Expression of the First N-Glycosylation Gene in the Dolichol Pathway, ALG7, Is Regulated at Two Major Control Points in the G1 Phase of the Saccharomyces cerevisiae Cell Cycle

The Saccharomyces cerevisiae ALG7 gene, which functions by initiating the dolichol pathway of protein N-glycosylation, displays properties of an early growth-response gene. To initiate studies of the involvement of ALG7 in cellular proliferation, we have now more precisely analyzed ALG7 expression in the G1 phase of cell cycle. We show that the rapid rate of ALG7 mRNA accumulation following growth stimulation was attenuated soon thereafter and that ALG7 growth induction occurred irrespective of α-factor. ALG7 growth induction was observed in mutants conditionally defective for reentry into the cell cycle from the stationary phase, indicating that the induction occurred prior to the performance of START. In addition, the steady-state levels of ALG7 mRNAs declined four-fold in response to START-I cell division arrest brought about by α-factor treatment later in G1. Importantly, deregulated expression of ALG7 resulted in an aberrant α-factor response. Our data not only indicate that ALG7 expression is regulated at two critical control points in G1 that determine the proliferative potential of cells, but also provide a link between ALG7 and START.     

TEC酪氨酸激酶基因是一种与肝再生调控相关的早期反应基因

 肝再生过程中立即早期反应基因的表达在成熟肝细胞由G0 期向G1期的转变中起着关键作用 .为探讨肝再生早期基因表达的变化 ,利用表达性差异显示分析 (RDA)技术研究了 2 3肝部分切除后 1h再生肝选择性基因表达 ,发现一株TEC酪氨酸激酶同源序列存在于差减产物中 ,RNA狭缝杂交证实确为差异表达基因 .从大鼠肝cDNA文库中分离其全长cDNA ,序列分析结果表明 ,该基因为小鼠 人TEC酪氨酸激酶的同源体 ,进而以该cDNA为探针 ,用Northern杂交证实 2 3肝部分切除后TEC酪氨酸激酶基因在 1h内呈现瞬间表达增加 ,其表达水平较基础水平增高 2 5倍 ;在原代培养大鼠肝细胞体系中 ,EGF可迅速诱导TEC基因表达 ,且不被蛋白合成抑制剂阻断 .结果表明 ,TEC基因是一种与肝再生调控密切相关的早期反应基因 .     

Rbf1-independent termination of E2f1-target gene expression during early Drosophila embryogenesis

The initiation and maintenance of G1 cell cycle arrest is a key feature of animal development. In the Drosophila ectoderm, G1 arrest first appears during the seventeenth embryonic cell cycle. The initiation of G1(17) arrest requires the developmentally-induced expression of Dacapo, a p27-like Cyclin E-Cdk2 inhibitor. The maintenance of G1(17) arrest requires Rbf1-dependent repression of E2f1-regulated replication factor genes, which are expressed continuously during cycles 1-16 when S phase immediately follows mitosis. The mechanisms that trigger Rbf1 repressor function and mediate G1(17) maintenance are unknown. Here we show that the initial downregulation of expression of the E2f1-target gene RnrS, which occurs during cycles 15 and 16 prior to entry into G1(17), does not require Rbf1 or p27(Dap). This suggests a mechanism for Rbf1-independent control of E2f1 during early development. We show that E2f1 protein is destroyed in a cell cycle-dependent manner during S phase of cycles 15 and 16. E2f1 is destroyed during early S phase, and requires ongoing DNA replication. E2f1 protein reaccumulates in epidermal cells arrested in G1(17), and in these cells the induction of p27(Dap) activates Rbf1 to repress E2f1-target genes to maintain a stable G1 arrest.     

Cell cycle analysis of Epstein-Barr virus-infected cells following treatment with lytic cycle-inducing agents

While Epstein-Barr virus (EBV) latency-associated gene expression is associated with cell cycle progression, the relationship between the EBV lytic program and the cell cycle is less clear. Using four different EBV lytic induction systems, we address the relationship between lytic cycle activation and the cell cycle. In three of these systems, G0 or G1 cell growth arrest signaling is observed prior to detection of the EBV immediate-early gene product Zta. In tetradecanoyl phorbol acetate-treated P3HR1 cultures and in 5-iodo-2'-deoxyuridine-treated NPC-KT cultures, cell cycle analysis of Zta-expressing cell populations showed a significant G1 bias during the early stages of lytic cycle progression. In contrast, treatment of the cell line Akata with anti-immunoglobulin (Ig) results in rapid induction of immediate-early gene expression, and accordingly, activation of the immediate-early gene product Zta precedes significant anti-Ig-induced cell cycle effects. Nevertheless, cell cycle analysis of the Zta-expressing population following anti-Ig treatment shows a bias for cells in G1, indicating that anti-Ig-mediated induction of Zta occurs more efficiently in cells traversing G1. Last, although 5-azacytidine treatment of Rael cells results in a G1 arrest in the total cell population which precedes the induction of Zta, cell cycle analysis of the Zta-expressing population shows a significant bias for cells with an apparent G2/M DNA content. This bias may result, in part, from activation of Zta expression following demethylation of the Zta promoter during S-phase. Together, these studies indicate that induction of Zta occurs through several distinct mechanisms, some of which may involve checkpoint signaling.     

The cell cycle dependence of thermotolerance. II. CHO cells heated at 45.0 degrees C

This report extends our investigations of the cell cycle dependence of the expression of thermotolerance to include tolerance expressed by Chinese hamster ovary (CHO) cells exposed to 45.0 degrees C hyperthermia. We examined the response of asynchronous cells following exposure at 45.0 degrees C. A maximum in thermotolerance under these conditions was reached approximately 12 hr after a 15-min exposure to 45.0 degrees C hyperthermia and progressively decreased thereafter. Cells were delayed in S and G2 phase for 24 hr, after which time cell growth resumed. We then characterized the response of CHO cell populations synchronized in G1 or early or late S phase. We observed that the expression of tolerance depended on the position of cells in the cell cycle and was modulated by changes in the sensitivity of cells as they progressed through the cell cycle subsequent to the tolerance induction dose. We measured the variation in the sensitivity of these cells to 45.0 degrees C hyperthermia throughout the cell cycle and found substantial changes as cells progressed through S phase. Cells in early S phase were the most sensitive to heat at this temperature, and as these cells progressed through S phase, they became progressively more resistant. In addition, G1 cells were delayed for approximately 15 to 18 hr by a 15-min, 45.0 degrees C heat pulse, whereas S-phase cells were delayed to a lesser extent. The data presented in this report suggest that the induction of thermotolerance is relatively non-cell-cycle specific, but the magnitude of expression of tolerance depends on the position of cells in the cell cycle at the time of the subsequent challenge heat dose.     

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.     

Non-local models for the formation of hepatocyte-stellate cell aggregates

Liver cell aggregates may be grown in vitro by co-culturing hepatocytes with stellate cells. This method results in more rapid aggregation than hepatocyte-only culture, and appears to enhance cell viability and the expression of markers of liver-specific functions. We consider the early stages of aggregate formation, and develop a new mathematical model to investigate two alternative hypotheses (based on evidence in the experimental literature) for the role of stellate cells in promoting aggregate formation. Under Hypothesis 1, each population produces a chemical signal which affects the other, and enhanced aggregation is due to chemotaxis. Hypothesis 2 asserts that the interaction between the two cell types is by direct physical contact: the stellates extend long cellular processes which pull the hepatocytes into the aggregates. Under both hypotheses, hepatocytes are attracted to a chemical they themselves produce, and the cells can experience repulsive forces due to overcrowding. We formulate non-local (integro-partial differential) equations to describe the densities of cells, which are coupled to reaction-diffusion equations for the chemical concentrations. The behaviour of the model under each hypothesis is studied using a combination of linear stability analysis and numerical simulations. Our results show how the initial rate of aggregation depends upon the cell seeding ratio, and how the distribution of cells within aggregates depends on the relative strengths of attraction and repulsion between the cell types. Guided by our results, we suggest experiments which could be performed to distinguish between the two hypotheses.     

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.     

Joint requirement for Rac and ERK activities underlies the mid-G1 phase induction of cyclin D1 and S phase entry in both epithelial and mesenchymal cells

Cyclin D1 gene induction is a key event in G1 phase progression. Our previous studies indicated that signaling to cyclin D1 is cell type-dependent because the timing of cyclin D1 gene expression in MCF10A mammary epithelial cells and mesenchymal cells such as fibroblasts and vascular smooth muscle cells is very different, with epithelial cells first expressing cyclin D1 in early rather than mid-G1 phase. In this report, we induced a mesenchymal phenotype in MCF10A cells by long-term exposure to TGF-beta and used the control and transitioned cells to examine cell type specificity of the signaling pathways that regulate cyclin D1 gene expression. We show that early-G1 phase cyclin D1 gene expression in MCF10A cells is under the control of Rac, whereas mid-G1 phase cyclin D1 induction requires parallel signaling from Rac and ERK, both in the control and transitioned cells. This combined requirement for Rac and ERK signaling is associated with an increased requirement for intracellular tension, Rb phosphorylation, and S phase entry. A similar co-regulation of cyclin D1 mRNA by Rac and ERK is seen in primary mesenchymal cells. Overall, our results reveal two mechanistically distinct phases of Rac-dependent cyclin D1 expression and emphasize that the acquisition of Rac/ERK co-dependence is required for the mid-G1 phase induction of cyclin D1 associated with S phase entry.     

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.     

MT1-MMP down-regulates the glucose 6-phosphate transporter expression in marrow stromal cells: a molecular link between pro-MMP-2 activation, chemotaxis, and cell survival

Bone marrow-derived stromal cells (BMSC) are avidly recruited by experimental vascularizing tumors, which implies that they must respond to tumor-derived growth factor cues. In fact, BMSC chemotaxis and cell survival are regulated, in part, by the membrane type-1 matrix metalloproteinase (MT1-MMP), an MMP also involved in pro-MMP-2 activation and in degradation of the extracellular matrix (ECM). Given that impaired chemotaxis was recently observed in bone marrow cells isolated from a glucose 6-phosphate transporter-deficient (G6PT-/-) mouse model, we sought to investigate the potential MT1-MMP/G6PT signaling axis in BMSC. We show that MT1-MMP-mediated activation of pro-MMP-2 by concanavalin A (ConA) correlated with an increase in the sub-G1 cell cycle phase as well as with cell necrosis, indicative of a decrease in BMSC survival. BMSC isolated from Egr-1-/- mouse or MT1-MMP gene silencing in BMSC with small interfering RNA (siMT1-MMP) antagonized both the ConA-mediated activation of pro-MMP-2 and the induction of cell necrosis. Overexpression of recombinant full-length MT1-MMP triggered necrosis and this was signaled through the cytoplasmic domain of MT1-MMP. ConA inhibited both the gene and protein expression of G6PT, while overexpression of recombinant G6PT inhibited MT1-MMP-mediated pro-MMP-2 activation but could not rescue BMSC from ConA-induced cell necrosis. Cell chemotaxis in response to the tumorigenic growth factor sphingosine 1-phosphate was significantly abrogated in siMT1-MMP BMSC and in chlorogenic acid-treated BMSC. Altogether, we provide evidence for an MT1-MMP/G6PT signaling axis that regulates BMSC survival, ECM degradation, and mobilization. This may lead to optimized clinical applications that use BMSC as a platform for the systemic delivery of therapeutic or anti-cancer recombinant proteins in vivo.     

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.     

Prolyl hydroxylase PHD3 enhances the hypoxic survival and G1 to S transition of carcinoma cells

Hypoxia restricts cell proliferation and cell cycle progression at the G1/S interface but at least a subpopulation of carcinoma cells can escape the restriction. In carcinoma hypoxia may in fact select for cells with enhanced hypoxic survival and increased aggressiveness. The cellular oxygen sensors HIF proline hydroxylases (PHDs) adapt the cellular functions to lowered environmental oxygen tension. PHD3 isoform has shown the strongest hypoxic upregulation among the family members. We detected a strong PHD3 mRNA expression in tumors of head and neck squamous cell carcinoma (HNSCC). The PHD3 expression associated with expression of hypoxic marker gene. Using siRNA in cell lines derived from HNSCC we show that specific inhibition of PHD3 expression in carcinoma cells caused reduced cell survival in hypoxia. The loss of PHD3, but not that of PHD2, led to marked cell number reduction. Although caspase-3 was activated at early hypoxia no induction of apoptosis was detected. However, hypoxic PHD3 inhibition caused a block in cell cycle progression. Cell population in G1 phase was increased and the population in S phase reduced demonstrating a block in G1 to S transition under PHD3 inhibition. In line with this, the level of hyperphosphorylated retinoblastoma protein Rb was reduced by PHD3 knock-down in hypoxia. PHD3 loss led to increase in cyclin-dependent kinase inhibitor p27 expression but not that of p21 or p16. The data demonstrated that increased PHD3 expression under hypoxia enhances cell cycle progression and survival of carcinoma cells.     

Correlating cell cycle with apoptosis in a cell line expressing a tandem green fluorescent protein substrate specific for group II caspases.

BACKGROUND: We describe a rapid flow cytometric assay that correlates cell cycle with apoptotic cell death in a cell line expressing a tandem green fluorescent protein (GFP). METHODS: A Jurkat cell line was transfected with a gene construct coding for constitutive expression of a tandem GFP molecule carrying a consensus cleavage site (DEVD) for group II caspases (C-2-Y). Cells were treated with CD95 antibody (Ab), then incubated with annexin V-phycoerythrin (PE), propidium iodide (PI), and Hoechst 33342. RESULTS: After CD95 treatment, the C-2-Y cell line had twice the number of nonapoptotic cells compared with both control cell lines. This proportion of viable, nonapoptotic cells after treatment was unaffected by the level of GFP (DEVD) expression in the cells, as confirmed by sorted populations. The early apoptotic cells in the C-2-Y cell line had an increased G0-G1 phase population compared with the control cell lines. CONCLUSIONS: Apoptosis is delayed in the C-2-Y cell line and the early apoptotic cells have a higher G0-G1 cell cycle frequency. The artificial substrate competes with the natural substrate(s), thereby slowing the apoptotic process. The expression level of DEVD-GFP does not alter the delayed induction of apoptosis. Caspase activation occurs prior to phosphatidylserine translocation.