Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.     

Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins

Epidermal growth factor (EGF) is a peptide which effects the growth and/or differentiated functions of many cell types. Several pieces of evidence indicate that EGF and its receptor may play a role in carcinogenesis. Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins are discussed. EGF has extensive homology with alpha-transforming growth factor (alpha-TGF), which may actually be an embryonic form of EGF. Nevertheless, both EGF and alpha-TGF elicit transformation-associated phenotypes in target cells under certain conditions. EGF effects are mediated by a receptor present on the plasma membrane. The EGF receptor is a highly complex protein having several functions in addition to binding EGF in a highly specific manner. One of these functions is to phosphorylate tyrosyl residues on certain proteins. This activity is similar to that expressed by the src family of oncogene-encoded proteins. Besides sharing functional homology the EGF receptor also exhibits structural homology to several oncogene-encoded proteins. The v-erb-B-transforming protein has a striking extent of homology (95%) to the cytoplasmic portion of the EGF receptor. These data support the concept that some aspect of EGF-stimulated metabolism is involved in cellular transformation.     

Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against alpha-transforming growth factor

Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human alpha-transforming growth factor (alpha-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting alpha-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native alpha-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of alpha-TGF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of alpha-TGF has a cellular role beyond that of an autocrine growth factor.     

Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions

A latent beta-TGF activity is spontaneously released into serum-free culture medium by chicken embryo fibroblasts. Anchorage-independent growth activity measured on NRK-49F indicator cells, of this latent beta-TGF can be revealed by four different treatments: acidification, alkalinisation, exposure to urea, and heating to 100 degrees C for 3 minutes. This lact activating treatment indicates that latent beta-TGF activation in vitro is non-enzymatic. Active beta-TGF exists in a low molecular weight form 16 Kd (apparent) in 1M acetic acid, which elutes on reverse phase (FPLC) between 33-35% acetonitrile. Under neutral conditions only a high molecular weight form excluded on Biogel P60 is observed. This form is poorly active on NRK-49F for anchorage independent growth but can be fully activated by prior acidification. Rechromatography of the latent beta-TGF-containing fractions under acidic conditions converts the high molecular weight form to an apparent 16 Kd active form. We suggest that the high molecular weight form may correspond to a complex of a beta-TGF associated with a carrier or binding protein.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Local effects of EGF, alpha-TGF, and EGF-like growth factors on lobuloalveolar development of the mouse mammary gland in vivo

Five-week-old female mice supplemented with estradiol and progesterone are able to respond to epidermal growth factor (EGF) and EGF-like growth factors (alpha-transforming growth factor [alpha-TGF] and crude mammary-derived growth factor) with local lobuloalveolar development when these growth factors are directly introduced into the mammary glands via slow-release cholesterol-based pellets. Contralateral glands receiving pellets containing only cholesterol showed no growth response. The local growth effect is maximal at 4-5 days of exposure to hormones and growth factors. The glands appear to be more sensitive to alpha-TGF than EGF, since local development is seen with one-fifth the level of the former vs. the latter growth factor and can be seen even in the absence of the systemic estrogen/progesterone supplement.     

Growth factor production by Creutzfeldt-Jakob disease cell lines.

Creutzfeldt-Jakob disease (CJD), a progressive dementia of humans, is caused by an infectious agent that is closely related to the scrapie agent of sheep. Although the molecular nature of these "unconventional" agents is still a matter of speculation and controversy, even less is known concerning the mechanism(s) of their effects on the central nervous system. To gain insight into the cellular effects of these agents, we have examined a series of cell lines derived directly from CJD-infected hamster brain or produced from nontransformed rodent lines by exposure to CJD infectious fractions in vitro. These cell lines appear transformed by a variety of criteria and secrete growth factors into the culture medium. All CJD lines produce a factor that is like alpha-transforming growth factor (alpha-TGF). Conditioned medium from these CJD lines also stimulates the synthesis of glial fibrillary acidic protein in normal astrocytic cells in vitro. This effect is mimicked by purified alpha-TGF and platelet-derived growth factors. Further study of CJD-induced growth factor production may elucidate fundamental properties of these unconventional agents.     

Chemically and virally transformed cells able to grow without anchorage in serum-free medium: evidence for an autocrine growth factor

BA10-IR transformed cells, obtained by treating Syrian hamster embryo fibroblasts (HEF) with 7-methylbenz(a)anthracene and cultivated for a long period, are highly tumorigenic and grow in suspension as aggregates (spheroids) (Levy et al., 1976). They also grow in attached form or as spheroids in serum-free (S-) synthetic medium, without insulin and transferrin, and form anchorage-independent (AI) colonies in this same, but semi-solid, medium. This exceptional phenotype was acquired stepwise, after other transformation parameters, and appears to be related to the capacity of the transformed cells to respond to a mitogenic growth factor which they secrete. The response to this autocrine factor is amplified by insulin and transferrin. Untransformed HEF, at late and early passages, and also mouse and rat embryo fibroblasts, secrete factors equally active on BA10-IR cells; but HEF do not respond, in S- medium, to their factor, or that of BA10-IR cells. Rat FR3T3 fibroblasts transformed by Kirsten murine sarcoma virus (FR3T3-Ki cells) also form AI colonies in semi-solid S- medium, secrete an autocrine factor potentiated by insulin and transferrin, and respond to the factors active on BA10-IR cells. However, they form far fewer colonies without additives, and respond as well to the mitogenic factors only in the presence of insulin and transferrin. BA10-IR cells and FR3T3-Ki cells also release beta-TGF, or a related factor, in an active and a latent form, activable by acidification, and HEF latent, activable beta-TGF. However, the factors shed by BA10-IR cells or HEF which stimulate AI growth of BA10-IR and FR3T3-Ki cells are proteins which seem unrelated to known transforming growth factors. Two major cellular alterations characteristic of the transformed phenotype in vitro are the ability to grow in the absence of anchorage, in semi-solid medium, and reduced dependence on serum growth factors (Hanafusa, 1977; Tooze, 1980). These alterations are often expressed together, and anchorage independence also appears to be the in vitro transformation parameter which correlates best with the tumorigenicity of the transformed cells (Pollack et al., 1975; Shin et al., 1975; Cifone and Fidler, 1980). However, this correlation is not constant (cf., Tooze, 1980). The cellular changes which confer anchorage independence remain unknown, but the culture conditions which allow anchorage-independent (AI) growth are better known. This growth occurs in the same media which permit the growth of attached cells, but generally requires serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fibronectin-associated transforming growth factor

We have studied the ability of fibronectins to induce anchorage-independent growth of NRK-49F cells in serum-free medium. Cells were seeded in soft agar in the presence of various concentrations of plasma fibronectins, and colonies were counted after 10 days. It was found that, with some exceptions, human plasma fibronectins induced anchorage-independent growth at concentrations in 20-100 micrograms/ml range. The ability of exogenously supplied fibronectins to promote anchorage-independent growth of NRK cells is attributed to a transforming growth factor (TGF) activity associated with gelatin-agarose affinity purified plasma fibronectins. This TGF activity required epidermal growth factor (EGF) in our serum-free assay system. The TGF-like activity appears to either co-purify or to be associated with fibronectin at neutral pH during molecular sieve chromatography and during ultracentrifugation through sucrose density gradients. The TGF activity "dissociates" from fibronectin at extremes of pH, however, and can be separated from fibronectin by molecular sieve chromatography in 1 M acetic acid. Under these conditions, the TGF-like activity chromatographed as a single peak with an apparent molecular weight of approximately 30 kDa. The physical-chemical properties, chromatographic behavior, and biological activity of this TGF suggest that it is type-beta transforming growth factor/growth inhibitor (beta-TGF/GI). The TGF activity has been observed in fibronectin isolated from fresh human plasma as well as in fibronectins from several other species obtained from commercial suppliers. Our results would suggest that caution be applied in the interpretation of experiments in which gelatin affinity purified fibronectins are used at micrograms/ml concentrations.     

Activated H-ras transforms rat intestinal epithelial cells with expression of alpha-TGF

We describe malignant transformation of cultured rat intestinal epithelial cells (IEC-18) by transfection with the activated human H-ras gene cloned from the EJ bladder carcinoma. Transformed cells showed a marked morphological change, expressed high levels of the transfected H-ras gene, were able to grow in agar and expressed antigenic markers identical with parental IEC-18 cells. When injected into syngeneic rats these cells formed rapidly growing tumors expressing the same intestinal-specific antigenic markers as the injected cells. Parallel to the high expression of H-ras mRNA in the transformants we document overexpression of rat alpha-TGF mRNA.     

Perisinusoidal stellate cells of the liver: important roles in retinol metabolism and fibrosis

In mammals, liver perisinusoidal stellate cells play an important role as a main store of body retinol (vitamin A). This fat-soluble vitamin is essential for vision, and regulates differentiation and growth of many cell types during embryonal development as well as in adult tissues. Thus, many cell types require a continuous supply of retinol. The storage of retinol (as retinyl esters) in stellate cells ascertains ample access of retinol to such cells also during periods with a low dietary intake. In lower vertebrates such as fish, vitamin A-storing stellate cells are found not only in the hepatic lobule, but also in the connective tissues of organs like intestine, kidney, ovaries, testes, and gills. Extrahepatic vitamin A-storing stellate cells are found in higher vertebrates when excessive doses of vitamin A are administered. It is not clear at present whether these cells also play a role in retinol metabolism under normal conditions. Stellate cells proliferate in a fibrotic liver, and they have been found to synthesize connective tissue compounds such as collagen. It was recently demonstrated that stellate cells are the principal cellular source of collagen and other extracellular substances in normal as well as fibrotic livers. Therefore, stellate cells, which seem to be a specialized type of pericyte, have a central role in the pathological changes observed during the development of liver fibrosis.     

Transforming growth factors and control of neoplastic cell growth

Transforming growth factors (TGFs) are peptides that affect the growth and phenotype of cultured cells and bring about in nonmalignant fibroblastic cells phenotypic properties that resemble those of malignant cells. Two types of TGFs have been well characterized. One of these, TGF alpha, is related to epidermal growth factor (EGF) and binds to the EGF receptor, whereas the other, TGF beta, is not structurally or functionally related to TGF alpha or EGF and mediates its effects via distinct receptors. TGF beta is produced by a variety of normal and malignant cells. Depending upon the assay system employed, TGF beta has both growth-inhibitory and growth-stimulating properties. Many of the mitogenic effects of TGF beta are probably an indirect result of the activation of certain growth factor genes in the target cell. The ubiquitous nature of the TGF beta receptor and the production of TGF beta in a latent form by most cultured cells suggests that the differing cellular responses to TGF beta are regulated either by events involved in the activation of the factor or by postreceptor mechanisms. The combined effects of TGF beta with other growth factors or inhibitors evidently play a central role in the control of normal and malignant cellular growth as well as in cell differentiation and morphogenesis. Since transforming growth factor as a concept has partially proven misleading and insufficient, there is a need to find a new nomenclature for these regulators of cellular growth and differentiation.     

Structures in focus--filopodia

Filopodia are thin cell surface extensions filled with tight parallel bundles of actin filaments. They are highly dynamic structures which rapidly extend and retract as well as sweep up and down and from side to side, and can be found at the leading edge of many types of motile cells such as fibroblasts and keratinocytes, as well as the growth cone tips of migrating axons. Cells appear to use filopodia to explore the extracellular matrix (ECM) and surfaces of other cells, identifying appropriate targets for adhesion or in the case of a migrating growth cone, for sensing guidance cues that enable the axon to navigate to it's appropriate target. As well as this sensory role, filopodia have also recently been shown to play an important mechanical role in epithelial adhesion, and are likely to be key players in developmental processes that require migrating epithelial sheets to zipper and fuse to one another. Their dynamic properties as well as their tendency to be damaged or lost after fixation mean they are best analysed using live imaging techniques. As this field improves, the number of tissues in which filopodia are seen to be playing key roles is fast increasing.     

Lymphocytes in cancer development: Polarization towards pro-tumor immunity

The classic view that the role of immune cells in cancer is primarily one of tumor rejection has been supplanted by a more complex view of leukocytes having both pro- and anti-tumor properties. This shift is due to the now well recognized capabilities of several myeloid cell types that foster pro-tumor programming of premalignant tissue, as well as the discovery that subsets of leukocytes also suppress development and effector functions of lymphocytes important for mediating anti-tumor immunity. In this review, we focus on the underappreciated role that T lymphocytes play in promoting tumor development. This includes, in addition to the role of T regulatory cells, a role for natural killer T cells and CD4⁺ T helper cells in suppressing anti-tumor immunity and promoting cancer growth and metastasis.     

Hsp70 in oncology

Hsp70 classes of molecular chaperones are highly conserved in all organisms and play an essential role in the maintenance of cellular homeostasis. Hsp70s assist nascent chain protein folding and denatured proteins, as well as the import of proteins to the organelles, and solubilization of aggregated proteins. ATPase function is required for Hsp70 function. Hsp70s use ATP hydrolysis driven mechanism for substrate protein binding and release. Various Hsps are unregulated in cancers but their significance for tumor growth is poorly understood. Studies have linked Hsp70 to several types of carcinoma. Human Hsp70s allow proliferation of cancer cells and suppress apoptotic and senescence pathways. This review presents Hsp70s role for growth of transformed cells and the current state of Hsp70 as a drug target along with recent patents in humans in this particular area.     

The carboxy-tail of connexin-43 localizes to the nucleus and inhibits cell growth

Gap junctions are plasma membrane intercellular communication channels that in addition to ensuring electrical coupling and coordinated mechanical activity, can act as growth suppressors. To define the role of a non-channel forming domain of connexin-43 (Cx43), the main constituent of cardiomyocyte gap junctions, on growth regulation, we expressed its C-terminal portion (CT-Cx43) in cardiomyocytes and HeLa cells. In addition to broad cytoplasmic localization, CT-Cx43 was also localized to the nucleus of both cell types, detected by immunofluorescence as well as immunoblotting of subcellular fractions. Furthermore, stable expression of CT-Cx43 in HeLa cells induced a significant decrease in proliferation. It is therefore suggested that plasma membrane localization and formation of channels are not required for growth inhibition by Cx43, and that nuclear localization of CT-Cx43 may exert effects on gene expression and growth.     

The IGF-I Receptor in Mitogenesis and Transformation of Mouse Embryo Cells: Role of Receptor Number

The type 1 receptor for insulin-like growth factors (IGF-IR) plays an important role in the growth and transformation of several types of cells. We have investigated the role of IGF-IR number in IGF-I-mediated mitogenesis and transformation of mouse embryo fibroblasts. We have used R⁻cells (3T3-like cells originating from mouse embryos with a targeted disruption of the IGF-IR genes) transfected with a plasmid expressing the human IGF-IR cDNA to generate clones with receptor numbers ranging from zero to 10⁶receptors per cell. In this model, between 15,000 and 22,000 receptors per cell are sufficient to render mouse embryo cells competent to grow in serum-free medium supplemented solely with IGF-I. For growth in soft agar, 30,000 receptors per cell seem to be the minimum requirement. These experiments indicate that a small increment in the number of receptors per cell, well within the physiological range, can modulate the mitogenic and transforming activities of the IGF-IR in 3T3-like cells.