Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation

Tumors arise from cells that have sustained genetic mutations resulting in deregulation of several of their normal growth-controlling mechanisms. Much of the research concerning the origins of cancer has focused on the genetic mutations within tumor cells, treating tumorigenesis as a cell-autonomous process governed by the genes carried by the tumor cells themselves. However, it is increasingly apparent that the stromal microenvironment in which the tumor cells develop profoundly influences many steps of tumor progression. In various experimental tumor models, the microenvironment affects the efficiency of tumor formation, the rate of tumor growth, the extent of invasiveness, and the ability of tumor cells to metastasize. In carcinomas, the influences of the microenvironment are mediated, in large part, by paracrine signaling between epithelial tumor cells and neighboring stromal fibroblasts. In this review, we summarize recent advances in understanding the paracrine signaling interactions between epithelial cancer cells and associated fibroblasts and examine the effects of these bidirectional interactions on various aspects of carcinoma formation. We note, however, that paracrine signaling between other cell types within the carcinomas, such as endothelial cells and inflammatory cells, may play equally important roles in tumor formation and we will refer to these heterotypic interactions where relevant.     

Differences between rat liver epithelial cells and fibroblast cells in sensitivity to 8-azaguanine

Summary Epithelial and fibroblast cells from adult rat liver were found to differ markedly in their sensitivities to the toxic effects of the purine analog, 8-azaguanine. Epithelial cells were rapidly killed by 8-azaguanine, whereas fibroblast cells suffered no observable toxicity. The resistance of fibroblast cells was not due to impermeability since it was shown by autoradiography that both cell types took up and utilized exogenous purines. Moreover, both cell types were sensitive to 6-thioguanine. The fibroblast cells, however, possessed a greater guanase activity than did the epithelial cells, measured by conversion of 8-azaguanine to 8-azaxanthine in the cell lines. Both cell types possessed hypoxanthine-guanine phosphoribosyl transferase for phosphoribosylating exogenous purines and thus making them metabolically available. Epithelial cell lysates convert 8-azaguanine to 8-azaguanosine-5′-monophosphate, the toxic metabolite of 8-azaguanine. Fibroblast cell lysates converted much more 8-azaguanine to 8-azaguanosine, an inactive metabolite, than did the epithelial cells. This conversion was presumably due to the much greater activity of 5′-nucleotidase in fibroblasts than epithelial cells; it degrades 8-azaguanosine-5′-monophosphate to 8-azaguanosine. These differences in purine metabolism suggest that fibroblast resistance to 8-azaguanine is due to the combination of a significant guanase activity that limits the amount of 8-azaguanine available and a high 5′-nucleotidase activity that would result in conversion of 8-azaguanosine-5-monophosphate, the toxic metabolite of 8-azaguanine, to 8-azaguanosine. This work was supported by Grant ES-01-724-01 from the National Institute of Environmental Health Sciences. C. T. is a recipient of the Young Environmental Scientist Health Research Grant Program, NIEHS.     

Differences between rat liver epithelial and fibroblast cells in metabolism of purines

Epithelial and fibroblast cells from adult rat liver were found to differ markedly in their metabolism of the purine hypoxanthine. Both cell types took up hypoxanthine and possessed hypoxanthine-guanine phosphoribosyl transferase for phosphoribosylating the purine. However, in the transferase assay, lysates from epithelial cells converted hypoxanthine predominantly to inosine monophosphate, with small amounts of the nucleoside inosine as product, whereas fibroblast cell lysates converted hypoxanthine predominantly to inosine. The inosine appeared not to be produced by direct ribosylation of the base, since fibroblast cell lysates had less purine nucleoside phosphorylase activity than epithelial cell lysates. Rather, the inosine produced by fibroblast lysates appeared to be derived from inosine monophosphate through catabolism of the mononucleotide by 5' nucleotidase. An inhibitor of 5' nucleotidase, thymidine triphosphate, reduced the amount of inosine formed.     

Vimentin-derived proteins : Differences between normal human fibroblasts and transformed human cells

Two-dimensional gel electrophoresis revealed a quantitative difference in a series of polypeptides ranging in MW from 45 000 to 51 000 and of lower isoelectric pH than vimentin, when comparing normal human fibroblasts with a virally transformed subline and with HeLa cells. Re-extraction of purified [³⁵S]vimentin with cold whole cell homogenates and peptide mapping showed that these polypeptides are derivatives of vimentin. They may be natural components of a normal fibroblast's architecture or they may arise from a pool of vimentin that is not structured within intermediate filaments at the time of extraction. Furthermore, we show that vimentin from the two transformed cell types is more resistant to proteolysis by whole cell homogenates than vimentin from normal fibroblasts. Structural alteration of vimentin may play an important role in the expression of transformation.     

Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells,fibroblasts, alveolar type II cells,airway epithelial cells and lymphocytes

Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC‐specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8⁺ T cells from bronchial lymph nodes (T‐BL) and CD8⁺ T cells from lungs (T‐LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up‐regulated and 70% down‐regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over‐expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types.     

Ventral stress fibers induce plasma membrane deformation in human fibroblasts

Interactions between the actin cytoskeleton and the plasma membrane are important in many eukaryotic cellular processes. During these processes, actin structures deform the cell membrane outward by applying forces parallel to the fiber’s major axis (as in migration) or they deform the membrane inward by applying forces perpendicular to the fiber’s major axis (as in the contractile ring during cytokinesis). Here we describe a novel actin–membrane interaction in human dermal myofibroblasts. When labeled with a cytosolic fluorophore, the myofibroblasts displayed prominent fluorescent structures on the ventral side of the cell. These structures are present in the cell membrane and colocalize with ventral actin stress fibers, suggesting that the stress fibers bend the membrane to form a “cytosolic pocket” that the fluorophores diffuse into, creating the observed structures. The existence of this pocket was confirmed by transmission electron microscopy. While dissolving the stress fibers, inhibiting fiber protein binding, or inhibiting myosin II binding of actin removed the observed pockets, modulating cellular contractility did not remove them. Taken together, our results illustrate a novel actin–membrane bending topology where the membrane is deformed outward rather than being pinched inward, resembling the topological inverse of the contractile ring found in cytokinesis.     

Cadherin-23 mediates heterotypic cell-cell adhesion between breast cancer epithelial cells and fibroblasts

In the early stages of breast cancer metastasis, epithelial cells penetrate the basement membrane and invade the surrounding stroma, where they encounter fibroblasts. Paracrine signaling between fibroblasts and epithelial tumor cells contributes to the metastatic cascade, but little is known about the role of adhesive contacts between these two cell types in metastasis. Here we show that MCF-7 breast cancer epithelial cells and normal breast fibroblasts form heterotypic adhesions when grown together in co-culture, as evidenced by adhesion assays. PCR and immunoblotting show that both cell types express multiple members of the cadherin superfamily, including the atypical cadherin, cadherin-23, when grown in isolation and in co-culture. Immunocytochemistry experiments show that cadherin-23 localizes to homotypic adhesions between MCF-7 cells and also to heterotypic adhesions between the epithelial cells and fibroblasts, and antibody inhibition and RNAi experiments show that cadherin-23 plays a role in mediating these adhesive interactions. Finally, we show that cadherin-23 is upregulated in breast cancer tissue samples, and we hypothesize that heterotypic adhesions mediated by this atypical cadherin may play a role in the early stages of metastasis.     

Percentage of epithelial cells, fibroblasts and endothelial cells in the dog thyroid

The proportions of various cells types were studied in the thyroids of a variety of dogs used for laboratory experiments. A remarkable constancy was found with epithelial cells accounting for 70% of the total number of cells. The relative proportions of luminal and epithelial cells spaces were also very similar in different animals (47% and 26% respectively).     

Defensins are mitogenic for epithelial cells and fibroblasts

Defensins are a family of structurally homologous peptides contained within phagocytic cells. Although these peptides are best known for their broad spectrum antimicrobial properties, they also inhibit ACTH (corticotropin) stimulated corticosterone production, chemoattract monocytes, and lyse mammalian cells. We now report that these peptides are potent mitogens in vitro in the same concentration range that they display potent antimicrobial activity in vitro. These concentrations are in the same range as those expected to be present in vivo during the wound healing process. All defensins tested were stimulatory for epithelial cells and fibroblasts and acted synergistically with insulin. These are the first data to disclose the strong growth-promoting effects of this unique family of peptides and point to another basic mechanism whereby the macrophage and neutrophil may participate in a variety of trophic, physiologic, and pathologic processes.     

Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts.

The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors.     

Rho-mediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts

Corneal keratocytes (stromal cells) are activated to fibroblasts and myofibroblasts during wound healing. Myofibroblast transdifferentiation is accompanied by the expression of α-smooth muscle actin (α-SMA) and the assembly of a robust stress fiber network and larger focal adhesions (FAs). The regulation of the assembly of stress fibers was evaluated in cultured corneal fibroblast and myofibroblast phenotypes. In both cell types, the inhibition of Rho GTPase activity by microinjecting C3 transferase into the cells resulted in the disassembly of stress fibers and FAs. However, the inhibition of the Rho-associated kinases ROKα and ROKβ with their inhibitor, Y27632, or by overexpression of their mutant kinase-dead forms resulted in only a partial loss of the stress fibers and FAs in myofibroblasts but a total loss in fibroblasts. ROK inhibitor-sensitive and -resistant stress fibers in myofibroblasts contained α-SMA, nonmuscle myosin II, tropomyosin, and calponin. The ROK inhibition-resistant stress fibers and FAs were lost upon the overexpression of the dominant-negative form of mDia1 (a mammalian homolog of Drosophila diaphanous protein). These findings indicated that while the assembly of stress fibers in fibroblasts critically involves both ROK and mDia1, in myofibroblasts, the assembly of α-SMA-containing stress fibers also occurs independently of ROK and involves Rho/mDia1.     

Formation, transport, contraction, and disassembly of stress fibers in fibroblasts

Swiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum-deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video-enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive manner at the cytoplasm-membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10-20 microns/h. When serum-deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post-stimulation.     

Bacillus anthracis internalization by human fibroblasts and epithelial cells

The current model for Bacillus anthracis dissemination in vivo focuses on macrophages as carriers. However, recent evidence suggested that other host cells may also play a role in the process. Here, we tested the possibility of B. anthracis being internalized by a human fibroblast cell line, HT1080 and an epithelial cell line, Caco-2. A combination of gentamicin protection assays, scanning and transmission electron microscopy (EM) and fluorescence microscopy was used. The results demonstrated for the first time that both spores and vegetative cells of B. anthracis Sterne strain 7702 were able to adhere to and be internalized by cultured HT1080 and Caco-2 cells. Spore adherence to and internalization by HT1080 cells were not affected by a germination inhibitor. This suggested that certain features on dormant spores were sufficient for these processes. Vegetative cell adherence to and internalization by both cell lines were growth phase-dependent. EM images suggested that vegetative cells may have the ability to escape phagocytic vacuoles. Finally, we showed that internalization of both spores and vegetative cells required active functions of the host cell cytoskeleton. These results raised the possibility that B. anthracis may disseminate in vivo by directly infecting non-phagocytic cells.     

Direct visualization of bipolar myosin filaments in stress fibers of cultured fibroblasts

The authors examined the molecular organization of myosin in stress fibers (microfilament bundles) of cultured mouse embryo fibroblasts. To visualize the organization of myosin filaments in these cells, fibroblast cytoskeletons were treated with gelsolin-like protein from bovine brain (hereafter called brain gelsolin), which selectively disrupts actin filaments. As shown earlier [Verkhovsky et al., 1987], this treatment did not remove myosin from the stress fibers. The actin-free cytoskeletons then were lightly sonicated to loosen the packing of the remaining stress fiber components and fixed with glutaraldehyde. Electron microscopy of platinum replicas of these preparations revealed dumbbell-shaped structures of approximately 0.28 micron in length, which were identified as bipolar myosin filaments by using antibodies to fragments of myosin molecule (subfragment 1 and light meromyosin) and colloidal gold label. Bipolar filaments of myosin in actin-free cytoskeletons were often organized in chains and lattices formed by end-to-end contacts of individual filaments at their head-containing regions. Therefore, after extraction of actin, it was possible for the first time to display bipolar myosin filaments in the stress fibers of cultured cells.     

Anti-fas induces apoptosis and proliferation in human dermal fibroblasts: Differences between foreskin and adult fibroblasts

Apoptosis, or programmed cell death, is a naturally occurring process mediated by extracellular signals. We studied anti-Fas (CD95/Apo-1) antibody-induced apoptosis in cultured human foreskin and adult dermal fibroblasts. Induction of apoptosis was identified by fluorescence in situ DNA end-labeling. Anti-Fas antibody induced apoptosis in fibroblasts in a dose- and time-dependent manner. Adult dermal skin fibroblasts were more susceptible to anti-Fas antibody-induced apoptosis than foreskin fibroblasts, with 21–52% dead cells in different strains. In foreskin fibroblasts, anti-Fas antibody (1.0 μg/ml) predominantly induced proliferation ([³H]thymidine incorporation increased to 115–165% of control level) and only low levels of apoptotic cell death after 48 hours of treatment. No induction of proliferation by anti-Fas was found in the adult fibroblasts. Addition of tumor necrosis factor-α (TNF-α) slightly augmented the anti-Fas antibody-induced apoptosis in both cell types. When we examined the levels of Fas expression using flow cytometry, we found two- to threefold higher Fas expression in adult fibroblasts. C6-ceramide treatment, which induces Fas-independent apoptosis, gave similar levels of cell death in both foreskin and adult fibroblasts. No proliferation was observed in C6-ceramide-treated fibroblasts. Thus, this difference in apoptosis between adult dermal and foreskin fibroblasts appears to be related to the level of Fas expression. When clones of foreskin fibroblasts were examined, there was heterogeneity of anti-Fas antibody-induced apoptosis and proliferation in the cloned fibroblast subpopulations, but this was not correlated with differences in Fas expression. Alterations in fibroblast populations during the process of differentiation and aging may result from selective loss of apoptosis-susceptible populations. J. Cell. Physiol. 175:19–29, 1998. © 1998 Wiley-Liss, Inc.     

Mechanisms of transforming growth factor-beta receptor endocytosis and intracellular sorting differ between fibroblasts and epithelial cells

Transforming growth factor-betas (TGF-beta) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-beta type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-beta receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.     

Differences in proteins secreted by human fibroblasts and muscle cells in culture

Cell cultures of human skin fibroblasts, myoblasts, and fused muscle cells were grown in the presence of [¹⁴C]leucine or a mixture of [¹⁴C]amino acids. The proteins synthesised and secreted or leaked into the culture medium during radio-labelling were separated by one and two-dimensional PAGE and detected by fluorography. Four major bands of M_r 54 kD, 52 kD, 51 kD, and 49 kD were present at greatly increased concentration in fibroblast media. These fibroblast-specific polypeptides can be readily detected in myoblast/fibroblast cocultures with fibroblast content as low as 5%.     

Calcyclin as a marker of human epithelial cells and fibroblasts

The distribution of calcyclin in human tissues was studied using polyclonal antibodies against this protein. In all organs examined (breast, heart, intestine, kidney, liver, ovary, placenta, stomach, thymus, and uterus) only epithelial cells and fibroblasts were stained. This suggests that calcyclin expression is related either to proliferation rate or secretion activity. The data show that calcyclin might be considered as a marker of some human epithelial cells and fibroblasts.     

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking- focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.