Growth-promoting effects of progesterone in a human endometrial cancer cell line (Ishikawa-Var I).

Significant growth responses to progesterone of human endometrial adenocarcinoma cells (Ishikawa-Var I) were observed under in vitro culture conditions. Progesterone affected both the rate of exponential proliferation and cell population densities after the exponential phase. In the presence of the hormone, the doubling time of exponentially proliferating cells was reduced from 44 to 35.6 h and cell densities were increased by as much as 2-3 times over those of controls during approx. 2 weeks in culture. The effects of progesterone on cell population growth were dose dependent. Estradiol (10(-8) M) and testosterone (10(-6) M) did not affect cell densities and the effects of dexamethasone (10(-6) M) were small. In contrast, both progesterone and estradiol stimulated colony formation under anchorage-independent conditions in soft agar. These results suggest the possibility that growth of sensitive cell clones in endometrial tumors could be enhanced in some patients during adjuvant progestin therapy.     

Cell differentiation and colony alteration of an edible terrestrial cyanobacteriumNostoc flagelliforme,in liquid suspension cultures

Morphological characteristics of an edible terrestrial cyanobacterium Nostoc flagelliforme in liquid suspension cultures under photoautotrophic conditions are presented. Different cell forms alternated in a regular manner during the experimentation period (30 d). N. flagelliforme exhibited a very complex life cycle in terms of colony morphology, including mainly 4 different colony morphological forms, viz. hormogonia, filaments, seriate colonies and aseriate colonies. Under laboratory conditions it formed spherical colonies on solid media but not threadlike colonies as it did under natural conditions. The overall life span of the alga was not altered by the existence of different nitrogen sources in the media despite the depression of some cell forms or colony morphologies. Compared with growth on the medium with urea and ammonium as nitrogen sources, the alga on standard medium had a short period of hormogonia and aseriate colony, suggesting that both ammonium and urea could stimulate the formation of hormogonia, at the same time inhibiting the formation of heterocystous cells. The new information on the growth and morphology of N. flagelliforme could be potentially used for the scale-up or field cultivation.     

Opposing effects of 12-o-tetradecanoylphorbol 13-acetate on human myeloid and lymphoid cell proliferation

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on human hematopoietic cells has been investigated. It was found that 1–10 ng/ml of TPA totally abrogated erythroid and granulocytic colony growth and, simultaneously in the presence of PHA, stimulated T-lymphocyte colony formation. TPA concentrations insufficient to inhibit myeloid colony growth also failed to stimulate lymphocyte colony formation. Optimal culture conditions for the growth of these colonies required the presence of TPA, PHA, and leukocyte-conditioned medium in the cultures. Cells within the colonies were 80–90% E-rosette positive and by monoclonal antibody characterization contained 45–66% OKT3-positive cells. Colony-forming cells were found in both E-rosette-positive and-negative fractions. Although by cell surface marker characterization the cells within the colonies had properties of T-cells, the exact relationship of cells forming colonies under these conditions to those detected in other T-cell colony assays remain to be determined.     

Fibronectin Fibrils and Growth Factors Stimulate Anchorage-Independent Growth of a Murine Mammary Carcinoma

Stromal cells are important regulators of mammary carcinoma growth and metastasis. We have previously shown that a 3T3-L1 adipocyte cell line secretes hepatocyte growth factor (HGF), which stimulates proliferation of a murine mammary carcinoma (SP1) in monolayer cultures (DNA Cell Biol.13, 1189–1897, 1994). We now examine the role of growth factors and the extracellular matrix protein fibronectin in stimulation of anchorage-independent growth of SP1 cells. Purified transforming growth factor-β (TGF-β) stimulated significant colony growth in soft agar cultures, whereas HGF had a lesser effect. Analysis by confocal microscopy revealed that carcinoma cell colonies contained extracellular microfibrils composed of fibronectin. Partial depletion of fibronectin from 7% FBS/agar cultures reduced the number of colonies; colony growth could be recovered by adding back exogenous fibronectin. Addition of the 70-kDa N-terminal fragment of fibronectin, which inhibits fibronectin fibril formation, reduced growth of SP1 cell colonies, but an 85-kDa fragment containing the cell binding domain did not inhibit colony growth. These findings indicate that deposition of extracellular fibronectin fibrils is necessary, but not sufficient, for anchorage-independent growth of SP1 mammary carcinoma cells; growth factors are also required. SP1 cells had less fibronectin mRNA and secreted less fibronectin protein under anchorage-independent conditions than under anchorage-dependent conditions, as determined by Northern blotting and immunoprecipitation analysis. Thus, both growth factors (HGF and TGF-β) and fibronectin may be important regulators of paracrine stimulation by stromal cells of anchorage-independent growth of mammary carcinoma cells.     

Identifying the factors and signal pathways necessary for anchorage-independent growth of Ha-ras oncogene-transformed NIH/3T3 cells

Ha-ras(Val 12) overexpression was positively correlated with colony formation by NIH/3T3 derivative "2-12" cells harboring an inducible Ha-ras(Val 12) transgene. The ras-farnesylation inhibitor, Lovastatin, completely suppressed colony formation at higher dosages. However, Ha-ras oncogene overexpression alone could not stimulate colony formation under serum-deprived conditions, suggesting that ras is required but not sufficient for supporting colony formation. Substituting cow colostrum (AC-2) for serum did not result in colony formation from 2-12 cells in soft agar, suggesting the colostrum lacked or contained insufficient amounts of factors that stimulate colony formation. Supplementation of AC-2-containing medium with growth factors, such as insulin-like growth factor-1 (IGF-1), partially restored the capability of anchorage-independent cell growth induced by Ha-ras overexpression. Consistently, antibodies specific for IGF-1 receptors only partially blocked colony formation from 2-12 cells. The data indicate that multiple factors, including IGF-1, are required for Ha-ras-dependent colony formation. Signal transduction studies revealed that, under Ha-ras overexpression conditions, IGF-1 utilizes phosphatidyl inositol 3-kinase and NF-kappaB to transduce colony formation-related signaling.     

浮游动物诱发藻类群体的形成

从研究蓝藻水华形成机理的需要出发,综述了浮游动物的牧食压力对藻类群体形成的诱发作用。指出诱发藻类群体形成的化合物来自牧食性浮游动物对藻类的有效牧食,是藻类群体形成的重要原因之一,而这些诱发性的化合物并不是有关生物体的组成成分,是种间相互作用的结果。藻类群体的形成方式有源于一个母细胞的分裂和业已存在的单细胞的聚合两种方式,栅藻的诱发性群体可能是来自一个母细胞的分裂,而在其它藻类的诱发性群体形成如铜绿微囊藻则可能是业已存在的单细胞的聚合。由于藻类形成群体后能显著降低浮游动物对其牧食速率,因此,这种诱发性群体形成的现象,可以解释为藻类对变化的牧食压力的一种有效的反牧食防御策略,也是两者协同进化的结果。浮游动物对藻类群体形成的重要作用,在研究模拟蓝藻群体及水华形成值得借鉴应用。作者还提出推测,水华蓝藻的群体形成,可能就是在富营养化条件下藻类快速生长,加上浮游动物的牧食压力共同作用下联合驱动的结果,而这种群体形成很可能在积累到一定程度后,结合特定的气象水文等理化因子,就会聚集于水表“爆发”出肉眼可见的水华。因此,开展浮游动物牧食作用对水华蓝藻早期群体形成诱发效应的研究不仅能加深对水华形成的全面认识,而且对于进一步认识藻类的诱发性反牧食防御适应机制、揭示生态系统中生物之间的复杂关系也具有重要的理论意义。     

Effects of five recombinant hematopoietic growth factors on enriched human erythroid progenitors in serum-replaced cultures

Erythroid progenitors from normal human marrow were purified by a two-step immune panning method permitting both the enrichment of erythroid progenitors (plating efficiency up to 10%) and the separation of CFU-E from BFU-E. The purified erythroid progenitors were grown in serum-replaced conditions; in some experiments at an average of one cell per well. Human recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL3), erythroid potentiating activity (EPA), and human erythropoietin (Epo) either recombinant or homogenous native were tested for their effect on CFU-E growth. Epo was an absolute requirement for CFU-E growth and was sufficient to obtain colony formation at the unicellular level whereas GM-CSF and IL3 did not further increase the plating efficiency. EPA potentiated the effect of Epo on this progenitor only in experiments performed at unicellular level. Human recombinant GM-CSF, IL3, Interleukin 1 alpha (IL1 alpha), and Epo were subsequently tested for their ability to promote BFU-E growth. GM-CSF and IL3 supported the growth of erythroid bursts in the presence of Epo, even at the unicellular level. However, IL3 promoted a higher number of bursts than GM-CSF under all conditions tested. These two growth factors have no or very small additive effects when tested in combination. IL1 alpha added to Epo alone had no effect on the growth of BFU-E whereas it potentiated the combined action of IL3 and GM-CSF on the primitive BFU-E. In conclusion, this study confirms at the unicellular level and under serum-free conditions that erythroid progenitors are regulated by multipotential growth factors in early phases of erythropoiesis and become sensitive only to Epo in later phases of differentiation.     

Influence of individual cell motility on the 2D front roughness dynamics of tumour cell colonies

The dynamics of in situ 2D HeLa cell quasi-linear and quasi-radial colony fronts in a standard culture medium is investigated. For quasi-radial colonies, as the cell population increased, a kinetic transition from an exponential to a constant front average velocity regime was observed. Special attention was paid to individual cell motility evolution under constant average colony front velocity looking for its impact on the dynamics of the 2D colony front roughness. From the directionalities and velocity components of cell trajectories in colonies with different cell populations, the influence of both local cell density and cell crowding effects on individual cell motility was determined. The average dynamic behaviour of individual cells in the colony and its dependence on both local spatio-temporal heterogeneities and growth geometry suggested that cell motion undergoes under a concerted cell migration mechanism, in which both a limiting random walk-like and a limiting ballistic-like contribution were involved. These results were interesting to infer how biased cell trajectories influenced both the 2D colony spreading dynamics and the front roughness characteristics by local biased contributions to individual cell motion. These data are consistent with previous experimental and theoretical cell colony spreading data and provide additional evidence of the validity of the Kardar-Parisi-Zhang equation, within a certain range of time and colony front size, for describing the dynamics of 2D colony front roughness.     

转录因子ACZ影响冠突曲霉色素及产孢数量的研究

本实验室前期在高渗条件下筛选得到8个转录因子,其中ACZ (SI65-00458)基因表达量最高,推测ACZ基因在冠突曲霉中参与响应渗透压及调控孢子的产生。因而本研究利用同源重组原理,构建了ACZ基因敲除载体,通过农杆菌介导转化,筛选获得了ACZ敲除菌株,将敲除株分生孢子液接种在含不同浓度NaCl的MYA培养基上,以野生型冠突曲霉为对照,观察敲除株与野生型菌株的区别。研究发现:培养基中不加NaCl时色素有明显变化,野生型为黄色,敲除株为褐黄色;在低渗透压下,菌落边缘不规则;在高渗透压下,敲除株产生的分生孢子数量为野生型的4倍多;无论是在27℃还是在37℃培养,敲除株在低渗及高渗条件下,菌落直径都较野生型小,菌丝较稀疏。表明敲除ACZ基因会影响冠突曲霉菌丝生长、色素的合成及孢子的产生。     

Hapten-specific murine colony-forming B cells. III. Delineation of functional B cell subpopulations grown under different conditions

The influence of anti-immunoglobulin M (IgM) and anti-IgD on the ability of fluorescein (FL)-specific B cells to proliferate in a colony-forming assay, and of their progeny to further differentiate in response to different FL-antigens was studied. Splenic FL-specific B cells were purified on FL-gelatin plates and were then cultured in semisolid agar in the presence or absence of anti-mu, and anti-delta, or both. Experiments were performed under conditions of either sheep red blood cell (SRBC)-potentiated or SRBC + lipopolysaccharide (LPS)-potentiated colony growth. The resulting colonies were then tested in secondary filler cell-dependent microcultures for the ability to be triggered by different classes of FL-antigens to yield plaque-forming cells (PFC). Anti-delta inhibited 47% of colony growth under both agar culture conditions. Anti-mu inhibited 55% of colony growth in SRBC + LPS-potentiated agar cultures, and inhibited 72% if only SRBC was present. If anti-delta and anti-mu were added together, inhibition was nearly additive. When anti-Ig-treated colonies were tested for PFC responses against FL-polymerized flagellin (POL), both normal and anti-delta resistant colonies, grown under both agar culture conditions, responded well. Anti-mu resistant colonies were refractory to FL-POL challenge. Only normal or anti-delta resistant colonies grown in SRBC + LPS agar cultures were able to respond well to FL-Ficoll, whereas even normal SRBC-potentiated colonies responded poorly. All except SRBC-potentiated, anti-mu treated colonies were able to respond to nonspecific signals present in cultures containing FL-KLH and activated T cell help. These data suggest that addition of specific anti-Ig antibodies, and variation of agar culture conditions, can select for B cell subpopulations responsive only to certain types of antigens.     

Genomic instability in Rhizobium phaseoli.

Experience from different laboratories indicates that Rhizobium strains can generate variability in regard to some phenotypic characteristics such as colony morphology or symbiotic properties. On the other hand, several reports suggest that under certain stress conditions or genetic manipulations Rhizobium cells can present genomic rearrangements. In search of frequent genomic rearrangements, we analyzed three Rhizobium strains under laboratory conditions that are not considered to cause stress in bacterial populations. DNAs from direct descendants of a single cell were analyzed in regard to the hybridization patterns obtained, using as probes different recombinant plasmids or cosmids; while most of the probes utilized did not show differences in the hybridization patterns, some of them revealed the occurrence of frequent genomic rearrangements. The implications and possible biological significance of these observations are discussed.     

Heterogeneous accessory cell requirement for human peripheral blood T lymphocyte activation by PHA into IL-2-responsive colony-forming cells

Mitogen-driven T cell proliferation in liquid culture requires accessory cells that cooperate in interleukin 2 production. We have investigated the accessory cell requirement for human lymphocyte colony formation under PHA stimulation. Semisolid medium limits cell-to-cell contact emphasizing the role of cooperating cells both in growth factor production and in triggering events. Culturing at high T cell density demonstrates that accessory cells can be substituted for colony formation by exogenous IL-2. Culturing at low T cell density in the presence of IL-2 also demonstrates that accessory cells are required for activation of a subset of progenitors into IL-2 responsive colony-forming cells. Consequently, T colony progenitors, contained in the E-rosetting cell fraction of peripheral blood, are heterogeneous in their triggering signals: a minor subset is directly inducible by PHA, and a major subset is inducible by PHA in the presence of accessory cells. We found that monocytes and some leukemic B cells support effective accessory function in both colony growth factor production and colony progenitor sensitization.     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

In vivo extraction of Arabidopsis cell turgor pressure using nanoindentation in conjunction with finite element modeling

Turgor pressure in plant cells is involved in many important processes. Stable and normal turgor pressure is required for healthy growth of a plant, and changes in turgor pressure are indicative of changes taking place within the plant tissue. The ability to quantify the turgor pressure of plant cells in vivo would provide opportunities to understand better the process of pressure regulation within plants, especially when plant stress is considered, and to understand the role of turgor pressure in cellular signaling. Current experimental methods do not separate the influence of the turgor pressure from the effects associated with deformation of the cell wall when estimates of turgor pressure are made. In this paper, nanoindentation measurements are combined with finite element simulations to determine the turgor pressure of cells in vivo while explicitly separating the cell‐wall properties from the turgor pressure effects. Quasi‐static cyclic tests with variable depth form the basis of the measurements, while relaxation tests at low depth are used to determine the viscoelastic material properties of the cell wall. Turgor pressure is quantified using measurements on Arabidopsis thaliana under three pressure states (control, turgid and plasmolyzed) and at various stages of plant development. These measurements are performed on cells in vivo without causing damage to the cells, such that pressure changes may be studied for a variety of conditions to provide new insights into the biological response to plant stress conditions.     

An osmotic model of the growing pollen tube

Pollen tube growth is central to the sexual reproduction of plants and is a longstanding model for cellular tip growth. For rapid tip growth, cell wall deposition and hardening must balance the rate of osmotic water uptake, and this involves the control of turgor pressure. Pressure contributes directly to both the driving force for water entry and tip expansion causing thinning of wall material. Understanding tip growth requires an analysis of the coordination of these processes and their regulation. Here we develop a quantitative physiological model which includes water entry by osmosis, the incorporation of cell wall material and the spreading of that material as a film at the tip. Parameters of the model have been determined from the literature and from measurements, by light, confocal and electron microscopy, together with results from experiments made on dye entry and plasmolysis in Lilium longiflorum. The model yields values of variables such as osmotic and turgor pressure, growth rates and wall thickness. The model and its predictive capacity were tested by comparing programmed simulations with experimental observations following perturbations of the growth medium. The model explains the role of turgor pressure and its observed constancy during oscillations; the stability of wall thickness under different conditions, without which the cell would burst; and some surprising properties such as the need for restricting osmotic permeability to a constant area near the tip, which was experimentally confirmed. To achieve both constancy of pressure and wall thickness under the range of conditions observed in steady-state growth the model reveals the need for a sensor that detects the driving potential for water entry and controls the deposition rate of wall material at the tip.     

Characteristics and differential T-cell dependency of human B-cell colony precursors

Studies were performed to characterize the human peripheral blood non-T cells forming colonies in semisolid cultures stimulated with Staph protein A (SpA). Negative selection experiments revealed that colony precursors largely consisted of cells bearing Fc receptors, complement receptors (CR), surface immunoglobulin (sIg), and Ia-like antigens. Most colony precursors expressed sIgM and sIgD, but not sIgG. Also, colony-forming cells were shown to be distinct from non-T cells proliferating in SpA-stimulated liquid cultures as evidenced by the greater sensitivity of colony precursors to anti-K,λ, or -Ia plus complement depletion. Two distinct categories of colony-forming cells could be distinguished by the expression of CR. CR-positive cells were responsible for greater than 85% of the colonies formed in the absence of optimal T cell numbers. Although under identical conditions CR^? cells demonstrated minimal colony growth, the addition of optimal T cell numbers significantly augmented colony responses. Thus, colony precursors express surface markers characteristic of B cells relatively advanced in the developmental pathway. However, less advanced cells are capable of colony growth in the presence of optimal T cell numbers.     

Colony,hanging drop,and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines

Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.     

Pattern formation of the coenobial algae Pediastrum biwae Negoro

It is suggested that flat colony patterns of the coenobial green algae Pediastrum biwae Negoro can be determined only by some properties of the zoospores without any other control system as a whole. These suggestions are made through observations of colony patterns and colony formation during asexual reproduction and digital electronic computer simulation. The zoospore can be regarded as a sphere which has two C-sites (presumptive sites for connection) and one H-site (presumptive site for horn formation) on its equator. This sphere swims rapidly at random in a transparent vesicle and undergoes a series of changes in its properties: (i) the regions along the equator might come to have a slight affinity for each other, which could cause the arrangement of the zoospores into a plane; (ii) the two C-sites might become connection sites and the zoospores, therefore, might form strings, leading to the characteristic pattern of the adult colony (e.g. a few concentric circular strings, a spiral string and so on); and finally, (iii) the H-site of the zoospore determines whether it grows to a horn or not, i.e. the zoospore grows into a horn cell or a triangular cell according to the absence or presence of other zoospores which prevent its growth.     

Control of MAPK specificity by feedback phosphorylation of shared adaptor protein Ste50

Many different signaling pathways share common components but nevertheless invoke distinct physiological responses. In yeast, the adaptor protein Ste50 functions in multiple mitogen-activated protein (MAP) kinase pathways, each with unique dynamical and developmental properties. Although Kss1 activity is sustained and promotes invasive growth, Hog1 activity is transient and promotes cell adaptation to osmotic stress. Here we show that osmotic stress activates Kss1 as well as Hog1. We show further that Hog1 phosphorylates Ste50 and that phosphorylation of Ste50 limits the duration of Kss1 activation and prevents invasive growth under high osmolarity growth conditions. Thus feedback regulation of a shared component can restrict the activity of a competing MAP kinase to ensure signal fidelity.