Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.     

Three-dimensional culture of human tumor cells under standardized medium conditions

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution.The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase.The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.     

Purification and characterization of lymphocytic clonal growth factor (LCGF) derived from human-human hybridoma SH-76 cells

Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma.     

Rat pleural mesothelial cells adapted to serum-free medium as a model for the study of growth factor effects

Mesothelial cells are the putative progenitors of mesotheliomas and cell lines have been used as tools to study the responses of these cells to various stimuli, including growth factors. The present study was undertaken to develop a rat mesothelial cell line capable of sustained growth under serum-free conditions with the object of avoiding the possible confounding effects of undefined serum components. Responses of mesothelial cells to epidermal growth factor were shown to differ under serum-free versus low-serum culture conditions. In contrast, a cell line, SFM1, adapted to growth in serum-free medium was characterized and found to exhibit responses to growth factors similar to the responses reported for human cell lines. This new line should prove to be a useful model for the study of these cells in vitro .     

Supplementary factors required for serum-free culture of rat kidney cells of line NRK-49F

Factors required as supplements to basal tissue culture medium for the multiplication of cells of the cloned rat fibroblast line called normal rat kidney 49F (NRK-49F) were identified as epidermal growth factor, fibronectin, insulin, and retinoic acid. The requirement for fibronectin was manifested on a clean glass surface but not on the polystyrene plastic surface tested. This set of required factors differs substantially from the factor sets required by the Madin-Darby canine kidney (MDCK) and LLC-PK1 pig kidney lines of epithelial cells and the baby hamster kidney 21 (BHK-21) line of fibroblasts. The serum-free medium supplemented with the four factors supported rapid growth of NRK-49F cells when the initial cell population density was about 8,000 cells/cm2 or greater. At lower initial densities, cell multiplication was markedly increased by adding serum-free medium that had been conditioned by NRK-49F cells. Cell growth rate in the defined serum-free medium stayed high through two serial passages but declined in the third serial passage unless the cell-conditioned medium was added.     

Growth factor effects on small cell lung cancer cells using a colorimetric assay: can a transferrin-like factor mediate autocrine growth?

A semiautomated colorimetric assay (MTT assay), based on the ability of live cells to reduce a tetrazolium-based compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), to a purplish colored formazan product that can be measured spectrophotometrically, has recently been adapted for use in drug sensitivity analysis of cultured human tumor cell lines. We report the application of this assay for the evaluation of the growth factor requirements of human small cell lung cancer (SCLC) cell lines. Specifically, the growth stimulation of each constituent of a previously reported serum-free defined medium system for SCLC including various concentrations of hydrocortisone, insulin, transferrin, 17 beta-estradiol, and selenium (HITES) was evaluated. The optimal concentrations for insulin, transferrin, and selenium derived in the previously reported experiments with direct counting of viable cells were similar to optimal concentrations determined for the growth of three SCLC cell lines (NCI-H82, NCI-N417, NCI-H526) using the MTT assay. In contrast to the previous report, the growth-stimulating effects of hydrocortisone and 17 beta-estradiol were negligible. Using the MTT we have shown that a SCLC cell line, NCI-H345 (which has been previously reported to produce a transferrin-like molecule), was growth-inhibited by an anti-transferrin receptor antibody, when grown in transferrin-free media. The conditioned media from this cell line is stimulatory to other transferrin-sensitive cell lines, suggesting the possibility of an autocrine role for this transferrin-like molecule at least in that cell line. With carefully defined conditions for a given cell line in which cell density and other parameters are within a range of constant MTT metabolism, the assay is well suited for precise analysis of growth factor effects.     

Growth modulation of human tumor cells by a growth-inhibiting activity derived from tumorigenic V79 chinese hamster cells

Summary A growth-inhibiting activity was identified in supernatants of the neoplastic V79 Chinese hamster cell line based on its ability to inhibit the proliferation of the same cell line. The partially purified activity, provisionally termed “growth inhibiting factor” (GIF) activity, inhibited the growth of a wide variety of human tumor cells, but not various normal human fibroblasts. This species-nonspecific activity was reversible, saturable, and highly potent in tumorigenic cell lines, and was noted in both monolayer culture and in soft agar. The inhibitory activity CIF was also exhibited in a chemically defined serum-free medium supplemented with insulin and transferrin. GIF activity was stable to acid, heat, trypsin, and dithiothreitol but sensitive to alpha-chymotrypsin. The pattern of growth modulation by GIF on V79 cells was apparently different from those exhibited by bifunctional peptides such as transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-1-alpha. In addition, GIF activity cannot be ascribed to these cytokines based on the physicochemical and immunologic properties. Although GIF has yet to be purified to homogeneity, these data suggest that GIF might be a novel growth regulator which has a critical role in regulating growth of V79 cells. The growth modulation of tumor cells by this tumor-derived growth inhibiting activity suggested the presence of an autocrine growth regulatory mechanism even in tumor cells.     

Continuous growth of human tumor cell lines in serum-free media

Summary Five human tumor cell lines were studied for growth factor requirements and for replication in serum-free media. Of the five tumor lines HT-29 (colon carcinoma), TWI (melanoma), A-549 (lung carcinoma), Panc-1, (carcinoma of the pancreas) and EJ, (bladder carcinoma) only HT-29 and TWI grew in the serum-free medium (SFM). In a series of additional experiments, a combination of transferrin (5 μg/ml), insulin (5 μg/ml), triiodothyronine (2×10⁻¹⁰ M), epidermal growth factor (20 ng/ml), and selenium (5 ng/ml) was added to Chee’s essential medium (CEM) without serum (C-TITES medium). The C-TITES modification of CEM was found to allow optimal replication of HT-29 and TWI cells. Both HT-29 and TWI cells have replicated continuously in C-TITES medium for periods of more than 15 mo. These cells replicate with slightly lower doubling times than in CEM supplemented with 10% fetal bovine serum. Deletion of insulin or transferrin from the C-TITES medium resulted in cessation of cell growth of HT-29 and TWI. HT-29 assumed a somewhat rounded morphology, whereas TWI grew with the characteristic fibroblastic morphology in C-TITES medium. Cell line EJ did not grow in C-TITES medium. The other two cell lines, A-549 and Panc-1, grew in C-TITES medium but their growth rate was much slower than that in SSM. Availability of cell lines that can be propagated in serum-free, hormone-supplemented medium may aid in the study of the mechanisms by which hormones influence cell growth. This work was supported by Veterans Administration Research Awards to two of the authors (Karimullah A. Zirvi and George J. Hill) and grant no. CA-37138 from the National Cancer Institute.     

Development of a serum-free medium which supports the long-term growth of human and nonhuman primate lymphoid cells

The ability to grow lymphoid cells in serum-free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, β-mercaptoethanol, L-α-phosphatidylcholine, and transferrin supported the long-term proliferation of a gibbon ape lymphoma T-cell line, MLA144. These cells continue to produce Interleukin 2 (IL-2, T-cell growth factor) constitutively in the serum-free medium. IL-2-dependent human T cells initiated and maintained in culture in serum-free medium containing IL-2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL-2-dependent marmoset T-cell line, OH-1, also proliferated on the serum-free medium when supplemented with IL-2. Several established primate B-cell lines which do not require IL-2 for growth were able to proliferate in the serum-free medium. These B-cell lines included B95-8, an Epstein-Barr virus (EBV)-transformed marmoset cell line, HuCo/R-H, a human cord B-lymphocyte line transformed with EBV, and Namalwa, an EBV-positive B-cell line established from a Burkitt's lymphoma. B95-8 cells grown on serum-free medium showed high levels of EBV antigen-positive cells after induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA).     

Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated. An activated Ha-ras expressed from a stably transfected plasmid is unable to overcome the inability of R- cells to grow in serum-free medium supplemented with purified clones. Nevertheless, even in the presence of serum, R- cells stably transfected with Ha-ras, alone or in combination with simian virus 40 large T antigen, fail to form colonies in soft agar. Reintroduction into R- cells (or their derivatives) of a plasmid expressing the human insulin-like growth factor I receptor RNA and protein restores their ability to grow with purified growth factors or in soft agar. The signaling pathways participating in cell growth and transformation are discussed on the basis of these results.     

Growth of NS0 cells in protein-free, chemically defined medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.     

Supplementary factors required for serum-free culture of rat kidney cells of line NRK-49F

Summary Factors requred as supplements to basal tissue culture medium for the multiplication of cells of the cloned rat fibroblast line called normal rat kidney 49F (NRK-49F) were identified as epidermal growth factor, fibronectin, insulin, and retinoic acid. The requirement for fibronectin was manifested on a clean glass surface but not on the polystyrene plastic surface tested. This set of required factors differs substantially from the factor sets required by the Madin-Darby, canine kidney (MDCK) and LLC-PK₁ pig kidney lines of epithelial cells and the baby hamster kidney 21 (BHK-21) line of fibroblasts. The serum-free medium supplemented with the four factors supported rapid growth of NRK-49F cells when the initial cell population density was about 8,000 cells/cm² or greater. At lower initial densities, cell multiplication was markedly increased by adding serum-free medium that had been conditioned by NRK-49F cells. Cell growth rate in the defined serum-free medium stayed high through two serial passages but declined in the third serial passage unless the cell-conditioned medium was added.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Polyvinyl formal surface promotes continuous growth of Vero cells in protein-free medium.

The effect of polyvinyl formal (PVF) culture surface on the growth of 10 mammalian continuous cell lines, including Swiss 3T6, NCTC clone 929 L, BHK-21 clone 13, CHO-K1, PK 15, A 431, HeLa, MDCK, LLC-MK2 and Vero in protein-free 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 supplemented with trace elements and L-ascorbic acid 2-phosphate, was investigated. Most of the cell lines showed only some initial proliferation on PVF similar to the polystyrene (PS) surface of commercially available culture flasks. In contrast, proliferation of monkey kidney cell line Vero was by far greater on PVF than on PS or poly-D-lysine treated culture surface. In addition, Vero cells on PVF could be subcultured in the protein-free medium without any significant decrease of growth rate in successive passages. These results showed that PVF provides a culture surface which selectively promotes continuous growth of Vero cells in protein-free, chemically defined medium.     

Lactoferrin induces growth arrest and nuclear accumulation of Smad-2 in HeLa cells

Lactoferrin (Lf) is a multifunctional glycoprotein. Due to its anti-inflammatory and anti-cancer properties and the resulting therapeutical potential, lactoferrin is at present focus of a variety of research areas. The regulation of cell growth represents one of the prominent performances of lactoferrin. In this study we found lactoferrin to inhibit proliferation of the human epithelial cancer cell line HeLa. The extent of this growth inhibition was comparable to the one induced by the transforming-growth-factor-beta-1 (TGFβ1). In contrast to other cell lines where lactoferrin stimulates growth, lactoferrin failed to activate the MAP kinases ERK1/2 or p38 in HeLa cells. However, by immunocytochemistry and cell fractionation experiments, we found that lactoferrin is capable of activating the TGFβ/Smad-2 pathway. The nuclear accumulation of Smad-2 induced by Lf was comparable in magnitude to the one induced by TGFβ1.     

Identification and characterization of a B cell growth inhibitory factor (BIF) on BCGF-dependent B cell proliferation

The culture supernatants of Con A-activated human peripheral blood mononuclear cells (PBM) contained at least two regulatory factors upon B cell proliferation. One was B cell growth factor (BCGF), which activated antigen-stimulated B cells to proliferation and clonal expansion, and the other was its inhibitory factor, arbitrarily named B cell growth inhibitory factor (BIF). This BIF inhibited the effect of BCGF on anti-mu-stimulated B cells or the monoclonal mature B cell line (CLL-T.H.) obtained from the peripheral blood lymphocytes of B cell-type chronic lymphocytic leukemia patients, which were activated only with BCGF and without adding other proliferating stimuli (e.g., anti-mu). BIF activity was detected in the 24 hr culture supernatants of Con A-activated human PBM in FCS containing medium and also in serum-free RPMI 1640 medium. This substance with BIF activity could not be derived from FCS. Con A-induced BIF (m.w. of 80,000 and an isoelectric point of pH 5.4) was analyzed by Sephadex G-200 gel filtration and chromatofocusing. BIF was stable at pH 2.0 and at 56 degrees C for 30 min. Partially purified BIF had no effect on cell viability and almost no interferon activity (less than 1 IU/ml). BIF with high titer had a slight but significant inhibition on TCGF-dependent T cell growth and on PHA or Con A responses, but the extent of these inhibitions was far less than that of BCGF-dependent B cell growth. Absorption of BIF with Con A blasts made its inhibition on T cell growth even less. On the other hand, BIF activity could not be absorbed with Con A blasts but was almost absorbed with large numbers of CLL-T.H. cells. BIF had almost no inhibitory effect on the proliferation of a mouse fibroblast cell line (NIH 3T3), a mouse myeloma cell line (NS-1), human lymphoid cell lines (MOLT-4, HSB-2, and Daudi), or a human myeloid cell line (K-562). BIF-producing cells were estimated to be T cells and were identified as T8+ T cells. On the other hand, Con A-induced BCGF was demonstrated to be produced predominantly by T4+ T cells. These results show that human B cell proliferation is regulated by interaction between T4+ and T8+ cells via soluble factors, namely BCGF and BIF, respectively.     

Cadmium uptake by Caco-2 cells. Effect of some milk components

The effect of some milk components on the cellular uptake of cadmium has been studied using a human intestinal cell line (Caco-2). Cadmium uptake by Caco-2 cells increased with the concentration of this metal in the culture medium, in a saturable way. These cells were exposed to different concentrations of cadmium and the synthesis of metallothionein was studied by a cadmium-saturation method. The levels of metallothionein increased with the cadmium concentration in the medium up to 20 μM of metal. Supplementation of the culture medium with 10% bovine milk caused a 25% decrease in the uptake of cadmium with respect to that internalized by the cells maintained in the culture medium alone. However, the uptake of cadmium from the medium supplemented with 10% human milk was similar to that with serum-free medium. β-Lactoglobulin interacted with cadmium when studied by equilibrium dialysis, showing a stoichiometric binding constant of 5 × 10⁴l/mol. Interaction of lactoferrin with cadmium, however, was negligible. When Caco-2 cells were incubated in culture medium containing lactoferrin, cadmium uptake decreased with respect to that observed incubating the cells in a medium containing β-lactoglobulin or in the free-protein medium. The inhibitory effect of lactoferrin on the uptake of cadmium might be due to a reduction of the cell surface charge, through its binding to the membrane.     

Isolation of myoblastic, fibro-adipogenic, and fibroblastic clonal cell lines from a common precursor and study of their requirements for growth and differentiation

C17-S1-D-T984 (to be referred to as T984) is a myogenic clonal cell line isolated from a mouse teratocarcinoma. T984 exhibits phenotypic instability since it gives rise not only to myogenic but also to fibro-adipogenic and fibroblastic clones. A cell line of each clone type has been established and studied with respect to (1) phenotypic expression and stability; and (2) growth and differentiation in serum-free and serum-supplemented media. In both respects, marked differences between the three cell lines were observed. All three cell lines respond by increased growth in serum-free media to insulin, transferrin, fibroblast growth factor (FGF) and the serum-spreading factor of Holmes. The fibroblastic and the fibro-adipogenic cell lines can both be grown indefinitely in a serum-free medium which contains the above factors. The fibro-adipogenic cell line, which differentiates in serum-supplemented medium, exhibits very limited differentiation in the absence of serum; the serum factor(s) required for adipogenic differentiation is (are) probably proteins of molecular weight superior to 10 000. In direct contrast, the myogenic cell line exhibits limited growth in serum-free medium but readily differentiates under these conditions. Moreover, myogenic differentiation could be obtained in the defined medium at very low densities and was not influenced by the addition of medium conditioned by cells seeded at high densities. Thus, in this system, muscular differentiation is apparently independent of diffusible endogenous or exogenous factors and is probably triggered by the arrest of growth. While our results do not explain the reason why T984 exhibits phenotypic instability, they do indicate that this clonal cell line and its clonal derivatives could be used to identify the factors that influence the growth and the differentiation of cells of different mesenchymal phenotypes. The possible relationship of phenotypic instability to muscular dystrophies is also discussed.     

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6–18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. β-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.     

Conditioned medium of human nasopharyngeal carcinoma epithelioid cell line CNE 1 contained the activities of transformed growth-inhibiting factor(s)

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE 1) was assaied by both the [3H] thymidine incorporation test and the soft agar test. It was found that the SFCM can stimulate the growth of long-term serum free cultured CNE 1 cells in accordence with the fact that the growth rate of long-term serum free cultured CNE 1 cells was directly proportional to the plating density. Alternatively 5% SFCM can inhibit the growth of short-term serum free cultured CNE 1 cells by 51% in which the indicator cell may remained the responsive state of growing in the serum supplemented medium to the effector of interest. Furthermore, SFCM resulted in inhibition of anchorage-independent growth of CNE 1 cells and A431 cells. Also in soft agar test, SFCM can reduce the colony formation of NRK-49F cells in the presence of EGF or EGF plus TGF-beta. These finding suggested that CNE 1 can autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum free medium, the latter can strongly reverse malignant phenotypies of CNE 1 and A431 cells in serum supplemented surrounding.