The effects of steroid hormones on a human colon cancer cell line in vitro.

Estrogen analogues, moxestrol (10(-8)-10(-5) M) and ethinyl estradiol (10(-8)-10(-6) M), produced a 30% and 15% inhibition of LoVo cell growth, respectively, in serum-free Ham's F-10 medium. Under the same conditions, no growth effects were observed on these cells following the addition of progesterone or testosterone (10(-8)-10(-6) M); however, metribolone (10(-8)-10(-6) M), a synthetic androgen with glucocorticoid receptor-binding properties, moderately stimulated cell growth (18%). The synthetic antiandrogen, RU 23908 (10(-6) M), did not reduce metribolone effects, and hydrocortisone (10(-9)-10(-7) M) stimulated LoVo cell growth by 31% in serum-free medium. In medium containing 10% charcoal-treated fetal bovine serum, the inhibitory effects of estrogens were not observed, and the lower concentrations (10(-11) M) of moxestrol and ethinyl estradiol facilitated cell growth (10 to 15%). The other steroid hormones produced the same results as observed with serum-free medium. These data suggest that estrogen and glucocorticoid hormones may play an important role in the growth of colon carcinoma cells. Androgen and progesterone hormones appear to be less significant in this regard. Serum factors alter the effects of estrogen, but not of glucocorticoids.     

Effects of epidermal growth factor,fibroblast growth factor,retinoic acid and serum on anchorage-dependent and anchorage-independent growth of HRRT cells

The effects of EGF, FGF, RA and serum on anchorage-dependent and anchorage-independent growth of HRRT cells were studied. The five different types of serum tested in the present work induced a dose dependent rise in anchorage-independent growth in aggregates. FCS, SBCS and RS also supported colony formation in soft agar, whereas BS and HS had no significant effect. EGF and FGF stimulated anchorage-dependent growth of HRRT cells in monolayers. The peptide growth factors were also found to induce phenotypic transformation of the nonneoplastic HRRT cells, as measured by anchorage-independent growth in soft agar as well as in aggregates. At equimolar concentrations EGF was much more effective than FGF. The stimulating effect of EGF and FGF on cell proliferation in the aggregate form was markedly inhibited by RA. Treatment of HRRT cells with the highest noncytotoxic concentration of RA, 2 × 10⁻⁷ M, reduced the stimulating effect of both growth factors by about 60%.     

Effects of various growth factors on growth of a cloned human esophageal squamous cancer cell line in a protein-free medium.

In order to investigate molecular mechanisms of the growth of human esophageal cancer in relation to growth factors, we have recently established a protein-free culture system [Ham's F-12: Eagle's minimum essential medium (1:1, v/v)] of TE-3-OS cells (a cloned cell line from human esophageal squamous cancer, TE-3). In the present study, we first examined effects of exogenous growth factors on the growth of TE-3-OS cells. The growth of TE-3-OS cells in the protein-free medium was significantly stimulated by insulin and insulin-like growth factor (IGF)-I or IGF-II, and less effectively stimulated by epidermal growth factor (EGF) or transforming growth factor (TGF)-alpha; platelet-derived growth factor, TFG-beta, acidic fibroblast growth factor (FGF) or basic FGF had no effects. TE-3-OS cells contained specific IGF-I binding sites (110,000 sites/cell), with a Kd value of 800 pM. Moreover, the growth induced by IGF-I, IGF-II or insulin was markedly and similarly (70-80%) inhibited by anti-IGF-I receptor antibody IgG. These data suggest that IGF-I, IGF-II and insulin, as well as EGF and TGF-alpha, are important mitogens for human esophageal cancer cells and that effects of IGFs and insulin are mediated predominantly via IGF-I receptors.     

Overexpression of epidermal growth factor and insulin-like growth factor-I receptors and autocrine stimulation in human esophageal carcinoma cells

The growth-stimulatory effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and insulin-like growth factor-I (IGF-I) on the human esophageal carcinoma cell line CE48T/VGH were evaluated. Under serum-free conditions, EGF, TGF-alpha, and IGF-I promoted 3.6- to 4.1-fold increased cell proliferation. Scatchard analyses and Northern blot hybridization revealed that both the EGF/TGF-alpha receptor and the IGF-I receptor were overexpressed in CE48T/VGH cells. Furthermore, ligand-dependent autophosphorylation of the EGF receptor and the IGF-I receptor was clearly detected using antireceptor and antiphosphotyrosine antibodies. Autocrine regulation was strongly indicated by the following evidence: (a) CE48T/VGH cells were found to express TGF-alpha and IGF-I genes, (b) serum-free conditioned medium promoted the growth of CE48T/VGH cells and stimulated the autophosphorylation of the EGF/TGF-alpha receptor and the IGF-I receptor, and (c) the addition of IGF-I receptor antibodies significantly suppressed CE48T/VGH cell growth under serum-free conditions. Our studies suggest that the overexpression of EGF and IGF-I receptors and autocrine growth regulation may concertedly control the proliferation of esophageal carcinoma cells.     

Mitogenic effect of neurotrophic factors on human IMR 32 neuroblastoma cells

The effect of growth factors with neurotrophic properties on proliferation of the human IMR 32 neuroblastoma (NB) cell line was studied. A colorimetric proliferation assay and an anchorage-dependent cell culture system were used. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), neurite-inducing factor (NIF), ciliary neurotrophic factor (CNTF), and a cell-free extract from selected embryonic chick eye tissues (CIPE) were assayed for their capacity to control proliferation. Basic FGF, NGF, and CIPE stimulated proliferation of IMR 32 NB cells in serum-containing culture conditions. The NIF and CNTF had no effect. The concentration of bFGF required to induce half-maximal cell growth was 4.6 +/- 1.8 ng/ml, but the half-maximally effective dose of NGF was 7.5 +/- 2.7 ng/ml. In combination these two growth factors were additive within a small concentration range. In serum-free culture conditions bFGF affected both proliferation and cell differentiation by promoting neurite growth and cell aggregate formation. In contrast, NGF induced cell neurite outgrowth only. These results, in conjunction with the evidence that bFGF-like molecules are present, in IMR 32 NB cells may support the notion that NB cells regulate their proliferation by an autocrine mechanism. Basic FGF and NGF, two distinct neurotrophic factors, appear to be involved in the regulation of NB cell proliferation.     

Stimulation of cell proliferation and inhibition of differentiation expression by tumor-promoting phorbol esters in dog thyroid cells in primary culture

12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4 beta-phorbol 12, 13-dibutyrate (PDBU) are potent tumor promoters and share several biological activities of epidermal growth factor (EGF). We have shown previously that EGF stimulates DNA synthesis and proliferation and inhibits TSH-induced markers of differentiation in dog thyroid follicle-derived primary cultures. Using this system, we have examined the biological action of TPA and PDBU in reference to that of EGF. Low concentrations (1.6-16 nM) and to a lesser extent higher concentrations (greater than 1.6 microM) of TPA and PDBU stimulated cell proliferation in a 1% serum, hormone-supplemented medium and triggered the DNA synthesis revealed by autoradiography in cells which were quiescent before stimulation in serum-free conditions. EGF, TSH, and dibutyryl cyclic adenosine 3':5'-monophosphate separately also induce DNA synthesis, but they produce little if any effects additive to those of TPA. In fact, TPA appeared to inhibit the mitogenic effects of EGF. Moreover like EGF, phorbol esters strongly inhibited in 2 days the morphological effects of TSH and basal and TSH-stimulated iodide transport capacity and thyroglobulin messenger RNA accumulation, two markers of thyroid differentiation. TPA also inhibited the expression of differentiation stimulated by dibutyryl cyclic adenosine 3':5'-monophosphate indicating a post-cyclic adenosine 3':5'-monophosphate site of action. TPA and EGF shared long-term morphological effects such as the induction of an elongated fusiform shape, but not acute effects. The thyroid cells progressively and spontaneously escaped both the mitogenic and differentiation-inhibiting effects of TPA and PDBU, while, as shown previously, these parameters are stably modified by continuous culture with EGF. This suggests specific desensitization processes to phorbol esters. As evidence is accumulating that phorbol esters act at least partly by stimulating the calcium-activated, phospholipid-dependent protein kinase C, our results shed light on the possible key role of this kinase in carcinogenesis and in the normal control of proliferation and expression of differentiation in the thyroid gland. Additionally they suggest that complex interactions occur between the mechanisms of action of EGF and of phorbol esters in the thyroid cell.     

The effect of schedule, protein binding and growth factors on the activity of suramin.

Suramin has been shown to have antiproliferative activity, either by blocking the binding of growth factors to their receptors or by inhibiting critical cellular enzymes. In 6 different cell lines from 5 tumour types (MCF-7, MCF-7/ADRR, PC3, HT-29, UM-SCC-11B and SW-1573/IR500), we studied the effect of scheduling of suramin, of FCS and of human serum albumin (HSA), of epidermal growth factor (EGF) and of the addition of growth factors in serum-free medium on the activity of suramin. The concentration of suramin which gave 50% growth inhibition (IC50) varied from 45 microM in SW-1573/IR500 to 153 microM in PC3 cells grown in medium supplemented with 5% FCS, after 6 days of continuous exposure. Exposure for more than 4 days did not enhance the sensitivity to suramin, except in PC3. At exposure to suramin for 1 day followed by 5 days recovery, high IC50 values (greater than 0.5 mM) were observed in MCF-7 cells. In medium with 1% and 0.5% FCS these values were 3 to 8 and 14 to 26 times lower respectively. Addition of HSA increased the IC50 in PC3 and MCF-7 cells. Suramin binding to protein was dependent on the concentration of protein and of suramin. Excess of EGF in medium with different FCS concentrations did not change the IC50 values of suramin in PC3 and MCF-7 cells. Addition of EGF, fibroblast growth factor or platelet-derived growth factor in PC3 cells cultured in serum-free medium did not increase the IC50 values. Suramin was active against these 6 cell lines at clinically achievable concentrations. This activity varied depending on the cell line, exposure time and suramin concentration. The most significant factor interfering with sensitivity to suramin was the amount of protein present in the culture medium.     

Effects of the liver tumor promoter ethinyl estradiol on epidermal growth factor-induced DNA synthesis and epidermal growth factor receptor levels in cultured rat hepatocytes

The objective of this study was to determine whether DNA synthesis induced in the livers of female rats treated with ethinyl estradiol (EE) was due to direct effects of this synthetic estrogen on hepatocytes. Hepatocytes, obtained by collagenase perfusion from female Lewis rats, were cultured in serum-free medium containing low or no phenol red and supplemented with insulin, transferrin, and selenium. When present at 10-15 microM for the initial 30 h of culture, EE caused a subsequent 2-2.7-fold increase in hepatocyte DNA synthesis. Pretreatment of the hepatocytes with EE during the first 30 h of culture caused an EE concentration-dependent enhancement of their subsequent DNA synthetic response to epidermal growth factor (EGF). Pretreatment with EE shifted the EGF dose-response curve, causing a dramatic enhancement of the response to EGF beginning at 2 ng EGF/ml. The response to a saturating (25 ng/ml) dose of EGF was also greatly enhanced. Determination of the effect of EE on hepatocyte surface EGF receptors revealed that the increased responsiveness of DNA synthesis to EGF was accompanied by a twofold increase in EGF receptor number per cell. These results indicate that EE has direct, growth-related effects on hepatocytes which may contribute to liver growth induced in vivo by this tumor promoter.     

Induction and modulation of anchorage-independent growth by platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta

Transforming growth factors (TGFs) reversibly induce the anchorage-independent growth of nontransformed cells. TGF activity is often monitored by the growth of normal rat kidney (NRK) fibroblasts in soft agar, and it is known that more than one growth factor is involved in the regulation of their soft agar growth. To more clearly define the growth factors responsible for the soft agar growth of NRK cells, the effects of four growth factors were examined: platelet-derived growth factor (PDGF); TGF-beta; epidermal growth factor (EGF); and fibroblast growth factor (FGF). This study determined that PDGF induces the soft agar growth of NRK cells, in both plasma-supplemented medium and serum-free medium supplemented with FGF, and neither TGF-beta nor an EGF-related growth factor is required for this effect. It was also determined that FGF, which alone does not induce the soft agar growth of these cells, potentiates the responses of NRK cells to various combinations of PDGF, TGF-beta, and EGF. Interestingly, the effect of TGF-beta was found to depend on the growth factor composition of the medium. In the absence of EGF, TGF-beta partially inhibits the soft agar growth response of NRK cells to PDGF, whereas, in the presence of EGF, TGF-beta increases their response to PDGF. These findings indicate that at least four unrelated growth factors regulate the anchorage-independent growth of NRK cells. These findings have important implications for the use of NRK cells to assay TGFs.     

尼卡地平和表皮生长因子对胶质瘤细胞生长的影响

目的：观察钙通道拮抗剂尼卡地平（ｎｉｃａｒｄｉｐｉｎｅ）和表皮生长因子（ｒｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＥＧＦ）对恶性胶质瘤细胞系Ｕ２５１ＭＧ生长的作用。方法：在含１０％小牛血清（ｆｅｔａｌ ｂｏｖｉｎｓ ｓｅｒｕｍ,ＦＢＳ）和无血清培养基中,予不同浓度的ＥＧＦ、尼卡地平和ＥＧＦ（１０ｎｇ／ｍｌ）干预,应用ＭＴＴ比色法观察它们的细胞生长效应。结果：在无血清培养基中,ＥＧＦ具有强大的     

Characterization of the epidermal growth factor receptor in human meningioma

We have characterized the epidermal growth factor (EGF) receptor in human meningioma (biopsy) microsomes, cellularly derived microsomes, and intact meningioma cells in culture. Scatchard analysis of competition studies reveals both high and low affinity EGF binding sites in the meningiomas tested [dissociation constant (Kd) = 0.9 nM, maximum number of binding sites (Bmax) = 280 fmol/mg protein; Kd = 5.0 nM, Bmax = 660 fmol/mg protein, respectively]. The binding of 125I-EGF is specific since it is abolished by excess unlabeled EGF but not by excess unlabeled platelet-derived growth factor or insulin. Meningioma cultures preincubated with platelet-derived growth factor (10 ng/ml) at 37 degrees C shifted the 125I-EGF competition curve to the right but did not affect receptor number (100,000 sites/cell) when compared to cultures preincubated at 4 degrees C. Cross-linking studies performed with ethyleneglycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveal a major band of specifically bound EGF (Mr approximately 150,000), although the normal (Mr approximately 170,000) and another putative proteolytic form (Mr approximately 125,000) can also be seen. These results indicate that human meningiomas contain a mixed population of EGF binding sites and exhibit properties of previously described EGF receptors.     

Transforming growth factor-alpha secretion by epidermal growth factor-dependent human tumor cell lines

Pairs of cell lines from spontaneous human tumors (cervical adenocarcinoma, melanoma, and synovial sarcoma) were established using serum-free culture conditions with and without exogenous epidermal growth factor (EGF). EGF-adapted cultures of melanoma and cervical adenocarcinoma origin secreted higher levels of bioactive transforming growth factor alpha (TGF-alpha) when compared to cultures maintained in the absence of EGF. Depletion of EGF for these EGF-adapted cultures resulted in growth arrest. In contrast, the sarcoma cell lines did not secrete TGF-alpha regardless of the culture conditions but EGF significantly stimulated proliferation of these cells in short-term assays. We show that exogenous EGF induces TGF-alpha production and supports proliferation of tumor cells of various tissue origin but is not essential for in vitro growth factor-deprived conditions.     

Role of tumor-derived fibroblasts in the growth of primary cultures of human breast-cancer cells: Effects of epidermal growth factor and the somatostatin analogue octreotide

In the present study we have investigated the role of human breast-cancer-derived fibroblasts in the proliferation of primary cultures of epithelial cells derived from the same tumor. For this purpose, a co-culture system, using Transwell tissue-culture inserts with microporous membranes was employed. Fibroblasts and epithelial cells were enriched according to differences in their density on Percoll density gradients. The co-culture system was first established using MCF-7 breast cancer cells and a human fibroblast line (HF cells). Insulin, 17β-estradiol, EGF and HF cells all significantly stimulated the growth of MCF-7 breast cancer cells. The stimulatory effects of insulin, E2 and EGF were additive to the stimulatory effect of HF cells. These data suggest that (unique) factor(s), other than the above-mentioned growth-promoting compounds, are responsible for the growth-promoting effects of fibroblasts. In half of the human breast cancers investigated, tumor-derived fibroblasts stimulated tumor-derived epithelial cell proliferation. EGF significantly stimulated epithelial cell proliferation in 4 out of 6 cultures. The stimulatory effects of fibroblasts and EGF were additive or synergistic, and were observed in the additional presence of FCS, again suggesting production of unique factor(s) by the fibroblasts, in one culture the fibroblasts significantly inhibited epithelial tumor-cell proliferation. Conversely, the epithelial cells significantly stimulated proliferation of fibroblasts in 3 out of 3 cultures. The somatostatin analogue octreotide significantly inhibited epithelial cell proliferation by 46% in one tumor-cell culture in the absence, but not in the presence, of fibroblasts. In one culture, octreotide significantly inhibited the proliferation of fibroblasts co-cultured with epithelial cells. © 1995 Wiley-Liss, Inc.     

Microbiological approach to the treatment of brain tumors

Growths factors, defined as polypeptides that stimulate cell proliferation, are major growth-regulatory molecules for cells in culture and probably also for cells in vivo. Evidence has been derived for autocrine system in which the cell produces its own growth factor. Several growth factors as well as their cellular receptors have been identified as productions of proto-oncogenes. Furthermore, these growth factors have been identified as mitogens in tumors of the central nervous system. The roles of growth factors including platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and its receptor. Insulin-like growth factors (IGFs), transforming growth factors (TGFs) and fibroblast growth factor (FGF) on the proliferation of brain tumors, especially glioma were reviewed. The activation of cellular proto-oncogenes resulting in the autocrine system of growth factors and their receptors offers the opportunity for therapeutic interference. Therapeutic efforts will be based on the concepts of neutralization of growth factors, antagonizing growth factors at their receptors, irreversibly blocking receptors, and interference with oncogene product synthesis. Specific antibody for growth factors or receptors will be able to inhibit the proliferation. Trapidil, an antagonist for PDGF, can inhibit the proliferation of a PDGF-producing glioma cell. We can assume that the further analysis of growth regulatory mechanism will allow the design of new therapeutic approaches.     

Purification and biological properties of type beta transforming growth factor from mouse transformed cells

Transforming growth factor type beta (TGF beta) has been purified from serum-free culture fluids of transformed mouse L-929 cells which are capable of continual growth in serum-free medium in the absence of any exogenously added polypeptide growth factors. TGF beta has been purified to homogeneity as judged by NH2-terminal amino acid sequence analysis. Analysis of the purified polypeptide by gel electrophoresis indicates that TGF beta is composed of two polypeptide chains of Mr 12,500 cross-linked by disulfide bonds. TGF beta was characterized by its ability to induce anchorage-dependent normal rat kidney (NRK) cells to grow in soft agar in the presence of epidermal growth factor (EGF). TGF beta was also able to enhance both EGF-induced DNA synthesis and cell proliferation on growth-arrested NRK cells in monolayer cultures under serum-free conditions. We also show that in mouse melanoma B-16 cells under serum-free conditions TGF beta stimulates both DNA synthesis in monolayer cultures and anchorage-independent growth in soft agar. Paradoxically, the anchorage-independent growth in the presence of serum of many human cell lines, including melanomas, and mammary, prostatic, vulvar, and lung carcinomas is inhibited by TGF beta at saturating concentrations similar to those that stimulate colony formation of the rodent cell lines L-929 and B-16 under serum-free conditions. The peculiar action of TGF beta is further revealed by the observations that while EGF and TGF beta synergize to induce inhibition of anchorage-independent growth of A-431 human vulvar carcinoma cells, their effects on the anchorage-independent growth of one human lung carcinoma cell line (A-549) and two human prostatic carcinoma cell lines (PC-3 and DU-145) are antagonistic. Moreover, we show that in the rodent and human cell lines TGF beta interacts with specific cellular receptors which may mediate the actions of TGF beta. We conclude that the expression of both TGF beta and TGF beta receptors by L-929 cells and the stimulation of growth of L-929 cells in serum-free medium by TGF beta suggests that TGF beta may be important for maintaining the transformed state of this tumor cell line, and the way in which a cell responds to TGF beta is dependent on the presence or absence of growth factors contained in the serum.     

Effects of suramin on cell proliferation of various types of human malignant cells]

Suramin, a polyanionic compound used clinically for the treatment of African trypanosomiosis and onchocerciasis, has been shown to inhibit the action of various growth factors such as platelet-derived growth factor, epidermal growth factor, fibroblast growth factor and transforming growth factor-beta to stimulate DNA synthesis of cells. Therefore, we investigated effects of suramin on cell proliferation of various types of human malignant cells in culture. Cell lines used were as follows: cervical cancer (HeLa), mammary cancer (MCF-7), bladder cancer (EJ), hepatoma (HuH-7, PLC/PRF/5), embryonal carcinoma (PA-1), and three in vitro transformed human fibroblast lines (KMST-6, SUSM-1, and VA-13). A serum-free defined medium, ASF103, was used when the effect of suramin on proliferation of cells was investigated. This culture medium contains only bovine serum albumin (0.1%), transferrin (5 micrograms/ml) and insulin (5 micrograms/ml) as peptide factors. On day 1, the drug was added to culture medium at the concentration of 25-100 micrograms/ml and 72-96 hr later, the number of cells was counted. The growth inhibition was expressed as the percentage of cells surviving after treatment of cells with suramin, with survival in the control condition representing 100 percent. Proliferation of HuH-7 cells was prominently inhibited and those of PA-1, PLC/PRF/5 and KMST-6 were moderately inhibited under the same conditions of treatment. On the other hand, other five cell lines were not responsive to up to 100 micrograms/ml suramin.     

Effects of androgen, fibroblast growth factors or other various growth factors on growth of Shionogi carcinoma cells in a protein-free medium

Shionogi carcinoma 115 (SC115) has been accepted for 20 years as an androgen-responsive mouse mammary tumor. We have recently established an androgen-dependent cloned cell line (SC-3) from a SC115 tumor. We found in the present study that the growth of SC-3 cells can be stimulated by 10(-8) M testosterone (up to 100-fold) even in a protein-free medium beginning from plating [Ham's F-12: Eagle's minimum essential medium (1:1, v/v)]. In the protein-free culture, the proliferation of SC-3 cells was also found to be stimulated by acidic or basic fibroblast growth factor (FGF) alone (up to 50-fold) among various growth factors examined such as FGFs, insulin, insulin-like growth factor (IGF)-I, IGF-II, nerve growth factor, platelet-derived growth factor, epidermal growth factor, transforming growth factor (TGF)-alpha and TGF-beta. The testosterone (10(-8) M)- or FGF (10 ng/ml)-induced growth of SC-3 cells was abolished only by TGF-beta (greater than or equal to 1 ng/ml) among various growth factors examined. We show for the first time in this study an androgen-dependent growth of cancer cells (SC-3) in a protein-free medium. In the protein-free medium, growth of SC-3 cells is also stimulated by FGFs, and the androgen- or FGF-induced growth is inhibited only by TGF-beta.     

Phenotypes of human esophageal squamous cell carcinoma based on matrix metalloproteinase production

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) on cell growth and on regulation of matrix metalloproteinases (MMPs) in four cell lines of human esophageal squamous cell carcinomas (TE8, TE9, TE10, and TE11). EGF stimulated the production of proforms of gelatinase B (MMP-9) by three cell lines that could synthesize EGF by themselves, with TE9 being the exception. Particularly, both the production of MMP-9 and DNA synthesis in TE10 were stimulated significantly by EGF. TGF-beta slightly stimulated DNA synthesis in two cell lines, TE9 and TE11, and TGF-beta secretion by TE9 was detected. The production of proforms of gelatinases A (MMP-2) and MMP-9 was gradually induced by TGF-beta in a concentration-dependent manner in all the cell lines except for TE9. Flow cytometric analysis revealed that all the lines expressed both EGF- and TGF-beta-receptors. In conclusion, our present results indicate that at least there are possibly two distinct phenotypes in human esophageal squamous cell carcinoma: one (TE10) depends on autocrine EGF production that enhances DNA synthesis and MMP-9 production; and the other (TE9) on autocrine TGF-beta that stimulates DNA synthesis but not in relation to gelatinase production.     

Autonomous proliferation of MeWo human melanoma cell lines in serum-free medium: secretion of growth-stimulating activities

The growth properties of a human melanoma cell line (MeWo) and of a variant (MeWo-LC1) endowed with higher metastatic potential in nude mice were compared using hormonally-defined serum-free media. The two cell lines failed to arrest in G0 following serum deprivation, and responded to INS and MSA but not to EGF, PDGF or FGF. Only MeWo-LC1 cells divided persistently in completely serum-free medium, and formed a high percentage of spheroids in agarose supplemented or not with serum or individual growth factors. The conditioned media of serum-free cultures of MeWo and MeWo-LC1 cells exhibited mitogenic activities. These were detected without prior concentration, fractionation or acid treatment. They stimulated DNA replication into sparse monolayers of autologous (MeWo, MeWo-LC1) or homologous (SK-MEL28 melanoma) cells and into NRK-49F normal rat fibroblasts, and acted in synergy with INS in a dose-dependent manner. Over a period of 5 days in culture, MeWo-LC1 cells produced bioactive material at a 2 to 3-fold higher rate than MeWo cells, in both the absence and presence of INS. MeWo-LC1-conditioned medium promoted or enhanced colony formation of MeWo and NRK-49F cells plated in serum-free (+/- INS) agarose. The two cell lines expressed the same amount of NGF and EGF receptors. On the basis of these results we suggest that: (i) MeWo and MeWo-LC1 melanoma cells have obviated allocrine stimulation of the division process characteristic of normal cells by responding to their own mitogens; (ii) some of these mitogens are akin to TGFs; (iii) the less efficient synthesis of autostimulatory factors by MeWo cells may account for their weaker proliferative capacity in vitro, and possibly in vivo.