Phospholipase Cδ1 regulates cell proliferation and cell-cycle progression from G1- to S-phase by control of cyclin E–CDK2 activity

2008 ◽  
Vol 415 (3) ◽  
pp. 439-448 ◽  
Author(s):  
Katherine A. Kaproth-Joslin ◽  
Xiangquan Li ◽  
Sarah E. Reks ◽  
Grant G. Kelley
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Cyclin E ◽  
Critical Role ◽  
S Phase ◽  
G1 Phase ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Cdk2 Activity

In the present study, we examined the role of PLCδ1 (phospholipase C δ1) in the regulation of cellular proliferation. We demonstrate that RNAi (RNA interference)-mediated knockdown of endogenous PLCδ1, but not PLCβ3 or PLCϵ, induces a proliferation defect in Rat-1 and NIH 3T3 fibroblasts. The decreased proliferation was not due to an induction of apoptosis or senescence, but was associated with an approx. 60% inhibition of [3H]thymidine incorporation. Analysis of the cell cycle with BrdU (bromodeoxyuridine)/propidium iodide-labelled FACS (fluorescence-activated cell sorting) demonstrated an accumulation of cells in G0/G1-phase and a corresponding decrease in cells in S-phase. Further examination of the cell cycle after synchronization by serum-starvation demonstrated normal movement through G1-phase but delayed entry into S-phase. Consistent with these findings, G1 cyclin (D2 and D3) and CDK4 (cyclin-dependent kinase 4) levels and associated kinase activity were not affected. However, cyclin E-associated CDK2 activity, responsible for G1-to-S-phase progression, was inhibited. This decreased activity was accompanied by unchanged CDK2 protein levels and paradoxically elevated cyclin E and cyclin E-associated CDK2 levels, suggesting inhibition of the cyclin E–CDK2 complex. This inhibition was not due to altered stimulatory or inhibitory phosphorylation of CDK2. However, p27, a Cip/Kip family CKI (CDK inhibitor)-binding partner, was elevated and showed increased association with CDK2 in PLCδ1-knockdown cells. The result of the present study demonstrate a novel and critical role for PLCδ1 in cell-cycle progression from G1-to-S-phase through regulation of cyclin E–CDK2 activity and p27 levels.

scholarly journals The PP2A-B56 Phosphatase Opposes Cyclin E Autocatalytic Degradation via Site-Specific Dephosphorylation

2017 ◽  
Vol 37 (8) ◽  
Author(s):  
Ryan J. Davis ◽  
Jherek Swanger ◽  
Bridget T. Hughes ◽  
Bruce E. Clurman
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Phosphorylation Site ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Cdk2 Activity ◽  
Interphase Cells

ABSTRACT Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCFFbw7 ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic.

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

1993 ◽  
Vol 13 (6) ◽  
pp. 3577-3587 ◽  
Author(s):  
E A Musgrove ◽  
J A Hamilton ◽  
C S Lee ◽  
K J Sweeney ◽  
C K Watts ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cell Cycle ◽  
Cyclin D1 ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Cell Cycle Progression ◽  
S Phase ◽  
G1 Phase ◽  
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.

scholarly journals HTLV-I p30 inhibits multiple S phase entry checkpoints, decreases cyclin E-CDK2 interactions and delays cell cycle progression

2010 ◽  
Vol 9 (1) ◽  
pp. 302 ◽  
Author(s):  
Hicham H Baydoun ◽  
Joanna Pancewicz ◽  
XueTao Bai ◽  
Christophe Nicot
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
S Phase ◽  
Cycle Progression ◽  
Phase Entry

scholarly journals A Critical Role for Cyclin C in Promotion of the Hematopoietic Cell Cycle by Cooperation with c-Myc

1998 ◽  
Vol 18 (6) ◽  
pp. 3445-3454 ◽  
Author(s):  
Zhao-Jun Liu ◽  
Takahiro Ueda ◽  
Tadaaki Miyazaki ◽  
Nobuyuki Tanaka ◽  
Shinichiro Mine ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Critical Role ◽  
S Phase ◽  
Early Gene ◽  
Cycle Progression ◽  
Cyclin C ◽  
Growth Properties

ABSTRACT Cyclin C, a putative G1 cyclin, was originally isolated through its ability to complement a Saccharomyces cerevisiae strain lacking the G1 cyclin geneCLN1-3. Unlike cyclins D1 and E, the other two G1 cyclins obtained by the same approach and subsequently shown to play important roles during the G1/S transition, there is thus far no evidence to support the hypothesis that cyclin C is indeed critical for the promotion of cell cycle progression. In BAF-B03 cells, an interleukin 3 (IL-3)-dependent murine pro-B-cell line, cyclin C gene mRNA was induced at the G1/S phase upon IL-3 stimulation and reached a maximal level in the S phase. Enforced expression of exogenous cyclin C in this cell line failed to alter its growth properties. In the present study, we examined whether cyclin C is capable of cooperating with the cytokine-responsive immediate-early gene products c-Myc and c-Fos in the promotion of cell proliferation. We found that cyclin C is able to cooperate functionally with c-Myc, but not c-Fos, to induce both BAF-B03 cell proliferation in a cytokine-independent fashion and the formation of cell clusters. Furthermore, cyclin C was primarily responsible for the induction of cdc2 gene expression. Our data define a novel role for cyclin C in the regulation of both the G1/S and G2/M phases of the cell cycle, and this effect appears to be independent of the activity of CDK8 in the control of transcription.

scholarly journals The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces Expression of E2F-1 and Other Proteins Involved in Cell Cycle Progression in Primary Keratinocytes and Gastric Carcinoma Cells

2002 ◽  
Vol 76 (24) ◽  
pp. 12543-12552 ◽  
Author(s):  
Amy Mauser ◽  
Elizabeth Holley-Guthrie ◽  
Adam Zanation ◽  
Wendall Yarborough ◽  
William Kaufmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Human Fibroblasts ◽  
Cell Types ◽  
S Phase ◽  
Cycle Progression ◽  
Stem Loop ◽  
Barr Virus ◽  
Early Protein

ABSTRACT The Epstein-Barr virus (EBV) immediate-early protein BZLF1 mediates the switch between the latent and lytic forms of EBV infection and has been previously shown to induce a G1/S block in cell cycle progression in some cell types. To examine the effect of BZLF1 on cellular gene expression, we performed microarray analysis on telomerase-immortalized human keratinocytes that were mock infected or infected with a control adenovirus vector (AdLacZ) or a vector expressing the EBV BZLF1 protein (AdBZLF1). Cellular genes activated by BZLF1 expression included E2F-1, cyclin E, Cdc25A, and a number of other genes involved in cell cycle progression. Immunoblot analysis confirmed that BZLF1 induced expression of E2F-1, cyclin E, Cdc25A, and stem loop binding protein (a protein known to be primarily expressed during S phase) in telomerase-immortalized keratinocytes. Similarly, BZLF1 increased expression of E2F-1, cyclin E, and stem loop binding protein (SLBP) in primary tonsil keratinocytes. In contrast, BZLF1 did not induce E2F-1 expression in normal human fibroblasts. Cell cycle analysis revealed that while BZLF1 dramatically blocked G1/S progression in normal human fibroblasts, it did not significantly affect cell cycle progression in primary human tonsil keratinocytes. Furthermore, in EBV-infected gastric carcinoma cells, the BZLF1-positive cells had an increased number of cells in S phase compared to the BZLF1-negative cells. Thus, in certain cell types (but not others), BZLF1 enhances expression of cellular proteins associated with cell cycle progression, which suggests that an S-phase-like environment may be advantageous for efficient lytic EBV replication in some cell types.

IGFBP7 Gene Promoting Cell Proliferation in Acute Myeloid Leukemia Through Activation of AKT3 and CCND1

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1447-1447
Author(s):  
Shaoyan Hu ◽  
Shui-yan Wu ◽  
Jian-nong Cen ◽  
Jian Pan ◽  
Xiaofei Qi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
K562 Cells ◽  
S Phase ◽  
G1 Phase ◽  
Rt Pcr ◽  
Cycle Progression ◽  
Childhood Aml

Abstract Abstract 1447 Insulin-like growth factor binding protein 7 (IGFBP7) has been ascribed properties of both tumor suppressor and enhancer of cell proliferation. In solid tumors the important role of IGFBP7 as a tumor suppressor was revealed in several studies. In acute T-lymphoblastic leukemia (T-ALL), high IGFBP7 expression is associated with a more immature phenotype of early T-ALL, inferior survival, and predicts primary chemotherapy resistance. In a separate study, IGFBP7 acts as a positive regulator of ALL and bone marrow stromal cells growth, and significantly enhances in-vitro resistance to asparaginase. Higher IGFBP7 mRNA levels were associated with lower leukemia-free survival (P=0.003) in precursor B-cell Ph negative ALL patients (n=147) treated with a contemporary polychemotherapy protocol. In acute myeloid leukemia, the role of IGFBP7 is largely unknown. In our previous published study [Hu et al, 2011], we demonstrated that IGFBP7 overexpressed in majority of childhood AML (n=66) at diagnosis and upon relapsed, but not at remission stage. We now further explore its mechanism in promoting AML cells proliferation. Compared with control, transfection of full length IGFBP7 in K562 cells [V-BP7] resulted in 23% increased of proliferation in 48 hours. Cell cycle analysis by flow cytometry showed decreased G0/G1 phase and increased S phase in V-BP7 comparing with control, suggesting enhanced cell cycle progression. While transfection of IGFPB7 siRNA produced an opposite effect of reducing the cell growth in K562 cells. In consistent with the nature of a secretory protein, the extracellular IGFBP7 level in the condition media from v-BP7 was significantly higher than that from vector control or parental K562 cells measured by ELISA. Incubation parental K562 cells in V-BP7 derived conditioned medium resulted in significant growth enhancement. Gene expression profiling (GEP) was performed on V-BP7 in contrast to parental K562 cells. Genes which were up-regulated or down-regulated more than 2 folds were regarded as significant difference. Among 10 verified genes, AKT3 showed the highest extent of up-regulation and IGFBP7 siRNA transfection reduced its expression. Cyclin D1 (CCND1) expression was also significantly up-regulated and validated by RT-PCR and Western blot. V-BP7 treated with an AKT inhibitor (Triciribine) at 2.5μM for 72 hours showed 50% reduction of cell viability. The cell cycle analysis indicated that triciribine reversed cell cycle progression in V-BP7, by increasing cells in G0/G1 phase and reducing cells in S phase. Western blot demonstrated that both phospho-AKT3 and CCND1 were down regulated after treatment with triciribine. Using real time RT-PCR, we further identified that IGFBP7 and AKT3 expression were significantly correlated (p=0.001; r=0.255) in 39 newly diagnosed childhood AML. Conclusions IGFBP7 aberrantly overexpressed in majority of childhood AML. IGFBP7 promotes proliferation of K562 cells and AML via overexpression/activation of AKT3 and CCND1. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The susceptibility of granulosa cells to apoptosis is influenced by oestradiol and the cell cycle

2006 ◽  
Vol 189 (3) ◽  
pp. 441-453 ◽  
Author(s):  
Susan M Quirk ◽  
Robert G Cowan ◽  
Rebecca M Harman
Keyword(s):  
Cell Cycle ◽  
Granulosa Cells ◽  
Cell Cycle Progression ◽  
Fas Ligand ◽  
S Phase ◽  
G1 Phase ◽  
Cycle Progression ◽  
Induced Apoptosis ◽  
Cell Proliferation Marker ◽  
D2 Protein

Experiments were conducted to test whether oestradiol (E2) protects granulosa cells from Fas ligand (FasL)-induced apoptosis and whether protection involves modulation of the cell cycle of proliferation. Treatment of cultured bovine granulosa cells with E2 decreased susceptibility to FasL-induced apoptosis. The effects of E2 were mediated through oestrogen receptor and were not mediated by stimulation of IGF production. E2 also increased the percentage of cells progressing from G1 to S phase of the cell cycle, and increased expression of cyclin D2 protein and the cell proliferation marker Ki67. Progression from G1 to S phase of the cell cycle was necessary for the protective effect of E2; blocking progression from G1 to S phase with the cdk2 inhibitor roscovitine, or blocking cells in S phase with hydroxyurea, prevented protection by E2. The stages of the cell cycle during which granulosa cells are susceptible to apoptosis were assessed. First, treatment with the G1 phase blocker, mimosine, protected cells from FasL-induced apoptosis, indicating that cells in G0 or early- to mid-G1 phase are relatively resistant to apoptosis. Secondly, examination of recent DNA synthesis by cells that became apoptotic indicated that apoptosis did not occur in S, G2 or M phases. Taken together, the experiments indicate that cells may be most susceptible to apoptosis at the transition from G1 to S phase. E2 stimulates transition from G1 to S phase and protects against apoptosis only when cell cycle progression is unperturbed.

scholarly journals Blocking the transcription factor E2F/DP by dominant-negative mutants in a normal breast epithelial cell line efficiently inhibits apoptosis and induces tumor growth in SCID mice.

1996 ◽  
Vol 183 (3) ◽  
pp. 1205-1213 ◽  
Author(s):  
R C Bargou ◽  
C Wagener ◽  
K Bommert ◽  
W Arnold ◽  
P T Daniel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Dominant Negative ◽  
Serum Starvation ◽  
Cycle Progression ◽  
Transcription Factor E2f

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.

scholarly journals Caveolin-1 Expression Negatively Regulates Cell Cycle Progression by Inducing G0/G1 Arrest via a p53/p21WAF1/Cip1-dependent Mechanism

2001 ◽  
Vol 12 (8) ◽  
pp. 2229-2244 ◽  
Author(s):  
Ferruccio Galbiati ◽  
Daniela Volonte' ◽  
Jun Liu ◽  
Franco Capozza ◽  
Philippe G. Frank ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Recombinant Expression ◽  
Critical Role ◽  
Primary Cultures ◽  
Serum Starvation ◽  
G1 Arrest ◽  
Cycle Progression ◽  
Caveolin 1

Caveolin-1 is a principal component of caveolae membranes in vivo. Caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). However, it remains unknown whether caveolin-1 plays any role in regulating cell cycle progression. Here, we directly demonstrate that caveolin-1 expression arrests cells in the G0/G1 phase of the cell cycle. We show that serum starvation induces up-regulation of endogenous caveolin-1 and arrests cells in the G0/G1 phase of the cell cycle. Moreover, targeted down-regulation of caveolin-1 induces cells to exit the G0/G1 phase. Next, we constructed a green fluorescent protein-tagged caveolin-1 (Cav-1-GFP) to examine the effect of caveolin-1 expression on cell cycle regulation. We directly demonstrate that recombinant expression of Cav-1-GFP induces arrest in the G0/G1 phase of the cell cycle. To examine whether caveolin-1 expression is important for modulating cell cycle progression in vivo, we expressed wild-type caveolin-1 as a transgene in mice. Analysis of primary cultures of mouse embryonic fibroblasts from caveolin-1 transgenic mice reveals that caveolin-1 induces 1) cells to exit the S phase of the cell cycle with a concomitant increase in the G0/G1 population, 2) a reduction in cellular proliferation, and 3) a reduction in the DNA replication rate. Finally, we demonstrate that caveolin-1-mediated cell cycle arrest occurs through a p53/p21-dependent pathway. Taken together, our results provide the first evidence that caveolin-1 expression plays a critical role in the modulation of cell cycle progression in vivo.

scholarly journals Golgi-associated RhoBTB3 targets Cyclin E for ubiquitylation and promotes cell cycle progression

2013 ◽  
Vol 203 (2) ◽  
pp. 233-250 ◽  
Author(s):  
Albert Lu ◽  
Suzanne R. Pfeffer
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Cell Cycle Control ◽  
S Phase ◽  
Cycle Progression ◽  
Ubiquitin Ligase Complex ◽  
Golgi Fragmentation ◽  
Ring E3 ◽  
Normal Cell Cycle

Cyclin E regulates the cell cycle transition from G1 to S phase and is degraded before entry into G2 phase. Here we show that RhoBTB3, a Golgi-associated, Rho-related ATPase, regulates the S/G2 transition of the cell cycle by targeting Cyclin E for ubiquitylation. Depletion of RhoBTB3 arrested cells in S phase, triggered Golgi fragmentation, and elevated Cyclin E levels. On the Golgi, RhoBTB3 bound Cyclin E as part of a Cullin3 (CUL3)-dependent RING–E3 ubiquitin ligase complex comprised of RhoBTB3, CUL3, and RBX1. Golgi association of this complex was required for its ability to catalyze Cyclin E ubiquitylation and allow normal cell cycle progression. These experiments reveal a novel role for a Ras superfamily member in catalyzing Cyclin E turnover during S phase, as well as an unexpected, essential role for the Golgi as a ubiquitylation platform for cell cycle control.

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