scholarly journals A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618 ◽  
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter

1993 ◽  
Vol 13 (3) ◽  
pp. 1610-1618
Author(s):  
J E Slansky ◽  
Y Li ◽  
W G Kaelin ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Phase Boundary ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Protein S ◽  
S Phase ◽  
Dependent Manner

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesis-dependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serum-stimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein E1A. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation.

scholarly journals An E-box-mediated increase in cad transcription at the G1/S-phase boundary is suppressed by inhibitory c-Myc mutants.

1995 ◽  
Vol 15 (5) ◽  
pp. 2527-2535 ◽  
Author(s):  
R J Miltenberger ◽  
K A Sukow ◽  
P J Farnham
Keyword(s):  
Cell Cycle ◽  
Phase Boundary ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Binding Activity ◽  
S Phase ◽  
Dependent Manner ◽  
Carbamoyl Phosphate ◽  
Dna Binding Activity ◽  
E Box

To better understand the signaling pathways which lead to DNA synthesis in mammalian cells, we have studied the transcriptional activation of genes needed during the S phase of the cell cycle. Transcription of the gene encoding a pyrimidine biosynthetic enzyme, carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (cad), increases at the G1/S-phase boundary. We have mapped the growth-dependent response element in the hamster cad gene to the extended palindromic E-box sequence, CCACGTGG, which is centered at +65 in the 5' untranslated sequence. Mutation of the E box abolished growth-dependent transcription, and an oligonucleotide corresponding to the cad sequence at +55 to +75 (+55/+75) restored growth-dependent regulation to nonresponsive cad promoter mutants when placed down-stream of the transcription start site. The same oligonucleotide conferred less G1/S-phase induction when placed upstream of basal promoter elements. An analogous oligonucleotide containing the mutant E box had no effect in either location. Nuclear proteins bound the cad +55/+75 element in a cell cycle-dependent manner in electromobility shift assays; antibodies specific to USF and Max blocked the DNA-binding activity of different growth-regulated protein-DNA complexes. Expression of c-Myc mutants which have been shown to dominantly interfere with the function of c-Myc and Max significantly inhibited cad transcription during S phase but had no effect on transcription from another G1/S-phase-activated promoter, dhfr. These data support a model whereby E-box-binding proteins activate serum-induced transcription from the cad promoter at the G1/S-phase boundary and suggest that a Max-associated protein complex contributes to the serum response.

scholarly journals Novel Regulatory Factors Interacting with the Promoter of the Gene Encoding the mRNA Cap Binding Protein (eIF4E) and Their Function in Growth Regulation

1998 ◽  
Vol 18 (10) ◽  
pp. 5621-5633 ◽  
Author(s):  
Kelly A. Johnston ◽  
Michael Polymenis ◽  
Shanping Wang ◽  
John Branda ◽  
Emmett V. Schmidt
Keyword(s):  
Protein Synthesis ◽  
Protein Interactions ◽  
Transcription Initiation ◽  
Growth Regulation ◽  
Cellular Proliferation ◽  
Binding Protein ◽  
Limiting Factor ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Regulate Protein

ABSTRACTRegulation of the mRNA cap binding protein (eIF4E) is critical to the control of cellular proliferation since this protein is the rate-limiting factor in translation initiation and transforms fibroblasts and since eIF4E mutants arrest budding yeast in the G1phase of the cell cycle (cdc33). We previously demonstrated regulation of eIF4E by altered transcription of its mRNA in serum-stimulated fibroblasts and in response to c-myc. To identify additional factors regulating eIF4E transcription, we used linker-scanning constructs to characterize sites in the promoter of the eIF4E gene required for its expression. Promoter activity was dependent on sites at −5, −25, −45, and −75; the site at −75 included a previously describedmycbox. Electrophoretic mobility shift assays identified DNA-protein interactions at −25 and revealed a binding site (TTACCCCCCCTT) that is unique to the eIF4E promoter. Proteins of 68 and 97 kDa bound this site in UV cross-linking and Southwestern experiments. Levels of 4E regulatory factor activities correlated with c-Myc levels, eIF4E expression levels, and protein synthesis in differentiating U937 and HL60 cells, suggesting that these activities may function to regulate protein synthesis rates during differentiation. Since the eIF4E promoter lacked typical TATA and initiator elements, further studies of this novel initiator-homologous element should provide insights into mechanisms of transcription initiation and growth regulation.

Role of EP1 and EP4 PGE2subtype receptors in serum-induced 3T6 fibroblast cycle progression and proliferation

2002 ◽  
Vol 282 (2) ◽  
pp. C280-C288 ◽  
Author(s):  
Teresa Sanchez ◽  
Juan Jose Moreno
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Tumor Development ◽  
Inhibitory Effect ◽  
S Phase ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Cycle Progression

Recent studies have suggested that prostaglandin E2 (PGE2) subtype receptors (EP) are involved in cellular proliferation and tumor development. We studied the role of EP1 and EP4PGE2 subtype receptor antagonists AH-6809 and AH-23848B, respectively, in serum-induced 3T6 fibroblast proliferation. This was significantly reduced in a dose-dependent manner (IC50∼100 and ∼30 μM, respectively) to an almost complete inhibition, without any cytotoxic effect. However, the effect of each antagonist on 3T6 cell cycle progression clearly differed. Whereas the EP1 antagonist increased the G0/G1population, the EP4 antagonist brought about an accumulation of cells in early S phase. These effects were associated with a decrease in cyclin D and E levels in AH-6809-treated 3T6 cells and lower cyclin A levels in AH-23848B-treated fibroblasts with respect to control cells. The G0/G1 accumulation caused by AH-6809 seems to be intracellular Ca2+ concentration ([Ca2+]i) dependent, because a 6-h 1 μM thapsigargin treatment allowed G0/G1-arrested cells to enter S phase. Similarly, treatment with 20 μM forskolin for 6 h allowed S-phase and G2/M progression of AH-23848B-treated cells. This study shows that the inhibitory effect of the EP1 and EP4 antagonists on serum-induced 3T6 fibroblast growth is due to their effect at various levels of the cell cycle machinery, suggesting that PGE2 interaction with its different subtype receptors regulates progression through the cell cycle by modulating cAMP and [Ca2+]i.

Murine dihydrofolate reductase transcripts through the cell cycle

1986 ◽  
Vol 6 (2) ◽  
pp. 365-371
Author(s):  
P J Farnham ◽  
R T Schimke
Keyword(s):  
Cell Cycle ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Untranslated Region ◽  
Cell Cycle Regulation ◽  
Leader Sequence ◽  
Reductase Gene ◽  
Opposite Strand ◽  
Polyadenylation Sites ◽  
Cycle Regulation

The murine dihydrofolate reductase gene codes for mRNAs that differ in the length of their 3' untranslated region as well as in the length of their 5' leader sequence. In addition, the dihydrofolate reductase promoter functions bidirectionally, producing a series of RNAs from the opposite strand than the dihydrofolate reductase mRNAs. We have examined the production of these RNAs and their heterogeneous 5' and 3' termini as mouse 3T6 cells progress through a physiologically continuous cell cycle. We found that all of the transcripts traverse the cell cycle in a similar manner, increasing at the G1/S boundary without significantly changing their ratios relative to one another. We conclude that cell-cycle regulation of dihydrofolate reductase is achieved without recruiting new transcription initiation sites and without a change in polyadenylation sites. It appears that the mechanism responsible for the transcriptional cell-cycle regulation of the dihydrofolate reductase gene is manifested only by transiently increasing the efficiency of transcription at the dihydrofolate reductase promoter.

scholarly journals Xanthohumol Induces Growth Inhibition and Apoptosis in Ca Ski Human Cervical Cancer Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Wai Kuan Yong ◽  
Sri Nurestri Abd Malek
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Morphological Changes ◽  
S Phase ◽  
Phase Cell ◽  
Cervical Cancer Cell Line ◽  
Dependent Manner ◽  
Cervical Cancer Cells

We investigate induction of apoptosis by xanthohumol on Ca Ski cervical cancer cell line. Xanthohumol is a prenylated chalcone naturally found in hop plants, previously reported to be an effective anticancer agent in various cancer cell lines. The present study showed that xanthohumol was effective to inhibit proliferation of Ca Ski cells based on IC50values using sulforhodamine B (SRB) assay. Furthermore, cellular and nuclear morphological changes were observed in the cells using phase contrast microscopy and Hoechst/PI fluorescent staining. In addition, 48-hour long treatment with xanthohumol triggered externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells. Additionally, xanthohumol mediated S phase arrest in cell cycle analysis and increased activities of caspase-3, caspase-8, and caspase-9. On the other hand, Western blot analysis showed that the expression levels of cleaved PARP, p53, and AIF increased, while Bcl-2 and XIAP decreased in a dose-dependent manner. Taken together, these findings indicate that xanthohumol-induced cell death might involve intrinsic and extrinsic apoptotic pathways, as well as downregulation of XIAP, upregulation of p53 proteins, and S phase cell cycle arrest in Ca Ski cervical cancer cells. This work suggests that xanthohumol is a potent chemotherapeutic candidate for cervical cancer.

scholarly journals Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

2012 ◽  
Vol 23 (21) ◽  
pp. 4203-4211 ◽  
Author(s):  
Dong-Hwan Kim ◽  
Deanna M. Koepp
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Charged Residues ◽  
Phase Progression ◽  
Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

scholarly journals Programmed cell death 2 protein induces gastric cancer cell growth arrest at the early S phase of the cell cycle and apoptosis in a p53-dependent manner

2014 ◽  
Vol 33 (1) ◽  
pp. 103-110 ◽  
Author(s):  
JIAN ZHANG ◽  
WEI WEI ◽  
HUI-CHENG JIN ◽  
RONG-CHAO YING ◽  
A-KAO ZHU ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cell Death ◽  
Cell Growth ◽  
Gastric Cancer Cell ◽  
Growth Arrest ◽  
S Phase ◽  
Dependent Manner ◽  
Cancer Cell Growth ◽  
Cell Growth Arrest

scholarly journals Evidence for DNA-PK-dependent and -independent DNA double-strand break repair pathways in mammalian cells as a function of the cell cycle.

1997 ◽  
Vol 17 (3) ◽  
pp. 1425-1433 ◽  
Author(s):  
S E Lee ◽  
R A Mitchell ◽  
A Cheng ◽  
E A Hendrickson
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Strand Break ◽  
S Phase ◽  
Dependent Manner ◽  
Severe Combined Immune Deficiency ◽  
Dsb Repair ◽  
G2 Checkpoint ◽  
G2 Phase

Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G1/early S phase and again at the G2 phase in wild-type cells. Interestingly, only the deficit of the G1/early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G2 phase may be required for exit from a DNA damage-induced G2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.

S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells

2001 ◽  
Vol 359 (2) ◽  
pp. 361-367 ◽  
Author(s):  
Elizabeth J. FOX ◽  
Stephanie C. WRIGHT
Keyword(s):  
Cell Cycle ◽  
Cellular Proliferation ◽  
Cultured Cells ◽  
S Phase ◽  
Proliferating Cells ◽  
Specific Expression ◽  
Related Function ◽  
Erythroleukemia Cells ◽  
A Cell ◽  
Pass Through

The Myc/Max/Mad transcription factor network plays a central role in the control of cellular proliferation, differentiation and apoptosis. In order to elucidate the biological function of Mad3, we have analysed the precise temporal patterns of Mad3 mRNA expression during the cell cycle and differentiation in cultured cells. We show that Mad3 is induced at the G1/S transition in proliferating cells; expression persists throughout S-phase, and then declines as cells pass through G2 and mitosis. The expression pattern of Mad3 is coincident with that of Cdc2 throughout the cell cycle. In contrast, the expression of Mad3 during differentiation of cultured mouse erythroleukemia cells shows two transient peaks of induction. The first of these occurs at the onset of differentiation, and does not correlate with the S-phase of the cell cycle, whereas the second is coincident with the S-phase burst that precedes the terminal stages of differentiation. Our results therefore suggest that Mad3 serves a cell-cycle-related function in both proliferating and differentiating cells, and that it may also have a distinct role at various stages of differentiation.

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