Showing papers on "Cell growth published in 1985"

Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vitro

Abstract: Modulation of the growth of human and murine cell lines in vitro by recombinant human tumor necrosis factor-alpha (rTNF-alpha) and recombinant human interferon-gamma (rIFN-gamma) was investigated. rTNF-alpha had cytostatic or cytolytic effects on only some tumor cell lines. When administered together with rIFN-gamma, rTNF-alpha showed enhanced antiproliferative effects on a subset of the cell lines tested. In contrast to its effects on sensitive tumor cells, rTNF-alpha augmented the growth of normal diploid fibroblasts. Variations in the proliferative response induced by rTNF-alpha were apparently not due to differences in either the number of binding sites per cell or their affinity for rTNF-alpha. These observations indicate that the effects of rTNF-alpha on cell growth are not limited to tumor cells, but rather that this protein may have a broad spectrum of activities in vivo.

Type beta transforming growth factor: a bifunctional regulator of cellular growth.

Abstract: Type beta transforming growth factor (TGF-beta) is a two-chain polypeptide of 25,000 daltons isolated from many tissues, including bovine kidney, human placenta, and human platelets. It has been characterized by its ability to stimulate reversible transformation of nonneoplastic murine fibroblasts, as measured by the formation of colonies of these cells in soft agar (ED50 = 4 pM TGF-beta for NRK fibroblasts). We now show that the response of cells to TGF-beta is bifunctional, in that TGF-beta inhibits the anchorage-dependent growth of NRK fibroblasts and of human tumor cells by increasing cell cycle time. Moreover, the anchorage-independent growth of many human melanoma, lung carcinoma, and breast carcinoma cell lines is inhibited by TGF-beta at concentrations in the same range as those that stimulate colony formation of NRK fibroblasts (average ED50 = 10-30 pM TGF-beta for inhibition). Whereas epidermal growth factor and TGF-beta synergize to induce anchorage-independent growth of NRK fibroblasts, their effects on the growth of A-549 human lung carcinoma cells are antagonistic. The bifunctional response of cells to TGF-beta is further demonstrated in Fischer rat 3T3 fibroblasts transfected with a cellular myc gene. In these cells TGF-beta synergizes with platelet-derived growth factor to stimulate colony formation but inhibits the colony formation induced by epidermal growth factor. The data indicate that the effects of TGF-beta on cells are not a function of the peptide itself, but rather of the total set of growth factors and their receptors that is operant in the cell at a given time.

Requirement for ras proto-oncogene function during serum-stimulated growth of NIH 3T3 cells.

Abstract: Human tumours often contain DNA sequences not found in normal tissues which are able to transform cultured NIH 3T3 cells. In some tumours the gene responsible for this transformation belongs to the cellular ras gene family1,2. A specific type of mutation is responsible for converting the cellular proto-oncogene into a ras oncogene capable of inducing transformation3–5. In a study of the function of a cellular ras gene, its protein product (produced in a bacterial cell) was microinjected into NIH 3T3 cells; the recipient cells became morphologically transformed and were induced to initiate DNA synthesis in the absence of added serum6, but only when cellular ras protein was injected at much higher concentrations than required with protein of the transforming ras gene6,7. To further analyse the function of the cellular ras gene, we have now injected monoclonal antibodies against ras proteins into NIH 3T3 cells. We report here that NIH 3T3 cells induced to divide by adding serum to the culture medium are unable to enter the S phase of the cell cycle after microinjection of anti-ras antibody, showing that the protein product of the ras proto-oncogene is required for initiation of the S-phase in NIH 3T3 cells.

The biology of cell reproduction

Abstract: PART I: BIOLOGY 1. The Cell Cycle Phases of the cell cycle Variability of the cell cycle 2. Nondividing Cells G0 cells Terminally differentiated cells 3. Populations of Cells Stem cells Transformed cells 4. Tissue Growth Growth in size Growth in number Doubling time Growth of tumors 5. Synchronization of Cells in the Cell Cycle Methods for synchronizing cells in culture Morphology of cells during the cell cycle PART II: CELL BIOLOGY 6. Temperature-Sensitive Mutants of the Cell Cycle Defining cell cycle mutants Conditional cell cycle mutants The execution point 7. Informational Content of Cells in Different Phases of the Cell Cycle Cell fusion Informational content of S phase cells Informational content of G0-G

An epithelial scatter factor released by embryo fibroblasts

Abstract: Medium conditioned by human embryo fibroblasts breaks structural junctions between several types of epithelial cells, leading to separation and scattering of the cells. An assay developed in MDCK cells shows activity up to a dilution of at least 1 in 64, equivalent to less than 100 ng of total protein. The activity is non-dialysable, heat-labile, and sensitive to trypsin, and it is assumed to be due to one or more proteins. After addition of the factor, separation of MDCK cells begins in about 15 min and is complete in 10 h. It increases migration of MDCK cells into wounds, and causes collapse of domes. Locomotion of isolated cells is not enhanced, but cell shape is affected by local membrane movement. Under the culture conditions used the factor, or an associated protein, causes a weak inhibition of cell growth without cytotoxic activity. The scattering factor has not been purified, nor has a physiological role been identified, but it might be concerned in the mobilization of epithelial cells.

Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts

Abstract: Differentiating mouse 3T3-L1 preadipocytes have been used as a model system to study the ability of type beta transforming growth factor (TGF-beta) to modulate cell development. We find that TGF-beta inhibits potently (ID50 approximately equal to 25 pM) the adipogenic conversion of 3T3-L1 cells. Inhibition is observed only when cells are exposed to TGF-beta before they become committed to differentiation. Even a transient (4 hr) exposure to TGF-beta immediately before the commitment point is sufficient to prevent differentiation. This point coincides with the time point immediately preceding the onset of coordinate expression of differentiation-specific proteins in 3T3-L1 cells. TGF-beta interacts with cell surface receptors in 3T3-L1 cells that have structural and binding properties similar to TGF-beta receptors in other cell types in which TGF-beta acts as a growth activator or a growth inhibitor. However, TGF-beta does not markedly alter differentiation-related mitosis in 3T3-L1 cells. The action of TGF-beta on 3T3-L1 cells does not involve changes in cAMP or prostaglandin E levels. These results suggest that TGF-beta is a unique modulator of adipogenic differentiation of fibroblasts.

[Establishment of a new human endometrial adenocarcinoma cell line, Ishikawa cells, containing estrogen and progesterone receptors].

Abstract: A new human endometrial adenocarcinoma cell line, Ishikawa cells, was established from an endometrial adenocarcinoma from a 39-year-old woman and has been maintained in vitro for more than 3 years. The cells were found to form a monolayer in a mosaic fashion and to tend to pile up. Population doubling time was calculated to be about 36, 29 and 27 hours at the 9th, 40th and 50th generations, respectively. The modal chromosomal number of the cells fell in a diploid range. Histology of the tumor induced in athymic nude mice showed it to be a well differentiated adenocarcinoma which closely resembled the original human tumor. Estrogen receptor and progesterone receptor were demonstrated to occur not only in the induced tumor in athymic nude mice but also in in vitro culture cells. From the fact that the cell growth was maintained in an estrogen-free medium, it appeared that the cells had no estrogen dependency.

Microinjected c-myc as a competence factor.

Abstract: While a number of oncogenes are expressed in a cell cycle-dependent manner, their role in the control of cell proliferation can only be established by a direct functional assay. The c-myc protein, upon microinjection into nuclei of quiescent Swiss 3T3 cells, cooperated with platelet-poor plasma in the stimulation of cellular DNA synthesis. This suggests that c-myc protein, like platelet-derived growth factor (PDGF), may act as a competence factor in the cell cycle to promote the progression of cells to S phase. The presence in the medium of an antibody against PDGF abolished DNA synthesis induced by microinjected PDGF; however, the microinjected c-myc protein stimulated DNA synthesis even when its own antibody was present in the medium. The c-myc protein may act as an intracellular competence factor, while PDGF expresses its biological activity only from outside the cells.

c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells

Abstract: Several lines of evidence suggest a role for the myc oncogene in cell proliferation. Most recently, mitogenic stimulation of quiescent lymphoid, fibroblast and epithelial cells has been demonstrated to lead to a sharp increase in c-myc RNA levels. To determine how c-myc expression is linked to the cell proliferative cycle, we have used centrifugal elutriation to enrich for populations of avian and human cells at different stages of the cell cycle. Centrifugal elutriation is a counterflow centrifugation method that separates cells on the basis of volume, a parameter correlating well with progression through the cell cycle. Using myc-specific anti-peptide antibodies, we show here that the synthesis, half-life and modification of c-myc proteins are constant throughout the cell cycle of normal and transformed cells.

Close link between reduction of c-myc expression by interferon and, G0/G1 arrest.

Abstract: It has recently been reported that c-myc is an inducible gene, regulated directly by growth signals which promote proliferation and expressed in a cell-cycle dependent manner Because various leukaemic cell lines express high levels of c-myc messenger RNA, it was of interest to discover whether the gene could be down-regulated in these cells by a growth inhibitor such as interferon (IFN) We show here that in Daudi Burkitt's lymphoma cells, IFN-alpha produces a five- to sevenfold reduction in c-myc mRNA through a decreased rate of c-myc gene transcription By isolating a growth-resistant Daudi cell variant that had escaped from this down-regulation, we provide the first clear link between reduction of c-myc mRNA and the IFN-mediated G0/G1 arrest characteristic of Daudi cells Furthermore, by screening other cell lines, we demonstrate the heterogeneity of human leukaemic cells with respect to these criteria Thus, IFN-alpha fails to reduce the c-myc mRNA and to change the cell-cycle distribution in HL-60 and U937 cells, although normal induction of other IFN-regulated activities takes place The latter group of cells shows a decline in c-myc gene expression when they become arrested in the G0/G1 phase as part of their terminal differentiation

Induction of c-fos during myelomonocytic differentiation and macrophage proliferation.

Abstract: Previous studies have suggested a role for c-fos in cellular differentiation in fetal membranes1–3, haematopoietic cells4,5 and teratocarcinoma stem cells6. In other cell types, such as fibroblasts, c-fos expression is normally very low, but is rapidly induced by peptide growth factors, implicating c-fos in growth control mechanisms7–10. Here, we show that the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced macrophage-like differentiation of HL60 human promyelocytic precursor cells11,12 is accompanied by the induction of both c-fos mRNA and protein within 15 min after treatment, suggesting a functional role for c-fos in this differentiation system. In quiescent terminally differentiated macrophages, expression of c-fos is inducible by the macrophage-specific growth factor colony-stimulating factor-1 (CSF-1)13. The kinetics of c-fos induction, however, are entirely different from those in growth factor-stimulated fibroblasts, supporting the view that the c-fos gene product may serve different functions in different cell types.

c-myc gene is transcribed at high rate in G0-arrested fibroblasts and is post-transcriptionally regulated in response to growth factors.

Abstract: There is increasing evidence that at least some of the cellular homologues to retroviral oncogenes (c-onc or proto-oncogenes) are directly linked to the control of cell growth (for a review see ref. 1). Among these, c-myc, the cellular homologue to the avian myelocytomatosis virus (MC29) oncogene, has been shown to express high levels of mRNA during early G0/G1 phase after mitogenic stimulation of T lymphocytes by concanavalin A or of fibroblasts by platelet-derived growth factor (PDGF) or serum. An attractive model proposed for this regulation is that the c-myc gene is strongly repressed in cells arrested in the G0 phase of the cell cycle by a growth factor-sensitive repressor. We have investigated an alternative model of post-transcriptional regulation. This latter model leads to two testable predictions. First, that c-myc mRNA should be unusually unstable, which we have confirmed. And second, that there would be a high level of constitutive expression, a situation opposite to that implied by the repressor model. Here we report that c-myc gene is indeed transcribed at a high rate in G0-arrested chinese hamster lung fibroblasts, although the level of mature c-myc mRNA is barely detectable. The early and dramatic increase in c-myc mRNA levels when these resting cells are stimulated by growth factors is not accompanied by any appreciable change in the transcription rate of c-myc gene. Taken together these findings support a model of post-transcriptional regulation of c-myc expression at the level of mRNA degradation.

Cyclosporin A binding to calmodulin: a possible site of action on T lymphocytes.

Abstract: Cyclosporin A, a potent immunosuppressive agent, has been widely used to treat patients with solid organ transplants. Although its precise mechanism of action is unknown, it appears to inhibit subsets of T lymphocytes at an early stage in cell activation. Fluorescent, fully active derivatives of cyclosporin A and calmodulin, a protein that binds calcium and is therefore essential to normal cell function, were utilized to demonstrate that cyclosporin A binds to calmodulin. Flow cytometry showed that the calmodulin inhibitors R24571 and W-7 competitively inhibited binding of cyclosporin A to cloned T lymphocytes. Cyclosporin A inhibited the calmodulin-dependent activation of phosphodiesterase in a dose-dependent manner. Binding of cyclosporin A to calmodulin may prevent the latter's role in the activation of the second messengers and enzymes required for effective cell proliferation and function in the immune response.

Stimulation and inhibition of growth by EGF in different A431 cell clones is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes.

Abstract: Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors.

Effect of heparin on vascular smooth muscle cells. I. Cell metabolism.

Abstract: Previous work from our laboratory has shown that heparin inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. The mechanism of action of this glycosaminoglycan is unknown. In this communication, we have examined the antiproliferative effect of heparin on smooth muscle and other cell types, and have investigated several aspects of heparin on smooth muscle cell metabolism. Smooth muscle and closely related cell types from several species, including human, were much more sensitive to heparin than any other cell type tested, including primary and established cell lines, normal and transformed cell pairs, fibroblasts, epithelial, and endothelial cells. Flow microfluorimetric analysis of cell cycle distribution indicated that heparin blocked either the G0----S transition or a very early S-phase event in smooth muscle cells. Heparin rapidly inhibited DNA and RNA synthesis, but did not affect the rate of protein synthesis. The decrease in nucleic acid synthesis could be accounted for by an inhibition of thymidine and uridine uptake. Interestingly, heparin did not block amino acid or glucose transport. Although no change in the overall rate of protein synthesis was observed in the presence of heparin, we noted at least two changes in the synthesis of specific proteins by smooth muscle cells: two 35,000-dalton proteins which appeared in the culture medium of heparin-treated cells, and the transient disappearance of a 48,000-dalton protein in the substrate attached material of smooth muscle cells exposed to heparin. The role of the observed changes in smooth muscle cell metabolism is yet to be determined, but they may provide valuable clues to the molecular mechanisms controlling the antiproliferative activity of heparin.

Regulation of cell proliferation and differentiation by interferons.

Abstract: The interferons are a widely studied group of proteins which were first identified by their ability to protect cells against virus infections (Stewart, 1979). They are synthesized and secreted by a variety of cell types in response to several classes of inducers (notably virus infections) and exert their effects in vivo as a result of interaction with other cells in distant parts of the body. In this sense the interferons are analogous to the polypeptide hormones. A functional similarity with hormones is further emphasized by the recent identification in target cells of specific membrane receptors for interferons and by the multiplicity of biological effects which result from the interactions of the interferons with these receptors. In recent years it has become clear that the interferons are capable of influencing cellular physiology and behaviour in a number of ways. Their effects include inhibition of cell growth and proliferation, regulation of the expression of specific genes, modulation of cell differentiation, and activation of certain cell types in the immune system (e.g. macrophages and natural killer cells). The mechanisms underlying the antiviral actions of the interferons have been widely studied and frequently reviewed (Baglioni, 1979; Hovanessian, 1979; Lengyel, 1982; Sen, 1982). In contrast, information concerning the effects of interferons on cell proliferation and differentiation has only been obtained during the last decade, as our understanding ofmechanisms ofgrowth regulation and gene expression in eukaryotes in general has increased. This review aims to summarize the current situation regarding these actions of the interferons and suggests areas in which our perception of such control of cell function may be expected to increase.

Growth control of activated, synchronized murine B cells by the C3d fragment of human complement.

Abstract: Three restriction points control the cell cycle of activated B lymphocytes. The first occurs directly after mitosis and is controlled by the occupancy of surface-bound immunoglobulin. The second is observed approximately 4 h after mitosis in the G1 phase of the cycle, that is, before DNA replication, and is controlled by growth factors that are produced by macrophages which we have previously classified as alpha-type factors. The third restriction point occurs in the G2 phase, 2-4 h before mitosis, and is controlled by beta-type growth factors probably produced by helper T lymphocytes. The third component of complement, C3, has long been implicated in the control of B-cell responses. C3 is secreted by monocytes and macrophages. We have found recently that crosslinked, but not soluble, human C3 stimulates activated, but not resting, murine B cells to thymidine uptake. Here we investigate the role of C3b and C3d in the progression of the cell cycle of activated, synchronized murine B cells. We find that crosslinked C3d replaces the action of alpha-factors within the cell cycle of these cells and allows entry into S phase. In contrast, soluble C3d inhibits the action of alpha-factors. This implies that a C3d-specific receptor, probably the murine analogue to the human complement receptor CR2, is a growth factor receptor on activated B cells that will give the cell a growth-positive signal when it is crosslinked, while occupancy by the soluble form of C3d will result in inhibition of the action of alpha-factors or of crosslinked C3b or C3d. A stretch of weak homology between the cDNA sequence of murine C3d and those of murine growth factors indicates that an insulin-like growth factor could be the active principle of C3d that controls the cell cycle of activated B cells.

Heparin affinity of anionic and cationic capillary endothelial cell growth factors: analysis of hypothalamus-derived growth factors and fibroblast growth factors.

Abstract: Bovine hypothalamus-derived growth factors (HDGF), pituitary fibroblast growth factor (FGF), and brain FGF were analyzed by chromatography on immobilized heparin and tested for the ability to stimulate the proliferation of capillary endothelial (CE) cells. Two distinct CE cell growth factors were found in hypothalamus, one anionic (aHDGF; pI of about 5) and one cationic (cHDGF; pI of about 8). Both aHDGF and cHDGF adhered tightly to immobilized heparin. They were eluted with 0.9-1.1 M NaCl and 1.3-1.5 M NaCl, respectively. Pituitary and brain FGF were also found to bind to immobilized heparin and to stimulate CE cell proliferation. Pituitary FGF was eluted at 1.4-1.6 M NaCl. The elution profile of brain FGF showed that two peaks of CE cell growth factor activity were eluted from the heparin column, one at 1.0 M NaCl and a second at 1.4-1.6 M NaCl. The tight binding of all of these growth factors to heparin (particularly aHDGF, whose binding is unexpected because of its negative charge) is presented as evidence that CE cell growth factors all share an affinity for heparin.

Small intensely fluorescent cells in culture: role of glucocorticoids and growth factors in their development and interconversions with other neural crest derivatives

Abstract: The neural crest gives rise to a number of adrenergic derivatives, including sympathetic neurons and adrenal chromaffin cells, which contain catecholamines (CAs) but differ in other morphological and functional characteristics Small intensely fluorescent (SIF) cells, which exist primarily as a minority cell population in autonomic ganglia, are a third cell type in the sympathoadrenal branch of the neural crest lineage In some respects these cells appear intermediate in phenotype between sympathetic neurons and adrenal chromaffin cells We established pure dissociated cell cultures of SIF cells from rat superior cervical ganglia (SCG) and used these to study the role of environmental factors in SIF cell development and the relationship of these cells to the other cell types of the sympathoadrenal lineage When cells from neonatal rat SCG were grown for 3 weeks in the presence of glucocorticoid and in the absence of nerve growth factor (NGF), pure cultures of SIF cells developed The properties of the cells included (i) small cell size and the occasional presence of short neurites, (ii) intense CA histofluorescence and immunoreactivity for CA synthetic enzymes, (iii) synthesis and storage of CA from radioactive precursors, and (iv) characteristic ultrastructure The concentration of the glucocorticoid and the presence or absence of non-neuronal cell factors influenced which types of SIF cells developed In micromolar glucocorticoid most of the cells resembled adrenal chromaffin or type II SIF cells: they displayed immunohistochemically detectable phenylethanolamine-N-methyltransferase (PNMT), synthesized and stored epinephrine, and contained large granular vesicles (100 to 300 nm) When SCG cells were grown in 10(-8) M hormone, many fewer SIF cells developed and a higher percentage of these lacked PNMT immunoreactivity, had neurites, and contained vesicles of smaller mean diameter (70 to 130 nm), similar to those of type I SIF cells in vivo In the presence of conditioned medium (medium conditioned by non- neuronal cells) as well as glucocorticoid, virtually all of the cells morphologically resembled type I SIF cells In the absence of glucocorticoid, no SIF cells were ever observed after 3 weeks in culture By following the development of CA histofluorescence and SIF cell ultrastructure in the cultures over time, we demonstrated that SIF cells were not present in large numbers in these cultures immediately after plating, but were induced from an apparently undifferentiated precursor by the hormonal environment, whereas most of the principal neurons died

Permissive role of interleukin 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture.

Abstract: We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.

Early events in Epstein-Barr virus infection provide a model for B cell activation.

Abstract: We have used Epstein-Barr virus (EBV) infection in vitro to delineate two distinct stages in B cell activation. Previous studies have shown that the BLAST-2 (EBVCS) (EBV cell surface) activation antigen is expressed on a small fraction of B cells within 24 h of stimulation with a variety of agents, including mitogens and EBV. In this study, we have been able to isolate the BLAST-2 (EBVCS)+ cells early after activation/infection with EBV. These cells are small B cells that are actively synthesizing RNA but not DNA, and are, therefore, clearly distinct from large proliferating lymphoblasts. In addition, they contain multiple copies of the EBV genome, express the viral nuclear antigen (EBNA) and, most importantly, proceed to undergo transformation when placed back in culture. By comparison, the BLAST-2 (EBVCS)- population does not undergo transformation, even though a fraction of these cells are activated for RNA synthesis and express EBNA. Thus, using the EBV system, we have been able to show directly that an activated B cell first expresses the BLAST-2 (EBVCS) antigen concomitant with an increase in RNA synthesis, and then subsequently proceeds to differentiate into a proliferating lymphoblast.

1α, 25‐Dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line

Abstract: The ability of the hormonally active vitamin D metabolite, 1 alpha, 25-dihydroxyvitamin D3, to affect cell growth, morphology and fibronectin production has been examined using the MG-63 human osteosarcoma cell line. Hormone treatment reduced cell growth rate, saturation density and [3H]thymidine incorporation. Inhibition was specific for 1 alpha, 25-dihydroxyvitamin D3 relative to other vitamin D metabolites (1 alpha, 25-dihydroxyvitamin D3 greater than 25-dihydroxyvitamin D3 greater than 24R,25-dihydroxyvitamin D3 greater than D3), antagonized by high concentrations of serum and readily reversed by removal of 1 alpha, 25-dihydroxyvitamin D3 from the culture medium. Hormone treatment also increased cell associated alkaline phosphatase activity up to twofold and altered morphology such that treated cells were more spread out on the culture dish and contained more cytoplasmic processes. Significantly, 1 alpha, 25-dihydroxyvitamin D3 increased cellular and medium concentrations of fibronectin, a glycoprotein known to be involved in cellular adhesiveness. MG-63 cells contain a specific 1 alpha, 25-dihydroxyvitamin D3 receptor which may mediate these responses.

Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line.

Abstract: The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.

Glucocorticoid Regulation of Alkaline Phosphatase in the Osteoblastic Osteosarcoma Cell Line ROS 17/2.8*

Abstract: Dexamethasone increased alkaline phosphatase levels up to 7-fold in the osteoblast-like rat osteosarcoma cell line ROS 17/2.8. This effect was associated with reduced cell growth and took place over several days in culture. The increase in enzyme activity was dose dependent, (half-maximum near 1 nm, with a hormone specificity suggesting glucocorticoid receptor mediation). Dexamethasone also increased enzyme activity in ROS 2/3 cells, but not in two nonosteoblastic osteosarcoma cell lines, indicating that among these cell lines, the effect is specific for osteoblast-like cells. Moreover, enzyme activity in both control and dexamethasone-treated cells correlated directly with levels of radioimmunoassayable bone-type isoenzyme. Increases in alkaline phosphatase activity in response to dexamethasone were detectable after about 5 h and were inhibited by both actinomycin D and cycloheximide. Thus glucocorticoids appear to increase de novo enzyme synthesis in ROS 17/2.8 cells. Finally, the cAMP-elevating agents ...

Oncogenes, ions, and phospholipids.

Abstract: Recent discoveries in tumor virology, lipid biochemistry, and ion transport studies promise to revolutionize our understanding of cell proliferation, differentiation, and tumorigenesis. A model is proposed, based on similar schemes presented recently by others, that incorporates these discoveries and provides a focus for future research on the functions of oncogene proteins. The model suggests that the early (competence) events in the initiation of cell proliferation are triggered by activation of phosphatidylinositol (PI) turnover, which releases two second messengers, 1,2-diacylglycerol (1,2-DG) and inositol-1,4,5-trisphosphate (IP3). PI turnover is proposed to be regulated by the oncogene protein kinases (src, ros, abl, fps) either directly (acting as PI kinases) or indirectly (as tyrosine kinases). The IP3 triggers Ca2+ release from internal stores, and the elevation of cytosolic Ca2+ acts synergistically with 1,2-DG to activate the Ca2+- and phospholipid-dependent kinase C. Kinase C copurifies with the receptor for the tumor-promoting phorbol esters. It is suggested that kinase C then activates the Na+-H+ exchange system, resulting in an elevation of cytosolic pH and Na+, and that these ionic signals (including the change in Ca2+), either in concert or individually, induce further events, including expression of the protooncogene c-myc, which together commit the cell to initiate replication. Evidences in support of this model are reviewed, together with complications indicating its present inadequacies, particularly recent data suggesting that 1,2-DG may activate tyrosine kinases independent of kinase C.