Action of phorbol esters in cell culture: Mimicry of transformation,altered differentiation,and effects on cell membranes

The carcinogenic process is usually multifactor in its causation and multistep in its evolution. It is likely that entirely different molecular mechanisms underlie the many steps in this process. In contrast t o initiating carcinogens, the action of the tumor-promoting phorbol esters does not appear t o involve covalent binding t o cellular DNA and they are not mutagenic. Recent studies in cell culture have revealed two interesting biologic effects of the phorbol esters and related macrocyclic plant diterpenes. The first is that at nanomolar concentrations they induce several changes that resemble those seen in cells transformed by chemical carcinogens or tumor viruses. These include altered morphology and increased saturation density, altered cell surface fucose-glycopeptides, decrease in the LETS protein, increased transport of deoxyglucose, and increased levels of plasminogen activator and ornithine decarboxylase. In transformed cells exposed to phorbol esters the expression of these features is further accentuated. Phorbol esters do not induce normal cells to grow in agar but they do enhance the growth in agar of certain transformed cells. The second effect of the phorbol esters is inhibition of terminal differentiation. This effect extends to a variety of programs of differentiation and is reversible when the agent is removed. With certain cell culture systems induction of differentiation, rather than inhibition, is observed. Both the transformation mimetic and the differentiation effects are exerted by plant diterpenes that have tumor-promoting activity but not by congeners that lack such activity. The primary target of phorbol esters appears to be the cell membrane. Early membrane-related effects include enhanced uptake of 2-deoxyglucose and other nutrients, altered cell adhesion, induction of arachidonic acid release and prostaglandin synthesis, inhibition of the binding of epidermal growth factor t o cell surface receptors, altered lipid metabolism, and modifications in the activities of other cell surface receptors. A model of “two stage” carcinogenesis encompassing the known molecular and cellular effects of initiating carcinogens and tumor promoters is presented. According to this model, initiating carcinogens induce stable alterations in the cellular genome but these are not manifested until tumor promoters modulate programs of gene expression and induce the clonal outgrowth of the initiated cell.     

Growth and secretion of erythropoietin of Chinese hamster ovary cells coexpressing epidermal growth factor receptor and erythropoietin genes: Design of cells for cell culture matrix

Chinese hamster ovary (CHO) cells were transfected withboth genes encoding erythropoietin (Epo) and epidermal growthfactor receptor (EGFR). The transfection of the Epo gene wasconfirmed by an enzyme-linked immunoassay. Overexpression ofEGFR was confirmed by Western blotting of EGFR. Thetransfected CHO cells were cultured in serum-free medium inthe presence of soluble epidermal growth factor (EGF) orimmobilized EGF. The CHO cells overexpressing EGFR grew in thepresence of less EGF than the cells not overexpressing EGFR.In addition, the growth of EGFR-overexpressing CHO cells wasenhanced in the presence of immobilized EGF more efficientlythan in the presence of soluble EGF. The amount of Eposecreted from the cells increased linearly with the increaseof growth rate. Consequently, culture of CHO cellscoexpressing Epo and EGFR on EGF-immobilized matrix was themost efficient for Epo production.     

Derivation and characterization of human embryonic stem cell lines from poor quality embryos

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.     

Three cell lines showing androgen-dependent, -independent,and-suppressed phenotypes,established from a single tumor of androgen-dependent shionogi carcinoma 115

Summary We investigated the heterogeneity of cells in terms of androgen responsiveness within a single tumor mass of Shionogi carcinoma SC-115 showing androgen-dependent growth. After cloning of the tumor by the limiting dilution method in the presence of androgen, we isolated 40 clones at random. Twenty-two clones required androgen for growth (androgen-dependent phenotype), 16 did not (androgen-independent phenotype), and the remaining two clones showed growth inhibition when androgen was added (androgen-suppressed phenotype). In addition, 22 androgen-dependent clones showed heterogeneity in growth factor sensitivity in the absence of androgen. All clones were sensitive to both acidic and basic fibroblast growth factor (FGF), 7 of 22 clones were sensitive to epidermal growth factor (EGS) and transforming growth factor (TGF)-α, and 2 of 22 clones were sensitive to TGF-β. This preexisting heterogeneity may be partly responsible for the growth of androgen-dependent tumor under hormone-deprived circumstances. Three typical clones, SC2G, SC1G, and SC4A, were selected from androgen-dependent, -independent, and-suppressed phenotypic groups, respectively. These clones, as well as original solid tumors, were found to produce heparin-binding growth factors of heterogeneous elution positions. The molecular nature of these growth factors is not yet known. Neither anti-basic FGF antibody nor anti-EGF antibody inhibited the cell growth when added in cell culture, suggesting the factors were distinct from basic-FGF and EGF.     

Mitogenic response to epidermal growth factor: Relationship to number,affinity, and down-regulation of EGF receptors in three murine embyro cell lines

Swiss 3T3 and C3H-M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H-10T1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10-nM EGF all three cell lines “down-regulate” their EGF receptors with the same time course, and to the same precentage of initial receptors.     

Proliferation and differnetiation of human squamous carcinoma cell lines and normal keratinocytes: Effects of epidermal growth factor,retinoids, and hydrocortisone

Summary Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca²⁺ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors. This work was partly supported by The Netherlands Cancer Foundation (Koningin Wilhelmina Fonds), grant IKW 85–71.     

In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: Inducing effects of brain-derived neurotrophic factor

Neural stem cells proliferate in vitro and form neurospheres in the presence of epidermal growth factor (EGF), and are capable of differentiating into both neurons and glia when exposed to a substrate. We hypothesize that specific neurotrophic factors induce differentiation of stem cells from different central nervous system (CNS) regions into particular fates. We investigated differentiation of stem cells from the postnatal mouse hippocampus in culture using the following trophic factors (20 ng/mL): brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial-derived neurotrophic factor (GDNF). Without trophic factors, 32% of stem cells differentiated into neurons by 4 days in vitro (DIV), decreasing to 10% by 14 DIV. Addition of BDNF (starting at either day 0 or day 3) significantly increased neuron survival (31–43% by 14 DIV) and differentiation. Morphologically, many well-differentiated neurons resembled hippocampal pyramidal neurons. 5′-Bromodeoxyuridine labeling demonstrated that the pyramidal-like neurons originated from stem cells which had proliferated in EGF-containing cultures. However, similar application of NT-3 and GDNF did not exert such a differentiating effect. Addition of BDNF to stem cells from the postnatal cerebellum, midbrain, and striatum did not induce these neuronal phenotypes, though similar application to cortical stem cells yielded pyramidal-like neurons. Thus, BDNF supports survival of hippocampal stem cell-derived neurons and also can induce differentiation of these cells into pyramidal-like neurons. The presence of pyramidal neurons in BDNF-treated hippocampal and cortical stem cell cultures, but not in striatal, cerebellar, and midbrain stem cell cultures, suggests that stem cells from different CNS regions differentiate into region-specific phenotypic neurons when stimulated with an appropriate neurotrophic factor. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 395–425, 1998     

Effect of lectin from the ascidian on the growth and the adhesion of HeLa cells

Cell adhesion molecules, some of which are lectins, play a key role in the control of normal and pathological processes of various living organisms. We found herein that NacetylDglucosaminespecific lectin, isolated from the ascidian Didemnum ternatanum (DTL), alters the growth properties of HeLa tumor cells depending on the anchorage. DTL was shown to increase the proliferation of HeLa cells grown in soft agar greatly (in anchorageindependent fashion). In contrast, DTL inhibits the proliferative activity of HeLa cells grown on solid substrate and acts as inductor of differentiation, slowing cell growth, increasing the cell attachment and spreading. Scanning electron microscopic data have demonstrated that DTL treatment resulted in pronounced changes of the shape and surface of HeLa cells. Changes of cellular morphology correlated with essential redistribution of actin microfilaments.     

Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor

Epidermal growth factor (EGF)–responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self‐renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF‐responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF‐generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain‐derived neurotrophic factor (BDNF) (5 ng in 0.5 μL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF‐responsive stem cell‐derived neurons possess limited intrinsic capability for long‐distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF‐responsive stem cell–derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 391–413, 1999     

IGF-I and IGF-II stimulate directed cell migration of bone-marrow-derived human mesenchymal progenitor cells

Insulin-like growth factors (IGFs) are known to be key regulators of bone growth, remodeling, and repair. Since all these processes depend on the recruitment of cells with the potential to be committed to the osteoblastic lineage, we studied possible effects of IGF-I and -II on migration of human mesenchymal progenitor cells (MPC) using a modified Boyden chamber assay. The results were compared to those of primary osteoblasts and in vitro-osteogenic-differentiated MPC. IGF-I and -II stimulated cell migration of all these cell populations in a dose-dependent manner from 1 to 100 ng/mL. The maximal chemotactic index (CI) was 4-5 for MPC and primary osteoblasts and about 3 for in vitro-differentiated MPC. Checkerboard analysis revealed that IGFs stimulated true directed cell migration (chemotaxis) and not simply chemokinesis. Addition of an antibody against the type I IGF receptor (αIR3) completely abolished (MPC) or markedly reduced (primary osteoblasts) the chemotactic effects of each of the IGFs. IGFBP-3 itself had no direct effect, while IGFBP-5 stimulated MPC migration at concentrations of 80 and 160 ng/mL. Parallel application of IGFBP-3 had borderline inhibitory effects while the addition of 40 ng/mL of IGFBP-5 enhanced the chemotactic effect of IGF-I on MPC. In conclusion, our results show that IGF-I and -II are chemotactic factors for MPC and indicate that IGFBP-5 both modulates the IGF-I effect and directly stimulates migration of human mesenchymal progenitor cells.     

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: an aspect of cell differentiation.

The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.     

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.