Isolation from animal tissue and genetic transformation of Coxiella burnetii are facilitated by an improved axenic growth medium

We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-β-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10(-5). Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.     

Isolation of clones of hamster embryo cells transformed by the bovine papilloma virus

Primary hamster embryo cells infected with bovine papilloma virus (BPV) or treated with BPV DNA-calcium phosphate precipitates showed striking morphological alterations characteristic of transformed cells. Long, spindle-shaped cells grew into dense foci, eventually overgrowing monolayers of normally shaped cells. Samples of these cells were tested for anchorage-independent growth in dilute agarose medium. Cells were able to grow in agarose to form colonies which, when removed from agarose and transferred to liquid medium, established clones. Mockinfected cultures inoculated with plain medium displayed normal cell morphology and growth properties. This is the first report of BPV-transformed cells demonstrating anchorage-independent growth in agarose and the establishment of BPV transformed clones.     

Clonal variation in colony morphology and growth of CHO cells cultured on agar

Single Chinese hamster ovary (CHO) cells plated on agar form macroscopic colonies with high efficiency. Colonies produced by cells from the uncloned cell line increase in diameter continuously for 10–12 days after plating to form mounds of cells about 1 mm in diameter. With further incubation, some of these colonies do not increase in diameter (arrested dome), some form an expanding annular monolayer of cells around the central mound (fried egg), and some grow by enlarging the central mound into a low multilayered disc (saucer).These colony types on agar appear to be clonal characteristics of the CHO cell line. Cloning the line gives two kinds of isolates: one forms a mixture of arrested dome and fried egg colonies in an inheritable ratio, and the other forms saucer colonies. Cells from saucer colonies form saucer colonies when replated on agar. Cells from all colony types replate with similar efficiency on plastic or agar, and exhibit the same growth rate and cell size in liquid suspension culture. On plastic substrate, all these CHO cells form colonies which increase continuously in diameter for as long as 21 days, and little clonal difference in the morphology of colonies or of single cells is observed.These observations reveal a previously unsuspected heterogenieity in an established line of cultured mammalian cells and provide a method for studying new classes of In vitro growth control phenomena. These control phenomena may help in the building an in vitro model for tumor growth.     

A New Way to Investigate Living Flagellated/Ciliated Cells in the Light Microscope: Immobilization of Cells in Agarose

A method for cell immobilization of living flagellated/ciliated cells in agarose has been developed that allows single cells to be viewed for prolonged periods of time using high resolution light microscopy. Embedding in ultralow gelling, soft agarose preserves cellular functions of various flagellated/ciliated protists including delicate species, marine dinoflagellates, cryptomonads, contractile ciliates, etc. for days. Cell division, morphogenesis of cell organelles and intracellular movements can thus be studied for the first time in great detail. The method may also be useful for the isolation of flagellated/ciliated protists from nature and for the establishment of axenic clonal cultures in a single step.     

Inhibition of the development of the cellular slime mould Dictyostelium discoideum by omega-aminocarboxylic acids.

Four omega-aminocarboxylic acids - epsilon-aminocaproic acid (EACA), trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA), p-aminomethylbenzoic acid (PAMBA) and omega-aminocaprylic acid (OACA) -- prevented fruiting body formation of the cellular slime mould Dictyostelium discoideum. At concentrations of 40 mM, 75 mM, 10 mM and 5 mM, respectively, they allowed aggregation but prevented all further development at 24 degrees C. At lower concentrations, EACA allowed fruiting body formation but with a reduced number of spores per fruiting body. Only t-AMCHA had a significant inhibitory effect on the growth of myxamoebae. EACA affected development only if it was present between 8 and 16 h after the cells were deposited on the filters. Its effect was enhanced by high salt concentrations and by higher temperature, and was also dependent on the manner in which the cells were grown. Only strains capable of axenic growth displayed this sensitivity to EACA, although strains carrying only one of the genetic markers for axenic growth (axe A) were partially sensitive.     

异源PDGF-A链表达对CHO细胞生长和转化的作用

用DNA磷酸钙盐沉淀方法把含人PDGF(血小板衍生生长因子)A链cDNA的表达质粒pSV_2neo-A转染CHO细胞(中国仓鼠卵巢细胞),然后经G 418(400-800 μg/ml)筛选分离20个转染细胞株。选出其中At_1和Aot7细胞株所进行的实验结果表明,这些细胞的形态和生长行为均发生明显的变化,PDGF-A链mRNA的表达水平比CHO细胞明显增高,胞质有强阳性的PDGF荧光反应,显示有PDGF样蛋白的合成。这些细胞不但生长速率加快,有高密度持续生长的特性,而且能在软琼脂培基上形成大集落和在裸鼠体内接种形成纤维肉瘤,提示外源PDGF-A链基因的表达有使CHO细胞生长失控和发生细胞恶性转化的作用。     

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.     

Morphological transformation, autonomous proliferation and colony formation by chicken heart mesenchymal cells infected with avian sarcoma, erythroblastosis and myelocytomatosis viruses

Normal chicken heart mesenchymal cells at low density in monolayer culture in plasma-containing medium have a polygonal shape and are proliferatively quiescent. The combination of epidermal growth factor and insulin at hyperphysiological concentration, an insulin-like growth factor surrogate, causes these cells to assume a fusiform shape and to increase 40-fold in number during four days of incubation. These mitogenic hormones do not, however, induce normal chicken heart mesenchymal cells to form colonies in agarose suspension culture. Chicken heart mesenchymal cells infected with the Schmidt-Ruppin or Prague-A strains of Rous sarcoma virus or with the Fujinami or Y73 avian sarcoma viruses assume spindle and round shapes, increase 50-100 fold in number during four days of monolayer culture in the absence of mitogenic hormones and form macroscopic colonies during 3-4 days of agarose suspension culture. The autonomous (mitogenic hormone-independent) proliferation, in monolayer culture, of cells infected with temperature-sensitive transformation mutants of Rous sarcoma virus (tsNY68, tsNY72, tsLA24, tsLA29) is temperature-sensitive. Chicken heart mesenchymal cells infected with avian erythroblastosis virus assume spindle shapes and proliferate in monolayer culture at a rate comparable to that of sarcoma virus-infected cells but do not, however, form colonies in agarose suspension culture. Cells infected with the myelocytomatosis virus MC29 assume stellate shapes and increase 18-fold in number during four days of monolayer culture. Cells infected with the myelocytomatosis virus MH2 assume fusiform shapes and increase fourfold in number during four days of monolayer culture. Neither MC29 nor MH2 renders chicken heart mesenchymal cells capable of colony formation in agarose suspension culture. Infection with avian leukosis viruses (RAV-1, RAV-2, RPL-42) or with transformation-defective mutants of Rous sarcoma virus (tdNY105, 107, 109) does not affect the morphology or proliferative behavior of chicken heart mesenchymal cells. Monolayer culture of chicken heart mesenchymal cells in plasma-containing medium appears, therefore, to define the ability of onc genes of acute transforming avian retroviruses to induce autonomous (mitogenic hormone-independent) cell proliferation, the essential characteristic of neoplasia. The differences in transformed morphology and rates of autonomous proliferation between cells infected with different acute transforming retroviruses probably reflects differences in the modes of action of the transforming proteins encoded by the onc genes of the respective viruses.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro cloning of tumor stem cells in semi-solid media containing agar and agarose

Summary The formation of tumor stem cell colonies in vitro has been studied by comparing the growth of three mouse teratocarcinoma derived cell lines and one human teratocarcinoma derived cell line in semi-solid media containing either agar or agarose. We show that agaroses should be used in higher concentrations than agar to obtain comparable results. The maximum number of colonies were obtained in agarose over a broader range of concentrations (1%–4% for SeaPrep and 0.5%–2% for SeaPlaque agarose) than in agar, which allowed anchorage-independent growth of tumor cells only over a narrow concentration range (0.3%–0.5%). Overall, the preparation of media containing agarose was less cumbersome than preparation of agar-containing media, primarily because agaroses gelled more slowly and remained liquid in the physiological temperature range. Furthermore, the transfer of colonies from semi-solid media containing agarose to solid surface tissue culture dishes was much more efficient than the transfer of colonies from agar. The stock solutions of SeaPrep agarose could be kept ready for use for extended periods of time. All these features show that the low melting point agarose has considerable advantages over agar for preparation of semi-solid media for anchorage-independent tumor cell growth.     

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

A Note on the Growth of Thiobacillus ferrooxidans on Solid Medium

An iron oxidizing strain of Thiobacillus ferrooxidans has been grown on solid medium using purified agar, carrageenan (Type 1) and agarose. This strain produces isolated and transferable colonies after 7 d incubation. Growth (increase in viable cells) by the direct plating method has been followed in relation to iron oxidation. Acidity, agar concentration and phosphate influenced colony development on solid medium.     

Physiological factors affecting transformation of Azotobacter vinelandii.

Cells of Azotobacter vinelandii (ATCC 12837) can be transformed by exogenous deoxyribonucleic acid towards the end of exponential growth. Transformation occurs at very low frequencies when the deoxyribonucleic acid is purified or when the transformation is carried out in liquid medium. Optimal transformation occurs on plates of Burk nitrogen-free glucose medium containing either high phosphate (10 mM) or low calcium (0 to 0.29 mM) content. Higher levels of calcium are inhibitory, whereas magnesium ions are essential for transformation and growth. Extracellular polymer and capsule are increasingly inhibitory to transformation and are most abundant when the calcium content of the medium is high. Transformation is optimal at pH 7.0 to 7.1 and at 30 C, conditions which also coincide with minimal extracellular polymer production. Nonencapsulated strains are excellent transformation recipients. Glycine-induced pleomorphism reduces the transformation frequency and the degree of inhibition is dependent on the phosphate concentration of the medium. Rifampin resistance and shifts from adenine, hypoxanthine, uracil, and nitrogenase auxotrophy to prototrophy can be achieved. Although single marker transfer is always greater than double marker transfer, the data suggest that rifampin resistance is linked to hypoxanthine, adenine and uracil protorophy at intervals of increasing distance. Rifampin resistance did not appear to be linked to nitrogenase.     

DIMETHYL SULFOXIDE-INDUCED REVERSION OF SEVERAL FEATURES OF POLYOMA TRANSFORMED BABY HAMSTER KIDNEY CELLS (BHK-21) : Alterations in Growth and Morphology

Cells of a polyoma virus transformed clonal line (Cl-I) of baby hamster kidney fibroblasts (BHK-21) were grown in medium containing 2 percent dimethylsulfoxide (DMSO). Unlike the untransformed BHK-21 cells, Cl-I cells adapted to replication in the presence of DMSO, and they exhibited a rapidly reversible phenotypic reversion of a number of properties characteristic of the transformed state. Restoration of density dependent growth inhibition with accumulation of cells in the G₁ phase of the cell cycle occurred and was associated with restoration of contact dependent behavior and with reversion of histological and ultrastructural features towards those which characterize untransformed cells. Concomitantly, Cl-I cells grown in 2 percent DMSO lost the ability to form colonies in semisolid medium. The data presented suggest that DMSO alters the expression of cellular functions which were altered as a result of viral transformation and which may be involved in cell tumorigenicity.     

Factors Controlling the Production of Lysogenic Cultures of B. megatherium

(1) The proportion of infected B. megatherium cells which develop lysogenic colonies depends on the number and kind of infecting virus particles and on the culture medium in which the cells are growing. (2) Cells infected with 100 or more T virus particles (from megatherium 899) in yeast extract peptone disintegrate, produce very few virus particles, and less than one lysogenic colony per 10⁷ infected cells. Cells infected with one or a few particles produce 500 to 1000 virus particles each and about 30 lysogenic colonies per 10⁷ infected colonies. (3) T phage obtained from lysogenic magatherium KM cultures produces many more lysogenic cells than does the original megatherium 899 virus. (4) Cells infected with megatherium 899 T virus in peptone medium and then transferred to asparagine medium give rise to 10⁶ lysogenic colonies per 10⁷ infected cells and this transformation will occur even after the infected cells have been in peptone for 60 to 90 minutes and are beginning to produce virus particles. (5) Continued growth of KM strain with either C or T virus from megatherium 899 for several hundred generations in the steady state apparatus results in a lysogenic strain which produces several different types of virus.     

A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5 encodes a functional homolog of yeast Vps34p. These observations indicate that in D. discoideum, different PI kinases regulate distinct cellular processes, including cell growth, development, and protein trafficking.     

外源血小板衍生生长因子(PDGF)B链基因在CHO细胞的表达

本文总结应用DNA-磷酸钙盐沉淀的基因转移技术,把含人c-sis cDNA的质粒pSM-1,以单独转染或与pSV_2neo共转染方法转入CHO细胞(中国仓鼠卵巢细胞),经低血清或G_(418)筛选分离得到多个有PDGF表达的细胞株。其中PDGF高表达件——FB_6,细胞形态和生长行为明显改变,可在软琼脂培基上形成集落,细胞生长速率加快,能在低血清(2%)培液中长期传代,细胞的条件培液有刺激静止的NRK细胞(大鼠肾成纤维细胞)DNA合成的活力。RNA点杂交和Southern Blot显示FB_5细胞有??PDGF mRNA的高表达和人c-sis基因的整合,而且,在连续传代7个月后,FB_5细胞基因组中仍然有人c-sis基因存在,说明CHO细胞的不正常生长和转化是由于PDGF基因的稳定整合和高表达所引起。这一稳定转化株(FB_5)是进一步研究PDGF对细胞生长控制和转化功能的理想模型。     

An Immobilization Antigen in Tetrahymena pyriformis Expressed Under Conditions of High Salt Stress*

SYNOPSIS. Cells of Tetrahymena pyriformis syngen 1 exposed to 200 mM NaC1 without previous exposure to a saline medium die within minutes. Lines that have been adapted to higher salt concentrations by growing in axenic peptone medium with 100 mM NaC1 are able to grow in previously intolerable concentrations. Acclimation to higher salt concentrations is accompanied by a transformation from the H serotype, expressed at room temperature in the standard peptone medium, to a new serotype, St. Complete transformation occurs at concentrations approaching the LD₅₀ for nonadapted cells, and the time required for the transformation is 3–4 days. The reverse transformation, St to H, involves a time interval during which the cells are refractory to both anti-St and anti-H. Among 12 of the 13 inbred families of T. pyriformis syngen 1, 2 different high salt serotypes are found. One of these, Sta, is found among families A, Al, A3, B, D, D1, E, and F. The other, Stc, occurs in families C, C1, C2, C3, and possibly B2.     

Induction of squamous differentiation of normal human bronchial epithelial cells by small amounts of serum

Abstract. Recently we reported a low calcium (110 μM) serum-free medium (LHC-1) for clonal growth of normal human bronchial epithelial (NHBE) cells. NHBE cells within colonies are small (mean surface area = 1,250 μ²) rarely migratory, have few tonofilaments, and multiply with an average population doubling time of 28 h. We have also noted that adding small amounts of blood-derived serum to LHC-1 medium (as little as 2%) significantly decreased the clonal growth rate. We have now found that the growth inhibiting effect of serum is due to the induction of squamous (terminal) differentiation. Serum quickly increases the size of the cells (mean surface area = 4,900 μ²). In addition, the cells acquire numerous desmosomal junctions and an extensive network of keratin bundles. In contrast, human lung carcinoma cells multiply rapidly at clonal density in LHC-1 medium containing as much as 8% serum.
Although high concentrations of calcium ions in the medium are known to induce squamous differentiation of epidermal keratinocytes in the absence of serum, high levels of Ca²⁺ (up to 1,000 μM) increased the number of desmosomal junctions, but did not significantly affect the clonal growth rate or size of the NHBE cells. However, high concentrations of calcium (above 450 μM) were found to potentiate serum differentiation-inducing activity. On the other hand, cholera toxin (10 ng/ml) inhibited the differentiation-inducing acitivy of serum. These results show that squamous differentiation of NHBE cells can be induced by serum and the potency of these serum factors can be modulated. In addition, the data show that lung carcinoma cells differ from their normal counterparts by not undergoing differentiation in the presence of serum.