Cell cycle dependent growth factor regulation of gene expression

The expression of the proto-oncogenes c-fos and c-myc is a rapid response of G0-arrested fibroblasts to serum and peptide growth factors; however, the role of the c-fos and c-myc gene products in subsequent cell cycle transit is not understood. We examined the expression of c-fos and c-myc mRNA in Balb/c 3T3 murine fibroblasts in response to platelet-derived growth factor (PDGF) and platelet-poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c-fos and c-myc mRNA in Balb/c 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence. c-fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c-fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G2 as well as in G0 arrest. In contrast, c-myc expression was both growth factor and cell cycle dependent. In G0 arrested cells, c-myc expression was PDGF-dependent and plasma-independent, and PDGF was required for maintenance of elevated c-myc levels during G1 transit. In cells transiting S phase, c-myc mRNA was induced in response to PDGF, but was also plasma-dependent in S phase cells that had been "primed" by exposure to PDGF during S phase.     

Role of c-myc and other genes in interleukin 2 regulated CT6 T lymphocytes and their malignant variants

The cloned murine cytotoxic T cell line CT6 solely requires interleukin 2 (IL 2) for viability and cell cycle progression. Treatment of G arrested cultures of CT6 cells with recombinant IL 2 induces the rapid sequential expression of the nuclear proto-oncogenes c-fos, c-myc, and c-myb but does not affect the expression of several cytosolic or membrane-associated proto-oncogenes. A comparison of early genes induced by growth factor treatment of quiescent NIH/3T3 fibroblasts and CT6 cells demonstrated that only c-fos and c-myc induction is shared in the two different lineages. Factor-independent lines derived from CT6 cells show no mitogenic response to IL 2, yet binding of IL 2 with its receptor in the cells was capable of inducing the expression of c-fos and c-myc. In factor-independent cell lines, c-myc was uniformly expressed at high constitutive levels, suggesting that c-myc abrogates growth factor requirements of these cells. The levels of c-myc expression in the factor-independent lines was not due to an autocrine production of IL 2 but may be a consequence of constitutively activated IL 2 receptors.     

Differential induction of the c-fos promoter through distinct PDGF receptor-mediated signaling pathways.

The multiple isoforms of PDGF induce fibroblastic mitogenesis through two distinct PDGF receptors, alpha and beta. The molecular mechanisms by which these alpha and beta PDGF receptors regulate gene expression are poorly understood. We present data which indicates that differential induction of c-fos gene expression by PDGF isoforms occurs through distinct PDGF alpha and beta receptor-mediated signaling pathways. Comparison of PDGF-AA with PDGF-BB stimulation showed that PDGF-BB induced prolonged expression of the c-fos gene in BALB/c-3T3 cells, but that PDGF-AA induced more potent activation of the serum response element (SRE) in transient transfection assays. PDGF-AA, which binds alpha but not beta PDGF receptors, could only induce the SRE through a protein kinase C (PKC)-dependent pathway, whereas PDGF-BB, which binds both alpha and beta PDGF receptors, could also induce the SRE through a PKC-independent pathway. These results suggest that PDGF alpha receptors activate the PKC-dependent signaling pathway while PDGF beta receptors also activate a PKC-independent pathway. In addition, we found that PDGF-BB could induce another c-fos promoter element within the -90 to +10 region, suggesting that the more potent mitogenic effect and prolonged c-fos gene expression induced by PDGF-BB may result from cooperativity between more than one c-fos promoter elements.     

Human cysteine-rich protein. A member of the LIM/double-finger family displaying coordinate serum induction with c-myc.

We previously reported the structure of the placentally derived human cysteine-rich (h crp) cDNA and demonstrated that it encodes a highly conserved and widely distributed zinc finger-like protein. We now report that the expression of both the mouse and human crp genes is induced as a primary response to serum in quiescent Balb/c 3T3 cells and in human fibroblasts. The profile of this primary response is remarkably parallel to that of c-myc in the Balb/c 3T3 cell line. The structure of the 23.2-kilobase h crp gene demonstrates that it is a member of a gene superfamily encoding proteins sharing a highly characteristic 52-amino acid "LIM/double-finger" motif. The evolutionarily conserved structure of cysteine-rich protein, its structural similarity to a number of developmentally critical proteins, its distinctive tissue distribution, and its primary response to early events in the cell cycle suggest that crp plays an important role in cell function.     

Expression of c-fos in NIH3T3 cells is very low but inducible throughout the cell cycle.

It has previously been shown that the c-fos proto-oncogene is rapidly and transiently induced following growth factor stimulation of quiescent NIH3T3 mouse fibroblasts. To investigate a possible role of c-fos in growth control mechanisms we have studied its expression and inducibility during the NIH3T3 cell cycle. Two major conclusions can be drawn from this analysis. First, expression of c-fos is not cell cycle-regulated, and is barely detectable in all phases of the cycle. Second, cells at different stages of the cell cycle (except for mitosis) are as sensitive to c-fos induction by growth factors as quiescent cells. These observations suggest that induction of the c-fos gene does not play a role during the continuous cycling of NIH3T3 cells, but they are fully compatible with the hypothesis that a function of c-fos may be associated with the induction of competence in fibroblasts. Through such a function c-fos may contribute to moving cells out of the quiescent state.     

Platelet-derived growth factor regulation of fos stability correlates with growth induction.

A brief exposure of quiescent, density-arrested Balb/c 3T3 fibroblasts to platelet-derived growth factor (PDGF) results in expression of the proto-oncogene c-fos; furthermore, the translation product of c-fos, p55fos, was shown to have increased stability in cells upon continued exposure to PDGF. The induction of competence or growth initiation requires a longer exposure to PDGF than that necessary for the induction of the immediate-early, growth-related genes. The need for the continued presence of PDGF for growth initiation beyond the time required for the induction of immediate-early gene expression may be due, in part, to PDGF-dependent post-translational stabilizations of gene products. We speculate that a PDGF-mediated event increases p55fos stability, resulting in a continued elevated level of Fos protein, which in turn allows a continued Fos-mediated activity required by Balb/c 3T3 cells to become competent to enter the cell cycle.     

Biochemical and functional discrimination of platelet-derived growth factor alpha and beta receptors in BALB/c-3T3 cells.

Three biologically active isoforms of platelet-derived growth factor (PDGF) exist: PDGF-AB, the predominant form in human platelets; PDGF-BB, the product of the c-sis protooncogene; and PDGF-AA. PDGF-BB and PDGF-AB interact with two distinct PDGF receptors (termed alpha and beta) of similar size, whereas PDGF-AA binds alpha receptors only. To dissect alpha and beta receptor-mediated signals, we compared the biological activities of PDGF-AA and PDGF-BB in density-arrested BALB/c-3T3 cells, which possess a 4:1 ratio of beta to alpha receptors, and assessed the contribution of alpha receptors to PDGF-BB- and PDGF-AB-induced responses. In addition, we describe a convenient method for resolving alpha and beta receptors on one-dimensional protein gels. This protocol involves treatment of cells with neuraminidase, a desialylating agent, and subsequent in vitro autophosphorylation of solubilized cells, and was used to monitor the presence or absence of alpha and beta receptors under various experimental conditions. Our data show that although higher concentrations were required, PDGF-AA stimulated DNA synthesis to the same extent as did PDGF-BB. Both isoforms induced inositol phosphate formation, epidermal growth factor transmodulation, and PDGF receptor autophosphorylation; PDGF-AA, however, was less effective than was PDGF-BB even at doses causing maximal mitogenesis. Pretreatment of cells with PDGF-AA for 30-60 min at 37 degrees C effectively down-regulated alpha receptors as verified by the absence of desialylated alpha receptor phosphorylation. Depletion of alpha receptors did not affect the capacity of PDGF-BB or PDGF-AB to activate the beta receptor tyrosine kinase, as assessed by tyrosine phosphorylation of an endogenous substrate, or stimulate the formation of inositol phosphates. We suggest that alpha and beta receptors independently mediate similar biological responses in BALB/c-3T3 cells, and that alpha receptors are not required for responses induced by PDGF-BB or PDGF-AB.     

Regulation of expression of c-fos and c-myc in rat lymphoma Nb-2 cells

This study examined the effects of the mitogen, prolactin and the cell cycle inhibitors, cyclosporin A and neomycin sulfate, on expression of the proto-oncogenes c-fos and c-myc in the rat lymphoma Nb-2 cell line. Stimulation of quiescent cultures with prolactin resulted in a 2-3-fold increase in the constitutive levels of c-myc mRNA which peaked at 4 h and declined thereafter. c-Fos mRNA was not detected in quiescent or prolactin-stimulated cultures. Cyclosporin A or neomycin sulfate reversibly blocked the mitogenic effect of prolactin on Nb-2 cells, but had little effect on constitutive levels of c-myc. However, the release of Nb-2 cells from a cyclosporin A or a neomycin sulfate block resulted in a rapid transient induction of c-fos which peaked at 0.5-1 h and declined rapidly thereafter. These results indicate that the rapid transient expression of c-fos following release from cell cycle blockage was not sufficient to elicit cell division, but these cells were competent to respond to prolactin. Prolactin allows progression through the cell cycle and enhances c-myc mRNA levels.     

Persistence of the competent state in mouse fibroblasts is independent of c-fos and c-myc expression

Induction of competence in quiescent fibroblasts by platelet-derived growth factor (PDGF) is accompanied by a dramatic increase in the expression of c-fos and c-myc genes. However, the maintenance of the competent state and progression through G1 does not require high expression of these proto-oncogenes. These results suggest that the induction of c-fos and c-myc by growth factors in quiescent fibroblasts may be required to render the cells competent for progression.     

Butyrate blocks the accumulation of CDC2 mRNA in late G1 phase but inhibits both the early and late G1 progression in chemically transformed mouse fibroblasts BP-A31

Sodium butyrate (6 mM) blocks the resumption of the cell division cycle in serum-deprived chemically transformed Balb/c-3T3 mouse fibroblasts (BP-A31). The inhibition of G1 progression by sodium butyrate is not restricted to a specific mitogenic signaling pathway and is equally effective when tetradecanoyl phorbol acetate (TPA), insulin, or fetal calf serum (FCS) is used as inducer. The inhibitor acts in early as well as late G1 phase as indicated by experiments in which inhibitor was added and withdrawn at different times after restimulation of quiescent cells by FCS. At the gene expression level, sodium butyrate does not affect the inducibility of early cell cycle-related genes (c-myc, c-jun) while blocking the induction of cdc 2 mRNA, a late G1 marker. We conclude that sodium butyrate does not interfere with the growth factor signaling pathways regulating the (early) cell cycle-related gene expression. However, the presence of sodium butyrate early in G1 phase inhibits the cascade of events leading eventually to the expression of late G1-characteristic genes such as cdc2. The antimitogenic activity of sodium butyrate may be related to its interference with an (unknown) process involved in the "mitogenic" cascade.     

Stimulation of protein kinase C or protein kinase A mediated signal transduction pathways shows three modes of response among serum inducible genes

Activation of the signal transduction pathways mediated by protein kinase A (PKA) or protein kinase C (PKC) led to different responses of several serum inducible genes including the jun gene family, c-fos, c-myc, krox 20 and krox 24. Whereas all of these genes were stimulated by the phorbol ester TPA, a chemical activator of protein kinase C, they were differently regulated upon cAMP stimulation of the PKA dependent pathway. The proto-oncogenes jun B, c-fos, and to a lesser extent jun D were stimulated by increasing the intracellular concentration of cAMP, whereas the TPA stimulation of c-jun and c-myc was inhibited under these conditions. Krox 20 and krox 24 were insensitive to this second messenger. This study allowed us to classify these growth stimulated genes into three distinct groups distinguished by their sensitivity to elevated concentrations of intracellular cAMP. The inhibition of c-jun and c-myc expression in the presence of increased cAMP levels may be at least partially responsible for the growth inhibitory effect of this agent in Balb/c-3T3 cells.     

Time of c-fos and c-myc expression in human diploid fibroblasts stimulated to proliferate after prolonged periods in quiescence

When human diploid fibroblasts such as WI-38 cells become crowded, they enter a viable state of quiescence (G0) in which they can remain for prolonged periods of time. These quiescent cells can be induced to re-enter the cell cycle by addition of fresh serum. However, cells held in G0 for long periods before stimulation require more time to enter DNA synthesis as compared to cells held in a quiescent state for short periods. We have used this model system to determine if a close temporal coupling exists between the time of expression of two proto-oncogenes associated with cell growth, c-fos and c-myc, and the time of entry into DNA synthesis. WI-38 cells were stimulated to enter DNA synthesis by the addition of fresh culture medium and serum at various lengths of time after plating, ranging from 7 to 34 days. At hourly intervals thereafter, cells were harvested and total RNA was isolated. These samples were then analyzed by RNase protection assay to determine the levels of c-fos and c-myc mRNA. Our results show that the time and pattern of c-fos and c-myc mRNA accumulation after stimulation is determined only by the time which the cells are treated with serum even when they exhibit a 19-h delay in the entry into DNA synthesis. In all of our experiments, c-fos could be detected 0.5 h after stimulation and remained detectable for approximately 2 h. Likewise, the peak of c-myc accumulation occurred at about 3 h after serum addition, regardless of how long it took to initiate DNA synthesis. These results suggest that the time of c-fos and c-myc induction clearly is not the only factor which determines the length of the prereplicative period and thus the ultimate time of initiation of DNA synthesis.     

Cell cycle dependent gene expression in quiescent stimulated and asynchronously cycling arterial smooth muscle cells in culture.

The expression of a set of cell cycle dependent (CCD) genes (c-fos, c-myc, ornithine decarboxylase (ODC), and thymidine kinase (TK)) was comparatively studied in cultured arterial smooth muscle cells (SMC) during exit from quiescence and exponential proliferation. These genes, which were not expressed in quiescent SMC, were chronologically induced after serum stimulation. c-fos mRNA were rapidly and transiently expressed very early in the G1 phase; c-myc and ODC peaked a few hours after serum stimulation and then remained at an intermediary level throughout the first cell cycle; TK mRNA and activity then appeared at the G1/S boundary and peak in G2/M phases. Except for c-fos, the other genes were also expressed in asynchronously cycling SMC (ACSMC); their expression was studied in elutriated subpopulations representative of cell cycle progression. c-fos mRNA were undetectable in any sorted subpopulations, even in the pure early G1 population. Despite a slight increase as the cell cycle advanced, c-myc and ODC genes were expressed throughout the ACSMC cell cycle. A faint TK activity was found in G1 subpopulations and increased in populations enriched in other phases; in contrast, TK mRNA remained highly expressed in all elutriated subpopulations. This study demonstrates significant modulations in CCD gene expression between quiescent stimulated and asynchronously cycling SMC in culture. This suggests that the events occurring during the emergence of SMC from quiescence are probably different from those in the G1 phase of ACSMC.     

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

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.