scholarly journals Differential Subcellular Distribution and Translocation of Seven 14-3-3 Isoforms in Response to EGF and During the Cell Cycle

2020 ◽  
Vol 21 (1) ◽  
pp. 318 ◽  
Author(s):  
Abdalla Abdrabou ◽  
Daniel Brandwein ◽  
Zhixiang Wang
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Mammalian Cells ◽  
Subcellular Fractionation ◽  
Cellular Functions ◽  
Broad Distribution ◽  
Hek 293 ◽  
Multiple Functions ◽  
Mcf 7

Multiple isoforms of 14-3-3 proteins exist in different organisms. In mammalian cells, 14-3-3 protein has seven isoforms (α/β, ε, η, γ, σ, θ/τ, and δ/ζ), with α and δ representing the phosphorylated versions of β and ζ, respectively. While the existence of multiple isoforms may represent one more level of regulation in 14-3-3 signaling, our knowledge regarding the isoform-specific functions of 14-3-3 proteins is very limited. Determination of the subcellular localization of the different 14-3-3 isoforms could give us important clues of their specific functions. In this study, by using indirect immunofluorescence, subcellular fractionation, and immunoblotting, we studied the subcellular localization of the total 14-3-3 protein and each of the seven 14-3-3 isoforms; their redistribution throughout the cell cycle; and their translocation in response to EGF in Cos-7 cells. We showed that 14-3-3 proteins are broadly distributed throughout the cell and associated with many subcellular structures/organelles, including the plasma membrane (PM), mitochondria, ER, nucleus, microtubules, and actin fibers. This broad distribution underlines the multiple functions identified for 14-3-3 proteins. The different isoforms of 14-3-3 proteins have distinctive subcellular localizations, which suggest their distinctive cellular functions. Most notably, 14-3-3ƞ is almost exclusively localized to the mitochondria, 14-3-3γ is only localized to the nucleus, and 14-3-3σ strongly and specifically associated with the centrosome during mitosis. We also examined the subcellular localization of the seven 14-3-3 isoforms in other cells, including HEK-293, MDA-MB-231, and MCF-7 cells, which largely confirmed our findings with Cos-7 cells.

scholarly journals E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

1997 ◽  
Vol 17 (12) ◽  
pp. 7268-7282 ◽  
Author(s):  
R Verona ◽  
K Moberg ◽  
S Estes ◽  
M Starz ◽  
J P Vernon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Critical Role ◽  
Biological Properties ◽  
Dependent Manner ◽  
Restriction Point ◽  
Cycle Dependent ◽  
Almost All

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

scholarly journals Subcellular localization of EEN/endophilin A2, a fusion partner gene in leukaemia

2004 ◽  
Vol 383 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Ngai CHEUNG ◽  
Chi Wai SO ◽  
Judy W. P. YAM ◽  
C. K. C. SO ◽  
Randy Y. C. POON ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Fusion Partner ◽  
Nucleocytoplasmic Shuttling ◽  
Cellular Functions ◽  
Protein Levels ◽  
Mixed Lineage Leukaemia ◽  
Partner Gene ◽  
M Phase ◽  
Endocytic Vesicles

EEN (extra eleven nineteen), also known as EA2 (endophilin A2), a fusion partner of the MLL (mixed-lineage leukaemia) gene in human acute leukaemia, is a member of the endophilin A family, involved in the formation of endocytic vesicles. We present evidence to show that EEN/EA2 is localized predominantly in nuclei of various cell lines of haemopoietic, fibroblast and epithelial origin, in contrast with its reported cytoplasmic localization in neurons and osteoclasts, and that EEN/EA2 exhibits nucleocytoplasmic shuttling. During the cell cycle, EEN/EA2 shows dynamic localization: it is perichromosomal in prometaphase, co-localizes with the bipolar spindle in metaphase and anaphase and redistributes to the midzone and midbody in telophase. This pattern of distribution coincides with changes in protein levels of EEN/EA2, with the highest levels being observed in G2/M-phase. Our results suggest that distinct subcellular localization of the endophilin A family members probably underpins their diverse cellular functions and indicates a role for EEN/EA2 in the cell cycle.

scholarly journals Nuclear localization of the ubiquitin-activating enzyme, E1, is cell-cycle-dependent

1994 ◽  
Vol 300 (3) ◽  
pp. 701-708 ◽  
Author(s):  
S J Grenfell ◽  
J S Trausch-Azar ◽  
P M Handley-Gearhart ◽  
A Ciechanover ◽  
A L Schwartz
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Initial Step ◽  
Immunoblot Analysis ◽  
Subcellular Fractionation ◽  
Cell Extract ◽  
Cell Fractionation ◽  
Differential Distribution ◽  
Cycle Dependent ◽  
Substrate Proteins

The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1. Previously we have described the subcellular localization of this enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.

The Akt isoforms are present at distinct subcellular locations

2010 ◽  
Vol 298 (3) ◽  
pp. C580-C591 ◽  
Author(s):  
Stacey A. Santi ◽  
Hoyun Lee
Keyword(s):  
Energy Metabolism ◽  
Subcellular Localization ◽  
Subcellular Location ◽  
T Antigen ◽  
Sv40 T Antigen ◽  
Cellular Functions ◽  
Hek 293 ◽  
Major Function ◽  
Akt Isoforms ◽  
Interfering Rna

Akt is involved in the regulation of diverse cellular functions such as cell proliferation, energy metabolism, and apoptosis. Although three Akt isoforms are known, the function of each isoform is poorly understood. To gain a better understanding of each Akt isoform, we examined the subcellular localization and expression of each isoform in transformed and nontransformed cells. Akt1 was localized in the cytoplasm, which is in agreement with the currently accepted model that cytoplasmic Akt is translocated and activated at the inner leaflet of the plasma membrane. Interestingly, HEK-293 and HEK-293T cells contained Akt1 in the nucleus and cytoplasm, respectively, suggesting that SV40 T-antigen plays a crucial role in the cytoplasmic localization and activation of Akt1 in HEK-293T. Akt2 was colocalized with the mitochondria, while Akt3 was localized in both the nucleus and nuclear membrane. The subcellular localization of the Akt isoforms was not substantially altered in response to ionizing radiation or EGF. Furthermore, the ablation of one Akt isoform by small interfering RNA (siRNA) did not alter the subcellular location of the remaining isoforms, suggesting that the major function of one isoform is not compensated for by other isoforms. Together, our data support the notion that Akt2 and Akt3 are regulated at the mitochondrial and nuclear membranes, respectively. The mitochondrial localization of Akt2 raises the possibility that this isoform may be involved in both glucose-based energy metabolism and suppression of apoptosis, two Akt functions previously identified with anti-pan-Akt antibodies.

scholarly journals Epanorin, a lichen secondary metabolite, inhibits proliferation of MCF-7 breast cancer cells

2019 ◽  
Vol 52 (1) ◽  
Author(s):  
Juan Palacios-Moreno ◽  
Cecilia Rubio ◽  
Wanda Quilhot ◽  
M. Fernanda Cavieres ◽  
Eduardo de la Peña ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Cell Line ◽  
Dna Fragmentation ◽  
Secondary Metabolite ◽  
Breast Cancer Cells ◽  
Mutagenic Activity ◽  
Hek 293 ◽  
Mcf 7

Abstract Background Epanorin (EP) is a secondary metabolite of the Acarospora lichenic species. EP has been found in lichenic extracts with antimicrobial activity, and UV-absorption properties have been described for closely related molecules; however, its antiproliferative activity in cancer cells has not yet been explored. It has been hypothesized that EP inhibits cancer cell growth. MCF-7 breast cancer cells, normal fibroblasts, and the non-transformed HEK-293 cell line were exposed to increasing concentrations of EP, and proliferation was assessed by the sulforhodamine-B assay. Results MCF-7 cells exposed to EP were examined for cell cycle progression using flow cytometry, and DNA fragmentation was examined using the TUNEL assay. In addition, EP’s mutagenic activity was assessed using the Salmonella typhimurium reverse mutation assay. The data showed that EP inhibits proliferation of MCF-7 cells, and it induces cell cycle arrest in G0/G1 through a DNA fragmentation-independent mechanism. Furthermore, EP’s lack of overt cytotoxicity in the normal cell line HEK-293 and human fibroblasts in cell culture is supported by the absence of mutagenic activity of EP. Conclusion EP emerges as a suitable molecule for further studies as a potential antineoplastic agent.

A casein kinase I isoform is required for proper cell cycle progression in the fertilized mouse oocyte

1997 ◽  
Vol 110 (24) ◽  
pp. 3083-3090 ◽  
Author(s):  
S.D. Gross ◽  
C. Simerly ◽  
G. Schatten ◽  
R.A. Anderson
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
T Antigen ◽  
Casein Kinase ◽  
Mouse Oocyte ◽  
Casein Kinase I ◽  
Sv40 Large T Antigen ◽  
Cycle Progression

Casein kinase I is a family of serine/threonine protein kinases common to all eukaryotes. In yeast, casein kinase I homologues have been linked to the regulation of growth, DNA repair and cell division. In addition, their subcellular localization to membraneous structures and the nucleus is essential for function. In higher eukaryotes, there exist seven genetically distinct isoforms: (alpha), ss, (gamma)1, (gamma)2, (gamma)3, (delta) and (epsilon). Casein kinase I(alpha) exhibits a cell cycle-dependent subcellular localization including an association with cytosolic vesicular structures and the nucleus during interphase, and the spindle during mitosis. casein kinase I has also been shown to modulate critical regulators of growth and DNA synthesis/repair in mammalian cells such as SV40 large T antigen and p53. These results suggest that casein kinase I may be involved in processes similar to those ascribed to the yeast casein kinase I homologues. To define a role for casein kinase I(alpha) in cell cycle regulation, the mouse oocyte was utilized because of its well-defined cell cycle and ease of micromanipulation. Immunofluorescence studies from meiosis I of maturation to the first zygotic cleavage demonstrated that the kinase was associated with structures similar to those previously reported. Microinjection of casein kinase I(alpha) antibodies at metaphase II-arrest and G2 phase, had no effect on the completion of second meiosis or first division. However, microinjection of these antibodies during the early pronucleate phase prior to S-phase onset blocked uptake of the kinase into pronuclei and interfered with proper and timely cell cycle progression to first cleavage. These results suggest that the kinase regulates the progression from interphase to mitosis during the first cell cycle.

Myricanol-9-acetate, a novel naturally occurring derivative of myricanol, induces ROS-dependent mitochondrial-mediated Apoptosis in MCF-7 cancer cells

2021 ◽  
Vol 21 ◽  
Author(s):  
Gazanfar Ahmad ◽  
Sameer Ahmad Mir ◽  
Loveleena Kour Anand ◽  
Faheem Hyder Pottoo ◽  
Neerupma Dhiman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Ros Generation ◽  
Cycle Arrest ◽  
Naturally Occurring ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Mcf 7

Background: Low therapeutic efficacy and drug-induced systemic toxicity of currently used anti-cancerous chemotherapeutic agents are major compelling factors for finding out clinically efficient molecules with high efficiency and less toxicity. Objective: The current research work was undertaken to evaluate the anticancer potential of Myricanol-9-acetate (MA), a novel naturally occurring derivative of myricanol. Methods: MCF-7, MiaPaCa-2, and HCT 116 were used for cytotoxicity determination of the MA and ML (Myricanol) by MTT assay. The mechanistic study involved the determination of cell cycle arrest, ΔΨm loss, ROS generation, western blot assay, and flow cytometry by reported methods on MCF-7 cells. Results: MA exhibited anticancer activity against all three cell lines; however, the molecule was found most active against the MCF-7 cell line. We observed IC50 20μM with MA treatment as compared to the IC50 of 42 μM for myricanol treatment. Detailed mechanistic studies revealed that MA induced apoptosis on MCF-7 cell line through ROS generation; a dose-dependent drop in mitochondrial membrane potential was found to be associated with cell cycle arrest at G0/G1 phase. Our results further demonstrated down-regulation of Bcl2 and activation of the caspase cascade as the events involved in the MA-induced apoptosis. Flow cytometry results indicated an increase in early and late apoptotic population in a dose-dependent manner with an apoptotic population of about 20% at 30 μM of MA, thus supporting our results. Conclusion: Present findings thus suggest that MA might serve as a promising novel drug candidate having high scope for further evaluation in preclinical and clinical studies.

scholarly journals Activation of JNK and p38 in MCF-7 Cells and the In Vitro Anticancer Activity of Alnus hirsuta Extract

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1073 ◽  
Author(s):  
Mina Ryu ◽  
Chung Ki Sung ◽  
Young Jun Im ◽  
ChangJu Chun
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Mammalian Cells ◽  
Biological Activities ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ros Generation ◽  
Cycle Arrest ◽  
Alnus Hirsuta ◽  
Mcf 7

JNK and p38 are important mitogen-activated protein kinases (MAPKs) that respond to stress stimuli. The stress-activated MAPKs associated with apoptotic cell death play vital roles in mammalian cells. Alnus hirsuta, which contains abundant diarylheptanoids derivatives, is a valuable medicinal plant. The CHCl3 extract (AHC) containing platyphyllenone (1) and platyphyllone (3) as main compounds showed in vitro anticancer effects. We report the biological activities of A. hirsuta extract associated with the regulation of apoptosis and JNK and p38 in MCF-7 breast cancer cells. Levels of phospho-JNK and phospho-p38 by AHC treatment were evaluated by enzyme-linked immunosorbent assay (ELISA). ROS production, apoptotic effect, and DNA contents of the cells were measured by flow cytometry. The two diarylheptanoids 1 and 3 and the AHC extract exhibited cytotoxic effects on MCF-7 cells in MTT assay, with IC50 values of 18.1, 46.9, 260.0 μg/mL, respectively. AHC induced ROS generation and elevated the endogenous levels of phospho-JNK and phospho-p38. AHC resulted in apoptosis and cell cycle arrest. We suggest that the antitumor effect of A. hirsuta extract is achieved by apoptosis promotion and cell cycle arrest mediated by the activation of JNK and p38 signaling pathway via ROS generation.

The Combination of 17-Estradiol and Bisphenol A Weaken their Respective Action

2010 ◽  
Vol 113-116 ◽  
pp. 2220-2223
Author(s):  
Lang Lang ◽  
Gaung Ming Zhang ◽  
Yu Li
Keyword(s):  
Cell Cycle ◽  
Bisphenol A ◽  
Cancer Cell ◽  
Endocrine Disrupting Chemicals ◽  
Estrogen Receptor Α ◽  
S Phase ◽  
Cancer Cell Growth ◽  
Endocrine Disrupting ◽  
Mcf 7

17-Estradiol (E2) and Bisphenol A (BPA) are important endocrine disrupting chemicals (EDCs). In this work, cell proliferation assay using cell cycle analysis method and determination of estrogen receptor α protein was used to investigated the mechanism and effects respectively and in mixture of the two EDCs in stimulating the growth of human breast cancer MCF-7 cell When the concentration of E2 and BPA were 10-12mol/L and 10-8mol/L respectively, E2 and BPA could reduce the express of ERα protein by activated ERα pathway, accelerated the cell cycle, decreased the percentages of cells in G0/1 phase, increased the percentages of cells in S phase at 48 h, and thus promoted the cancer cell growth. However, the combination effect of these two compounds did not induce the growth of cancer cell and the mixture reduced the ratio in S phase compared with E2 or BPA alone, which lead to weakening of the proliferation ability. There was a significant interaction between BPA and E2 and such a weakening effect had not been reported before.

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