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  • 1
    Electronic Resource
    Electronic Resource
    A serum-free [3H]thymidine incorporation assay for the detection of transforming growth factors (1986)
    Springer
    Methods in cell science 10 (1986), S. 117-123 
    Details
    ISSN: 1573-0603
    Keywords: growth factors ; DNA synthesis ; cancer ; transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A rapid mitogenic assay suitable for the detection of transforming growth factors in the extracts of tissues or cells or in the medium conditioned by tumor cells in vitro is described. The method utilizes a nontumorigenic mouse embryo cell line (AKR-2B cells) maintained in serum-free conditions. Three classes of growth factors can be distinguished using this assay.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01404602
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  • 2
    Electronic Resource
    Electronic Resource
    Serum-free culture of normal human melanocytes: Growth kinetics and growth factor requirements (1989)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 140 (1989), S. 565-576 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Normal human epidermal melanocytes were selectively propagated from mixed (keratinocyte-melanocyte) cultures and primary epidermal cell suspensions in serum-free medium, MCDB 153 containing insulin, bovine pituitary extract (BPE), phorbol-12-myristate-13-acetate (PMA), ethanolamine, phosphoethanolamine, and hydrocortisone. Neonatal foreskin melanocytes (NFMs) replicated more readily than adult melanocytes in culture. Early passage NFMs grown in serum-free medium exhibited a population generation time of 24-48 hours. NFMs assumed a less dendritic appearance and were less pigmented than adult melanocytes. PMA or other protein kinase C-activating phorbol esters significantly enhanced mitogenesis of NFMs; however, cAMP-elevating agents were not required for efficient replication of NFMs. Basic fibroblast growth factor (bFGF) was a potent mitogen for NFMs and replaced the requirement for BPE in the culture medium. NFMs expressed a single class of specific, high-affinity receptors for bFGF, exhibiting a Kd = 3 × 10-11 M and approximately 76,500 receptors/cell. Neither EGF nor TGF-α were mitogenic for NFMs, and TGF-β reversibly inhibited NFM growth. Rapidly growing, early passage NFMs were shown to have cell cycle times of 19.5, 7.5, and 9 hours for G1, S, and G2 /M phases of the cell cycle, respectively. Culture of NFMs to confluence or depletion of growth factors from the culture medium caused reversible, G1 phase-specific, cell cycle growth arrest. Senescence of NFMs was associated with irreversible growth arrest in the G1 phase after 40-45 population doublings in culture. Our data demonstrate that basal medium MCDB 153 can be supplemented with defined factors to cultivate selectively two major constituent cell types of the epidermis, the melanocyte and the keratinocyte.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041400323
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  • 3
    Electronic Resource
    Electronic Resource
    Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 153 (1992), S. 103-111 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrin growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-indendent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-RGF receptors antibody to the culture medium, implication AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-α) mRNA revealed coordinate expression of AR and TGF-α in these cells. These data suggest that both AR and TGF-α mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs. © 1992 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041530114
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  • 4
    Electronic Resource
    Electronic Resource
    Differential effects of a heparin antagonist (hexadimethrine) or chlorate on amphiregulin, basic fibroblast growth factor, and heparin-binding EGF-like growth factor activity (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 163 (1995), S. 418-429 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF) are two recently identified members of the EGF family. Both AR and HB-EGF share with EGF the ability to interact with the type-1 EGF receptor; however, AR and HB-EGF differ from EGF in that both of these mitogens bind to heparin while EGF does not. To determine whether interactions with heparin-like molecules on the cell surface influence binding of AR and HB-EGF with EGF receptors and the subsequent mitogenic activity exerted by these growth factors, murine AKR-2B and Balb/MK-2 cells were treated with either an inhibitor of proteoglycan sulfation (chlorate) or a heparin antagonist (hexadimethrine). As expected, neither treatment significantly altered the specific binding of 125I-EGF on AKR-2B cells. Interestingly, treatment with either chlorate or hexadimethrine inhibited the ability of AR to compete with 125I-EGF for cell surface binding and also attenuated AR-mediated DNA synthesis. Thus, as has been suggested for other heparin-binding growth factors such as basic fibroblast growth factor (bFGF), the interaction of AR with an EGF-binding receptor appears to be facilitated by interaction with cell-associated sulfated glycosami-noglycans or proteoglycans. Unexpectedly, however, neither chlorate nor hexadimethrine treatment caused an inhibition of HB-EGF-induced mitogenic activity. Chlorate treatment did not significantly alter the ability of HB-EGF to compete with 125I-EGF for cell surface binding sites, however, heparin and hexadimethrine reduced the ability of HB-EGF to compete for 125I-EGF binding. These results suggest that, in AKR-2B cells, HB-EGF may mediate its mitogenic response at least in part through a receptor which appears to be selective for HB-EGF and permits HB-EGF-mediated mitogenic responses in the presence of hexadimethrine or heparin. Finally, hexadimethrine inhibited the specific binding and mitogenic activity of bFGF, suggesting that this cationic polymer can function as an antagonist of heparin-binding mitogens other than AR. © 1995 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041630222
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  • 5
    Electronic Resource
    Electronic Resource
    Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses, growth kinetics, and cell cycle studies (1984)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 121 (1984), S. 31-44 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G1, S, G2, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (〉 30 population doublings) and neonatal prokeratinocytes (〉 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041210106
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  • 6
    Electronic Resource
    Electronic Resource
    Differential expression of mRNA coding for heparin-binding growth factor type 2 in human cells (1988)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 136 (1988), S. 297-304 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The proliferation of normal human fibroblasts, keratinocytes, and melanocytes in vitro can be controlled by purified polypeptide growth factors and serum. We have studied the cellular expression of the heparin-binding growth factor type 2/basic fibroblast growth factor (HBGF-2/bFGF) gene to determine whether these cell types synthesize mRNA for this mitogen. Our results indicate that normal human fibroblasts synthesize four distinct mRNAs of 7.0, 3.7, 2.2, and 1.5 kilobases, which hybridize to a specific HBGF-2/bFGF cDNA probe. In fibroblasts, the level of all four of these transcripts increases dramatically (more than tenfold) within 4 hours of treatment of quiescent cells with fresh fetal bovine serum. Of the purified growth factors tested, transforming growth factor type-beta also increased HBGF-2/bFGF mRNA abundance, but not to the levels attained by serum treatment. Treatment of fibroblasts with cycloheximide before and during serum treatment blocked the ability of serum to induce the expression of the HBGF-2/bFGF gene. The gene is expressed at low levels in human fibroblasts rapidly growing in serum-free medium and at higher levels in cells rapidly growing in serum-containing medium. In contrast to fibroblasts, mRNA coding for HBGF-2/bFGF is undetectable in proliferating normal human keratinocytes, melanocytes, or mammary epithelial cells. Because keratinocytes and melanocytes proliferate in response to purified HBGF-2/bFGF, our results suggest that HBGF-2/bFGF may mediate the proliferation of epidermal cells through paracrine mechanisms involving stromal fibroblasts. Moreover, we have shown that a human squamous cell carcinoma cell line (SCC-25) expresses mRNA coding for HBGF-2/bFGF, suggesting that the gene may become activated in some carcinomas.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041360212
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  • 7
    Electronic Resource
    Electronic Resource
    Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor (1989)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 138 (1989), S. 511-518 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 μg/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041380310
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  • 8
    Electronic Resource
    Electronic Resource
    Growth factor-independent proliferation of normal human neonatal keratinocytes: Production of autocrine- and paracrine-acting mitogenic factors (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 146 (1991), S. 277-289 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 × 103/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGFα, or aFGF did not significantly alter growth rate. Heparin sulfate, TGFβ, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGFα, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGFα peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H] thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H] thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by kerati-nocytes modiated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041460213
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  • 9
    Electronic Resource
    Electronic Resource
    Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells (1987)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 132 (1987), S. 143-148 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Suramin, a polyanionic compound, has previously been shown to dissociate platelet-derived growth factor (PDGF) from its receptor. In the present study suramin was found to inhibit the growth of sparse cultures of AKR-2B cells in fetal bovine serum (FBS)-supplemented medium in a dose-dependent, reversible fashion. Suramin also inhibited the ability of FBS, transforming growth factor β (TGFβ), heparin-binding growth factor type-2 (HBGF-2), and epidermal growth factor (EGF) to stimulate DNA synthesis in density-arrested cultures of AKR-2B cells. The inhibition of growth factor-stimulated mitogenicity was directly correlated to the dose of suramin required to inhibit the binding of 125I-labeled TGFβ, HBGF-2, and EGF to their cell surface receptors. Suramin affected TGFβ and HBGF-2-related events at a 10-15-fold lower dose than that required for EGF-related events. It was also noted that suramin inhibited TGFβ-stimulated soft agar colony formation of AKR-2B (clone 84A) cells as well as the spontaneous colony formation of AKR-MCA cells, a chemically transformed derivative of AKR-2B cells. This demonstrates that suramin's spectrum of action for growth factors and their receptors should be extended to include TGFβ, HBGF-2, and EGF as well as PDGF. The data further suggest that the spontaneous growth of AKR-MCA cells in soft agar is dependent on growth factor binding to cell surface receptors.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041320120
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  • 10
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    Control of entry of Swiss 3T3 cells into S phase by fibroblast growth factor under serum-free conditions (1983)
    Elsevier
    Details
    Publication Date: 1983-07-01
    Print ISSN: 0014-4827
    Electronic ISSN: 1090-2422
    Topics: Biology , Medicine
    Published by Elsevier
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