Skin stem cell hypotheses and long term clone survival--explored using agent-based modelling.

Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation.

Phenotypic alterations in fibroblasts and fibrosarcoma cells that overexpress latent transforming growth factor-beta 1.

Mouse embryo-derived AKR-2B fibroblasts and murine fibrosarcoma cells (the 1591 cell line) were transfected with a murine transforming growth factor-beta 1 (TGF beta 1) cDNA under the transcriptional control of either the simian virus-40 early promoter or the cytomegalovirus promoter/enhancer. Selected clones secreted 2- to 4-fold more TGF beta-competing activity into their media than the parental cell line or neomycin-transfected controls. The TGF beta 1 released into the cell-conditioned medium was latent. Despite the latency of the overexpressed TGF beta 1, TGF beta 1-transfected cells exhibited phenotypic features of TGF beta 1-treated cells. When confluent, the TGF beta 1-transfected cells had the morphological characteristics of the parental cells that have been treated with active TGF beta 1. AKR-2B cells that expressed higher levels of TGF beta 1 also expressed high levels of c-sis and c-myc mRNAs and decreased TGF beta 2 and TGF beta 3 mRNAs in the same manner as parental AKR-2B cells that had been treated with active TGF beta 1. The transfected 1591 cells that overexpressed TGF beta 1 bound less [125I]TGF beta 1 than did parental 1591 cells, but after a mild acid wash demonstrated an increase in [125I]TGF beta 1 binding. Our results suggest that these TGF beta 1-transfected fibroblast and fibrosarcoma cells have the capacity to activate TGF beta; however, as very little activated TGF beta is detected in the medium, it is hypothesized that these cells activate latent TGF beta 1 and bind the activated TGF beta 1, thus acquiring a phenotype consistent with TGF beta 1-treated cells.

Identification and analysis of transforming growth factor beta receptors on primary osteoblast-enriched cultures derived from adult human bone.

Primary human osteoblast-enriched (PHO) cultures derived from adult trabecular bone were analyzed to determine the presence or absence of transforming growth factor beta (TGF-beta) receptors. Saturation binding studies were performed with 125I-TGF-beta in the absence or presence of 200-fold excess cold TGF-beta. Cross-linking experiments utilizing 125-I-TGF-beta were performed to identify specific cell surface binding proteins for TGF-beta. The saturation binding studies demonstrated saturable binding for TGF-beta on PHO cells. TGF-beta was cross-linked to cell surface binding proteins of 50 to 110 KDa and a high molecular weight component. Thus, these receptors appear to be similar in affinity, number per cell, and molecular weight to those previously identified with other cell types. The potential biological effects of TGF-beta on the growth of PHO cultures were evaluated by both 3H-thymidine incorporation and cell number determination. Growth of PHO cells in the presence of TGF-beta resulted in an approximately two-fold stimulation in cell number as compared to control cells while the 3H-thymidine experiments demonstrated a two to four-fold increase in thymidine uptake in the presence of TGF-beta. Radiographic emulsion studies revealed that the alkaline phosphatase positive and negative cell populations were responsive to the TGF-beta mitogenic stimulation. The cumulative findings of saturable binding, specific cell surface binding proteins, and biological effects suggest that functional TGF-beta cell surface receptors are present on primary osteoblast-enriched cultures derived from adult human trabecular bone.

TGF beta 1 and TGF beta 2 are potential growth regulators for low-grade and malignant gliomas in vitro: evidence in support of an autocrine hypothesis.

Low-grade astrocytomas, anaplastic astrocytomas and glioblastomas in vitro were found to ubiquitously produce the mRNA of transforming growth factor-beta (TGF beta). TGF beta 1 and TGF beta 2 mRNA were expressed to a lesser degree among the hyperdiploid malignant gliomas. By radioreceptor assay of conditioned medium, TGF beta was secreted predominantly in latent form, in both latent and active form, or only in active form within a panel of low-grade and malignant gliomas. The TGF beta receptor (types I, II, and III) was evident among the glioma lines. Many near-diploid gliomas were growth-inhibited by TGF beta 1 and TGF beta 2 in vitro. Most hyperdiploid glioblastomas showed a positive mitogenic response to exogenous TGF beta 1 and TGF beta 2. A synergistic or additive mitogenic interaction with epidermal growth factor and insulin was observed among some. Under serum-free conditions, anti-TGF beta antibody neutralized the expected growth-regulatory effect of endogenous TGF beta, thus establishing the specificity of the response in vitro. TGF beta 1 also enhanced the clonogenicity of certain gliomas which had been growth-stimulated in monolayer. Thus, basic elements in support of an autocrine hypothesis have been demonstrated: TGF beta mRNA was expressed among low-grade and malignant gliomas, TGF beta was secreted in latent and/or active form into conditioned media and appeared to serve as an endogenous regulator of glioma proliferation in vitro. The mitogenic response, either positive or negative, correlated with the degree of anaplasia and karyotypic divergence.

Modulation of growth-related gene expression and cell cycle synchronization by a sialoglycopeptide inhibitor.

When an 18-kDa cell surface sialoglycopeptide (SGP), isolated from intact bovine cerebral cortex cells, was incubated with exponentially growing Swiss 3T3 cells, cell proliferation was efficiently arrested. The inhibition was totally reversible since after removal of the SGP the arrested cells resumed their progress in the cell cycle in a synchronized manner for at least two divisions. Readdition of the GSP 4 h after reversal of the inhibition did not, however, affect the commitment of the cells to advance through metaphase, although progress through the cell cycle was once again inhibited after the cells reentered the G1 phase. The efficient nature of the SGP-mediated cell cycle arrest in G1 provided us with a basis to examine potential changes in the expression of several competence genes, and genes associated with mid and late G1, that have been implicated in cell cycle progression. Upon serum stimulation of quiescent Swiss 3T3 cells, the induction of c-myc and c-fos expression was not influenced by the SGP at concentrations highly inhibitory to cell cycling. Expression of JE was induced by serum, and the presence of the SGP had little effect on the expression of this growth-related gene. KC expression was not appreciably stimulated by serum although, surprisingly, the addition of the SGP resulted in a significant increase in expression. In addition, we learned that the SGP did not alter expression of ornithine decarboxylase, c-ras, or thymidine kinase, which are induced later than the genes associated with the initial stages of competence.

Altered regulation of protein disulfide isomerase in cells resistant to the growth-inhibitory effects of transforming growth factor beta 1.

A murine keratinocyte cell line that is resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF beta 1) was examined for differential gene expression patterns that may be related to the mechanism of the loss of TGF beta 1 responsiveness. Cells that were resistant to the growth-inhibitory effects of TGF beta 1 (KCR cells) were derived from K-ras-transformed BALB/MK keratinocytes (KC cells). Using a subtractive hybridization procedure with KC and KCR mRNAs, we isolated a complementary DNA clone for murine protein disulfide isomerase (PDI). The mRNA for PDI is inhibited by TGF beta 1 treatment in the parental KC cells, but not in the TGF beta 1-resistant KCR cells. Similar PDI down-regulation also occurs in other TGF beta-sensitive cells, but not in a human pancreatic carcinoma cell line which is insensitive to the growth-inhibitory effects of TGF beta 1. The results suggest that misregulation of PDI, an important component of co- and posttranslational modification systems, may be involved in the mechanism by which some cells escape from the growth-inhibitory effects of TGF beta 1.

Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors beta 1 and beta 2.

Regulation of transforming growth factor beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 mRNAs in murine fibroblasts and keratinocytes by TGF beta 1 and TGF beta 2 was studied. In quiescent AKR-2B fibroblasts, in which TGF beta induces delayed stimulation of DNA synthesis, TGF beta 1 autoregulation of TGF beta 1 expression was observed as early as 1 h, with maximal induction (25-fold) after 6 to 12 h. Increased expression of TGF beta 1 mRNA was accompanied by increased TGF beta protein production into conditioned medium of AKR-2B cells. Neither TGF beta 2 nor TGF beta 3 mRNA, however, was significantly induced, but both were apparently down regulated at later times by TGF beta 1. Protein synthesis was not required for autoinduction of TGF beta 1 mRNA in AKR-2B cells. Nuclear run-on analyses and dactinomycin experiments indicated that autoregulation of TGF beta 1 expression is complex, involving both increased transcription and message stabilization. In contrast to TGF beta 1, TGF beta 2 treatment of quiescent AKR-2B cells increased expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs, but with different kinetics. Autoinduction of TGF beta 2 mRNA occurred rapidly with maximal induction at 1 to 3 h, enhanced TGF beta 3 mRNA levels were observed after 3 h, and increased expression of TGF beta 1 occurred later, with maximal mRNA levels obtained after 12 to 24 h. Nuclear run-on analyses indicated that TGF beta 2 regulation of TGF beta 2 and TGF beta 3 mRNA levels is transcriptional, while TGF beta 2 induction of TGF beta 1 expression most likely involves both transcriptional and posttranscriptional controls. In BALB/MK mouse keratinocytes, minimal autoinduction of TGF beta 1 occurred at only the 12- and 24-h time points and protein synthesis was required for this autoinduction. The results of this study provide an example in which TGF beta 1 and TGF beta 2 elicit different responses and demonstrate that expression of TGF beta 1, and TGF beta 3 are regulated differently. The physiological relevance of TGF beta 1 autoinduction in the context of wound healing is discussed.

Regulation of transforming growth factor beta 1 action by multiple transducing pathways: evidence for both G protein-dependent and -independent signaling.

The effects of cholera toxin (CT) on transforming growth factor beta 1-stimulated protooncogene expression, [gamma-35S]GTP binding, GTPase activity and growth under anchorage-independent and -dependent conditions were studied in AKR-2B fibroblast cells. CT was shown to inhibit TGF beta 1-stimulated c-sis and c-myc mRNA expression. Actinomycin D decay and nuclear runon experiments demonstrated that this inhibition was not due to an increased decay of protooncogene message, but to a decreased transcriptional activation. These inhibitory effects were not secondary to changes in the ability of TGF beta 1 to bind to its receptor(s) since radioreceptor assays and affinity labeling studies demonstrated that CT had no effect on TGF beta 1 binding. ADP ribosylation of AKR-2B plasma membranes with [alpha-32P]NAD+ revealed a Mr 45,000 protein as the major CT substrate. The labeling of this Mr 45,000 protein in membranes could be inhibited by prior pretreatment of the cells with increasing concentrations of CT. Treatment of membranes with nanogram concentrations of CT abolished the increase in [gamma-35S]GTP binding following addition of TGF beta 1 as well as decreased basal binding. Similarly, CT pretreatment of membranes inhibited TGF beta 1-stimulated GTPase activity. Unexpectedly however, the stimulatory effects of TGF beta 1 on anchorage-independent growth in soft agar were unaffected by CT. Only pertussis toxin was able to inhibit TGF beta 1-induced colony formation in soft agar in a dose-dependent manner. Furthermore, differential effects of both CT and pertussis toxin were observed on TGF beta 1-stimulated monolayer growth; CT was inhibitory, whereas pertussis toxin was without effect. These results suggest that the diverse biological effects of TGF beta 1 are mediated through multiple intracellular pathways distinguishable by their toxin sensitivities.

Transforming growth factor beta in the control of epidermal proliferation.

Transforming growth factor beta is a polypeptide growth factor with a multiplicity of diverse biologic effects. Increasingly, data support a role for TGF beta in the autocrine regulation of normal epithelial cell growth (Figure 1). Definition of the normal pathways for growth stimulation and inhibition of epithelial cell growth by autocrine peptides like TGF beta and TGF alpha undoubtedly will increase understanding of normal growth and development, embryogenesis, wound repair, and tumorigenesis.

Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta.

Transforming growth factor beta (TGF beta) is a potent inhibitor of epithelial cell proliferation. A nontumorigenic epidermal growth factor (EGF)-dependent epithelial cell line, BALB/MK, is reversibly growth arrested by TGF beta. TGF beta will also abrogate EGF-stimulated mitogenesis of quiescent BALB/MK cells. Increased levels of calcium (greater than 1.0 mM) will induce differentiation in BALB/MK cells; in contrast, TGF beta-mediated growth inhibition does not result in induction of terminal differentiation. In the present study, the effects of TGF beta and calcium on growth factor-inducible gene expression were examined. TGF beta markedly decreased c-myc and KC gene expression in rapidly growing BALB/MK cells and reduced the EGF induction of c-myc and KC in a quiescent population of cells. TGF beta exerted its control over c-myc expression at a posttranscriptional level, and this inhibitory effect was dependent on protein synthesis. TGF beta had no effect on c-fos gene expression, whereas 1.5 mM calcium attenuated EGF-induced c-fos expression in quiescent cells. Expression of beta-actin, however, was slightly increased in both rapidly growing and EGF-restimulated quiescent BALB/MK cells treated with TGF beta. Thus, in this system, TGF beta selectively reduced expression of certain genes associated with cell proliferation (c-myc and KC), and at least part of the TGF beta effect was at a posttranscriptional level.

Growth modulation of mouse keratinocytes by transforming growth factors.

The effects of exogenously added transforming growth factor (TGF alpha and TGF beta on the growth of BALB/MK cells were examined. TGF alpha supplanted the epidermal growth factor (EGF) requirement in these cells. In contrast, TGF beta reversibly inhibited the growth of BALB/MK cells by abrogating the stimulatory actions of EGF or TGF alpha. The inhibitory effects of TGF beta appeared to be mediated by events distal to EGF ligand-receptor interactions. Growth inhibition of BALB/MK cells by TGF beta did not result in the induction of differentiation. This finding is different from the growth inhibition of these cells induced by elevated calcium levels (1.5 mM) which was tightly coupled to terminal differentiation. The BALB/MK cells were found to express TGF alpha mRNA, as well as TGF beta mRNA and protein. In addition, TGF alpha, as well as EGF, enhanced TGF alpha gene expression. These studies suggest a role for endogenous TGFs in regulating BALB/MK proliferation. TGF alpha provides a positive growth signal, while TGF beta is a potent inhibitor of growth even in the presence of such positive modulators as TGF alpha and EGF.

Inhibition of epidermal growth factor-stimulated DNA synthesis by a bovine sialoglycopeptide inhibitor occurs at an intracellular level.

The control of cell proliferation involves the complex interaction between growth factors and growth inhibitors. We have examined this interaction with the mitogen epidermal growth factor (EGF) and a recently purified 18 kD, pI 3, sialoglycopeptide that reversibly inhibits cellular metabolism of a variety of cells. The sialoglycopeptide was a very potent inhibitor of EGF action; 0.22 nM of the inhibitor completely blocked the mitogenic effect of 1.60 nM of EGF. The sialoglycopeptide, however, did not affect the binding of EGF to 3T3 cells. Neither the mixed affinities (0.11-1.9 nM) of binding nor the total number of receptors (50,000 receptors/cell) for EGF were altered by the addition of the sialoglycopeptide. In addition, competitive binding experiments demonstrated the specificity of inhibitor binding to 3T3 cells and also showed that EGF and the sialoglycopeptide did not share the same receptor, suggesting that the inhibitor blocked EGF action at a postreceptor, intracellular event in the signal cascade. We further demonstrated that the sialoglycopeptide had to be added within 2.5 hr after EGF to block effectively the stimulation of DNA synthesis by the growth factor, suggesting that the inhibitor blocked EGF stimulation at a relatively early step in the signal transduction mechanism.

A unique sialoglycopeptide growth regulator that inhibits mitogenic activity of a phorbol ester tumor promoter.

The ability of a naturally occurring cell surface sialoglycopeptide growth inhibitor to antagonize the induction of DNA synthesis by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied with mouse 3T3 cells. The bovine sialoglycopeptide was shown to be a potent antagonist of TPA-induced DNA synthesis in confluent 3T3 cell cultures. Kinetic studies demonstrated that inhibition of TPA-induced DNA synthesis required the addition of the sialoglycopeptide within 15 min of TPA treatment. Addition of the sialoglycopeptide 30 min or longer after the cells were exposed to TPA did not block stimulation of DNA synthesis by TPA. The inhibition of TPA action was shown not to be restricted to DNA synthesis in 3T3 cultured cells since the sialoglycopeptide also inhibited TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxylase, EC 4.1.1.17) activation in suspensions of mouse epidermal and 3T3 cells.

Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis.

We have isolated from bovine cerebral cortex cells and purified to homogeneity an 18,000 dalton, pl 3.0 sialoglycopeptide that inhibits protein synthesis and DNA synthesis of nontransformed but not transformed cells without affecting uptake of radiolabeled precursors. In this paper, we examine the relationship between the binding of the sialoglycopeptide inhibitor to 3T3 cells and inhibition of protein synthesis. Binding of the sialoglycopeptide to 3T3 cells was rapid at 37 degrees C and reached a maximum at 30 min; the binding at 37 degrees C was shown to be saturable and specific. Scatchard analysis of the binding indicated that 3T3 cells contained about 2 X 10(4) receptors/cell with a dissociation constant of 1.0-1.5 nM. Several lines of evidence indicated that receptor occupancy on 3T3 cells correlated with the protein synthesis inhibitory activity of the sialoglycopeptide. A comparison of the kinetics of inhibitor binding with the kinetics of protein synthesis inhibition demonstrated that binding directly correlated with the inhibition of protein synthesis, concentration-dependent inhibition of protein synthesis directly correlated with concentration-dependent receptor occupancy, and a direct correlation was also observed between the kinetics of inhibitor dissociation from its specific cell surface receptor and the kinetics of recovery from protein synthesis inhibition.

Cell agglutination by a novel cell surface sialoglycopeptide inhibitor and the relationship between its protease and biological activities.

A bovine sialoglycopeptide, purified to homogeneity and capable of inhibiting cellular protein synthesis and proliferation, was shown to agglutinate a wide variety of nontransformed and transformed cells. The cell agglutination activity was shown to be independent of the biological inhibitory action and most likely related to a protease activity that could not be physically separated during purification of the sialoglycopeptide. Samples that were completely biologically inactivated retained full protease activity and their ability to agglutinate target cells. Balb/c 3T3 cells were not agglutinated by the sialoglycopeptide and they elicited a protein that interfered with the agglutination reaction and even redispursed cells that already had been aggregated by the inhibitor.

The effects of a calcium and a sodium ionophore on protein synthesis inhibition by a bovine cell surface sialoglycopeptide.

The ability of the calcium ionophore A23187 and the sodium ionophore Monensin to antagonize the inhibition of 3T3 cell protein synthesis by a bovine cell surface sialoglycopeptide was measured. A23187, when added before and shortly after the sialoglycopeptide, significantly reduced the biological activity of the inhibitory glycopeptide. In contrast, Monensin had little, if any, influence on protein synthesis inhibition by the sialoglycopeptide. The ability of A23187 to circumvent the inhibitory action of the bovine glycopeptide was shown to be independent of the time the ionophore was incubated with the cells and the binding of the sialoglycopeptide to the 3T3 target cells. Neither the total amount of sialoglycopeptide bound to the cells, nor its affinity to the cell surface receptor, was influenced by the presence of A23187.

Cell surface interaction is sufficient for the biological activity of a bovine sialoglycopeptide inhibitor.

A sialoglycopeptide inhibitor, isolated from bovine cerebral cortex cells, that reversibly inhibits protein and DNA synthesis, was coupled to either Sepharose or polyacrylamide beads. Whereas over 1 ng of the inhibitor was released from Sepharose beads after 30 min at 37 degrees C, less than 0.2 ng of the sialoglycopeptide was released from the polyacrylamide beads. When added to 3T3 cells, the immobilized sialoglycopeptide efficiently inhibited protein synthesis. No detectable sialoglycopeptide inhibitor was released into the assay medium in the presence or absence of 3T3 cells. Addition of [125I]sialoglycopeptide, coupled to acrylamide P100 beads, to 3T3 cells also demonstrated that the sialoglycopeptide was not internalized by the cells. Thus we conclude that an interaction of the sialoglycopeptide at the cell surface is sufficient for biological inhibitory activity.

Purification and characterization of a bovine cerebral cortex cell surface sialoglycopeptide that inhibits cell proliferation and metabolism.

A sialoglycopeptide from bovine cerebral cortex cells was purified to apparent homogeneity by a procedure that included chloroform/methanol extraction, diethylaminoethyl ion exchange chromatography, wheat germ agglutinin affinity chromatography, size-exclusion HPLC, and hydrophobic interaction chromatography. The cell surface inhibitor had a molecular weight of approximately 18,000, no subunit composition was detectable on reduction and polyacrylamide gel electrophoresis analysis, and the glycopeptide apparently contained sialic acid, as illustrated by its ability to bind to Limulus polyhemus lectin. Deglycosylation of the molecule, however, did not reduce its protein synthesis inhibitory activity. As little as 20 ng of the sialoglycopeptide was capable of inhibiting protein synthesis in a wide variety of fibroblast cell lines but not in transformed cells. Mice immunized with the sialoglycopeptide produced antibodies that, when bound to protein A-agarose gel, removed the inhibitory activity from solution. The antibodies were used to identify a single isoelectric focused band and to establish the pI of 3.0 for the molecule.