Serial propagation of adult human prostatic epithelial cells with cholera toxin

Summary Reproducible subculture of adult human prostatic epithelial cells from normal, benign hyperplastic and malignant tissue has been achieved. Cholera toxin is the key component in the culture system, but use of an optimal basal medium (PFMR-4) supplemented with a high level of serum in collagen-coated dishes also improves growth and serial propagation. Editor’s statement The critical first step in the development of an optimized culture system for any cell type is to obtain enough growth to perform detailed growth-response studies. Multiplication of human prostatic epithelial cells in primary culture has been possible for several years, but scarcity of tissue specimens and inability to subculture have severely limited the usefulness of such cultures. The procedures described by Peehl and Stamey in this communication make it possible to expand the original inoculum substantially and to use subcultures to perform precisely controlled replicate experiments. This will open the way for rapid progress in the development of practical cell culture model systems for research on normal, hypertrophic, and malignant human prostatic cells. Richard G. Ham     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. I. Evidence for reversible binding and inactivation of the platelet-derived growth factor by heparin

Summary We have investigated the effects of interactions between growth factors and heparin-like glycosaminoglycans on untransformed human arterial smooth muscle cells (hASMC) in vitro. The results indicate that heparin in the presence of serum mitogens prevents the cells from entering the S phase of the cell cycle by binding and inactivating reversibly some serum mitogen(s). Our results suggest that platelet-derived growth factor (PDGF) is one of them and that it is the most potent stimulator of hASMC growth in vitro. Thymidine incorporation as well as increase in DNA content was inhibited not only by the presence of heparin in serum-containing medium but also when serum was chromatographed on Heparin-Sepharose at physiologic salt concentrations before exposure to the cells. The mitogenic activity of the unretained serum fraction was restored by the addition of PDGF AA, AB, or BB dimers or of a fraction (RF I) that dissociated from Heparin-Sepharose at 0.2 to 0.6M NaCl. Radiolabeled recombinant PDGF (c-sis) dissociated from Heparin-Sepharose within a concentration range of NaCl similar to that of RF I. Neither the unretained material nor the RF I or PDGF dimers were effective alone. The effect of RF I was significantly decreased by the addition of an anti-PDGF IgG that is known to neutralize the PDGF mitogenic activity partially. Addition of heparin abolished DNA-synthesis when the PDGF dimers or RF I were combined with the unretained fraction. A second fraction (RF II) bound strongly to Heparin-Sepharose and eluted between 1.1 and 1.6M NaCl. The RF II also induced DNA synthesis but was neither as efficient as RF I nor depending on other serum fractions for growth promotion and it was not inhibited by anti-PDGF IgG. A similar strong affinity for Heparin-Sepharose was found for labeled basic fibroblast growth factor and we cannot exclude the possibility that RF II represent fibroblast growth factor. Under these culture conditions, inhibition of hASMC proliferation was directly correlated with the expression of smooth muscle specific alpha actin isoforms in stress fibers and the suppression of a proliferating cell-specific nuclear antigen. Conversely, stimulation of hASMC proliferation was associated with the opposite phenomenon. We conclude that heparin-like glycosaminoglycans influence growth and phenotype of hASMCs in vitro by binding and inactivating PDGF. Inasmuch as heparin-like substances constitute a significant proportion of the proteoglycan-associated glycosaminoglycans of the arterial wall, such mechanisms might be important for the development of atherosclerotic lesions.     

Serum-free growth of adult human prostatic epithelial cells

Summary Proliferation of adult human prostatic epithelial cells in serum-free medium occurs upon the addition of cholera toxin, epidermal growth factor, pituitary extract, and hydrocortisone to basal medium PFMR-4A. Insulin and selenium enhance proliferation and permit growth at lower cell densities. Reducing the level of calcium in the medium dramatically alters morphology and also seems to increase proliferation. Mortal strains of cells derived from normal central or peripheral zone, benign hyperplasia, or cancer respond similarly to growth factors and calcium, but two populations of cancer cells which have been long-lived and may be immortal lines behave differently. GKC-CA cells require serum proteins or high levels of pituitary extract for optimal growth, and neither GKC-CA cells or cells of another cancer line, WB-CA, proliferate well in medium containing reduced levels of calcium. These observations may, however, be a reflection of attachment phenomena rather than of growth responses per se. Growth of cells in serum-free medium has allowed definitive studies of the effects of androgens, and regardless of cell type no response to androgens of prostate epithelial cells under any experimental conditions has been seen.     

An optimized culture medium for human vascular endothelial cells from umbilical cord veins

Various polypeptide growth factors, culture substrates, basal media, sera and further supplements were assayed for improvement of growth of human vascular endothelial cells from umbilical cord veins. The resulting optimized medium consisted of gelatinized culture substrates, a mixture (1:1) of Iscove's MDM and Ham's F12 basal media supplemented with 20% newborn calf serum, 500 ng/ml crude fibroblast growth factor, 20 ng/ml epidermal growth factor, 5 g/ml transferrin, 5 g/ml insulin and 10 g/ml heparin. The medium allowed long term cultivation of HUVEC up to 45 generations with maximal cell densities of about 10⁵ cells per cm² and a minimal doubling time of about 14 hours at low cell densities.Abbreviations HUVEC Human Endothelial Cells From Umbilical Cord Veins - FGF Fibroblast growth factor - EGF Epidermal Growth Factor - FCS Fetal Calf Serum - NCS Newborn Calf Serum - HBS HEPES-Buffered Saline - ECM Extracellular Matrix - LHM Peptide PyroGlu-His-Ser-Phe-Thr-Ile-Lys-Ile-ThrCONH₂ - IF 1:1 mixture of Iscove's MDM and F12 basal media     

Optimized medium for clonal growth of human microvascular endothelial cells with minimal serum

Summary An optimized basal nutrient medium, MCBD 131, has been developed that supports clonal growth of human microvascular endothelial cells (HMVEC) with as little as 0.7% dialyzed fetal bovine serum (dFBS) when also supplemented with 10 ng/ml epidermal growth factor (EGF) and 1 μg/ml hydrocortisone. An extensive initial survey of available media showed that MCDB 402, a medium optimized for low-serum growth of Swiss 3T3 cells, supported the best clonal growth of HMVEC with 10% dFBS. Quantitative adjustment of the composition of MCDB 402 for improved clonal growth of HMVEC with reduced amounts of dFBS resulted in development of MCDB 131. Although many different adjustments contributed to the optimal properties of MCDB 131 for growth of HMVEC, the most unusual feature of this medium is its high magnesium concentration. A major benefit was achieved by increasing Mg²⁺ from 0.8 mM in MCDB 402 to 10.0 mM in MCDB 131. In the absence of defined supplements, MCDB 131 supports good clonal growth of HMVEC with 2% dFBS. This can be reduced to 0.7% by adding EGF and hydrocortisone, which act synergistically to improve growth with low levels of dFBS. This research was supported by grant CA 15305 from the National Cancer Institute, Bethesda, MD.     

Clonal growth characteristics of adult human prostatic epithelial cells

Summary The ability of human epithelial cells derived from adult prostatic tissues to undergo clonal growth in culture was examined. In a previously described serum-free culture system, such cells exhibited a density-dependent growth requirement. It was found that raising the level of one of the constituents of the culture medium, bovine pituitary extract, to 100 μg/ml permitted excellent clonal growth when as few as 100 cells were inoculated/60-mm² dish. Raising the levels of supplements other than pituitary extract (cholera toxin, epidermal growth factor, hydrocortisone, or insulin) did not produce this result. The average colony-forming efficiency of cells derived from primary or early passage cultures was approximately 25%. When single cell suspensions were prepared from tissue isolates and directly analyzed for clonal growth, colony-forming efficiencies were approximately 5%, perhaps indicating the proportion of stem cells with proliferative potential in the original isolates. The colony-forming efficiency of a cell population derived from cancer tissue was not significantly different from those of populations derived from normal tissues.     

Functional role of PKC in contraction of cultured human prostatic stromal cells

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI-17 was over-expressed, PDBu induced a large contraction, but the agonist-induced contraction did not become larger than expected. In alpha-toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI-17 over-expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI-17 and also that the PKC-CPI-17 pathway does not appear to play a major role in the agonist-induced contraction even when CPI-17 was over-expressed.     

Heterologous regulation of EGF receptors in fibroblastic cells

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.     

Cooperative interaction of autocrine and paracrine mitogens for airway epithelial cells

Mitogens of the EGF family may play an important role in regulating the proliferation of airway epithelial cells (AEC). We examined the production of autocrine mitogenic activity by mouse AEC cultured from explants of tracheal tissue. DNA synthesis by growth-arrested AEC was stimulated by conditioned media from cells maintained in serum-free culture without exogenous growth factors. The mitogenic activity was blocked by a specific inhibitor of the EGF receptor tyrosine kinase. Furthermore, conditioned media from AEC contained molecular species that could compete with radiolabeled EGF in a receptor binding assay. However, mitogenic activity was not blocked by neutralizing antibodies to EGF or to transforming growth factor-, but was partly inhibited by co-incubation with heparin, suggesting that it might be due to a heparin-binding member of the EGF family. The activity was potentiated by co-incubation with IGF-1, analogous to the potentiation by IGF-1 of the mitogenic activity of EGF for AEC. Moreover, the autocrine mitogen produced by AEC exhibited cooperative interaction with the mitogenic activity in conditioned media from growth factor-deprived mouse lung fibroblasts, consistent with the hypothesis that interactions with mesenchymal cells could influence the proliferation of AEC in vivo.     

The enhancement of growth and differentiation of rat adrenal nerve cells by the addition of conditioned medium from human fibroblast cultures

The growth of rat adrenal nerve cells was remarkably enhanced by supplementing the cultured medium from the human fibroblast cell line, Hs 68. Maximum specific growth rate and length of the neurites were observed as 0.076 (1/hr) and 0.026 mm, respectively in 20% supplement of five day old medium. In adding more than 20% of the cultured medium both cell and neurite growth was severely decreased. It was interesting that the cultured medium from Hs 68 cells could play a role in the extension of the neurites rather than in the growth of neurite cells. It was also found that molecules lower than 50,000 daltons in the conditioned medium could improve the growth of neurite bearing cells and the extension of the neurites than larger molecules. The efficacy of the proteins (<50,000 MW) was similar to that of human nerve growth factor and much better than that of basic fibroblast growth factor which was mainly secreted from human fibroblast cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane

We have demonstrated previously that adult human synovial membrane-derived mesenchymal stem cells (hSM-MSCs) have myogenic potential in vitro (De Bari, C., F. Dell'Accio, P. Tylzanowski, and F.P. Luyten. 2001. Arthritis Rheum. 44:1928-1942). In the present study, we have characterized their myogenic differentiation in a nude mouse model of skeletal muscle regeneration and provide proof of principle of their potential use for muscle repair in the mdx mouse model of Duchenne muscular dystrophy. When implanted into regenerating nude mouse muscle, hSM-MSCs contributed to myofibers and to long term persisting functional satellite cells. No nuclear fusion hybrids were observed between donor human cells and host mouse muscle cells. Myogenic differentiation proceeded through a molecular cascade resembling embryonic muscle development. Differentiation was sensitive to environmental cues, since hSM-MSCs injected into the bloodstream engrafted in several tissues, but acquired the muscle phenotype only within skeletal muscle. When administered into dystrophic muscles of immunosuppressed mdx mice, hSM-MSCs restored sarcolemmal expression of dystrophin, reduced central nucleation, and rescued the expression of mouse mechano growth factor.     

Suppression of extracellular signals and cell proliferation through EGF receptor binding by (−)-epigallocatechin gallate in human A431 epidermoid carcinoma cells

Tea polyphenols are known to inhibit a wide variety of enzymatic activities associated with cell proliferation and tumor progression. The molecular mechanisms of antiproliferation are remained to be elucidated. In this study, we investigated the effects of the major tea polyphenol (−)-epigallocatechin gallate (EGCG) on the proliferation of human epidermoid carcinoma cell line, A431. Using a [³H]thymidine incorporation assay, EGCG could significantly inhibit the DNA synthesis of A431 cells. In vitro assay, EGCG strongly inhibited the protein tyrosine kinase (PTK) activities of EGF-R, PDGF-R, and FGF-R, and exhibited an IC₅₀ value of 0.5–1 μg/ml. But EGCG scarcely inhibited the protein kinase activities of pp60^v-src, PKC, and PKA (IC₅₀ > 10 μg/ml). In an in vivo assay, EGCG could reduce the autophosphorylation level of EGF-R by EGF. Phosphoamino acid analysis of the EGF-R revealed that EGCG inhibited the EGF-stimulated increase in phosphotyrosine level in A431 cells. In addition, we showed that EGCG blocked EGF binding to its receptor. The results of further studies suggested that the inhibition of proliferation and suppression of the EGF signaling by EGCG might mainly mediate dose-dependent blocking of ligand binding to its receptor, and subsequently through inhibition of EGF-R kinase activity. J. Cell. Biochem. 67:55–65, 1997. © 1997 Wiley-Liss, Inc.     

Cholera toxin stimulation of human mammary epithelial cells in culture

Summary Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system. This work was supported by USPHS Grant CA-24844 from the National Cancer Institute and Grant CD-61B from the American Cancer Society.     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.