Requirement of conditioned medium or a high concentration of serum for anchorage-independent growth of adenovirus type 12 E1a-transformed rat cells (HY1)

Adenovirus type 12 (Ad12) Ela-transformed rat cells (HY1) grew in methocel medium containing 9% fetal calf serum (FCS), but the frequency of colony formation was very low (the order of 10(-4)). The addition of conditioned medium or a high concentration of serum (20% FCS) to the methocel medium accelerated colony formation, and plating efficiency increased 10- to 100-fold. In contrast, in stationary culture, HY1 cells grew well even in 1%-FCS medium. These results indicate that HY1 cells require high concentrations of growth factors for anchorage-independent growth. The effects of conditioned medium or FCS also were demonstrated in several transformed cell lines induced by transfection of combined sets of Ad12-transforming genes (E1a, E1b and E4). These growth behaviors suggest that the first step in cell transformation with adenovirus 12 is the acquisition of responsiveness to growth factors in methocel culture, which must be the function of the Ad12-E1a gene products. The function of the other two Ad12-transforming genes was discussed.     

Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum

The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium.     

Serum inhibits the immunosuppressive function of myeloid-derived suppressor cells isolated from 4T1 tumor-bearing mice

As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.     

Vitronectin is the major serum protein essential for NGF-mediated neurite outgrowth from PC12 cells.

The rat pheochromocytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf serum (FCS). Using a novel assay procedure we have developed a purification protocol which has allowed the isolation of the protein in serum responsible for neurite outgrowth after NGF treatment. FCS has been fractionated using four chromatographic procedures and in each case the peak of biological activity copurified with vitronectin. We have concluded, therefore, that vitronectin is the protein present in FCS which can mediate NGF-dependent neurite outgrowth in PC12 cells. Vitronectin and fibronectin from FCS have been chromatographically separated and only the former is capable of inducing neurite outgrowth. We have also shown that vitronectin utilizes the RGD amino acid sequence in binding to the surface of PC12s.     

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.     

Suppression of endocytosis in polyclonally activated Con A-stimulated T lymphocytes by 25-hydroxycholesterol

A new high quality young-calf serum, Hy-clone calf serum (HcCS), was tested for use in hybridoma culture. This calf serum alone had little growth promoting activity and was much inferior to fetal calf serum (FCS). Red cell lysate (RCL) used in combination with the young-calf serum showed very good growth promoting activity. Growth was increased about threefold over that in the presence of FCS. However, HcCS and RCL could not substitute for feeder cells when hybridomas were cultured as single cells under conditions of limiting dilution. It is thought likely that the potent growth promoting factor in red cell lysate is hemoglobin.     

Preadipocyte stimulating factor in rat serum: evidence for a discrete 63 kDa protein that promotes cell differentiation of rat preadipocytes in primary cultures

In primary cultures of rat preadipocytes (PA) isolated from epididymal or perirenal depots, rat serum is more effective than other animal sera (fetal calf, newborn calf, human, horse, rabbit, cat, sheep, goat, dog, pig) in promoting adipogenic conversion, biochemical differentiation, and mitogenesis. Only mouse serum is comparable to rat serum. This activity is attributable to a specific growth factor (preadipocyte stimulating factor, PSF). An assay for PSF in rat serum was devised using PA from perirenal fat of 3-month-old Fischer 344 rats grown first to confluence in FCS for 8 days and then for the next 3 days in test serum, followed by measurement of triglyceride (TG) and glycerol-3-phosphate dehydrogenase (GPDH). Rat serum induces dose-dependent rapid cell division, which coincides with accumulation of TG and increase of GPDH; for routine quantitation, TG is assayed. The biochemical characteristics of PSF in serum are as follows: stable at 4 degrees C for up to 1 year; inactivated at 100 degrees C (80% loss, 30 min) but stable at 56 degrees C for 1 hr; stable at pH 2-12; non-dialyzable; completely resistant to pepsin, trypsin, and chymotrypsin but destroyed by pronase and subtilisn; stable to DTT and periodate; and m.w. between 68 kDa (Sephacryl-300) and 58 kDa (Sephacryl-300 in 5 M urea). PSF activity is greater in serum from Wistar than from Fischer 344 rats, while activity of serum from Zucker obese (fa/fa) rats is at least as great as that from Wistar rats and, like serum of rats made obese by feeding a high-fat, high-carbohydrate diet, is not suppressed. PSF activity is not due to insulin, insulin-like growth factor-1 (IGF-1), growth hormone, glucocorticoids, or combinations of these hormones. PSF activity was not seen with a number of growth factors including colony-stimulating factor (CSF-1), GM-CSF, interleukins 1, 2, and 3, neuroleukin, tumor necrosis factor, and others. PSF is distinct from the low molecular weight (4-8 kDa) differentiation factor present in rat serum, FCS, and human serum that promotes the adipogenic conversion and cellular differentiation of 3T3-L1, 3T3-F442A, and Ob17 cells. PSF appears to be a new differentiation factor for rat preadipocytes, has properties suggestive of a highly glycosylated protein, and may be highly species specific.     

Culture medium enhances semicarbazide-sensitive amine oxidase activity

Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells. Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro. Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC). In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO. In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media. Semicarbazide-sensitive amine oxidase activity in FCS (5-20 microl) was significantly enhanced (approximately 1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement. Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages. Activity in rat plasma was reduced by approximately 25% in the presence of DMEM. The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine.HCl, and L-glutamine, were not correlated with enhancement. This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.     

Differential response of cultured rabbit articular chondrocytes (RAC) to transforming growth factor beta (TGF-beta)-evidence for a role of serum factors

We show that addition of TGF-beta (0.01-10 ng/ml) to proliferating rabbit articular chondrocytes in presence of low level of fetal calf serum (FCS, 2%) results in a sustained decrease of cell number and DNA synthesis up to 72 h. In contrast, incubation with high serum concentration (10% FCS) induces a transient increase of cell number after 48 h without elevation of DNA synthesis. Moreover, when the factor is added in 10% FCS-containing medium, a differential effect is observed at 48 h (either increase or decrease of cell number) depending on the serum level (2 or 10%) present between 24 and 48 h. Recruitment of cells in late S-phase occurred under TGF-beta-treatment in both 2 and 10% FCS. These arrested cells may then be released by further exposure to 10% FCS-containing medium. The data show that factor(s) from the serum modulate(s) the action of TGF-beta on chondrocyte proliferation. Addition of epidermal growth factor (EGF) to the cultures in presence of 2% FCS mimicks the effects observed with 10% serum, suggesting that the serum component(s) involved in the mechanism could be of EGF type.     

Differential effects of transforming growth factor-beta and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

We examined the effect of transforming growth factor (TGF-beta) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]-thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF-beta at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S-phase (SQ or G2Q) in either 2% or 10% FCS-containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF-beta addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose-dependence inhibition of cell growth was detected, in contrast, for 2% FCS-treated cells. Presence of TGF-beta during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet-poor plasma at 10% produced the same effects as FCS, suggesting that platelet-derived factors, such as platelet-derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF-beta to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases were observed. When combined, TGF-beta and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]-thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-beta than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell-number increases at 48 h in 10% FCS-containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF-beta induces first a recruitment of chondrocytes in noncycling SQ- or G2Q-blocked cells. The, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS).(ABSTRACT TRUNCATED AT 400 WORDS)     

Atrial natriuretic factor inhibits proliferation of vascular smooth muscle cells stimulated by platelet-derived growth factor

The effect of atrial natriuretic factor (Isoleucine-ANF 101-126) on basal and platelet-derived growth factor (PDGF)-stimulated proliferation of rat aortic vascular smooth muscle cells (VSMC) was assessed by microscopy and measurement of incorporation of tritiated thymidine by cells cultured with or without addition of PDGF in the presence of various concentrations (10(-8)-10(-6) molar) of ANF. ANF had little effect on proliferation of cells grown in media supplemented with 2% fetal calf serum (FCS) alone but exhibited clear dose-related inhibition of PDGF-stimulated thymidine incorporation.     

Effect of serum and 12-O-tetradecanoyl-phorbol-13-acetate on FSH-stimulated conversion of 4-androstene-3,17-dione to oestrogens in cell and organ cultures of suckling mouse ovaries

The effect of serum and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the FSH-stimulated oestrogen production was studied in both cell and organ cultures. Ovaries were removed from (WB X C57BL/6)F1 mice at 9-days of age, and the conversion of 4-androstene-3,17 -dione to oestrogens was stimulated by the addition of FSH in vitro. Either 10% serum (fetal calf, mouse, rat and horse) or 0.1 microM TPA markedly inhibited the FSH-stimulated oestrogen production by dispersed and cultured ovarian cells. In contrast, neither serum nor TPA influenced the oestrogen production in the organ culture. This suggests that the presence of tissue architecture may prevent the inhibitory effect of serum and TPA on the FSH-stimulated oestrogen production.     

The growth-regulated gene 1B6 is identified as the heavy chain of calpactin I

The expression of 1B6, a growth-regulated sequence isolated from a Syrian hamster fibroblast cDNA library, was studied in BALB/c 3T3 cells. The level of cytoplasmic 1B6 mRNA (1600 bases) was low in quiescent cells and plateaued in mid/late G1 after the cells were stimulated with 15% fetal calf serum (FCS). Protein synthesis was not required for the induction of 1B6 mRNA; therefore, the expression of 1B6 is a primary response to serum stimulation. The induction of 1B6 mRNA was also observed after stimulation with insulin, epidermal growth factor, and fibroblast growth factor but not with platelet-derived growth factor. When quiescent cells were serum-stimulated, the percentage of cells that became committed to enter DNA synthesis was proportional to the length of their incubation with serum. To determine if 1B6 expression was also correlated with the time of exposure to serum, quiescent cells were stimulated with a pulse of 15% FCS and the abundance level of 1B6 induced by that pulse was determined. The amount of 1B6 mRNA increased with increasing time of exposure to serum and paralleled the increase in the percentage of nuclei that were induced into DNA synthesis by the serum pulse. Comparison of the nucleotide sequence of the p1B6 cDNA to the GenBank database revealed a striking identity of 1B6 to the 3' end of p36, the heavy chain of calpactin I. The previous characterization of p36 as a substrate for tyrosine kinases suggests a possible role for 1B6/p36 in cell proliferation.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

c-fos reduces growth factor requirements for mitogenic stimulation of L6 rat myoblasts

Addition of fetal calf serum (FCS) to serum-deprived L6J1 rat myoblasts increases fos-like immunoreactivity. The nuclear immunoreactivity reached a maximum 2 h after serum addition. Effects of the c-fos protein on myoblast proliferation were analyzed in L6J1 rat myoblasts transfected with the murine c-fos gene under control of a metallothionein promoter. L6J1 myoblasts with elevated expression of transfected c-fos reached higher cell densities than neo transfected control myoblasts when approaching a stationary phase in normal culture conditions (5% FCS). The differences in cell densities were even more pronounced at low serum concentrations (0.5% FCS). c-fos transfected cells also had a faster growth rate than did control cells in serum-free medium supplemented with calcium chloride, lithium chloride, sodium selenite, hydrocortisone, and insulin. The cell morphology of c-fos transfected L6J1 myoblasts was not affected compared to control myoblasts. These results suggest that c-fos protein expression in L6J1 myoblasts is activated by serum and that mitogenic stimulation of L6J1 myoblasts is facilitated by the presence of elevated amounts of c-fos protein.     

Mitogen-mediated protein phosphorylation in Werner's syndrome fibroblasts

DNA synthesis of WF-1 fibroblasts derived from a patient with Werner's syndrome was stimulated by fetal calf serum and adult human serum but not by various mitogens including epidermal growth factor, platelet-derived growth factor (PDGF), fibroblast growth factor, insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). To clarify the cause of nonresponsiveness to these mitogens, we compared the rate of protein phosphorylation in normal fibroblasts HF-O and Werner's WF-1 cells. PDGF and TPA enhanced the phosphorylation of a Mr 80 K protein, which is known to be a substrate for protein kinase C, both in HF-O and WF-1 cells. This indicates that the pathway involving PDGF receptor, phosphatidylinositol turnover and protein kinase C activation is operational in WF-1 cells. Several species of phosphoproteins of Mr 250 K, 135 K, 110 K, 78 K and 42 K were detected in normal HF-O cells by immunoprecipitation using an anti-phosphotyrosine antibody. The same species of phosphoproteins were detected in Werner's WF-1 cells at passage 6, but only when treated with various mitogens and were not detected in WF-1 cells at passage 10 even after the PDGF- or TPA-treatment. These results suggest that the reduction of phosphorylation of these target proteins may be in part responsible for the diminished mitogenic responsiveness of Werner's fibroblasts.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.     

Interaction of epidermal growth factor with initiators and complete carcinogens in the C3H10T1/2 cell culture system

Unlike 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor (EGF) could not promote the appearance of type III foci from initiated C3H10T1/2 cells. At appropriate concentrations, EGF induced the formation of type II colonies in the absence of any initiator. At higher concentrations, EGF suppressed the induction of both type II and type III colonies elicited by methylcholanthrene.