Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation

The differentiation of murine mesenchymal stem cells occurs in nonterminal and terminal phases. In previous reports we established the characteristics of nonterminally differentiated cells and showed that transition from the nonterminal to the terminal state of differentiation can be induced by human plasma. We also showed that this transition is blocked by protein synthesis inhibitors and other pharmacological agents. In this paper, we have employed two-dimensional gel electrophoresis to evaluate changes in specific polypeptides that are induced when cells lose proliferative capacity associated with the terminal event in differentiation. Using silver staining procedures for analysis of electrophoretograms, we detected only seven major polypeptide differences between nonterminally differentiated and terminally differentiated cells. Six polypeptides were expressed only in preparations of terminally differentiated cells; these included two polypeptides identified in cytosolic fractions and four polypeptides identified in nuclear fractions. One polypeptide was also found to be selectively expressed only in nuclear fractions of nonterminally differentiated cells. Based on these observations we conclude that the loss of proliferative potential that occurs during the terminal event in mesenchymal stem cell differentiation is associated with changes in the composition of a limited number of specific polypeptides. We suggest that one or more of these polypeptides may be important in the regulation of cellular proliferation.     

Regulation of the terminal event in cellular differentiation: biological mechanisms of the loss of proliferative potential

Human plasma has been demonstrated to contain factors that induce the sequential expression of nonterminal and terminal adipocyte differentiation in 3T3 T mesenchymal stem cells. We now report the development of methods for the isolation of purified populations of nonterminally differentiated cells and terminally differentiated cells, and we show that it is possible to experimentally induce transition from the nonterminal to the terminal state of differentiation. With this model system it is therefore now possible to examine the biological and molecular processes associated with the terminal event in differentiation, i.e., the irreversible loss of proliferative potential. In this regard, we demonstrate that transition from the nonterminal to terminal state of differentiation is a complex metabolic process that consists of at least two steps and that this process can be triggered by pulse exposure to an inducer for approximately 12 h but that approximately 24-48 h is required for the process to be completed. The data also establish that induction of the terminal event in differentiation requires protein synthesis but not RNA and DNA synthesis. These and additional results suggest that loss of proliferative potential associated with the terminal event in cellular differentiation is a distinct regulatory process, and we suggest that defects in this regulatory process may be of etiological significance in the pathogenesis of specific human diseases, especially cancer.     

Differential mitogenic effects of methyl isobutyl xanthine and a tumor growth factor on G1-arrested 3T3 T proadipocytes at the predifferentiation GD state and the growth-factor deficiency GS state

Two growth-states exist in the G1 phase of the 3T3 T proadipocyte cell cycle. GD is the arrest state at which proadipocytes must growth-arrest prior to differentiation. GS is the arrest state at which proadipocytes growth-arrest following deprivation of serum or growth factors. In an attempt to further distinguish these arrest states, we have compared the relative ability of a variety of mitogens to induce GD- and GS-arrested cells to initiate DNA synthesis. The data show that GD-arrested cells at both high and low densities can be induced to proliferate by methyl-isobutyl-xanthine (MIX), whereas high and low density GS-arrested cells are not. The data also show that a tumor growth factor can stimulate the proliferation of both high and low density GD- and GS-arrested cells, whereas other agents are poor mitogens for high density GD-arrested cells. We conclude that MIX and a tumor growth factor (TUGF) can serve as probes to study the characteristics of the GD arrest state.     

Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO)

Abstract. The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 nonterminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation-inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growtharrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth-arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation-inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage-dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.     

Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation

Experimental evidence is presented that supports a cell cycle model showing that there are five distinct biological processes involved in proadipocyte differentiation. These include: (a) growth arrest at a distinct state in the G1 phase of the cell cycle; (b) nonterminal differentiation; (c) terminal differentiation; (d) loss of the differentiated phenotype; and (e) reinitiation of cell proliferation. Each of these events is shown to be regulated by specific human plasma components or other physiological factors. At two states designated GD and GD', coupling of growth arrest and differentiation is shown to occur. We propose that these mechanisms for the coupling of growth arrest and differentiation are physiologically significant and mimic the regulatory processes that control stem cell proliferation in vivo.     

Three distinct effects of SV40 T-antigen gene transfection on cellular differentiation

SV40 large T-antigen-induced transformation has been reported to block differentiation, but the mechanism(s) of this effect has not been established. The results presented here show that stable transfection of the SV40 T-antigen gene, via the pSV3neo plasmid, has at least three distinct effects on 3T3T adipocyte differentiation. Cells first show a decreased ability to undergo predifferentiation growth arrest, which is a prerequisite for in vitro 3T3T adipocyte differentiation. However, if predifferentiation growth arrest is accomplished by use of stringent differentiation-inducing culture conditions, adipocyte differentiation can occur with high frequency. The pSV3neo-transfected cell clones also show other modifications of the adipocyte differentiation process, including changes in nonterminal (reversible) and terminal (irreversible) steps of adipocyte differentiation. When compared to nontransfected 3T3T cells, the cell clones containing pSV3neo require markedly reduced growth factor concentrations to restimulate proliferation of nonterminally differentiated adipocytes and the terminal step of differentiation is also blocked. These results suggest that integration of the T-antigen gene, through pSV3neo transfection, has multiple effects on the cellular mechanisms of differentiation. It does not block the differentiation process per se; rather it appears to make cells highly sensitive to proliferation signals, thereby making differentiation more difficult.     

Reduction in DNA repair capacity following differentiation of murine proadipocytes

It has been suggested that terminally differentiated mammalian cells have a decreased DNA repair capacity, compared with proliferating stem cells. To investigate this hypothesis, we have examined gamma-ray-induced DNA strand breaks and their repair in the murine proadipocyte stem cell line 3T3-T. By exposure to human plasma, 3T3-T cells can be induced to undergo nonterminal and then terminal differentiation. DNA strand breaks were evaluated using the technique of alkaline elution. No difference was detected among stem, nonterminally differentiated, and terminally differentiated cells in the initial levels of radiation-induced DNA strand breaks. Each of the strand break dose response increased as a linear function of gamma-ray dose. The strand breaks induced by 4 Gy rejoined following biphasic kinetics for each cell type. At each time point examined after irradiation, however, the percentage of strand breaks that had not rejoined in terminally differentiated cells was three to six times greater than in stem cells. The rate of strand break rejoining in nonterminally differentiated cells was of an intermediate value between that of the stem and of the terminally differentiated cells. These results indicate that, at least for 3T3-T cells, differentiated cells have a reduced capacity for DNA repair.     

A rapid decrease in epidermal growth factor-binding capacity accompanies the terminal differentiation of mouse myoblasts in vitro

Specific mitogens stimulate the proliferation and repress the differentiation of mouse myoblasts (MM14). When mitogens are depleted, MM14 cells cease proliferation, commit to terminal differentiation, and become refractory to growth stimulation. The behavior of mitogen receptors during the transition from a proliferative to a permanently postmitotic state was examined using the epidermal growth factor receptor (EGFR) as a model system. Whereas proliferating myoblasts bound substantial amounts of EGF, their binding capacity declined rapidly upon exposure to low-mitogen medium. The decline became irreversible when a cell differentiated. Within 24 h, less than 5% of the original EGF binding capacity remained. Since the ability to internalize and degrade bound EGF was unaffected, the change presumably reflected a decrease in EGFR availability. Several observations indicated that loss of EGFR following mitogen removal is related to differentiation rather than the result of starvation or cell-cycle arrest. First, the decline is correlated with the absence of a single mitogen (fibroblast growth factor) and is independent of serum concentrations. Second, myoblasts that are either cycling through G1 or arrested at G0, but prevented from differentiating, all bind large amounts of EGF. These findings suggest that specific reduction in mitogen receptors could be part of a mechanism whereby terminally differentiating cells become refractory to mitogenic stimulation.     

Evidence for c-myc in the signaling pathway for TGF-beta in well-differentiated human colon carcinoma cells

Previously, we described a model culture system for comparing responsiveness of poorly differentiated and well-differentiated human colon carcinoma cells to exogenous growth factors. While polypeptide growth stimulators elicited an up-regulation of c-myc, as well as a mitogenic response in the well-differentiated cells, the poorly differentiated cells were insensitive to exogenous growth stimulators. We now show, by thymidine incorporation experiments and autoradiographic analysis, that transforming growth factor beta 1 (TGF-beta) abrogated the mitogenic responses to the growth factors epidermal growth factor + insulin + transferrin (IC50 = 0.8 ng/ml), as well as to nutrients (basal medium; IC50 = 0.2 ng/ml) in the well-differentiated cells. The poorly differentiated cells did not respond to TGF-beta. Moreover, TGF-beta (10 ng/ml) completely abrogated the growth factor-stimulated up-regulation of c-myc in the TGF-beta responsive, well-differentiated colon carcinoma cells. Addition of TGF-beta to the TGF-beta-responsive, well-differentiated cells, at a time after c-myc had been transiently up-regulated in response to growth stimulatory factors, resulted in a loss of responsiveness to TGF-beta. Addition of TGF-beta to these cells at increasing time periods after EIT stimulation also resulted in a loss of the TGF-beta-induced repression of c-myc. The results suggest an important role for c-myc in the mechanism of action of TGF-beta in well-differentiated human colon carcinoma cells.     

Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with various combinations of transforming growth factor (TGF)-beta, fibroblast growth factor (FGF), and insulin-like growth factor I (IGF-I). In serum-free defined medium the following observations were made: TGF-beta depressed proliferation and inhibited differentiation; FGF stimulated proliferation and depressed differentiation; IGF-I stimulated proliferation to a small degree but demonstrated a more pronounced stimulation of differentiation. In evaluating combinations of these three factors, the differentiation inhibiting effect of TGF-beta could not be counteracted by any combination of IGF-I or FGF. The proliferation-depressing activity of TGF-beta, however, could not inhibit the mitogenic activity of FGF. Maximum stimulation of proliferation was observed in the presence of both FGF and IGF-I. The highest percentage fusion was also observed under these conditions, but differentiation with minimal proliferation resulted from treatment with IGF-I, alone. By altering the concentrations of TGF-beta, FGF, and IGF-I, satellite cells can be induced to proliferate, differentiate, or to remain quiescent.     

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states

Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G1 phase of the cell cycle. These include the predifferentiation arrest state (GD) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (GS), and the nutrient deficiency arrest state (GN). Cells at states of reversible nonterminal differentiation (GD') and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at GD, GS and GN states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [35S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the GS, GD, GD' and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the GD' and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation.2+he identification of differential     

The reactivity of frozen B lymphocytes to B cell mitogens and human B cell growth factor: A study of step 1 and step 2 activators

Human B lymphocytes, purified from the peripheral blood of several different donors can be pooled, frozen, and stored in liquid nitrogen to provide an easy and reproducible source of cells for mitogenic assays. These B cell preparations did not show any reactivity to T cell mitogens, but responded to Staphylococcus aureus Cowan strain 1 (SAC) and anti-IgM antibodies to the same extent as freshly purified B cells. When stimulated with either anti-IgM antibodies or SAC, these B cells became responsive to B cell growth factor (BCGF), allowing a quantitative measurement of this important lymphokine activity. In addition, we have studied the reactivity of frozen B lymphocytes to various combinations of activators. We have confirmed that phorbol myristate acetate (PMA) was a very potent mitogenic agent for preactivated human B cells and shown that bacterial lipopolysaccharide (LPS), although not mitogenic by itself, could synergize with anti-IgM antibodies to yield increased levels of stimulation. Furthermore experiments using the lysosomotropic agent leucine methyl ester showed that the action of LPS on anti-IgM-stimulated B cells did not require the presence of functional monocytes. Neither PMA nor LPS could induce BCGF responsiveness and thus these two compounds can be considered exclusive step 2 activators for human peripheral blood B cells.     

Cell senescence and hypermitogenic arrest

A diverse range of conditions, from mitogenic stimuli to cytotoxic stress, can induce cell senescence. Here, I propose that simultaneous stimulation of mitogen-activated pathways and downstream inhibition of cyclin-dependent kinases leads, ultimately, to cell senescence. This model distinguishes between two types of growth arrest: first, exit to G0 phase, which is caused by the withdrawal of mitogens and can lead to apoptosis; and second, hypermitogenic arrest, which is stimulated by mitogens and can lead to senescence. The concept of hypermitogenic arrest defines cell senescence as a functionally active, stable and conditionally reversible state.     

Control of proliferation in the retina: temporal changes in responsiveness to FGF and TGF alpha.

Proliferation in the rat retina, as in other parts of the nervous system, occurs during a restricted period of development. In addition to regulating cell number, the mechanisms that control proliferation influence the patterning of tissues, and may affect the determination of cell type. To begin to determine how proliferation is controlled, several growth factors found in the retina were tested for effects on progenitor cell division in culture. Proliferation was enhanced by TGF alpha, bFGF and aFGF, and many of the dividing cells later differentiated into cells with the antigenic phenotypes of retinal neurons and glial cells. The mitotic response of retinal cells to these factors changed during development: progenitor cells from younger retinas (embryonic day 15 to 18; E15-E18) were more responsive to FGF's, while progenitor cells from older retinas (greater than E20) were more responsive to TGF alpha. Progenitor cells stopped dividing in vitro, even when treated with excess mitogen. These observations suggest that proliferation in the retina may be stimulated by multiple mitogenic signals provided by TGF alpha, FGF, or related factors, and that proliferation is not controlled by limiting concentrations of mitogen alone. Rather, these data demonstrate that retinal cells change during development in their responsiveness to mitogenic signals. Such changes may contribute to the regulation of proliferation.     

Cell senescence: hypertrophic arrest beyond the restriction point

Withdrawal of mitogens (growth factors) arrests normal cells in G0 (quiescence). All other stresses and factors arrest cell cycle beyond the restriction point in G1 and G2 (non-G0 arrest), in the presence of mitogenic stimulation. Strong mitogenic stimuli by themselves cause non-G0 arrest. Unlike G0, arrest beyond restriction point is characterized by both high levels of cyclins and CDK inhibitors, activated mitogenic pathways with a secondary GF resistance, and continuous mass growth (cell hypertrophy). Prolonged hypertrophic arrest culminates in cell senescence. This review discusses that quiescence and senescence are two opposite, mutually exclusive conditions and that cell senescence can be reversed and prevented.     

Regulation of differentiation of the BC3H1 muscle cell line through cAMP-dependent and -independent pathways

Serum mitogens, fibroblast growth factor (FGF), and type beta transforming growth factor (TGF-beta) suppress differentiation of the mouse muscle cell line BC3H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC3H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC3H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E1, and cholera toxin, prevented up-regulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC3H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-beta. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC3H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-beta inhibit differentiation through a mechanism independent of cAMP or protein kinase C.     

Growth arrest of proadipocytes at a distinct predifferentiation G1 state associated with cytotoxic responsiveness to 8-bromo cyclic AMP

The control of cell proliferation can result from the coupling of growth arrest and differentiation. In this regard, we recently demonstrated that growth arrest which precedes the differentiation of 3T3 T proadipocytes must occur at a distinct state in the G1 phase of the cell cycle (GD). Cells arrested at GD differ in several biological parameters from cells arrested in G1 at other states induced by either serum deprivation (GS) or nutrient deficiency (GN). Specifically, GD-arrested cells can differentiate in the absence of DNA synthesis and GD-arrested cells can be induced to proliferate when stimulated with 1-methyl-3-isobutylxanthine; GS- and GN-arrested cells cannot. In addition, GD-, GS- and GN-arrested cells reside at topographically distinct states in G1. We now report that GD-arrested proadipocytes are also distinct in that they are highly sensitive to a cytotoxic effect of 8-bromocyclic AMP, whereas GS- and GN-arrested cells are not.     

C-fos expression is hypersensitive to serum-stimulation in cultured cystic kidney cells from the C57BL/6J-cpk mouse.

Cystic kidneys of the C57BL/6J-cpk murine model of polycystic kidney disease show a marked overexpression of the proto-oncogenes c-fos, c-myc, and c-Ki-ras, consistent with an increased rate of cell proliferation and an altered state of differentiation. To determine if cystic cells have increased responsiveness to stimulation with mitogenic agents, quiescent primary cultures from normal and cystic cpk kidneys were treated with fetal bovine serum (FBS), 8-bromo-cAMP (cAMP), or epidermal growth factor (EGF). The level of c-fos induction following stimulation by FBS was found to be dramatically higher in cystic cells than in normal cells; whereas induction by cAMP or EGF was essentially the same in both cell types and much less than that seen in FBS-stimulated cells. To determine if this serum hypersensitivity reflects an increased proliferative state in vivo, c-fos induction was examined in cultures derived from normal kidneys stimulated to regenerate by folic acid-induced acute renal injury. As with cystic kidneys, the folic acid-injured kidneys showed increased c-fos responsiveness to FBS in cell culture. These experiments suggest that cystic and regenerating kidneys have an altered phenotypic state in vivo that is manifested in cell culture by serum hypersensitivity. However, whereas the folic acid-injured kidneys ultimately reestablish normal kidney function, cystic kidneys further progress to renal failure, suggesting that cystic epithelial cells are locked in this altered state of differentiation.     

Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts.

The effect of various growth factors on the synthesis of hyaluronan in human fibroblasts was investigated. When tested in medium containing 0.5% fetal calf serum, platelet-derived growth factor (PDGF)-BB was found to stimulate hyaluronan synthesis; the maximal response was equal to or higher than that obtained with 10% fetal calf serum. PDGF-AA gave only a limited effect, indicating that the stimulatory effect of PDGF on hyaluronan synthesis was mainly transduced via the B-type PDGF receptor. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta 1 also stimulated hyaluronan synthesis; their effects were less than that of PDGF-BB, but combinations of factors produced potent stimulatory effects on hyaluronan synthesis. All factors stimulated hyaluronan synthesis in sparse as well as dense cultures. The effects of the factors on hyaluronan synthesis did not correlate with their mitogenic activities; PDGF-BB, EGF and bFGF are equipotent mitogens, but PDGF-BB had a much more potent effect on hyaluronan synthesis, and TGF-beta actually inhibits the growth of fibroblasts under the conditions of the assay.