Cyclic AMP and c-myc gene expression in PY815 mouse mastocytoma cells

The possibility was examined that inhibition of growth of PY815 mouse mastocytoma cells by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (DB cyclic AMP) results from inhibition of c-myc gene expression. Temporary increases in c-myc RNA which occurred soon after DB cyclic AMP treatment and upon removal of the drug were not consistent with direct inhibition of c-myc gene expression by DB cyclic AMP. The increases in c-myc RNA coincided with the passage through, or accumulation of cells in late G1-early S phase. It is proposed that cyclic AMP may stimulate c-myc gene expression which normally occurs only in late G1-early S phase in PY815 cells and that cyclic AMP prevents c-myc expression in cells at other phases of the cell cycle by inhibiting their progression past a cyclic AMP-sensitive restriction point in early G1 phase.     

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.     

Prostaglandins A and J arrest the cell cycle of cultured vascular smooth muscle cells without suppression of c-myc expression.

The effects of prostaglandins (PGs) A and J, which are anti-tumor eicosanoids, on the proliferation of cultured vascular smooth muscle cells were investigated. Serum-stimulated DNA synthesis was potently inhibited by PGA1, PGA2, PGJ2, and delta 12-PGJ2 in similar dose-dependent fashions. The effects of PGA1 and PGA2 were reversible when they were removed from the culture media, whereas recoveries were only partial in the cells treated with PGJ2 and delta 12-PGJ2. PGs were effective even if they were added immediately before entry into S phase. Inhibition of DNA synthesis was sustained when hydroxyurea, which blocks cell cycle at the G1/S border, was added after the removal of PGA2, and vice versa; PGs blocked DNA synthesis when they were added after the removal of hydroxyurea. Levels of c-myc mRNA formed two peaks during the G1 phase, at 1-2 h and at 8-12 h. The PGs did not affect the first elevation, but enhanced the second and sustained it up to 18-24 h, whereas in controls, c-myc mRNA decreased quickly after entry into S phase. The rate of degradation of c-myc mRNA was much smaller in PG-treated cells than in nontreated cells. We conclude, therefore, that PGA and PGJ inhibit a crucial event(s) in the cell cycle occurring at the G1/S border, but that this inhibition is not accompanied by the reduction in c-myc gene expression in contrast with some types of tumor cells treated with PGs.     

A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary

A novel cell cycle blocking agent profoundly suppressed the proliferation of mitogen-stimulated T lymphocytes. The carboxythiazole derivative arrested cells in the G1 phase of the cell cycle but did not inhibit the induction of cell surface receptors for either interleukin-2 or transferrin. The uncoupling of transferrin receptor expression from DNA synthesis indicated that a previously undefined restriction point in the cell cycle has been identified which occurs after transferrin receptor expression in late G1 and just prior to the initiation of DNA replication in S phase. T cells incubated in an inhibitory dose of the carboxythiazole derivative resumed cell cycle progression subsequent to its removal, indicating that the compound reversibly arrests cells at the late G1 restriction point. In contrast to other techniques which have been inefficient in achieving T cell synchronization, T cells released from the block mediated by the carboxythiazole compound progress through S phase with a considerable degree of synchrony.     

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression.

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.     

IFN-gamma inhibits human airway smooth muscle cell proliferation by modulating the E2F-1/Rb pathway

Elucidating the factors that inhibit the increase in airway smooth muscle (ASM) mass may be of therapeutic benefit in asthma. Here, we investigated whether interferon-gamma (IFN-gamma), a potent inducer of growth arrest in various cell types, regulates mitogen-induced ASM cell proliferation. IFN-gamma (1-100 U/ml) was found to markedly decrease both DNA synthesis and ASM cell number induced by the mitogens epidermal growth factor (EGF) and thrombin. Interestingly, IFN-gamma had no effect on mitogen-induced activation of three major mitogenic signaling pathways, phosphatidylinositol 3-kinase, p70(S6k), or mitogen-activated protein kinases. Mitogen-induced expression of cell cycle regulator cyclin D1 was increased by IFN-gamma, whereas no effect was observed on degradation of p27(Kip1). Expression array analysis of 23 cell cycle-related genes showed that IFN-gamma inhibited EGF-induced increases in E2F-1 expression, whereas induction of c-myc, cyclin D2, Egr-1, and mdm2 were unaffected. Induction of E2F-1 protein and Rb hyperphosphorylation after mitogen stimulation was also suppressed by IFN-gamma. In addition, IFN-gamma decreased activation of cdk2 and expression of cyclin E, upstream signaling molecules responsible for Rb hyperphosphorylation in the late G1 phase. IFN-gamma also increased levels of IFI 16 protein, whose mouse homolog p202 has been associated with growth inhibition. Together, our data indicate that IFN-gamma is an effective inhibitor of ASM cell proliferation by blocking transition from G1-to-S phase by acting at two different levels: modulation of cdk2/cyclin E activation and inhibition of E2F-1 gene expression.     

Equilibrium between cell division and apoptosis in immortal cells as an alternative to the G1 restriction mechanism in mammalian cells

Starvation arrests cultured mammalian cells in the G(1) restriction point of the cell cycle, whereas cancer cells generally lose the regulatory control of the cell cycle. Human lymphocytes, infected with Epstein-Barr virus (EBV), also lose their cell cycle control and produce immortal lymphoblastoid cell lines. We show that during starvation, EBV-lymphoblasts override the cell cycle arrest in the G(1) restriction point and continue cell division. Simultaneously, starvation activates apoptosis in an approximately half of the daughter cells in each cell generation. Continuos cell division and partial removal of cells by apoptosis results in stabilization of viable cell numbers, where a majority of viable cells are in the G(1) phase of the cell cycle. In contrast to starvation, anticancer drug etoposide activates apoptosis indiscriminately in all EBV-lymphoblasts and convertes all the viable cells into apoptotic. We conclude that the removal of surplus cells by apoptosis may represent a survival mechanism of transformed (i.e., cancer) cell population in nutrient restricted conditions, whereas nontransformed mammalian cells are arrested in the G(1) restriction point of the cell cycle.     

A reversible arrest point in the late G1 phase of the mammalian cell cycle

The effects of two different cell cycle inhibitors on the proliferation of human lymphoblastoid cells have been analyzed by flow cytometric techniques. Mimosine, a plant amino acid, reversibly blocks the cell cycle at a point which occurs roughly 2 h before the arrest mediated by aphidicolin, an inhibitor of DNA polymerase alpha activity, which defines the G1/S phase boundary. The levels of thymidine kinase mRNA, which increase at the onset of S phase, are higher in cells blocked with aphidicolin than in cells treated with mimosine whereas the opposite results are obtained in the case of p53 mRNA levels, which are known to be maximal in the late G1 phase. These results indicate that mimosine inhibits cell cycle traverse in the late G1 phase prior to the onset of DNA synthesis and identifies a previously undefined reversible cell cycle arrest point.     

Cyclin A but not cyclin D1 is essential for c-myc-modulated cell-cycle progression

The proto-oncogene c-myc is a key player in cell-cycle regulation and is deregulated in a broad range of human cancers and cell proliferation disorders. Here we reported that overexpression of c-myc in human embryonic lung fibroblasts (HEL) that have low endogenous c-myc enriched S phase cells with increased expression of cyclin D3, E, A, Cdk2, and Cdk4, and decreased expression of p21 and p27. To the opposite, using RNAi to downregulate c-myc expression in A549 cells that have high endogenous c-myc enriched G1 phase cells with decreased expression of cyclin D3, E, A, Cdk2, Cdk4, and increased expression of p21 and p27. We found that cyclin A expression was the most susceptive to changes in c-myc levels and essential in c-myc-modulated cell cycle pathway via co-transfection, however, cyclin D1 showed no change between treated and control groups in either HEL or A549 cells. Our results indicated that upregulation of c-myc expression promotes cell cycling in HEL cells, whereas downregulation of c-myc expression causes G1 phase arrest in A549 cells, and the c-myc-mediated cell-cycle regulation pathway was dependent on cyclin A and involved cyclin D3, E, Cdk2, Cdk4, p21, and p27, but not cyclin D1.     

Equilibrium Between Cell Division and Apoptosis in Immortal Cells as an Alternative to the G1 Restriction Mechanism in Mammalian Cells

Starvation arrests cultured mammalian cells in the G1 restriction point of the cell cycle, whereas cancer cells generally lose the regulatory control of the cell cycle. Human lymphocytes, infected with Epstein-Barr virus (EBV), also lose their cell cycle control and produce immortal lymphoblastoid cell lines. We show that during starvation, EBV-lymphoblasts override the cell cycle arrest in the G1 restriction point and continue cell division. Simultaneously, starvation activates apoptosis in an approximately half of the daughter cells in each cell generation. Continuos cell division and partial removal of cells by apoptosis results in stabilization of viable cell numbers, where a majority of viable cells are in the G1 phase of the cell cycle. In contrast to starvation, anticancer drug etoposide activates apoptosis indiscriminately in all EBV-lymphoblasts and convertes all the viable cells into apoptotic. We conclude that the removal of surplus cells by apoptosis may represent a survival mechanism of transformed (i.e. cancer) cell population in nutrient restricted conditions, whereas non-transformed mammalian cells are arrested in the G1 restriction point of the cell cycle.     

Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12-16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.