pre-T cell phenotype

We have studied the phenotypic characteristics and growth properties of murine T lymphoma cell lines derived from primary x-ray-induced thymic lymphomas at the earliest stage at which they can be detected, and well before spreading to other organs has occurred. These cell lines serve as model systems for the earliest events in T cell lymphoma induction, before tumor cell progression and spreading to other organs. We find that primary x-ray-induced T cell lymphoma lines have phenotypic characteristics of thymic pre-T cells and show no proliferative response to any of the IL tested nor to other hematopoietic growth factors. However, they do proliferate in response to insulin-like growth factor I (IGF-I) and to a small autocrine peptide distinct from IGF-I, which we term lymphoma growth factor. One of the earliest lesions in T cell lymphoma induction may therefore be an inhibition of differentiation at one of several specific points. In its early stages, T lymphoma cell growth may be restricted to an environment where local concentrations of specific growth factors such as IGF-I or lymphoma growth factor are sufficiently high.     

New method of synthesis of Vinca alkaloid derivatives

Vinblastine and vinorelbine analogues have been synthesised by reacting new versatile electrophilic vindoline derivatives with various 3-substituted indoles. The resulting compounds have been evaluated for their antimitotic properties, but exhibited less potent activities in comparison with the standard binary Vinca alkaloids.     

ULTRASTRUCTURE OF MAIZE ENDOSPERM SUSPENSION CULTURES

Suspension cultures derived from developing maize (Zea mays L.) endosperm were examined by electron microscopy, after both glutaraldehyde-OsO₄ and KMnO₄ fixation, and compared with intact endosperm. Tissue clumps consisted of interconnected cell clusters without any organization of the different cell types. The cultures were comprised of cells with dense cytoplasm and small vacuoles, large vacuolate cells, and cells in which storage products (starch, protein bodies, or lipid) accumulated. The endomembrane system of cultured cells was more highly developed than that of cells of the intact endosperm. In particular, arrays of smooth endoplasmic reticulum were seen only in the cultured cells. An abundance of endoplasmic reticulum, dictyosomes, and ribosomes is consistent with the recently reported extracellular secretion of enzymes by these cultures. Cell wall ingrowths, a characteristic of basal endosperm transfer cells, were observed occasionally in cultured cells, but cells with ingrowths had no histological organization. Some of the observed features may have resulted from perturbation of normal cellular events caused by the conditions of in vitro growth. These cultures are a useful tool for studying cellular mechanisms of protein secretion and storage product accumulation in developing maize endosperm.     

Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering

Catharanthus roseus plants produce many pharmaceutically important indole alkaloids, of which the bisindole alkaloids vinblastine and vincristine are antineoplastic medicines and the monoindole alkaloids ajmalicine and serpentine are antihypertension drugs. C. roseus cell cultures have been studied for producing these medicines or precursors catharanthine and vindoline for almost four decades but so far without a commercially successful process due to biological and technological limitations. The research thus focused on the one hand on engineering the bioreactor process on the other engineering the cell factory itself. This review mainly summarizes the progress made on biochemical engineering aspects of C. roseus cell cultures in bioreactors in the past decades and metabolic engineering of indole alkaloid production in recent years. The paper also attempts to highlight new strategies and technologies to improve alkaloid production and bioreactor performance. Perspectives of metabolic engineering to create new cell lines for large-scale production of indole alkaloids in bioreactors and effective combination of these up- and down-stream processing are presented.     

Indole alkaloids from cell suspension cultures of Rauwolfia serpentina benth

Twelve indole alkaloids belonging to the Ajmaline-, Sarpagine-, Yohimbine-, and Heteroyohimbine-type have been isolated and identified from cell suspension cultures of Rauwolfia serpentina. Ten of the alkaloids were found for the first time in cultured R. serpentina cells. The yield of the main alkaloid vomilenine was 51 times more than that of differentiated plants. Crude enzymes isolated from this cell suspension culture completely metabolize the biogenetic precursor strictosidine under formation of several alkaloidal compounds.     

Development of a new method for isolation and long-term culture of organ-specific blood vascular and lymphatic endothelial cells of the mouse

Endothelial cells are indispensable components of the vascular system, and play pivotal roles during development and in health and disease. Their properties have been studied extensively by in vivo analysis of genetically modified mice. However, further analysis of the molecular and cellular phenotypes of endothelial cells and their heterogeneity at various developmental stages, in vascular beds and in various organs has often been hampered by difficulties in culturing mouse endothelial cells. In order to overcome these difficulties, we developed a new transgenic mouse line expressing the SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary expression system based on Cre/loxP recombination. tsA58T Ag-positive endothelial cells in primary cultures of a variety of organs proliferate continuously at 33 degrees C without undergoing cell senescence. The resulting cell population consists of blood vascular and lymphatic endothelial cells, which could be separated by immunosorting. Even when cultured for two months, the cells maintained endothelial cell properties, as assessed by expression of endothelium-specific markers and intracellular signaling through the vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3, as well as their physiological characteristics. In addition, lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an organ-specific endothelial characteristic was maintained. These results show that our transgenic cell culture system is useful for culturing murine endothelial cells, and will provide an accessible method and applications for studying endothelial cell biology.     

Insect tissue culture systems: models for study of hormonal control of development

Summary The regulation of growth and development of insects is under endocrine control and involves both juvenile hormones and ecdysteroids. Neuropeptides are master regulators which control the secretion of these hormones. Most experiments in insect endocrinology have been conducted in vivo, but tissue culture methodology is playing an increasing role due to the great interest in simpler model systems for the study of complex processes that occur in vivo. The availability of appropriate media has allowed the culture of a variety of insect organs and cell lines of defined origin which have kept certain properties of the parent tissues. Tissue culture approaches have been useful for studying hormonal control of morphogenetic processes. Cell lines are particularly suited to the study of hormonally regulated mechanisms of macromolecular biosynthesis and gene expression. Thus, the value of in vitro analysis in studies of regulation of hormone production is now recognized. Results obtained from tissue culture allow more precise definition of the hormonal requirements of insect cells and tissues for growth and differentiation and might make possible the discovery of new growth regulators.     

Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones

We examined stimuli which are required for the induction of in vitro proliferation of follicular lymphoma cells, a low grade non-Hodgkin's B cell lymphoma characterized by a specific chromosomal translocation, t(14;18)(q32;q21), and by in vivo growth of the lymphoma cells in germinal center-like follicles infiltrated with CD4+ T cells. The purified follicular lymphoma cells, which are morphologically uniform, small, and dense, did not respond to stimulation with soluble lymphokines in the absence of T cells. Vigorous in vitro proliferation of follicular lymphoma cells was induced, however, when the follicular lymphoma cells were cultured with a CD4+ T cell clone which recognized alloantigens expressed by the lymphoma cells. This response required B-T cell contact, and was inhibited by anti-class II but not by anti-class I MHC mAb, indicating that these neoplastic B cells behaved as normal B cells and responded to normal activation and differentiation signals from T cells. After the cognate B lymphoma-T cell interaction occurred in culture, addition of IL-2 or IL-4 enhanced the proliferation of the tumor cells. These results, with a monoclonal and homogeneous population of B cells, affirm the idea that cognate interaction between B cells and Th cells is required for the effective activation of resting B cells. Moreover, these results suggest that a critical host-tumor interaction occurs in vivo, and that the polyclonal CD4+ T cells that infiltrate follicular lymphomas play a role in sustaining rather than inhibiting tumor growth in vivo. If so, therapies directed not only against the neoplastic cell but also against specific T cells and their cognate interactions with tumor cells may have a rationale.