Source and target cell specificities of a conditioned medium factor that increases choline acetyltransferase activity in cultured spinal cord cells

A macromolecular factor(s) in muscle conditioned medium (CM), when applied to spinal cord (SC) cells in culture, causes large increases in the activity of choline acetyltransferase (CAT), the enzyme which synthesizes the neurotransmitter acetylcholine. We have found apparent specificity of both species and cell type for the production, release, or action of this CAT stimulation component (CSC). Rat and mouse muscle CMs contained CSC which was active in mouse SC cells; chick muscle CM did not. In addition to muscle CM, the CM from cell cultures of mouse heart, liver, and kidney contained CSC. However, CM from secondary cultures of liver cells contained little if any CSC. These apparent specificities were not due to differences in the protein content of either the cells providing CM or of the CM itself. There was also apparent specificity of response to CSC among cholinergic cells in culture. Cultures of cells from only two of four regions of the mouse central nervous system, and from one of five neuronal cell lines tested, had increased CAT activity after treatment with muscle CM. The response in NG108-15 neuroblastoma-glioma hybrid cells was further characterized, and was used to develop a more convenient and rapid assay for CSC.     

Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. I. Effects of muscle-conditioned medium

The enzymatic machinery for neurotransmitter synthesis and breakdown have been compared in sister cultures of newborn rat sympathetic neurons grown for 12-28 days either in the presence (CM+ cultures) or in the absence (CM- cultures) of a culture medium conditioned by rat skeletal muscle cells. Neuron numbers, total protein, and lactate dehydrogenase activities were identical in CM+ and CM- cultures. Choline acetyltransferase activity was 27- to 100-fold higher in homogenates of CM+ than CM- cultures, whereas acetylcholinesterase activity was 2.5-fold lower. The activities of tyrosine hydroxylase (TOH), DOPA decarboxylase, and dopamine beta-hydroxylase were all about twofold lower in homogenates from CM+ cultures. All these effects were also observed in homogenates of sympathetic neuron cultures grown with and without a macromolecular factor partially purified from CM (Weber, J. (1981). Biol. Chem. 256, 3447-3453.). Experiments of mixing homogenates from CM+ and CM- cultures suggested that the differences in each of the enzyme activities did not result from differences in the concentrations of hypothetical reversible enzyme activators and/or inhibitors. In addition, the deficit in TOH activity in CM+ cultures resulted from a decrease in the enzymatic Vmax with no significant variation in the apparent Km's for the substrate and the cofactor. An identical decrease in the Vmax was observed if TOH was assayed under phosphorylating or nonphosphorylating conditions, suggesting that this decrease did not result from differences in the state of enzyme phosphorylation. Immunoprecipitation curves of TOH activity by an anti-TOH antiserum were parallel when performed on homogenates from CM+ and CM- cultures, suggesting a difference in the number of enzyme molecules without detectable alteration of their kinetic properties.     

Comparison of the effects of elevated K+ ions and muscle-conditioned medium on the neurotransmitter phenotype of cultured sympathetic neurons

Neuronal depolarization and culture media conditioned by certain nonneuronal cells (CM) are known to exert opposite effects on the expression of cholinergic and noradrenergic traits in cultured rat sympathetic neurons. We have compared their effects on the developments of choline acetyltransferase (CAT), tyrosine hydroxylase (TOH), dopa decarboxylase (AADC) and acetylcholinesterase (AcChE) in these cultures. A macromolecular factor which was partially purified from CM increased CAT development in a dose-dependent manner and depressed the development of TOH and AADC by 5- to 10-fold. In the presence of intermediate concentrations of this partially purified factor, both CAT and catecholamine synthesizing enzymes developed to high levels, whereas high concentrations caused a long-lasting, but not total, impairment of TOH development. The effects of CM on both CAT and AADC activities resulted from variations in the number of immunotitratable enzyme molecules. Conversely, K+ ions (30-40 mM) depressed the development of CAT by 90% and stimulated TOH development 2.5-fold. Cultures grown with CM in high K+ medium had similar CAT and TOH activities as compared to those cultures grown without CM in low K+ medium suggesting that CM and K+ ions had antagonistic effects on the expression of these enzymes. However, K+ ions did not affect the development of AADC in these cultures. CM suppressed in a reversible manner the development of the 16 S form of AcChE. In the presence of 40 mM K+, the rate of development of AcChE was reduced. In particular, the development of 16 S AcChE was strikingly impaired, although not totally suppressed. The effect of elevated K+ ions on the percentage of 16 S AcChE was rapidly reversible. It is concluded that CM and elevated K+ ions have antagonistic effects on CAT and TOH, but not on AADC development; AcChE, in particular its asymmetric 16 S form, is regulated independently of the cholinergic/noradrenergic status of sympathetic neurons.     

Increase of Choline Acetyltransferase by Colchicine in Primary Cell Cultures of Spinal Cord

Abstract: Colchicine (5–10 μ M ) increased choline ace-tyltransferase (ChAT) activity 5–10-fold and suppressed acetylcholinesterase (AChE) and glutamate decarboxylase (GAD) activities to 30% and 50%, respectively, of the levels of control cells in mouse spinal cord cells cultured for several days. The synthesis of radiolaheled acetylcholine (ACh) from [¹⁴C]choline was also enhanced 4.6-fold, although the uptake of [¹⁴C]choline into cells was decreased to 80% of control level. Neither the incorporation of [³H]Ieucine into protein nor the total amount of protein was increased by colchicine. Vinblastine also increased ChAT activity while cytochalasin B was not effective. Immunochemical titration study revealed that the increase of ChAT activity by colchicine was due to the accumulation of ChAT molecules. Co-culture of spinalcord cells with skeletal muscle markedly stimulated ChAT activity, and the addition of colchicine to the co- cultures showed greater than additive effect. These observations indicate that colchicine increases ChAT molecules in a specific manner, that the stimulatory effect of colchicine on ChAT activity is possibly mediated via the interaction with microtubules, and that the increase of ChAT activity is based on a mechanism different from that of co-cultures with skeletal muscle cells.     

INHIBITION OF MALIGNANT CELL PROLIFERATION BY CULTURE MEDIA CONDITIONED BY CARDIAC OR SKELETAL MUSCLE

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G-50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS-PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.     

Action mechanisms of physiological doses of androgen or pharmacological doses of estrogen in growth stimulation of Shionogi carcinoma 115 in mice

Shionogi carcinoma 115 (SC115) had been accepted for 20 yr as an androgen-dependent mouse mammary tumor. However, we recently found that the growth of SC115 tumors in vivo is also stimulated by pharmacological doses of estrogen through estrogen receptor. In the present study, action mechanisms of androgen or high doses of estrogen in the growth stimulation of SC115 were examined using a cloned cell line (SC-3) derived from the SC115 tumor. In serum-supplemented [2% steroid-free fetal calf serum-Eagle's minimum essential medium (MEM)] and serum-free [HAM F-12: MEM (1:1, v/v) containing 0.1% bovine serum albumin] media, testosterone (Test, 10(-9)-10(-6) M) significantly increased both cell number and DNA synthesis of SC-3 cells (by up to 10-fold), whereas oestradiol-17 beta (10(-12)-10(-6) M) had no such effects; the Test-induced growth was completely inhibited by the addition of a 100-fold molar excess of cyproterone acetate (CA). The serum-free medium cultured with SC-3 cells in the presence or absence of 10(-8) M Test was collected [conditioned medium (CM) or conditioned medium without Test (CM-)], and then Test in CM was removed by Gel filtration using Sephadex G-100 or inactivated by the addition of a 100-fold molar excess of CA. In the serum-free culture system, the addition of the CM without Test activity significantly enhanced both number of SC-3 cells and DNA synthesis in the cells, whereas CM(-) had no such effects. The present findings suggest that growth-stimulatory activities of androgen and high doses of estrogen on SC115 cells are mediated by growth factor(s), secreted from SC115 cells through androgen receptor and from some of nontransformed cells through estrogen receptor, respectively.     

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro

We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations. Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.     

Multilineage, non-species specific hematopoietic growth factor(s) elaborated by a feline fibroblast cell line: enhancement by virus infection

In studies designed to determine the role of feline leukemia virus (FeLV) in the pathogenesis of marrow failure in the cat, we tested medium conditioned by uninfected and FeLV-infected feline embryonic fibroblasts (FEA) for its effect on hematopoietic colony growth in culture. As opposed to an inhibitory effect, we found that the conditioned medium (CM) from FEA or FEA/FeLV increased the in vitro growth of multiple hematopoietic progenitor cell types including erythroid burst-forming cells (BFU-E), granulocyte/macrophage colony-forming cells, megakaryocytic colony-forming cells, and mixed-cell colony-forming cells. Furthermore, CM enhanced the growth of progenitors in cultures of mouse or human marrow cells, as well as cat marrow cells. Stimulation of feline BFU-E was most marked with an increment in growth of 400% over control. The human burst promoting activity (BPA) of the CM was equivalent or better than other CM available in our laboratory. The evidence suggest that the growth-promoting activity is a constitutive product(s) released by FEA which was enhanced eightfold with virus infection. Studies with non-adherent and T-lymphocyte-depleted human marrow cells and human peripheral blood cells suggest that the growth factor(s) acts directly on progenitor cells and not through readily identified accessory cells. These findings are consistent with the concept that mesenchymal cells such as fibroblasts have the capacity to release hematopoietic growth factor(s) capable of acting on primitive hematopoietic progenitors. The results provide an example of how injury of such cells, through virus infection, may enhance growth factor(s) release and influence the hematopoietic microenvironment.     

Sciatin: a myotrophic protein increases the number of acetylcholine receptors and receptor clusters in cultured skeletal muscle

Factors present in neural extracts or in media conditioned by neurons have been shown by others to increase both the number of acetylcholine receptors (AChRs) and the number of receptor clusters in cultures of embryonic skeletal muscle. We have recently shown that the glycoprotein, sciatin, exerts trophic effects on developing muscle in vitro. In the present study, we investigated the effect of sciatin on AChRs in aneural cultures of chick skeletal muscle. Sciatin caused a significant increase in the number of AChRs/dish as measured by binding of ¹²⁵I-α-bungarotoxin (α-Btx) and in acetylcholinesterase (AChE) activity/dish in differentiating muscle cells. The increase in AChRs elicited by sciatin was due solely to increased receptor synthesis and incorporation. The rate of AChR synthesis in sciatin-treated cultures was as much as five times the control rate and was significantly reduced by cycloheximide (10 μM). AChR degradation was unaffected by the myotrophic protein. Although the number of AChRs/dish was increased by sciatin during myogenesis, AChR specific activity, expressed as picomoles ¹²⁵I-α-Btx bound/mg cell protein, was only transiently increased by the myotrophic protein. This contrasted with AChE specific activity in sciatin-treated cultures which remained elevated throughout differentiation. Autoradiographs of ¹²⁵I-α-Btx-labeled cultures showed that sciatin caused an increase in the number and size of AChR “hot spots” and maintained the integrity of these AChR clusters in aneural muscle cultures for up to 5 weeks. At this time control cultures had completely degenerated. The mechanism by which sciatin enhanced the synthesis of AChRs appeared to be distinct from that of tetrodotoxin (TTX), an agent which abolishes muscle activity. However, like theophylline, sciatin might evoke increased synthesis of AChRs via regulation of cyclic AMP since the myotrophic protein increased cAMP both in cells and in conditioned medium. The results of this study suggest that sciatin may be related to the diffusible factor(s) from motor neurons described by others which has trophic effects on AChRs. Furthermore, we suggest that this myotrophic protein may be responsible for the clustering of AChRs and maintenance of receptor clusters at neuromuscular junctions in developing avian muscle.     

Assays for cholinergic properties in cultured rat Schwann cells

Cultured rat Schwann cells did not contain detectable levels of choline-acetyltransferase (less than 0.5 pmol ACh min-1 mg-1 of protein) or of acetylcholinesterase and nicotinic acetylcholine receptors. After adding Schwann cells to primary rat myotube cultures, the level of cholineacetyl-transferase in the co-cultures increased after three weeks to as high as 5 pmol ACh min-1 mg-1. The activity appearing in co-cultures sedimented at approximately 4S, and was inhibited 50% by 4(1-napthylvinyl)pyridine in the concentration range of 10-50 microM. After treatment of co-cultures with anti rat neural antigen-1 (RAN-1) and complement, 70-80% of the activity was lost, suggesting that it is induced in the Schwann cells. Attempts to obtain the effect by exposure of Schwann cells to medium conditioned by the myotube cultures, or by co-culture with fibroblasts gave levels of activity at or below the limit of detection.     

Head and trunk neural crest in vitro: Autonomic neuron differentiation

Isolated cultures of premigratory neural crest cells were used to study the initial stages of autonomic neuron development. Autonomic neurons are phenotypically characterized on the basis of their neurotransmitter synthetic enzymes, dopamine β-hydroxylase (DBH) and choline acetyltransferase (CAT). DBH converts dopamine to norepinephrine in noradrenergic neurons while CAT synthesizes acetylcholine from choline in cholinergic neurons. Activities of both enzymes were detected in isolated cultures of trunk neural crest and head neural crest. DBH was detected at all culture ages examined (from 1 to 20 days) whereas CAT activity was first detected only after 5 days in vitro. While specific enzyme activity of DBH peaks on Day 6 and specific enzyme activity of CAT peaks on Day 10, absolute activity for both enzymes increases throughout the 20-day culture period. DBH and CAT develop in vitro without any spinal presynaptic input, without typical target tissue interactions (such as blood vascular elements or heart tissue), and without addition of conditioned medium factors.     

Production of GABA by cultured hippocampal glial cells

Medium conditioned by cultured hippocampal glial contains an inhibitory factor that can hyperpolarize and suppress neuronal activity. Using biochemistry, electrophysiology, pharmacology, and mass spectrometry, we have identified the inhibitory factor as GABA (gamma-aminobutyric acid). Like GABA, the inhibitory factor increases chloride and potassium currents in neurons, which can be blocked by bicuculline. Mass spectrometry analysis of conditioned medium reveals peaks that are identical to that for GABA. Up to 500 micromolar GABA is found in conditioned medium from glial cultures. No GABA is found in conditioned medium from neuronal cultures. Hippocampal glia make much more GABA than cortical glia or glia from other brain regions. It is not clear how hippocampal glia synthesize GABA. Although they express GAD mRNA and adding glutamate to the culture medium increases the amount of GABA produced, other data suggest that glia do not use GAD to make GABA. Identifying the mechanism(s) by which GABA is produced by hippocampal glia would help clarify its role in modulating neuronal activity in the brain.     

Role of electrical activity and trophic factors during cholinergic development in dissociated cultures

The role of electrical activity in the developmental regulation of cholinergic neurons was investigated in dissociated spinal cord--dorsal root ganglion (SC-DRG) cultures. Application of tetrodotoxin (TTX) during the first 6 days after plating had no effect on choline acetyltransferase (CAT) activity. Suppression of electrical activity during the 7th day decreased CAT to 68% of control. These decreases in CAT activity were still apparent 2 weeks after removal of the TTX. GABAergic neurons, as indicated by glutamic acid decarboxylase activity and high affinity [3H]GABA uptake, were not affected by TTX treatment. Addition of 8-bromo-cAMP or conditioned medium obtained from SC-DRG cultures at certain developmental periods produced dose-dependent increases in CAT levels on TTX-treated cultures as compared with those treated with TTX alone. Similar studies with 8-bromo-cGMP revealed no significant effects on CAT activity. Vasoactive intestinal peptide (VIP) produced a dose-dependent increase in CAT activity when added to cultures between days 12 and 14. Similar studies conducted on younger cultures (days 5-7) or older cultures (days 21-23) revealed no increases in CAT activity. Addition of 0.1 nM VIP to TTX-treated cultures resulted in CAT levels which were not significantly different from those of electrically active controls. These data suggest that cyclic AMP, VIP, and trophic substances in conditioned medium may have roles in the mechanism of cholinergic toxicity produced by electrical blockade of developing spinal cord neurons.     

Regulation of neurotransmitter synthesis: from neuron to gene.

We are interested in the molecular mechanisms of the regulation of neurotransmitter related gene expression by neurotrophic factors and neuronal activity. Previous work has shown that conditioned medium of muscle cells induces choline acetyltransferase (CAT) activity and represses tyrosine hydroxylase, dopamine-beta-hydroxylase and aromatic L-amino acid decarboxylase (AADC) activities in cultured sympathetic neurons. Here, we show that a new muscle-derived cell line secretes two factors which induce CAT activity in spinal cord cultures; one of those is related to LIF, a CAT inducing factor for sympathetic neurons. Preliminary data are presented on the structure of the human AADC and CAT genes. Putative promoter regions have been coupled to reporter genes; transient transfection experiments will allow us to determine the promoter elements responsible for the regulation by neurotrophic factors. We also summarize the distribution of AADC-immunoreactive cells in rat and cat brain, which will be used as a reference for the study of the specificity of expression of AADC promoter in transgenic mice.     

Acetylcholine sensitivity of innervated and noninnervated Xenopus muscle cells in culture

The chemosensitivity of Xenopus muscle cells grown in culture to iontophoretically applied acetylcholine (ACh) in the presence or absence of neurons was examined. Muscle cells grown without nerve cells are sensitive to ACh over their entire surface (2.4 mV/pC) with occasional spots of high chemosensitivity (“hot spots”). In cultures containing neural tube cells, the ACh sensitivity of muscle cells increased by approximately 50% regardless of the presence of nerve contacts or functional synapses. A similar increase in the ACh sensitivity was observed in muscle cells cultured in medium conditioned by neural tube cells. The ACh sensitivity of the extrajunctional region in functionally innervated muscle cells was not different from that of noninnervated cells growing in the same cultures. However, the chemosensitivity at the junctional region was about fivefold higher than that of the extrajunctional area. This increase in junctional chemosensitivity may well account for the increase in miniature endplate potential amplitude which has previously been reported to occur during nerve-induced ACh receptor accumulation.     

Neuronal and glial enzyme studies in cell culture

Summary Newborn BALB/c mouse brain was cultured as disaggregated cells after serial trypsin dissociations. The ontogeny of the cultures was followed by assays of cell number, deoxyribonucleic acid, and protein content and by the activities of three enzymes considered to be markers of neuronal differentiation. Aliquots of the freshly dissociated cells were assayed for choline acetylase, acetylcholinesterase, and glutamic acid decarboxylase activities and compared with intact brain. The percentages of recovery of activities, expressed as¹⁴C product formed per mg of protein per 10 min, at pH 6.8 and 37°C, were 37% for choline acetylase, 54% for acetylcholinesterase, and 24% for glutamic acid decarboxylase. The remainder of the freshly dissociated cells were placed into culture; enzyme assays were performed as the cells multiplied and then when the cultures became static. Choline acetylase activity increased as the cells rapidly divided, and glutamic acid decarboxylase activity increased only after the cultures became confluent. Under the culture conditions, acetylcholinesterase was not induced, despite active synthesis of acetylcholine. Neuroblastoma clone N18, C1300 cell line, was grown in cell culture, and the activity of acetylcholinesterase was measured as the cells multiplied and came to confluency. The specific activity of mouse neuroblastoma acetylcholinesterase increased 25-fold when the rate of cell division was restricted. The rate of cell division could be regulated by adjusting the serum concentration. By removing fetal calf serum during the growth period, cell division ceased, and acetylcholinesterase activity was significantly and rapidly induced. Choline-O-acetyltransferase specific activity was measured in rapidly dividing and in static cultures. Its specific activity was highest in nondividing cultures, compared to cultures containing actively dividing cells (6-fold), and the specific activity of thymidylate synthetase was increased 2.5-fold in actively dividing cultures, compared to static cultures. Glioblastoma cells obtained from the rat astrocytoma, clone C6, were grown in culture, and glucose metabolism was measured in control cultures, and in cultures containing norepinephrine (0.017 mg per ml). Norepinephrine produced a 50% inhibition in the incorporation ofd-[¹⁴C]glucose. Cells incubated for 2 hr in the presence ofd-[¹⁴C]glucose, washed and then incubated in control medium or in medium containing norepinephrine, resulted in the release of greater than 50% of radioactive metabolites in the norepinephrine treated plates. Norepinephrine caused a 50% increase in¹⁴CO₂ production in glioblastoma cells incubated withd-[1-¹⁴C]glucose. Norepinephrine, under similar conditions, did not affect the metabolism of glucose in clone C46, C1300 mouse neuroblastoma cells. Portions of this work were supported by a research grant (6-444946-58605) from the American Cancer Society.     

Chlorella vulgaris (Chlorellaceae) does not secrete autoinhibitors at high cell densities

Filtrates (conditioned medium) from high-density Chlorella vulgaris cultures in photobioreactors were obtained and tested for autoinhibitory activity under different conditions. Exponentially growing cells were inoculated at low initial cell concentration (2 × 10⁵ cells/ml) in 90% conditioned medium (CM) supplemented with 10% fresh medium (FM) at low (atmospheric) CO₂ levels. The time sequence of DNA histograms of cells in CM cultures showed that there is an accumulation of cells with two and four DNA equivalents in the culture over a period of time, signifying a blockage of cells at the division stage of the cell cycle. Examination of the chemical composition of CM showed the presence of high concentrations (> 10 mM) of bicarbonate. Adding similar bicarbonate concentrations to FM were found to have similar effects as CM cultures, causing blockage of cell division, though the intensity of the blocking effect was lower. The bicarbonate-free CM did not show any cell cycle modulating or inhibitory activity. The growth of cells cultivated at high (5%) CO₂ levels in 90% CM supplemented with 10% FM was comparable to 10% FM cultures, indicating nutrient limitation in 90% CM culture. When the 90% CM culture was supplemented with 100% nutrients, the growth rate and final cell concentration was similar to 100% FM culture. Based on these results we conclude that C. vulgaris does not secrete any autoinhibitor(s) or cell cycle modulating compound(s) under the conditions from which the CM was obtained.     

THE RELATIONSHIP BETWEEN CELL-SUBSTRATE ADHESIVENESS AND CELL GROWTH: A STUDY ON CHONDROCYTES CULTURED IN VITRO WITH CONDITIONED MEDIUM

Sparsely cultured chondrocytes from the sternae of 14-day chick embryos grew vigorously with a liquid medium using Falcon tissue culture dishes, when the conditioned medium (CM) prepared from the high-density cultures of neural retinae was used as a growth medium. In CM most cells adhered to the culture substrate, while in the fresh medium, in which cell growth was very slow, many viable cells remained unattached to the substrate. Enhancement of cell growth by CM was much less marked in cultures of chondrocytes suspended in agarose. The main effect of CM in enhancing cell growth is thus considered to be related to the increase of cell adhesiveness to the substrate. The main active factor in CM is non-dialyzable and heat-stable.     

Growth control of androgen-dependent Shionogi carcinoma cells by growth factor(s) produced from androgen-independent Shionogi carcinoma cells in a paracrine mechanism

Androgen-dependent (SC3) and -independent (CADO21) cloned cell lines were established from androgen-dependent mouse mammary tumor (Shionogi carcinoma 115). The effects of conditioned medium (CM) collected from SC3 and CADO21 cells on the anchorage-independent growth of SC3 cells in soft agar were studied. CM prepared from SC3 cells in the absence of testosterone was unable to stimulate the growth of SC3 cells, whereas CM prepared from SC3 cells in the presence of 10(-8) M testosterone stimulated the growth of SC3 cells in a concentration-dependent manner (21 colonies at 10% and 48 colonies at 20%) and this growth-stimulatory effect was not inhibited by 10(-6) M cyproterone acetate. CM prepared from CADO21 cells in the absence of testosterone was also able to stimulate the SC3 cell growth in a concentration-dependent manner (9 colonies at 10% and 19 colonies at 20%). These results suggest that the growth of androgen-dependent SC3 cells is stimulated by androgen-induced growth factor(s) produced from the same cells (autocrine mechanism) and is also regulated by autonomous growth factor(s) produced from androgen-independent cancer cells formed from the dependent cancer cells (paracrine mechanism). A suggested possible mechanism of the progression from androgen-dependent to -independent growth of cancer cells is also discussed.