Interface-mediated death of unconditioned Tetrahymena cells: effect of the medium composition

ABSTRACT We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic. The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68. Here, we report that cell death can also be caused by the medium. The specific effects of several medium constituents were tested in the presence and absence of an interface. Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca²⁺ (68 μM) and Mg²⁺ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death. The effect of the amino acids and the salt ions Ca²⁺ and Mg²⁺ can be abolished by the addition of insulin (10^-6 M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions. On the basis of our findings, a new recipe for a chemically-defined medium has been formulated. Single cells can grow in this medium in the presence of medium-air interface without any supplements.     

Effects of steroid hormones on five functional parameters of Tetrahymena: evolutionary conclusions

The unicellular Tetrahymena pyriformis was studied for chemotaxis, chemotactic selection, phagocytosis, growth and body shape changes in the presence of water soluble (beta-cyclodextrin-coupled) steroid hormones (testosterone, estradiol, progesterone, hydrocortisone and dexamethasone). Testosterone was chemoattractant over a wide range of concentrations, while progesterone and dexamethasone were active only at one concentration (10(-5) and 10(-6) mg ml(-1) respectively) and were either neutral or repellent at other concentrations. Hydrocortisone and estradiol were unambiguously chemorepellent. Chemotactic selection enhanced the effect of testosterone and estradiol, while in the case of hydrocortisone the action was reversed. The other parameters were mildly influenced by the steroid hormones. The results call attention to the fine molecular recognition capacity of Tetrahymena and to the possible rapid effects of steroid hormones at membrane receptors at a very low evolutionary eukaryotic level.     

Modulation of tumor cell susceptibility to cytokine-induced cell death by hormones, growth factors, and cell density.

The mechanisms controlling "spontaneous" cellular death rates in normal and tumorigenic tissues are largely unknown. An important parameter in this respect is the susceptibility of the target cell to induction of the lytic pathway by appropriate signals. In the present article it is demonstrated in a serum-free in vitro system that the susceptibility of human tumor cells (TC) to induction of lysis by cytokine signals generated during interaction of TC with elutriated human monocytes (MO) is a highly dynamic parameter subject to modulation by hormones, growth factors, and tumor cell density. It was found that growth stimulatory signals such as insulin, and especially epidermal growth factor (EGF), increase lytic susceptibility, whereas hydrocortisone, which does not exert significant growth modulatory effects in these examples, protects TC against the induction of lysis. Increasing TC density above confluence dramatically enhances lytic susceptibility, suggesting interactions between TC to be involved in the induction of their death. In conjunction with previous data demonstrating the insusceptibility of TC, which are forced out of the cell cycle into the quiescent state (G0), the hypothesis is put forward that growth stimulatory factors increase a TC's lytic susceptibility by preventing its transit from G1 to G0 in response to growth inhibitory signals generated during MO/TC interaction. The data support the concept that TC susceptibility to the induction of cell death is a consequence of simultaneously activated growth stimulatory and growth inhibitory signalling pathways.     

Effect of hormones and growth factors on the proliferation of adult cricket neural progenitor cells in vitro

In the adult cricket brain, a cluster of neuroblasts produces new interneurons that integrate into the mushroom body (MB), the main associative structure for multisensory information of the insect brain. In previous study we showed the antagonist role of the two morphogenetic hormones, juvenile hormone (JH) and ecdysone, on the regulation of adult MB neurogenesis in vivo. In order to examine whether these hormones act directly on neural progenitor cells, we developed an organotypic culture of MB cortices. Cell proliferation was assessed by 5-bromo, 2'-deoxyuridine (BrdU) incorporation. We showed that JH increased mushroom body neuroblast (MBNb) proliferation, confirming the mitogenic effect of JH observed in vivo. By contrast, ecdysone did not affect the amount of BrdU-labeled nuclei, suggesting that the inhibitory effect observed in vivo probably proceeded from an indirect pathway. We then examined the role of growth factors known to stimulate neural stem cell/progenitor cell proliferation in vertebrates. As shown by calcium imaging, MBNb only expressed functional receptors for insulin whereas mature interneurons responded to IGF-I and bFGF. Both insulin (10 microg/ml) and IGF-I (10 ng/ml) enhanced MB progenitor cell proliferation in culture, although the insulin effect was more pronounced. This effect was abolished when an inhibitor of polyamine biosynthesis was present in the medium, suggesting a link between polyamines and the insulin signaling pathway. By contrast, bFGF (20-200 ng/ml) failed to stimulate MBNb proliferation. Our results point to conserved and divergent mechanisms between vertebrates and invertebrates in the regulation of adult neural progenitor cell proliferation.     

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.     

Effect of hormones and related compounds on the multiplication of Tetrahymena.

The vertebrate hormone triiodothyronine, the commonly occurring animal and plant hormone serotonin, and the plant alkaloid gramine, chemically related to the latter, stimulated the multiplication of Tetrahymena. Neither epinephrine nor gibberellin had such an effect. These experimental observations support the previous suggestion that unicellular animals possess structures capable of responding to certain hormones, which do not seem to elicit specific responses of the unicellular animal, but rather act through the activation of certain general functions.     

Effect of hormones and growth factors on in vitro development of sheep preantral follicles

The present investigation attempts to improve the frequency of in vitro maturation of oocytes by culturing small (150–250 μm) and large (>250–400 μm) preantral follicles (PFs) of sheep for 6 days in various combinations/sequences of thyroxin (T₄), FSH, LH, transforming growth factor alpha (TGF-), epidermal growth factor (EGF) and heat-treated foetal calf serum (FCS). Bicarbonate-buffered tissue culture medium 199, supplemented with 50 μg ml⁻¹ gentamicin sulphate, served as the control medium. In vitro development was initially assessed by the proportion of PFs exhibiting an increase in size, mean increase in diameter and antrum formation. Nuclear maturation to the metaphase II stage of the oocytes isolated from cultured PFs, after an additional 24-h in vitro maturation, indicated success. A total of 15% of oocytes from small PFs and 55% from large PFs, cultured in T₄ + FSH, matured to metaphase II. Culture of PFs in other combinations/sequences of hormones and growth factors, including the control medium, supported a significantly lower proportion of oocytes maturing to metaphase II stage. It is concluded that 6-day in vitro culture of sheep PFs in thyroxin and FSH greatly improves the frequency of oocyte maturation to metaphase II stage.     

Growth factors and hormones which affect survival, growth, and differentiation of the MCF-7 stem cells and their descendants

The human breast tumor cell line was separated by Percoll density gradient centrifugation into six different subpopulations, A to F, one of which (E) appears to contain the stem cells on the basis of several criteria (M. Resnicoff et al. 1987, Proc. Natl. Acad. Sci. USA 84, 7295. We now analyzed the response of the isolated subpopulations to insulin, thrombin, PGF2 alpha, estradiol, and 13-cis-retinal. We demonstrate that the first two growth factors stimulate [3H]thymidine incorporation in the more differentiated subpopulations (D and F), while PGF2 alpha has mitogenic activity in subpopulations C and D. In the absence of any added growth factor, estradiol has the extreme and transient capacity of allowing the stem cell to detach from the tissue culture dish and to grow in suspension as multicellular aggregates (MCF-7/SE cells). 13-cis-Retinal acts as a negative modulator of differentiation and protects the cells from the inhibitory and differentiation activity of Na-butyrate.     

The effect of hormones and growth factors on the proliferation of cultured mouse granulosa cells

A method of obtaining granulosa cell culture reacting to the action of gonadotropins and growth factors is described. The efficiency of cell cloning is enhanced under influence of insulin, epidermal growth factor (EGF) and fibroblast growth factors (FGF). Stimulation of proliferation by the latter two factors is seen in the medium with the low serum concentration. Luteinizing and follicle-stimulating inhibit the cell growth in culture. The role of growth factors and gonadotropins in regulation of granulosa proliferation in mammalian ovarian follicles is discussed.     

Hormonal interactions in Tetrahymena: effect of hormones on levels of epidermal growth factor (EGF)

Tetrahymena pyriformiswas treated with insulin, histamine or serotonin for 30 min and epidermal growth factor (EGF) level was studied inside the cells using specific antibodies and flow cytometry as well as confocal microscopy. The EGF concentration was highly significantly elevated after hormone treatment, regardless of the hormone used. EGF was localized mainly in the cortical region (mucocysts) and in vesicles and this localization did not differ in untreated and treated cells. The results call attention to the possibility of interactions between hormones at unicellular level and points to the presence of a hormonal system in Tetrahymena that includes receptors, hormones and signal transduction pathways as well as hormonal interactions. This could be the basis of further evolution to the hormonal system of multicellulars.     

Influence of physical factors on the growth of insect cells in vitro

Summary The tolerances of a cell line (IMC-HZ-1) from a moth,Heliothis zea, for the monovalent cations Na⁺ and K⁺ were defined. Cells shifted to media containing more than 70mm of K⁺ showed decreased growth rates. No evidence was obtained for Na⁺ toxicity. The osmotic pressure tolerances were influenced by the K⁺ concentration of the medium. The richer the medium was in K⁺, the narrower was the spectrum of osmotic pressure tolerance. Once the limit of K⁺ tolerance was exceeded, the rate of decline of growth was linear with respect to further increases in K⁺. This rate of decline was independent of osmotic pressure. The initial responses of cells during one subculture (2 to 4 population doublings) in media differing from the standard medium (used to maintain the cell line) were not reliable indicators of the growth potential of the cells. Continued subculture in such media resulted in an upward trend in population growth rates in most cases. This investigation was supported by U. S. Public Health Service Research Grant no. AI 09914 from the National Institute of Allergy and Infectious Diseases. This is Paper no. 8637, Scientific Journal Series, Minnesota Agricultural Experiment Station. The material is part of the dissertation of T. J. K. presented for the Ph.D. degree at the University of Minnesota.