An Autocrine Activity Capable of Substituting for Serum in Cell Cultures

Abstract

Provided that high cell densities (above 10⁶/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-PI) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-PI cells permits the serum-free survival of FDC-PI cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-PI for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-PI enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-PI cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-PI-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.     

Multiple autocrine factors including an extracellular matrix protein are required for the proliferation and spreading of human colon carcinoma cells in vitro

The human colon carcinoma cell line LIM1215 proliferates and changes morphology (spread) in a cell density-dependent manner in response to epidermal growth factor (EGF). At high density, production of autocrine transforming growth factor-alpha enables the cells to proliferate and spread in the absence of exogenous EGF or serum. At low cell density (< 1 x 10(4)/cm2) EGF alone fails to elicit a mitogenic or morphological response and requires the presence of conditioned medium (derived from high cell density serum-free culture of the same cells) to exert its effects. This synergy between EGF and LIM1215 conditioned medium was investigated further. Using a low cell density assay and fractionated LIM1215 conditioned medium, we show that EGF-mediated mitogenic and morphological responses are separable. These responses are dependent on the synergistic action of a low molecular weight autocrine survival factor and an extracellular matrix-like spreading factor(s) secreted into the culture medium respectively. We find that under low cell density, serum-free conditions, EGF alone is insufficient to rescue LIM1215 from rapid apoptotic death. Catalase or LIM1215 autocrine survival factor prevent the death of LIM1215 cells and restore their proliferative (but not morphological) response to EGF, suggesting that cell death under these conditions may be the result of oxidative stress. Combination of EGF, partially purified autocrine survival and spreading factors induced proliferation and spreading of low density LIM1215 cells similar to that observed with EGF and unfractionated conditioned medium. GRGDS peptides strongly inhibited the spreading of LIM1215 cells in the presence of EGF and the partially purified autocrine spreading factor, demonstrating that integrin receptors are involved in the spreading process. Comparison of the spreading response of LIM1215 and Colo 526 cells on ASF and various adhesion proteins indicate that ASF is not collagen-I, collagen-IV, fibronectin or vitronectin. Taken together, these results support the concept that the autonomous growth of colon carcinoma cells in vitro is dependent on the synergistic interaction between several autocrine systems.     

Growth factor responsiveness declines during adulthood for human skin-derived cells

Keratinocytes and fibroblasts were obtained from upper arm biopsies of young (22-27 years) and old (60-82 years) adult donors. Keratinocytes were grown in serum-free medium containing variable quantities of either epidermal growth factor (EGF) or a bovine hypothalamic extract known to contain keratinocyte growth factor (KGF). Fibroblasts were grown in serum-free medium containing variable quantities of EGF or insulin. Paired keratinocyte cultures were plated in serum-free medium containing 20% newborn keratinocyte-conditioned medium (NM) or 20% control conditioned medium (CM). Newborn foreskin keratinocytes were plated in 20% conditioned media derived from newborn, young adult or old adult keratinocyte cultures. In spite of large inter-donor variability, keratinocyte growth significantly decreased with age (0.05 greater than P greater than 0.01). Cell yield at 7 days showed an 8-fold increase for young adults over the KGF dose range treated, but only a 4-fold increase for old adults. Young adult cells in varying concentrations of EGF achieved 3-fold to 5-fold higher densities than old, although EGF was not stimulatory for either adult age group. Donor age-associated loss of growth factor responsiveness was confirmed with dermal fibroblasts derived from the same biopsies. Newborn but not adult keratinocytes were stimulated by NM, while newborn cells were not stimulated by either young or old adult conditioned media (YM or OM). An epidermal proliferation index, incorporating both donor cell yield and cell yield of newborn cells in donor conditioned medium, was significantly different (P less than 0.01) for newborn vs. young or old adult cells. Our findings confirm that a decreased proliferative capacity is measurable within adulthood, and suggest that this decrease may be due to a reduced ability to synthesize or respond to mitogens, including autocrine factors.     

Stimulation of diploid fibroblast growth with serum-free medium conditioned by mezerein-treated monocytic leukemia cells

Medium conditioned by mezerein-treated human acute monocytic leukemia cells (THP-1) stimulated human fibroblast replication. Maximum mitogenic activity was elaborated by THP-1 cells with a 24-hr incubation in 10(-7) M mezerein (activator phase) followed by a 36-hr incubation in insulin-supplemented serum-free Roswell Park Memorial Institute (RPMI)-1640 medium (effector phase). Growth stimulation was not due to the presence of residual mezerein. We previously reported that leukemia cells also produced a growth inhibitor. Fibroblast stimulation was resolved by isoelectrofocusing into several active fractions separate from the growth inhibitory activity for malignant mammary cells. Conditioned medium was mitogenic for fibroblasts in the presence of high concentrations of fetal bovine and human whole blood sera. Growth stimulation was observed in plasma-derived serum only when supplemented with exogenous platelet-derived growth factor. Thus, this THP-1 cell product does not fulfill the role of a competence factor.     

The Fibroblast Mitogenic Activity Released from Human Basophilic Cell Line KU812 is Separate from Tryptase and PDGF Expression

The human leukaemia cell line KU812 has previously been used to study basophil differentiation. In this study the authors analysed the capacity of KU812 to produce the mast cell proteinase tryptase and to synthesize factor(s) mitogenic for fibroblasts. KU812 cells were treated with tetradecanoyl-phorbol-13-acetate (TPA), conditioned medium from the human T-cell line Mo (Mo-CM), or cultured under serum free conditions. After 4 days the cells were analysed for cell growth, differentiation, content of tryptase, and secretion of fibroblast mitogenic activity. Mo-CM and serum starvation increased the expression while TPA treatment down-regulated the expression of FcεRI-α chain. An increase in tryptase content in cell extracts was detected after 4 days of culture in serum-free medium or in the presence of Mo-CM. KU812 conditioned media was found to have a baseline expression of mitogenic activity on normal human foreskin fibroblasts that was increased after serum starvation or after treatment with TPA. Mast cell-derived tryptase has previously been reported to be mitogenic for fibroblasts, but in this study the expression of tryptase did not correlate with the expression of fibroblast mitogenic activity in KU812 cells. Furthermore, affinity-purified lung tryptase did not show any mitogenic activity. Platelet-derived growth factor was also excluded. Although the factor(s) from KU812 cells stimulating fibroblast proliferation have not been identified, our results indicate that basophils may be potential producers of growth factors inducing fibroblast proliferation.     

Removal of phytohemagglutinin from conditioned medium by affinity chromatography

Supernatants of lymphocytes cultured with phytohemagglutinin (PHA-induced conditioned medium) are known to contain residual lectin. Studies with T cells specifically sensitized against a given antigen and maintained in culture by the T cell growth factor in conditioned medium may be hampered by the presence of PHA since the lectin could induce polyclonal activation of T cells.We developed a procedure for removing lectin from conditioned medium by affinity adsorption on porcine thyroglobulin-Sepharose. The affinity method was capable of removing detectable amounts of lectin since the mitogenic capacity for peripheral blood lymphocytes was lost after adsorption. In contrast, thyroglobulin-Sepharose adsorbed CM retained good mitogenic activity and growth-supporting capacity for human cultured T cells.     

Growth suppression of hybrids between transformed cells and normal fibroblasts in serum-free medium: Correlation with retention of human chromosomes

Somatic cell hybrids formed by crossing PG19 mouse melanoma cells with mouse embryo fibroblasts have a reduced ability to proliferate in growth factor-unsupplemented serum-free medium relative to the parental melanoma cells. The suppression of growth of the hybrid cells in serum-free medium is attributable to a strict requirement of these cells for polypeptide growth factors (insulin plus platelet-derived growth factor, fibroblast growth factor, or epidermal growth factor). In contrast, the parental melanoma cells are able to grow without exogenously added growth factors. Fifteen hybrids derived from crosses between mouse L cells and normal human skin fibroblasts also have been tested for ability to grow in growth factor-unsupplemented serum-free medium. Depending on which human chromosomes are retained, growth of these hybrids in serum-free medium is also suppressed relative to growth of the L cell parent. There appear to be several genes on different chromosomes that are involved in suppression of serum-free growth of the fibroblast × L cell hybrids. One weak suppressor gene appears to be on the human X chromosome.     

The matrix of human breast tumor cells is mitogenic for fibroblasts.

The basis of the scirrhous reaction to human breast carcinoma was investigated. When normal human skin fibroblasts were plated on the preformed extracellular matrix of human breast tumor cells, a remarkable series of changes was observed. The matrix of the tumor cells was mitogenic for the fibroblasts. An increased growth rate and a fourfold increase in cell density was observed. There was also a change in cell morphology and in the pattern in which the cells grew, with an apparent loss of contact inhibition. The spindle-shaped fibroblasts became more elliptical and grew in a series of whorls and dense ridges with spaces between them. These observations were made with the use of newborn foreskin fibroblasts and the matrix of an established line of human breast cancer cells, ZR75-1. No such effect was seen when fibroblasts were plated on their own preformed matrix, on the matrixes of other cell types, on various type-specific collagen gels, or on a combination of collagen and fibronectin or when fibroblasts were grown in media conditioned by the ZR75-1 cells. A floating tumor cell matrix added to the cell media also did not provide the mitogenic stimulus. Apparently, fibroblasts required direct contact with the tumor cell matrix for the mitogenic response to occur. In vivo, the matrix of breast tumor cells may modulate the growth and the morphology of host stromal cells. Collagen is a major synthetic product of fibroblasts. The stimulation of stromal cells to proliferate by adjacent breast tumor matrix may be the basis of the desmoplastic reaction, the intense fibrotic response associated with human breast cancer.     

Hepatocyte growth inhibitory factor derived from HTLV-I(+) T cell lines: effect on the epidermal growth factor-dependent proliferation of rat hepatocytes

A human T cell leukemia virus-I infected T cell line, ATL-2, produces an interleukin-2 receptor inducing factor, adult T cell leukemia (ATL)-derived factor (ADF). In the conditioned medium (CM) of ATL-2, we found an inhibitory activity on the epidermal growth factor (EGF)-dependent proliferation of primary cultured rat hepatocytes, measured by cell number and [3H]thymidine incorporation. ATL-2 CM dose-dependently inhibited hepatocyte proliferation. This activity was fractionated by gel filtration at a molecular size of 15,000 to 40,000 and was tentatively called hepatocyte growth inhibitory factor (HGI). Further fractionation with the ion-exchange column indicated that HGI was separable from ADF. Nevertheless, there was a positive correlation between HGI and ADF production, because the HGI activity was also detected in the CM of another ADF producer cell line (HUT102), while no significant HGI activity was detected in the CM of low ADF producer cell lines, ED and MOLT4.     

Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal growth factor (EGF), insulin, cholera toxin, and endothelial cell growth supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding growth factors, and in this report we have compared the effect of ECGS to purified heparin-binding growth factors. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding growth factors, alpha-endothelial cell growth factor (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic fibroblast growth factor (FGFa) stimulated [3H]thymidine incorporation by 16-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding growth factors for both endothelial cells and fibroblasts but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding growth factors had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dog mastocytoma cells secrete a growth factor for fibroblasts.

It is suspected that mast cells play a part in the pathogenesis of fibrotic diseases, but the mediators that might be involved in induction of fibrosis have not been identified. We asked whether cultured dog mast cell lines produced growth factor(s) for fibroblasts. Three mastocytoma cell lines were found to secrete proliferative activity for human, hamster, and rabbit fibroblasts. Both mastocytoma cell-conditioned medium and cell extract served as competence factors for induction of DNA synthesis in confluent mouse Swiss 3T3 fibroblasts. The mitogenic activity in the conditioned medium was stable to heat, acid, and high concentrations of chaotropic agents or organic solvents but was decreased by treatment with proteases or reducing agents. The activity had an apparent molecular mass of 10 kD and did not bind to heparin. Activity eluted in a single peak from reverse-phase HPLC, and retention time differed from that of typical mesenchymal mitogens. We offer the hypothesis that mast cells produce growth factors for fibroblasts, possibly including a novel growth factor, and that this may contribute to pathologic fibrosis.     

Urokinase and the mechanism of osteoblastic metastases by prostate cancer

We have examined the pathogenesis of osteoblastic metastases by attempting to identify growth factors for cells of the osteoblast phenotype which are produced by prostatic cancer tissue. In initial studies, surgical specimens of human prostate cancer (CA) and benign prostatic hyperplasia (BPH) tissue were employed as a source of prostatic tissue, and mitogenic activity for osteoblastic cells was assessed in primary cultures of fetal calvarial cells and in osteosarcoma cells. These studies identified the presence of acid stable peptide mitogens for osteoblasts in both CA and BPH. To pursue these findings, the prostate cancer cell line PC-3 was employed. Biochemical analysis of serum-free conditioned PC-3 medium, using mainly reverse-phase high pressure liquid chromatography, disclosed the presence of a mitogen for osteoblastic cells with a molecular weight of approximately 15 kDa. Amino acid sequencing indicated that this was an amino-terminal fragment of urokinase (UK). Further studies demonstrated abundant plasminogen activator activity in PC-3 conditioned medium and substantial messenger RNA encoding UK in PC-3 cells. High molecular weight (HMW) UK but not low molecular weight (LMW) UK was mitogenic and increased cell numbers in osteoblastic cultures but not in control fibroblast cultures. Specific, competitive binding of HMW but not LMW UK to osteoblastic cells was observed. These studies indicated that the mitogenic activity of UK indeed resides in the amino-terminal region. This was confirmed by demonstrating mitogenic activity for osteoblastic cells using a peptide containing the ‘growth factor domain’ of UK. In summary, these studies have shown that an amino-terminal fragment of UK produced by PC-3 cells has growth factor activity for cells of the osteoblast phenotype. UK fragments may therefore be of importance in the pathogenesis of osteoblastic metastases produced by prostatic cancer cells.     

Dermatophagoides extract-treated confluent type II epithelial cells (cA549) and human lung mesenchymal cell growth.

BACKGROUND: Chronic severe persistent asthma is associated with damaged epithelial cells with discontinuous tight junctions that contribute to dysregulated fibroblast and endothelial cell (mesenchymal) growth. Dermatophagoides species-derived proteases have been shown to cause damage to epithelial cell tight junctions. OBJECTIVE: To determine whether Dermatophagoides species can stimulate confluent A549 (cA549), a cell type with discontinuous tight junctions that approximate differentiated type II cells, to undergo altered growth and secrete putative soluble factors that affect the growth of human lung fibroblasts and microvascular endothelial cells. METHODS: Dialyzed Dermatophagoides pteronyssinus or Dermatophagoides farinae extracts (0, 300, 600, and 1000 AU/mL) were cultured with and without cA549 in serum-free media for 24 hours. After changes in cA549 growth were recorded, conditioned media from extracts with cA549 (CM) and without cA549 (control media [CTLM]) were transferred to fibroblasts and endothelial cells for 24 hours. Fibroblast and endothelial cell growth responses to CM and CTLM were observed and measured. RESULTS: All conditions showed greater than 95% cell viability. Confluent A549 showed dose-dependent growth changes characterized by increased aggregation when incubated with 300, 600, and 1000 AU/mL of D pteronyssinus in serum-free media relative to control. The CM, but not the CTLM, induced dose-dependent aggregation by fibroblasts and endothelial cells. Fibroblasts also showed decreased adhesion when incubated with CM. Dermatophagoides farinae-treated cA549 showed similar but weaker results. The use of serum, boiled CM, or boiled extract inhibited these findings. CONCLUSIONS: Dialyzed Dermatophagoides species extracts altered cA549 growth and stimulated the secretion of factors that dysregulate mesenchymal cell growth in vitro.     

Stimulation of the differentiation of osteogenic rat bone marrow stromal cells by osteoblast cultures

The effect of conditioned medium (CM) from rat calvaria (RC) cel cultures on the growth and differentiation of osteogenic cells in rat bone marrow stromal cell (BMSC) cultures was investigated. Control cultures received either CM from periodontal ligament fibroblast cultures or fresh medium. RCCM stimulated the formation of nodules of bonelike tissue in bone marrow stromal cell cultures in a dose-dependent manner,and the maximal stimulation was associated with the osteoblast-enriched cell populations of the RC cultures. Ultrafiltration demonstrated that activity was confined to a CM fraction of 10- to 30-kilodalton molecular size. The activity was sensitive to boiling and trypsin treatments, but was not affected by neutralizing antibodies to transforming growth factor beta or insulin-like growth factor I or II. RCCM was found to initially increase the number and proportion of cells that expressed alkaline phosphatase activity, although the proportion of alkaline phosphatase-positive cells subsequently declined. These data were consistent with an initial stimulation of proliferation of a subpopulation of osteoprogenitor cells within the cultures, followed by their differentiation. The results suggest that mature osteoblasts may produce a paracrine growth factor that can stimulate the differentiation of osteoblasts from precursor cells.     

Macrophage-derived growth factor for fibroblasts and Interleukin-1 are distinct entities

P388D1, a mouse macrophagelike cell line, was adapted to grow continuously in an unsupplemented, serum-free culture medium and continued to elaborate substances that were mitogenic for quiescent mouse fibroblasts (BALB/c 3T3 cells) and for thymocytes suboptimally stimulated with lectins. We have previously described [37] the fibroblast mitogenic activity as a macrophage-derived competence factor (MDCF). Serum-free, macrophage-conditioned culture medium was concentrated 1,000-fold by a combination of ultrafiltration (hollow fiber) and lyophilization. Concentrates of medium were subjected to gel filtration (Sephadex G-75 or G-150), and the fractions were assayed for mitogenic activity (MDCF) on density-arrested BALB/c 3T3 cells and for Interleukin-1 (IL-1) activity in suboptimally stimulated (Con A) mouse thymocytes. The apparent molecular weight (MW) of MDCF activity was estimated at 56,000 daltons, whereas the peak of IL-1 chromatographed at an apparent MW of 14-16K daltons. There was no detectable IL-1 activity in the MDCF fractions and no detectable MDCF in the IL-1 fractions. These data indicate that P388D1 cells produce both MDCF and IL-1 activities under continuous serum-free conditions and that the two activities are not identical. Stimulation of responsive mononuclear phagocytes with lipopolysaccharide and/or lymphokine-rich supernates resulted in a differential modulation of MDCF and IL-1 activities. Finally, antibody-purified IL-1 had no significant ability to stimulate DNA synthesis in quiescent fibroblasts at concentrations that were mitogenic for thymocytes. However, IL-1 did augment the mitogenic activity of suboptimal amounts of platelet-derived growth factor (PDGF), another competence factor. Further studies revealed that neither the generation nor the activity of MDCF was modulated by the presence of various inhibitors of proteolytic enzymes.     

Effects of platelet-derived growth factor and epidermal growth factor on antigen-induced proliferation of human T-cell lines.

When the serum content of tissue culture medium is reduced from 10% to 1%, the capacity of T cells to proliferate in response to antigen within that medium is dramatically reduced. Physiological concentrations of platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) are able to partially replace the requirement for serum, in that they are able to increase antigen-driven T-cell proliferation at a serum concentration of 1%. Neither growth factor is mitogenic for T cells in the absence of antigen, and neither is able to act synergistically with T-cell growth factor (TCGF) or IL-2) in the absence of antigen. Antigen-presenting cells (APC) pulsed with antigen in the presence of PDGF or EGF are able to stimulate antigen-specific T-cell proliferation to a greater extent than antigen-presenting cells pulsed in the absence of exogenous PDGF or EGF. Both growth factors increase the expression of MHC Class II antigens on antigen-presenting cells.     

IGF-I and IGF-binding proteins: stimulatory and inhibitory factors secreted by human prostatic adenocarcinoma cells.

Deregulation of growth observed in malignant cell cultures has been assumed to be the result of increased secretion by these cells of autocrine growth factors, as well as the decreased sensitivity of these cells to inhibitory molecules which are diffused from normal or transformed cells. Our results show that PC-3 cells secreted into the medium, factors having stimulatory and inhibitory activities. We found an IGF-like molecule in medium conditioned by PC-3 cells. Its concentration was less than 1 ng/ml of conditioned medium. We demonstrated that PC-3 cells have receptors for IGF-I and are stimulated by this growth factor. However, the dose response curve shows that 1 ng/ml of IGF-I is not sufficient to indicate autocrine growth regulation by IGF of prostatic carcinoma cells. IGF-binding proteins of 90,000, 45,000, 34,000 and 28,000 molecular weight were also secreted by PC-3 cells. It is noteworthy that the secreted proteins which had the greatest inhibitory effect on chick embryo fibroblast growth also has the strongest IGF-binding activity. The probability that the IGF-binding protein secreted by PC-3 cells inhibited serum stimulation of DNA synthesis by preventing stimulation induced by IGF present in the serum is discussed. It is of interest that these IGF-binding proteins inhibited chick embryo fibroblast proliferation but did not inhibit PC-3 cells. This is in agreement with the assumption that IGF present in the medium is not an autocrine growth factor for these cells.