Production of urokinase-type plasminogen activator by normal and transformed rat thyroid cells in culture

We studied the relationship between differentiation, transformation, and uPA production in a system of rat thyroid cells in vitro. The fully differentiated FRTL5 cells did not produce detectable amounts of uPA, even after stimulation with phorbol esters, potent inducers of uPA expression. All the other cell lines (i.e., FRT, cells which have lost the characteristics of the differentiated thyroid cells; 1-5 G and FRA, transformed cells derived from rat thyroid tumors) produced uPA, the 1-5 G line being the highest producer. Also the FRTL line became positive for uPA production after viral transformation (clone KM4). The lack of uPA expression in FRTL5 cells was not due to the presence of inhibitors and these cells did not produce an inactive molecule, as shown by immunoprecipitation with anti-uPA antibody. However, in FRTL5 cells Northern analysis showed the presence of a small amount of uPA-specific mRNA that increased appreciably after phorbol ester stimulation. In conclusion, in our system uPA expression was a property of undifferentiated and transformed cells; in fully differentiated cells uPA expression was switched off by a still unclear mechanism.     

Dissociation between transformed and differentiated phenotype in rat thyroid epithelial cells after transformation with a temperature-sensitive mutant of the Kirsten murine sarcoma virus.

Differentiated rat thyroid epithelial cells, infected in vitro with a temperature-sensitive mutant of the Kirsten murine sarcoma virus, expressed at the permissive temperature (33 degrees C) some phenotypic properties typical of transformed cells, including morphological features, colony formation in agar, and induction of tumors in newborn animals. Specific functional markers of these differentiated cells, i.e., synthesis/secretion of thyroglobulin, synthesis of thyroglobulin mRNA and iodide uptake, were blocked during growth at 33 degrees C. Normal morphology, failure to grow in agar, and the requirement of hormones for optimal growth were all restored after shifting to the temperature nonpermissive for transformation (39 degrees C), though the typical differentiated functions remained blocked. Infection with a leukemia helper virus clone (Moloney or Kirsten murine leukemia virus) did not lead to the loss of the differentiated phenotype of rat epithelial thyroid cells, thus demonstrating that the loss of the differentiated phenotype is caused by the sarcoma virus component. These results indicate that the expression of some of the phenotypic properties of transformed differentiated rat thyroid epithelial cells is under the direct control of the p21 thermosensitive activity, whereas the block in the expression of two typical differentiation markers of thyroid epithelial cells is irreversible and probably controlled by different mechanisms.     

Expression of neuronal markers in chick and quail embryo neuroretina cultures infected with Rous sarcoma virus

Cultures of neuroretina (NR) cells from 7-day chick and quail embryos were infected with ts NY-68, a thermosensitive mutant of Rous sarcoma virus (RSV) which transformed NR cells at 36 degrees C. The following differentiation markers for neurones were studied: tetanus toxin-binding sites at the cell surfaces, presence of synapses, and the specific activity of the enzymes choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Appearance of synapses and expression of CAT were similar in control and transformed cultures. Tetanus toxin-binding cells were observed in transformed primary cultures and also in quail NR subcultures. GAD-specific activity was markedly stimulated in chick and quail primary cultures transformed by ts NY-68 and further increased in subcultures of ts NY-68-transformed quail NR cells. Stimulation of GAD activity is controlled by the transforming (src) gene of RSV since it was not observed in cultures infected with RAV-1, a leukosis virus which lacks the src gene. These data show that infection of chick and quail NR cultures with RSV results in the transformation of cells with neuronal markers.     

Stimulation by thyrotropin and cyclic AMP of the proliferation of quiescent canine thyroid cells cultured in a defined medium containing insulin

We have developed serum-free primary cultures of differentiated follicular dog thyroid cells which allow the study of the hormonal control of cell proliferation. The cooperation of insulin and increasing cellular cyclic AMP by thyrotropin triggers the DNA synthesis and the proliferation. Dog thyroid cells are an example of a system in which cyclic AMP is a sufficient signal to stimulate the proliferation in quiescent cells.     

Characteristics of arachidonic acid metabolism of human endothelial cells in culture

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E₂ and thromboxane A₂ in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum.After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E₂ and thromboxane A₂ production showed no variations during the study.     

A novel regeneration system for a wild passion fruit species (<Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 μM 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog’s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.     

Immunohistochemical study of the C-cell complex of dog thyroid glands with reference to the reactions of calcitonin,C-thyroglobulin and 19S thyroglobulin

Summary Continued from the previous study in fetal animals (Kameda et al. 1980), the development and maturation of C-cell complexes in postnatal dogs from newborn to adult were investigated by use of an immunoperoxidase method using antisera to calcitonin, C-thyroglobulin (C-Tg) and 19S thyroglobulin, respectively. The younger the animals were, the more numerous were undifferentiated cells and high columnar epithelial cells in the complexes. With increasing age, the constituent elements of the complexes progressively differentiated. In one type of complex there are a large number of C-cells in various developmental stages, as well as undifferentiated cells and cysts. C-cell complexes composed mostly of mature C-cells were regarded as the more highly differentiated structures of this type. A second type contains follicular cells in various stages of differentiation in addition to undifferentiated cells and C-cells, i.e., 19S-positive cell masses not yet organized into follicles, primordial follicles with small lacunae and comparatively larger follicles. The follicular cells in the complexes were similar with respect to immunoreaction and folliculogenesis to the cells of fetal thyroids, but they developed very slowly. In conclusion, the present study indicates that follicular thyroid cells can differentiate within C-cell complexes, i.e., they develop from cells of ultimobranchial body origin.     

Expression of receptors for VEGFs on normal human thyroid follicular cells and their role in follicle formation

Human thyroid follicular cells in culture expressed the mRNAs for the receptors for vascular endothelial growth factors (VEGFRs). The relative expression was neuropilin1 = neuropilin2 = VEGFR2 > VEGFR1 > VEGFR3. Western blotting for VEGFR2 showed labeling of proteins ~200-230 kDa. Clonal follicular thyroid cell lines (FRTL5 and FTC133) also expressed mRNAs for the VEGFR1 and 2 obviating concerns of endothelial cell contamination. In the primary cultures, TSH, which is essential for expression of differentiated function, reduced VEGFR2 mRNA levels by 60%. Immunostaining for VEGFRs and neuropilin2 (NRP2), showed expression on the plasma membrane but with the exception of neuropilin1 (NRP1), all VEGFRs were also found in the cytoplasm and nucleus. Antibody specific for phosphotyrosine 1214 in VEGFR2 showed that the receptor was phosphorylated in the primary cultures and the cell lines. When VEGFR signaling was blocked with a specific inhibitor, follicle formation in the primary cultures was enhanced suggesting that VEGFR activation was detrimental to follicle formation. Immunostaining of sections of normal thyroids and various pathologies showed staining for VEGFR2 and pVEGFR2. We conclude that normal thyroid follicular cell express VEGFRs. For VEGFR2 its subcellular localization suggests functions additional to that of a cell surface receptor and a role in follicular integrity.     

Changes in the proportion of endogenous osmotic solutes accumulated by Chlorella emersonii in the light and dark

Abstract. The type of endogenous osmotic solute accumulated by Chlorella emersonii grown at high external osmotic pressure (π_ext) depended on the light/dark conditions: proline accumulated to high concentrations in cells in the light, while sucrose accumulated to high concentrations in the dark. These findings were made during the alternating light dark cycles used to obtain synchronized cultures, i.e. cultures containing cells at only one stage of development at any one time. Similar decreases in proline and increases in sucrose in the dark were found for cells previously grown in continuous light to obtain non-synchronized cultures, i.e. cultures containing cells at all stages of development.
In cultures synchronized at 200 mol m ⁻³ NaCl (π_ext= 1.01 MPa), recently divided 'daughter cells' at the beginning of the light periods contained 60 mol m⁻³ proline and 100mol m⁻³ sucrose, while mature cells towards the end of light periods contained 130 mol m proline and 20 mol m⁻³ sucrose. The changes in proline and sucrose which occurred in synchronized cultures were due mainly to light/dark conditions and to a much lesser extent to different stages of cell development. The proportion of proline to sucrose in daughter cells collected from non-synchronized cultures in continuous light was not different from the proportion in heterogeneous populations of cells.
Results are discussed in relation to the accumulations of two, rather than one, endogenous osmotic solute and to growth reductions of C. emersonii exposed to high external osmotic pressures.     

Umasking large and persistent reductions in proliferation rate of aging cells

Summary We have reported that nontransformed sublines of NIH 3T3 cells that are incubated under the growth constraint of confluence for 10 d or longer exhibit heritable reductions of growth rate upon serial subculture at low density, which simulate the effects of aging in vivo on cell growth. There is also a marked increase in the likelihood of neoplastic transformation. After switching to a new batch of calf serum (CS), we found the reduced growth rate was no longer produced within the previously established timeframe. However, substitution of fetal bovine serum (FBS) for CS during the period of recovery from confluence or the following tests of growth rate resulted in profound inhibition of growth in cells serially subcultured from confluent cultures. In some cases, fewer than one in a thousand cells from subcultures of confluent cultures formed colonies in FBS although they cloned at relatively high efficiency in CS. The reduced growth in FBS was retained in the postconfluent subcultures after many generations of multiplication at low density in CS. Generally, similar results with individual variations were obtained with three other batches of FBS. The numbers of cells per 3-d colony initiated from subcultures of confluent cultures were lower than those of control cultures that had never been confluent. Supplementation of FBS-containing medium with CS fully restored the growth of the postconfluent subcultures to the rate in CS medium, indicating that there is a deficiency of growth factor(s) in FBS rather than the presence of an inhibitor. The results show that prolonged incubation at confluence induces a populationwide heritable increase in requirement for growth factor(s) in short supply in FBS. Because clonal studies have shown that the reduction in growth rate is irreversible and varies in degree from clone to clone, we propose it arises from damage to DNA at any of many different genetic loci or from chromosome aberrations. Such genetic damage is also consistent with the increased tendency for neoplastic transformation in subcultures from the long-term confluent cultures.     

Development of the Kranz structure during leaf growth in C4 Euphorbia maculate

The development of the Kranz structure was investigated in leaves of C₄ Euphorbia maculata using electron microscopy. Four leaf stages, i.e., primordial, immature, young, and mature, were examined, based on the photosynthetic tissue that surrounded the veins. The examination revealed how cells differentiated into distinct bundle sheath cells (BSCs) and mesophyll cells (MCs). Specialization of the BSCs was invariably associated with the development of the veins as well as the MCs. Precursors for BSC and MC were recognizable fairly early, at the immature stage, according to their position and differential enlargement Once these precursors were delimited from the procambial area, differentiation into each cell type occurred synchronously, in a coordinated manner. All cells enlarged as they were displaced from the Kranz precursor area, but the BSC precursors were initially larger and remained relatively larger than the other cell types throughout leaf development The developmental changes sharply distinguished BSCs from the adjacent MCs at the onset of Kranz formation and continued until maturity. Chloroplast enlargement also occurred during cell displacement, but the rate of enlargement was greater in BSCs, resulting in larger chloroplasts at later stages. However, no significant structural differences were detected among the chloroplasts of BSC and MC in the early stages. Most of the specialized features appeared at the young-leaf stage; structural dimorphism became prominent at the later stages. This enhanced development of the BSC chloroplasts was correlated with asymmetric distribution of cellular components. In addition, the BSC formed thin primary pit fields with numerous plasmodesmata. Peripheral reticulum was present, but generally was not conspicuous. We also discuss the characteristics of leaf anatomy and ultrastructure inE. maculata as they relate to the C₄ photosynthetic pathway.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.     

The development of a kitten lung cell strain and its chromosomes

Summary The development of a line of epithelial cells derived from lung tissue of a 4-week old kitten (KL strain) with evidence regarding its chromosomal changes in vitro is described. The outgrowing cells from fragments in primary cultures in Eagle's medium plus 10% horse serum were scraped with a rubber policeman and dispersed with a syringe fitted with a No. 15 needle. The cell suspension was transferred into a T 30 flask. The floor of the T 30 flask was covered with avian plasma clot which was allowed to set for 10 minutes before adding the cell suspension. The appearance of the cells was epithelial-like at all stages of cultivation. The most frequent chromosome number in 6-day primary culture preparations was 38 (68%). Counts made from cells in the 4th, 9th and 33rd subcultures (64th, 83rd and 165th days from the date of primary culture) showed a spread of the chromosome numbers. In the latest observation, the 84th subculture (411th day), the most frequent chromosome numbers were 90 (22%) and 92 (26%). In addition, the mitotic activity of cells in the strain cultures was observed by phase microscopy.Tobacco Industry Research Committee Fellow.     

A diploid rat liver cell culture

Summary Chronic exposure of a cloned rat hepatocyte culture (RL-PR-C) to a subtoxic, sublethal dose of aflatoxin B₁ resulted in malignant transformation. Continuous exposure to aflatoxin B₁ caused increasing tumorigenic potential as tested by back injection into isogenic animals. Control cultures exhibited spontaneous transformation, although approximately 20 passages beyond the chemically induced event. Neither aflatoxin-treated nor control cultures exhibited cytopathological morphology, formation of cell foci, growth in soft agar, or irregular fibroblast-like growth patterns that could be specifically related to the onset of tumorigenic potential. In general, those parameters commonly used to monitor fibroblast cultures for transformation in vitro were not applicable for assessing the tumorigenic potential of these epithelial cells. Karyotypic analyses revealed no specific chromosomal aberrations associated with aflatoxin treatment; however, chromosomal instability was a property of the tumorigenic cell populations. Injection of both aflatoxin-treated and control cultures at passage 56 resulted in tumors indicative of both carcinoma and sarcoma indicating to us the multipotency of these epithelial cells transformed in vitro.     

The influence of the genotype on the process of ageing of chick lens cells in vitro

We reported previously that changes in crystallin expression in differentiating long-term primary cultures of lens cells from five different chick genotypes are similar to those which occur in vivo between hatching and the 8-week-old adult. These changes followed a similar program in all genotypes but occurred more rapidly in cells from the fast-growing than from the slow-growing genotypes. The present study examines ageing changes in lens cell populations from the same five genotypes, over a 4-6 month period, using long-term serial subcultures. The capacity for lentoid differentiation was progressively lost, but the rate of loss was inversely related to the intrinsic growth rate of the cells of these genotypes, occurring at the first passage in the slowest-growing strain, while fifth passage cells of the fastest-growing strain still retained some lentoid-forming capacity. The rate of loss of crystallin expression was also inversely related to the genetic growth rate, but the sequence of changes appears to be nonrandom, since it was broadly similar in all genotypes, starting with a preferential loss of delta-crystallin, as occurs in vivo; although alpha- and beta-crystallins were undetectable in late dedifferentiated cultures, the capacity of the cells for their synthesis was still present. Cultures from both fast-growing genotypes eventually showed senescence, but those from all three slow-growing genotypes underwent transformation. The major cell component in late cultures of all genotypes was actin.     

A virus-producing cell line developed by transformation of human parapharyngeal cells with SV40.

Normal cultures of epithelial appearance were initiated by trypsinization of a surgically resected, histologically normal branchial cyst. Cellular morphology was consistent with derivation from the stratified squamous epithelium of the cyst or from vascular endothelium, although electron micrographs of the cultured cells failed to show any junctional complexes. Infection with SV40 produced transformants which were also epithelioid in appearance. These grew vigorously for 22 to 50 population doublings (about 23 to 32 subcultures, depending upon regimen) and then became quiescent. During this evolution, virus was detectable at all stages by both direct isolation (cell extracts) and cocultivation with permissive cells. In two sublines in which selection for rapidly growing cell types occureed, virus was detected only by cocultivation. The work confirms that of others in the finding that normal human epithelial cells are susceptible to transformation by oncogenic viruses, but are apparently less responsive than are fibroblasts to such transforming agents. It also suggests that subcultivation techniques that maintain the populations of transformed cells at low density tend to select against cell strains that are continous producers of infectious virus.     

Thyrotropin-induced formation of functional follicles in primary cultures of ovine thyroid cells

In primary cultures of ovine thyroid cells, TSH induced the expression of several differentiated functions including the formation of follicles, and synthesis and storage of iodinated thyroglobulin in the follicular lumen. In the present report, these follicles were shown by transmission (TEM) and scanning electron microscopy (SEM) to be intact, comprised of two or more cells and to possess numerous microvilli on the inner cell membranes facing the follicular lumen. The TSH-induced formation of follicles was reversible and dynamic, with the kinetics of formation preceding that of iodination. The follicles were further demonstrated to be functional in terms of thyroglobulin storage and iodination.     

Dynamics of morphological and anatomical changes in leaf tissues of an interspecific hybrid of oil palm during acquisition and development of somatic embryogenesis

Oil palm is an economically important plant species due to its high oil production per unit area. Large-scale clonal propagation of the species’s elite specimens is only possible through somatic embryogenesis, although methodology is partially still unknown and insufficiently understood. Current study characterizes in morphological and anatomical terms the acquisition and development stages of somatic embryogenesis of the oil palm’s immature leaves. The respective embryogenic stages were analyzed and characterized: immature leaves (initial explants); leaves with calli formation; leaves which failed to respond to calli formation; leaves with formation of root structures; primary calli; primary calli with differentiation of embryogenic calli; embryogenic calli; pro-embryogenic calli; calli with differentiated somatic embryos; somatic embryos at globular and torpedo stage; and mature fruit zygotic embryos. Cell masses emerged after approximately 60 days of cultivation through the proliferation of cells associated to initial explants´ vascular bundles. Consequently, the formation of two different types of calli was identified, namely, primary and embryogenic, respectively consisting partially and completely of meristematic cell clusters. After 420 days of cultivation, the propagules formed somatic embryos with no connection to source tissues, initially composed (globular stage) of a very characteristic ground meristem and protoderm. After 480 days of cultivation, as the cultures matured (torpedo stage), procambial strands, a structural characteristic also observed in mature zygotic embryos, were reported. The results provide an in-depth understanding of somatic embryogenesis of immature leaves of oil palm. Further, current analysis develops morphological markers at different stages of development obtained during the process.     

A virus-producing cell line developed by transformation of human parapharyngeal cells with SV40

Summary Normal cultures of epithelial appearance were initiated by trypsinization of a surgically resected, histologically normal branchial cyst. Cellular morphology was consistent with derivation from the stratified squamous epithelium of the cyst or from vascular endothelium, although electron micrographs of the cultured cells failed to show any junctional complexes. Infection with SV40 produced transformants which were also epithelioid in appearance. These grew vigorously for 22 to 50 population doublings (about 23 to 32 subcultures, depending upon regimen) and then became quiescent. During this evolution, virus was detectable at all stages by both direct isolation (cell extracts) and cocultivation with permissive cells. In two sublines in which selection for rapidly growing cell types occurred, virus was detected only by cocultivation. The work confirms that of others in the finding that normal human epithelial cells are susceptible to transformation by oncogenic viruses, but are apparently less responsive than are fibroblasts to such transforming agents. It also suggests that subcultivation techniques that maintain the populations of transformed cells at low density tend to select against cell strains that are continuous producers of infectious virus. This study was supported by Grant CA 13494 from the National Cancer Institute.