Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium

Summary An improved procedure has been developed for clonal growth of normal human epidermal keratinocytes (HK) without feeder cells or conditioned medium. The use of medium 199, supplemented with 0.4 μg/ml hydrocortisone (HC) and 20% (v/v) whole fetal bovine serum (wFBS) and conditioned overnight by 3T3 cells, eliminated the need for a feeder layer of lethally irradiated 3T3 cells for HK growth. Several other media with equivalent conditioning and supplementation failed to support satisfactory multiplication of HK, including Dulbecco's modified Eagle's medium, which is normally used for growth of HK with a feeder layer. Increasing the concentration of HC to 10 μg/ml (2.8×10⁻⁵ M) made possible clonal growth of HK without any conditioning of the medium. The addition of 10⁻⁵ M putrescine, 10⁻⁵ M vitamin B₁₂, or 3.7×10⁻⁶ M β-estradiol further enhanced growth in unconditioned medium. Substantially greater improvement was obtained by the addition of pituitary extract or fractions prepared from pituitary extract. In medium 199 supplemented with 10 μg/ml HC, 20% (v/v) wFBS, and 0.15 mg/ml each of two pituitary fractions, single HK attach with a colony-forming efficiency equal to that in conditioned medium and form stratified, keratinized colonies that grow to confluency and can be subcultured. These results make it clear that HK do not require special “conditioning factors” from fibroblasts for clonal growth and differentiation in culture. Thus, factors directly involved in growth and the expression of differentiation can be analyzed without the interfering effects of any other type of cell. Preliminary studies with epidermal growth factor (EGF), which stimulates growth and extends life span of HK grown in the presence of fibroblasts, have shown that, in the absence of fibroblasts, EGF has no effect either on clonal growth or on cumulative multiplication potential of HK. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Tyrosinase activities of cell-free extracts and living cells of cultured melanoma cells

Melanogenesis in the course of monolayer culture of a stably melanotic clonal line C₂M, derived from a mouse melanoma B 16, was investigated. Tyrosinase activity per cell of cell-free extracts was highest when the extract was prepared from cells in the mid-exponential phase of growth, when it was more than 6 times the activity of that prepared from a fully grown culture or a culture in the very early phase. On the other hand, the enzyme activity per cell of living cells in culture was highest in the early phase of culture and decreased rapidly to a level of less than one tenth of the maximum activity, in the stationary phase.The upper limit of population density of cultured melanoma cells permissive for melanin synthesis (2 to 3 × 10⁵ cells/cm²) was much higher than that of normal (nonneoplastic) melanocytes, which had been reported to produce melanin only under conditions of clonal growth.The relative efficiency of tyrosinase activity in situ, expressed by the ratio of tyrosinase activity in culture to that of cell-free extract, decreased rapidly in the exponential phase of growth. This decrease correlates to the cell density in the culture, and little if at all to the division rate, and suggests a suppressing mechanism of melanin synthesis working at the enzyme level.     

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses,growth kinetics,and cell cycle studies

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G₁, S, G₂, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (> 30 population doublings) and neonatal prokeratinocytes (> 50 population doublings) is associated with senescence and the G₁ arrest of noncycling cells.     

Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. Differential effects of hydrocortisone and tissue extracts

Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin. Prolactin was measured immunologically in culture medium and within cells by complement fixation. Rates of prolactin production varied from 6.6 to 12 µg/mg cell protein per 24 hr in four different cell strains. In these cultures ratios of production of prolactin to growth hormone varied from 1.0 to 4.1. A fifth clonal strain produced growth hormone but no detectable prolactin. Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1–2 hr. Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 x 10^-6 M), which stimulated the production of growth hormone 4- to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25% of that in control cultures. Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50%. We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.     

Effects of growth factors, hormones, bacterial lipopolysaccharides, and lipotechoic acids on the clonal growth of normal ureteral epithelial cells in serum-free culture.

In vitro tissue culture techniques were employed to study the effects of bacterial endotoxins on the growth of normal epithelial cells from the human ureter (NHU). Primary cultures of NHU cells were initiated from explant outgrowth cultures of human ureteral tissue and cultured on collagen gel in F-12* medium containing 1% fetal calf serum (FCS). Optimal clonal growth of secondary cultures of NHU cells seeded at relatively low seeding cell densities, directly on plastic dishes, was achieved in F-12* medium containing bovine pituitary extract (0.5% BPE) and 0.05% BSA. Results indicated that insulin in the F-12* medium could be replaced by three orders of magnitude less IGF-1. Further clonal growth experiments demonstrated that PGE1 is growth stimulatory and can replace BPE as a growth factor requirement. This finding was in agreement with the fact that BPE growth requirement could be replaced by cholera toxin or dibutyryl cAMP. These results suggested that both BPE and cholera toxin operated by activation of a cAMP-dependent mitogenic pathway. Seven gram-negative bacterial lipopolysaccharides (LPS) and three gram-positive bacterial lipotechoic acids (LT) were tested for their effects on NHU clonal growth. Three out of the five LPS derived from Escherichia coli (strains 055:B5, 0128:B12, and 0127:B8), LPS from Klebsiella pneumoniae, and LPS from Pseudomonas aeruginosa all showed significant growth inhibitory effects at minimally effective doses ranging from 5 to 25 micrograms/ml. LPS derived from E. coli strain (0111:B4) had no growth effects at the highest concentration tested (100 micrograms/ml). In contrast, LT derived from Streptococcus pyogenes, S. faecalis, Staphylococcus aureas, and Bacillus subtilis all markedly enhanced clonal growth at concentrations ranging from 1 microgram/ml less than [LT] less than 50 micrograms/ml. LT from Strep. pyogenes was inhibitory to clonal growth at 100 micrograms/ml. The growth inhibitory effects of LPS were shown to be sensitive to the presence of hydrocortisone in the growth medium, indicating that LPS effects on growth are mediated via the arachidonic acid cascade. We speculate that these results indicate a link between the susceptibility of uroepithelial tissue to the pathogenic microflora seen in urinary tract diseases and the differential sensitivity of proliferation-competent uroepithelial cells to growth inhibition by LPS produced by gram-negative bacteria. However, further studies with uropathogenic serotypes will be necessary to corroborate this possibility. The growth-stimulating activity of LTs produced by gram-positive bacteria may be due to their ability to bind to cell-associated fibronectin and to activate the fibronectin receptor as part of ligand receptor-induced mitogenic transmembrane signalling pathway.     

Proliferation of epithelial cells derived from rat dorsolateral prostate in serum-free primary cell culture and their response to androgen

Summary Primary cultured epithelial cells derived from the rat dorsolateral prostate proliferated in serum-free nutrient medium WAJC 404 supplemented with mitogens: insulin (650 nM), cholera toxin (120 pM), epidermal growth factor (EGF) (2.5 nM), dexamethasone (300 nM), and bovine pituitary extract (25 μg/ml). The culture consisted of two types of epithelial cell colonies: one originated from single cells or small cell aggregates and the other was epithelial cell outgrowth from small tissue fragments attached to a substratum. There were differences in requirements for the mitogens between the two types of colonies. Requirements for cholera toxin, bovine pituitary extract, and dexamethasone were higher in the former type of colonies, and those for EGF were higher in the latter type of colonies. Proliferation of the epithelial cells in either type, of colony was suppressed more than 50% by 1 nM dihydrotestosterone. This suppressive effect was not mediated by stromal component in the tissue fragments, and was counteracted by cyproterone acetate, indicating specific and direct action of the androgen on prostate epithelial cells. The results suggest that there is discrete participation of polypeptide growth factors and androgen in proliferation and differentiation, respectively, of prostate epithelial cells in vivo.     

Regulation of glycoconjugate expression on cloned mammalian cells. Studies on the growth dependence of membrane-associated/blood group H angigen.

Studies of HeLa S-3 cell clones derived from suspensions of single cells were carried out to determine the relationship of clonal growth patterns to blood group H antigen expression. Cloning efficiency of these cells was excellent in the presence of growth medium which contained pooled human serum. At the end of the growth period of 9 to 11 days, three quarters of the clones were medium sized (cell counts ranged from 100–500), or large (greater than 500 cells), and approximately one fourth were classified as small (less than 100 cells). A mixture of H positive and H negative cells was observed in medium and large clones; therefore the composition of such clones resembled the cellular blood group composition of the original suspension (50% H positive, 50% H negative). Small clones contained all H-negative cells or only a few H-positive cells. Optimal expression of H antigen thus appeared to depend upon a growth pattern similar to that observed in exponentially growing monolayer cells. Clones at the 2 to 8 cell stage were mostly H-negative; this finding was consistent with the view that alterations in culture technique may have caused transient cellular and enzyme alterations which in the case of H antigen was reflected by a temporary loss of phenotype.     

The origin of chicken hematopoietic colonies as assayed in semisolid agar.

This investigation was undertaken to determine whether primitive stem cells and/or fully differentiated macrophages were the source of in vitro colonies derived from hematopoietic tissues. The chicken colony-forming cell (CFC) present in uncultured yolk sac was a nonadherent, presumably undifferentiated cell. The efficiency of colony formation in this case was approximately 0.08%. In contrast to uncultured yolk sac, the CFC present in one-week old yolk sac cultures was evidently a macrophage. Yolk sac cultures, which consisted of greater than 99% macrophages, produced colonies with an efficiency of 1-5% while cultures derived from peritoneal macrophages produced colonies with an efficiency of 10%. Silica selectively destroyed macrophages and reduced the colony forming efficiency of cells derived from yolk sac cultures.     

Stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor

Viable cells of Micrococcus luteus secrete a proteineous growth factor (Rpf) which promotes the resuscitation of dormant, nongrowing cells to yield normal, colony-forming bacteria. When washed M. luteus cells were used as an inoculum, there was a pronounced influence of Rpf on the true lag phase and cell growth on lactate minimal medium. In the absence of Rpf, there was no increase in colony-forming units for up to 10 days. When the inoculum contained less than 10⁵ cells ml^–1, macroscopically observable M. luteus growth was not obtained in succinate minimal medium unless Rpf was added. Incubation of M. luteus in the stationary phase for 100 h resulted in a failure of the cells to grow in lactate minimal medium from inocula of small size although the viability of these cells was close to 100% as estimated using agar plates made from lactate minimal medium or rich medium. The underestimation of viable cells by the most-probable-number (MPN) method in comparsion with colony-forming units was equivalent to the requirement that at least 10⁵ cells grown on succinate medium, 10³ cells from old stationary phase, or approximately 10–500 washed cells are required per millilitre of inoculum for growth to lead to visible turbidity. The addition of Rpf in the MPN dilutions led to an increase of the viable cell numbers estimated to approximately the same levels as those determined by colony-forming units. Thus, a basic principle of microbiology –“one cell-one culture”– may not be applicable in some circumstances in which the metabolic activity of “starter” cells is not sufficient to produce enough autocrine growth factor to support cell multiplication. Received: 7 December 1998 / Accepted: 7 April 1999     

Growth regulation of serum-free cultures of epithelial cells from normal human buccal mucosa

Summary Human buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factorβ-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca²⁺ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia. This work was supported in part by grants from the Swedish National Board of Laboratory Animals, the Swedish Medical Research Council, the Swedish Natural Science Research Council, the Swedish Fund for Scientific Research Without Animal Experiments, the Swedish Cancer Society, the Swedish Tobacco Company, and Lions Club International, Djurg?rden, Stockholm, Sweden.     

Accumulation of secretory granules in pituitary clonal cells derived from the epithelium of Rathke's pouch

Summary 2A8 clonal cells derived from the epithelium of Rathke's pouch of the fetal rat are essentially agranular when grown in vitro, in spite of their active secretion of hormones, i.e., ACTH, prolactin and growth hormone. These cells do not produce detectable amounts of thyrotrophic or gonadotrophic hormones in vitro. When the growth medium (Ham's F10) was supplemented with rat median eminence extract (MEE), l-thyroxin and fresh serum from hypophysectomized rats, some of the 2A8 cells accumulated and stored secretory granules which were characteristic of the cells of the intact pituitary gland. Typical thyrotrophic and gonadotrophic cells also became recognizable and all six anterior pituitary hormones were released into the culture medium. Growth of 2A8 cells in this modified culture medium resulted in an increased production of all hormones with increasing time in culture.These results indicate that the processes that lead to the accumulation of typical mature secretory granules which characterize the individual pituitary cell types are initiated or promoted by some unidentified factor or factors which are present in fresh rat serum. It is also apparent that fresh rat serum can promote the differentiation of gonadotrophs and thyrotrophs in vitro.Supported by USPHS Grant Am 12583 and Institutional Research Grant of The University of Texas Health Science Center at San Antonio, TexasThe authors wish to thank Mrs. Pauline Polette for her skillful technical assistance     

Modulation of TGF-β-inducible hypermotility by EGF and other factors in human prostate epithelial cells and keratinocytes

Keratinocytes migrating from a wound edge or initiating malignant invasion greatly increase their expression of the basement membrane protein Laminin-322 (Lam332). In culture, keratinocytes initiate sustained directional hypermotility when plated onto an incompletely processed form of Lam332 (Lam332′) or when treated with transforming growth factor beta (TGF-β), an inducer of Lam332 expression. The development and tissue architecture of stratified squamous and prostate epithelia are very different, yet the basal cells of both express p63, α6β4 integrin, and Lam332. Keratinocytes and prostate epithelial cells grow well in nutritionally optimized culture media with pituitary extract and certain mitogens. We report that prostate epithelial cells display hypermotility responses indistinguishable from those of keratinocytes. Several culture medium variables attenuated TGF-β-induced hypermotility, including Ca⁺⁺, serum, and some pituitary extract preparations, without impairing growth, TGF-β growth inhibition, or hypermotility on Lam322′. Distinct from its role as a mitogen, EGF proved to be a required cofactor for TGF-β-induced hypermotility and could not be replaced by HGF or KGF. Prostate epithelial cells have a short replicative lifespan, restricted both by p16^INK4A and telomere-related mechanisms. We immortalized the normal prostate epithelial cell line HPrE-1 by transduction to express bmi1 and TERT. Prostate epithelial cells lose expression of p63, β4 integrin, and Lam332 when they transform to invasive carcinoma. In contrast, HPrE-1/bmi1/TERT cells retained expression of these proteins and normal TGF-β signaling and hypermotility for >100 doublings. Thus, keratinocytes and prostate epithelial cells possess common hypermotility and senescence mechanisms and immortalized prostate cell lines can be engineered using defined methods to yield cells retaining normal properties.     

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas

Summary Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar¹,Ile⁸-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.     

Proliferation and differentiation of Abelson virus-infected murine myeloid leukemia cell lines does not involve an autocrine mechanism

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus-infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony-forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth-factor independent replication nor the spontaneous differentiation of Abelson virus-infected myeloid cells involves autocrine secretion of growth regulators.     

Lipopolysaccharide effects on sensitive and resistant variant chinese hamster ovary cell lines

Chinese hamster ovary (CHO · K1 · PRO) cell growth was inhibited by addition of a gram-negative bacterial lipopolysaccharide (LPS) to the cell culture medium. Growth inhibition began after three or four days of incubation, was dose-dependent up to a maximum at an LPS concentration of 500 μg/ml and was accompanied by cell shape changes and enhanced cytoplasmic vacuolization. Formation of bizarre CHO · K1 · PRO cell shapes and vacuole formation were most pronounced after seven days of incubation with LPS and could be observed by light and electron microscopy. An LPS-resistant cell population was obtained by intermittent in vitro exposure to high levels of LPS; these variant cells or clones derived from them failed to display growth inhibition in the presence of LPS. A clone from the LPS-resistant variant population showed altered cell properties compared to the parental cell line which included changes in cell morphology, adhesion, and endocytosis. Parental cells were markedly density-inhibited, whereas the variant clone exhibited considerable growth after confluency. The LPS-resistant variant cells showed a more elongated morphology than the parental line. No significant differences were observed between rates of detachment of parental and variant cells when sparse cultures of either line were removed from tissue culture dishes by ethylenediaminetetracetate (EDTA). However, at confluency approximately 100% of the variant cells versus 35% of the parental cells were removed by EDTA in one hour. Measurements of ¹²⁵I-ferritin uptake by parental and variant cells showed approximately twenty-fold and twofold increases, respectively, in uptake induced by LPS when compared to untreated control cultures.     

The effect of culture conditions on colony morphology and proliferative capacity in human prostate cancer cell lines

Primary cultures and cell lines form three types of colonies, termed holoclones, meroclones and paraclones by Barrandon and Green (Proc Natl Acad Sci U S A 84:2302-2306, 1987). They suggested that the three types correspond to colonies derived from stem, transit-amplifying and terminally differentiated cells. We determined the effect of culture conditions (seeding density, serum concentration, type of medium and substrate) on the proportion of each colony type and the cell number of individual colonies, using three prostate cancer cell lines, DU145, LNCaP and PC-3. In less favourable culture conditions, stem cell (SC) colonies tended to be lost; but in more favourable conditions, only modest increases in the proportion of SC colonies were observed. Under some conditions, cell number, but not colony-forming ability, was altered, indicating that colony cell number is controlled, at least in part, by different factors to colony formation. Colony-forming ability of individual cell lines is remarkably stable and there is little evidence for clonal evolution in culture, which might be expected and would result in more aggressive, faster-growing cells. Better understanding of how colony-forming efficiency is controlled could lead to the identification of drug targets that control SC growth and modify the progression of cancer.     

Derivation of the clonal‐cell lines from Siberian sturgeon (Acipenser baerii) head‐kidney cell lines and its applicability to foreign gene expression and virus culture

This study was conducted to establish and characterize the clonal‐cell lines from Siberian sturgeon Acipenser baerii head‐kidney tissues and to evaluate its applicability as a research tool. From the culture of A. baerii head‐kidney derived cells, 10 cell lines were established first and then eight clonal‐cell lines were derived from clonal growth and colony expansion of two cell lines that showed significant high colony‐forming ability. All eight clonal‐cell lines were morphologically similar and grew stably under monolayer culture but their growth rates were significantly different. They possessed diploid DNA contents, expressed epithelial cell‐related genes and showed strong anchorage dependency to substrates. When a clonal‐cell line was transfected separately with three plasmid vectors including fluorescent reporter genes driven by cytomegalovirus, marine medaka Oryzias dancena β‐actin or A. baerii β‐actin promoter, the cell lines expressed fluorescent signals regardless of promoter types. The cells harbouring foreign genes could be expanded to stable cell lines under drug selection and then they additionally could form the extensively proliferating colonies at low‐density culture. Finally, the clonal‐cell lines showed the susceptibility to viral haemorrhagic septicaemia virus (VHSV). Collectively, the clonal‐cell lines from A. baerii head kidney were established and these cell lines will be able to provide an excellent in vitro system for various biological studies in this fish species.     

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.