Proteins excreted by primary human colon carcinoma cells (SW 480) promote spreading and growth of metastatic human colon carcinoma cells (SW 620) in serum-free medium

The growth behavior of the two human colon tumor cell lines (SW 480, primary and SW 620, metastatic), originating from the same patient, was studied in six different serum-free media (SFM) [GF3, Chee's essential medium plus insulin, transferrin and selenium; GF3F, GF3 plus fetuin; GF4, GF3 plus linoleic acid-BSA; GF5, GF4 plus fetuin; GF5E, GF5 plus EGF; GF5T, GF5 plus triiodothyronine]. SW 480 grew in all of the SFM. In contrast, SW 620 grew only in four SFM. The cells did not grow in GF3 and GF4. When grown in SFM, SW 480 attached much more firmly to the dishes than SW 620 as determined by the time required to detach the cells with trypsin-EDTA (SW 480, greater than 20 min and SW 620, less than 5 min). It was speculated that SW 480 cells excrete proteins in SFM which influence attachment and growth of the cells. Growth behavior of SW 480 cells which did not grow in GF3, was studied using GF3 medium and SW 480 substratum dishes. SW 620 cells readily attached to the SW 480 substratum dishes and grew. Furthermore, when SW 620 cells were grown on substratum prepared from serum-supplemented medium incubated in the absence of cells (serum substratum), the cell growth was comparable to the cell growth on SW 480 substratum in GF3. Substratum from SW 480 cells and the serum substratum were compared for their components using SDS-PAGE system. The SW 480 substratum contains many more components than serum substratum. A protein band at 60 kD appears to be common in both SW 480 and serum substrata.     

N-linked oligosaccharide processing and autocrine stimulation of tumor cell proliferation

Somatic mutations which impair complex-type N-linked oligosaccharide processing and chemical inhibitors of processing have been shown to reduce metastatic potential in several experimental tumor models. In this report, we demonstrate that glycosylation mutants of the metastatic MDAY-D2 tumor cell line with either truncated glycans lacking sialic acid and galactose or a mutant with less branched N-linked oligosaccharides grow more slowly in serum-free medium (SFM) than do MDAY-D2 cells. In medium containing fetal calf serum, growth rates of the cell lines were similar. A revertant of the former mutation showed a return to a more rapid growth rate in SFM. The N-linked processing inhibitor swainsonine also reduced cell growth rate in SFM but not in serum-containing medium. One of five randomly selected clones of the MDAY-D2 tumor cell line showed a slower growth rate in SFM and also showed decreased expression of branched N-linked oligosaccharides. These observations suggest that in MDAY-D2 cells, optimal factor-independent stimulation is dependent upon expression of branched complex-type N-linked oligosaccharides. The growth rate of MDAY-D2 cells in SFM was dependent on the initial seeding density of the cultures, and medium conditioned by the cells accelerated the growth of low-density cultures, suggesting that the cells respond to an autocrine factor. Culture supernatants conditioned by mutant and wild-type cells had similar levels of growth-stimulating activity. However, both mutants and swainsonine-treated cells were less responsive to this growth-stimulating activity. The growth rates of the MDAY-D2 tumor cell lines in vivo as subcutaneous tumors correlated with their relative growth rates in SFM in vitro. The results suggest that branched complex-type N-linked oligosaccharides commonly expressed in malignant cells are required for optimal autocrine-dependent growth in vitro and may be a significant factor in tumor progression in vivo.     

Dab2 is pivotal for endothelial cell migration by mediating VEGF expression in cancer cells

Although angiogenesis is crucial for tumor growth and metastasis, the molecular mechanisms controlling this process are not clearly understood. Here, we explore the role of Dab2 in tumor angiogenesis. We found that Dab2 is expressed in several cancer cells, including A549 lung cancer cells, but it is hardly detectable in SW480 colon cancer cells. Migration and Erk phosphorylation were enhanced in human umbilical vein endothelial cells (HUVECs) treated with the conditioned medium obtained from Dab2-overexpressing SW480 stable cells. In addition, vascular endothelial growth factor (VEGF) protein was strongly detected in conditioned medium derived from Dab2-overexpressing SW480 cells, and Erk phosphorylation enhanced by Dab2(+) CM was restored by VEGF inhibition. Moreover, Dab2 depletion in A549 cells led to a decrease in HUVEC migration and Erk phosphorylation. Furthermore, we show that Dab2 is required for the TGFβ-induced gene expression of angiogenic factors such as VEGF and FGF2. Taken together, these results suggest that Dab2, which is expressed in cancer cells, is pivotal for endothelial cell migration by affecting VEGF expression.     

Neurite outgrowth induced by the substrate associated material from nonneuronal cells

Cultured embryonic heart cells release a powerful inducer of neurite outgrowth into the surrounding medium. The present report demonstrates that these cells also deposit material which induces neurite outgrowth directly onto their culture substratum. Thus, embryonic heart cells condition both the culture medium and the culture substratum with respect to neurite outgrowth. Conditioned substrata were prepared by incubating heart cell monolayers in EDTA until the cells released from the substratum and were discarded. When dissociated neurons from ciliary or sympathetic chain ganglia were plated in fresh medium onto a conditioned substratum, neurite outgrowth was initiated in 80–95% of the neurons within 60 min. The neurite-inducing activity is trypsin sensitive, but is not inactivated by antibodies to the cell attachment protein fibronectin, by the membrane-solubilizing detergent Triton X-100, or by the enzymes collagenase, RNase, or DNase. The factor in conditioned medium which also induces neurite outgrowth depends for its activity on attachment to an artificial polyornithine substratum, under which condition it appears to promote adhesion of neuronal filopodia to the substratum. Thus, neurite outgrowth in these two culture systems occurs only if the substratum is conditioned by the appropriate extracellular materials: conditioned either directly by the deposition of heart cell products or indirectly by the binding of a conditioned medium factor to the polyornithine substratum. These substratum-conditioning factors may be related to those components of the extracellular matrix which support neurite outgrowth in vivo.     

Adipose tissue-derived stem cells differentiate into carcinoma-associated fibroblast-like cells under the influence of tumor-derived factors

Carcinoma-associated fibroblasts (CAF) are considered to contribute to tumor growth, invasion and metastasis. However, the cell type of origin remains unknown. Since human adipose tissue-derived stem cells (hASCs) are locally adjacent to breast cancer cells and might directly interact with tumor cells, we investigated whether CAFs may originate from hASCs. We demonstrated that a significant percentage of hASCs differentiated into a CAF-like myofibroblastic phenotype (e.g., expression of alpha smooth muscle actin and tenascin-C) when exposed to conditioned medium from the human breast cancer lines MDAMB231 and MCF7. The conditioned medium from MDAMB231 and MCF7 contains significant amounts of transforming growth factor-beta 1 (TGFβ1) and the differentiation of hASCs towards CAFs is dependent on TGFβ1 signaling via Smad3 in hASCs. The induction of CAFs can be abolished using a neutralizing antibody to TGFβ1 as well as by pretreatment of the hASCs with SB431542, a TGFβ1 receptor kinase inhibitor. Additionally, we found that these hASC-derived CAF-like cells exhibit functional properties of CAFs, including the ability to promote tumor cell invasion in an in vitro invasion assay, as well as increased expression of stromal-cell-derived factor 1 (SDF-1) and CCL5. Taken together, these data suggest that hASCs are a source of CAFs which play an important role in the tumor invasion.     

Human fibroblast conditioned media contains growth-promoting activities for low density cells

Normal diploid human fibroblasts, cultured at high density (1–2 × 10⁵ cells per cm²) release two growth promoting activities into the culture medium. The fibroblast proliferation activity-conditioned medium facilitates the attachment of low density cells to the substrate. That activity resides in a non-dialyzable material that is sensitive to proteolytic inactivation. A second activity is dialyzable and can be recovered in the dialysate. In the presence of serum it stimulates cell growth. After 168 hours of incubation conditioned medium cultures contain five times more cells than are present in comparable cultures without conditioned medium. A reproducible biological assay for each activity is described.     

Production of factors required for cell attachment and spreading is a constitutive property in mouse A9 cells.

It is demonstrated here that cells in a suspension culture of an established mammalian cell line release non-dialyzable factors into their growth medium. These factors are capable of promoting the adhesion and spreading of these cells on a generally non-attachable substratum and also promote spreading on an adhering substrate. Evidence is presented which demonstrates that the spreading promotion activity of the condition medium is dependent on the cell density of the culture from which it was derived. Dilution of the conditioned medium results in a proportionate dilution of the spreading promotion activity. The results clearly demonstrate that the production of this spreading promotion factor is continued even in the absence of cell to substrate attachment.     

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

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

微管与肿瘤细胞运动——免疫细胞化学研究

运用免疫细胞化学方法对三种不等恶性程度人结肠腺癌细胞株HT-29分化较好,呈上皮样,细胞排列紧密,呈聚集性生长;免疫细胞化学显示;微管不甚明显。SW480及SW620细胞呈梭形。细胞间松散,常呈单个生长,免疫细胞化学显示微管十分丰富,该种形态特征与原发肿瘤恶性程度一致,前为高分化结肠腺癌,后为高恶性度结肠腺癌,具较强转移潜能,加入TGFβ1后,三株细胞均呈生长加速,多见分裂相,但微管未见明显改变,说明细胞快速分裂并非肿瘤浸润的唯一原因,加入道诺红菌素作用后,SW480及SW620微管结构出现明显改变,表现为断裂,卷曲或呈颗粒状。提示道诺红菌素能破坏细胞微管成分,微管的破坏可能抑制肿瘤细胞的转移。     

Evidence for a FSH dependent secretion of a receptor reactive transforming growth factor beta-like material by immature Sertoli cells in primary culture

Type beta Transforming Growth Factor (TGF beta)-like activity was identified in conditioned medium obtained from immature porcine Sertoli cell-enriched cultures using the following criteria: (i) stimulation of anchorage independent growth of mesenchymal cell lines, (ii) competition with pure human TGF beta in a radioreceptor assay. The secretion of the receptor reactive TGF beta-like material in Sertoli cell conditioned medium is decreased to very low or undetectable levels by Follicle Stimulating Hormone, one of the major hormones involved in the physiological testicular activities. The effects of this factor are probably exerted in the context of the local control of the male gonad functions.     

Enhanced production and extracellular deposition of the endothelial- type plasminogen activator inhibitor in cultured human lung fibroblasts by transforming growth factor-beta

Cultured human embryonic lung fibroblasts were used as a model to study the effects of transforming growth factor-beta (TGF beta) on the plasminogen activator (PA) activity released by nontumorigenic cells into the culture medium. The cells were exposed to TGF beta under serum- free conditions, and the changes in PA activity and protein metabolism were analyzed by caseinolysis-in-agar assays, zymography, and polypeptide analysis. Treatment of the cells with TGF beta caused a significant decrease in the PA activity of the culture medium as analyzed by the caseinolysis-in-agar assays. The quantitatively most prominent effect of TGF beta on confluent cultures of cells was the induction of an Mr 47,000 protein, as detected by metabolic labeling. The Mr 47,000 protein was a PA inhibitor as judged by reverse zymography. It was antigenically related to a PA inhibitor secreted by HT-1080 tumor cells as demonstrated with monoclonal antibodies. The induced Mr 47,000 inhibitor was deposited into the growth substratum of the cells, as detected by metabolic labeling, immunoblotting analysis, and reverse zymography assays of extracellular matrix preparations. TGF beta also decreased the amounts of urokinase-type and tissue-type PAs accumulated in the conditioned medium, as detected by zymography. Epidermal growth factor antagonized the inhibitory effects of TGF beta by enhancing the amounts of the PAs. These results indicate that growth factors modulate the proteolytic balance of cultured cells by altering the amounts of PAs and their inhibitors.     

A serum-free medium for clonal growth and serial subculture of diploid rat liver epithelial cells

Summary Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free “attachment medium” was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation. This research was sponsored by the National Cancer Institute, DHHS, under Contract NO1-CO-23909 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U. S. Government.     

Growth regulatory peptide production by human breast carcinoma cells

The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.     

Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro

Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF-2, TGF beta 1, or FGF-2/TGF beta 1). Cells were then induced to redifferentiate in 3D pellets using serum-supplemented medium (SSM), serum-free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGF beta 1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real-time PCR. FGF-2/TGF beta 1-expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR-expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF-, PDGFbb- or FGF-2-expanded chondrocytes. Upon supplementation of SFM with TGF beta and dexamethasone (SFM TD), CII/CI ratios decreased 4.4-fold for EGF- and PDGFbb-expanded chondrocytes, but increased 96-fold for FGF-2/TGF beta 1-expanded cells. Chondrocytes expanded with FGF-2/TGF beta 1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment.     

Manipulation of cell-cell and cell-substratum interactions in mouse mammary tumor epithelial cells using broad spectrum antisera

Two antisera were raised in goats against material shed by two different mammary epithelial cell lines into serum-free culture medium. These antisera, when added to the medium of intact, growing mouse mammary tumor cells in the absence of complement, cause distinct and dramatic alterations in cell morphology and adhesiveness. One antiserum (anti-SFM I) causes mouse mammary tumor epithelial cells to round and detach from the substratum. Treatment with the other antiserum (anti- SFM II) does not affect cell-substratum interactions, but causes the cells to convert from an epitheloid to a fibroblastic morphology. Statistical analysis of transmission electron micrographs of control and antibody-treated cells indicates that treatment with anti-SFM II is associated with a substantial reduction in the extent of intercellular junctions, particularly desmosomes. To identify the components with which the two antisera interact, nonionic detergent extracts of mouse mammary tumor cells were fractionated, and the ability of various fractions to block the morphological effects of either antiserum was determined. The whole Nonidet P40 (NP40) extract of the epithelial cells blocked the effects of both antisera. After the extract was subjected to ion exchange and lectin affinity chromatography, two separate fractions were obtained. One fraction blocks and anti-SFM I induced rounding and detachment of cells from the substratum. The second fraction blocks the effects of both antisera. The isolation of the former fraction, which has highly restricted number of components, represents a significant first step toward identifying the surface membrane molecule(s) involved in cell-substratum adhesion in epithelial cells.     

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

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

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.     

HIF1A/miR-20a-5p/TGFβ1 axis modulates adipose-derived stem cells in a paracrine manner to affect the angiogenesis of human dermal microvascular endothelial cells

Angiogenic cytokines secreted by the adipose-derived stem cells (ADSCs) might promote the angiogenesis of endothelial cells. In the present study, we hypothesize that miR-20a targets TGFB1 to modulate the transforming growth factor β1 (TGFβ1) secretion by ADSCs, therefore affecting the angiogenesis. We found that hypoxia-inducible factor 1A (HIF1A) and TGFβ1 expressions were increased by hypoxia, accompanied with promoted ADSC cell viability. Incubation with conditioned medium from ADSCs treated with hypoxia significantly enhanced the angiogenesis capacity of human dermal microvascular endothelial cells (HDMECs), while TGFB1-silenced ADSCs medium significantly reverses HDMECs angiogenesis. miR-20a suppresses the expression of TGFB1 and secretion of TGFβ1 by ADSCs via binding to its 3′untranslated region, therefore modulating the HDMEC angiogenesis via affecting the paracrine from ADSCs; the effects of miR-20a-overexpressed conditioned medium on HDMEC angiogenesis were significantly reversed by TGFB1-overexpressed conditioned medium. Finally, HIF1A suppressed the expression of miR-20a via targeting its promoter region, subsequently promoting the paracrine from ADSCs and HDMEC angiogenesis.     

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.