Cancer Research Meeting Calendar Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwak, I. H.
Right arrow Articles by Lim, I. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwak, I. H.
Right arrow Articles by Lim, I. K.
[Cancer Research 64, 572-580, January 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Nuclear Accumulation of Globular Actin as a Cellular Senescence Marker

In Hae Kwak, Hong Seok Kim, Ok Ran Choi, Min Sook Ryu and In Kyoung Lim

Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Korea


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated the nuclear actin accumulation as a new marker of cellular senescence, using human diploid fibroblast (HDF), chondrocyte primary cultures, Mv1Lu epithelial cells, and Huh7 cancer cells. Nuclear accumulation of globular actin (G-actin) and dephosphorylated cofilin was highly significant in the senescent HDF cells, accompanied with inhibition of LIM kinase (LIMK) -1 activity. When nuclear export of the actin was induced by 12-O-tetradecanoylphorbol-13-acetate, DNA synthesis of the senescent cells increased significantly, accompanied with changes of morphologic and biochemical profiles, such as increased RB protein phosphorylation and decreased expressions of p21WAF1, cytoplasmic p-extracellular signal-regulated kinase 1/2, and caveolins 1 and 2. Significance of these findings was strengthened additionally by the fact that nuclear actin export of young HDF cells was inhibited by the treatment with leptomycin B and mutant cofilin transfection, whose LIMK-1 phosphorylation site was lost, and the old cell phenotypes were duplicated with nuclear actin accumulation, suggesting that nuclear actin accumulation was accompanied with G1 arrest during cellular senescence. The aforementioned changes were observed not only in the replicative senescence but also in the senescence induced by treatment of HDF cells, Mv1Lu, primary culture of human chondrocytes, or Huh7 cells with H-ras virus infection, hydroxyurea, deferoxamine, or H2O2. Nuclear actin accumulation was much more sensitive and an earlier event than the well-known, senescence-associated ß-galactosidase activity.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proliferation of normal diploid cells is arrested irreversibly after a certain number of divisions, called replicative senescence, which is a paragon of the general process of cellular senescence (1, 2, 3) . Once senescent, cells continue to function metabolically but will not respond to mitogens. Decreased growth rate, limited cell division, flat and large cell shapes, and tight binding of the cells to a culture dish are well-known characteristics of cells entered into senescence (4 , 5) . Replicative senescence also is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is much evidence to indicate that escape from senescence is important for malignant transformation. Citing a few, cellular senescence is thought to be tumor suppressive. Immortality increases greatly the susceptibility to malignant transformation in culture and in vivo (6 , 7) . Many tumors contain immortal cells or cells with an extended replicative life span (8 , 9) . Some oncogenes primarily immortalize or extend the life span of cells (10 , 11) . The tumor suppressors p53 and pRb, which suffer commonly from loss-of-functional mutations in human cancers, are critical for regulation of cellular senescence (12 , 13) . Sharpless and DePinho (14) have reviewed recently the complicated correlation between tumorigenesis and p53-regulated organismal aging. Most human tumors overcome the lack of telomerase activity, which is silent in senescent cells and most somatic cells, except germ cells, stem cells, and activated leukocytes (15) . Analysis of the various aspects and types of senescence has been informative about numerous in vivo processes, particularly regarding carcinogenesis (16, 17, 18) .

It has been known that premature senescence was accelerated in response to constitutive mitogen-activated protein/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase mitogenic signaling, suggesting that the various changes of senescent cells might be related closely to actin cytoskeleton organization, focal adhesions between the cells and cells to extracellular matrix, and the related signal transductions (19) . Rho family proteins are regulators of signaling pathways that regulate the organization of the actin cytoskeleton and are members of the Ras superfamily of small GTPases (20) , and premature senescence can be induced in primary cells by oncogenic ras expression (21) . Furthermore, cells need a tightly regulated and highly coordinated actin polymerization and depolymerization system for cell division (22 , 23) , thereby providing cell motility and adhesion and typical cell morphology. Because cell growth rate is decreased dramatically and arrested irreversibly at the G1 phase with double nuclei in the senescent cells, it is highly probable that actin plays an important role in induction of growth arrest during cellular senescence. Actin is involved in the nuclear division not only in amoeba (24) but also in eukaryotic cells (25) , forming a constriction ring at the middle of dividing nucleus.

Globular actin (G-actin) can be polymerized to fibrous actin (F-actin) by profilin (26 , 27) , and F-actin is depolymerized rapidly by dephosphorylated cofilin, an actin-severing protein, in various mammalian cell lines and in yeast and Drosophila (28, 29, 30) . Phosphorylation of serine-3 residue by LIM kinase (LIMK) prevents cofilin from severing actin filaments (31) . In addition to cofilin, CapG, an actin-capping protein, also plays an actin-modifying role after release from phosphatidylinositol-4,5-bisphosphate (32 , 33) . Actin-binding proteins can translocate to the nucleus in a phosphorylation-dependent manner (34, 35, 36, 37, 38) . Recent study of serum response factor-dependent gene transcription revealed that a serine-3 mutant (S3A) of cofilin was able to inhibit serum response factor reporter activation by LIMK and to block LIMK-induced formation of actin aggregates in the cytosol, indicating that the role of cofilin in signal transduction to serum response factor response elements is solely to increase G-actin levels in cells (39) . Although it is not clear whether the role of cofilin or actin is cytoplasmic entirely, evidence argues strongly that actin plays a role in the nucleus.

We reported previously that during the replicative senescence and H-ras mutant-induced senescence of human diploid fibroblast (HDF) cells, a large fraction of actin molecules were accumulated in the nucleus (40) and that significantly high levels of reactive oxygen species generated in the senescent HDF cells inactivated protein phosphatases 1 and 2A and mitogen-activated protein kinase phosphatase 3, accompanied by sequestration of p-extracellular signal-regulated kinase (p-Erk) in cytoplasm (41) . These observations have been supported strongly by two recent reports that actin polymerization was essential for nuclear translocation of p-Erk, which occurred in a caveolae compartment of rat cardiomyocytes through the stretch-induced RhoA and Rac1 activation (42) , and that senescent phenotype was reversed by reduction of caveolin expression (43) .

In this study, we attempted to elucidate the biological significance of nuclear accumulation of actin molecules in the replicative senescence and induced senescence of various cell types. We also aimed to test the applicability of nuclear actin accumulation as a valuable and robust marker for cellular senescence and the causal link between senescence and nuclear G-actin accumulation in various normal cells and H2O2-treated cancer cells. We observed that G-actin accumulation in the nucleus and perinucleus of the senescent cells was accompanied by nuclear accumulation of dephosphorylated cofilin. Immunoprecipitation of LIMK-1 and its in vitro cofilin phosphorylation activity were decreased markedly in the senescent cells compared with that in the young cells. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment could induce nuclear export of actin, cofilin phosphorylation concomitant with increased LIMK activity, and induction of DNA synthesis only in the old cells. Moreover, the aforementioned phenotypes almost could be reproduced in the induced senescence of various cell types, especially H2O2-induced senescence. Because nuclear actin accumulation was a more sensitive and earlier event than the appearance of senescence-associated ß-galactosidase (SA-ß-gal) activity, we suggest strongly nuclear actin accumulation as a new marker of cellular senescence.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures and Induction of Cellular Senescence.
HDF primary culture was prepared in our laboratory from the foreskin of a 4-year-old boy and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum at 37°C in 5% CO2 incubator (40) . To induce premature senescence, different methods were applied to various cells: H-ras mutant virus infection (V12C40, V12G37, and V12S35) to HDF cells, treatments of HDF cells with H2O2 (0.2 x 10-3 M) every 24 h, or Mv1Lu mink lung epithelial cells with hydroxyurea (1 x 10-3 M). The cells were maintained in DMEM (low glucose) with 10% fetal bovine serum. Young, mid-old, and old cells used in the present experiments were based on their doubling time of ~24 h, 7–10 days, and >2 weeks, respectively. For replicative senescence of human chondrocytes, female knee cartilage was obtained from the operation room at Ajou University Hospital with informed consent, and its primary culture was split 1:5 when the cells became 75% confluent. Number of the passage was counted from 0 to 6. Cancer cell senescence was induced by daily treatment of Huh7 human hepatoma cells with H2O2 (0.2 x 10-3 M) or once with 0.5 x 10-3 M deferoxamine (44 , 45) .

Preparation of Nuclear Matrix and Immunocytochemistry.
Nuclear matrix was prepared using the method described by Clubb and Locke (46) . Cells were cultured on glass coverslips, and soluble proteins then were extracted by incubation for 1.5 min with cytoskeleton (CSK) buffer [0.01 M HEPES (pH 6.8), 0.1 M NaCl, 0.3 M sucrose, 3 x 10-3 M MgCl2, 1 x 10-3 M EGTA, and 0.5 x 10-3 M phenylmethylsulfonyl fluoride] containing 0.5% Triton X-100. To remove chromatin, the aforementioned Triton X-100-treated cell residues were treated additionally with 100 units/ml DNase I in CSK buffer for 30 min and rinsed with 0.25 M (NH4)2SO4 for 5 min. Coverslip was immersed in fresh CSK buffer, and an equal volume of 4 M NaCl was added gradually to remove the residual protein. The preparation was fixed subsequently in 4% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature, permeabilized in 0.5% Triton X-100 in PBS for 5 min, and incubated with 3% BSA. The aforementioned preparation was incubated with primary antibodies against antiactin (Sigma Chemical Co., St. Louis, MO) and anticofilin (Cytoskeleton, Denver, CO; 1:200) overnight at 4°C in a humidified chamber, and then with secondary antibody conjugated with FITC, Cy3 (1:200), or Texas red (Jackson ImmunoResearch Laboratories, West Grove, PA) and Hoechst 33258 (0.1 µg/ml) for 1 h at 4°C. For assessing F-actin dynamics, cultured cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature and then permeabilized in PBS containing 0.1% Triton X-100 and 3% BSA. F-actin was stained with 5 units of rhodamine phalloidin in methanol/PBS (Molecular Probes, Eugene, OR) for 1 h at room temperature and then observed after mounting the coverslip.

Cell Synchronization, Fluorescence-Activated Cell Sorter, and IEF Analyses.
To harvest S-phase-specific cells, young HDF cells were treated with 2 x 10-3 M thymidine for 24 h, followed by the second treatment for 16 h after an 8-h interval. The cells were harvested at 4 h and 6 h after incubation in the thymidine-free medium. To harvest mitotic cells, the cells were first synchronized twice with thymidine and then treated with nocodazole (100 ng/ml) for 16 h after 4 h of thymidine release. Mitotic cells were harvested by shake-off at 0 h and then 3 h after removal of nocodazole. To harvest G1-phase cells, young cells were incubated with serum-free medium for 24 h and then stimulated with 10% serum-containing medium for 7, 16, and 20 h. Each cell cycle was confirmed with 2 x 105 cells by fluorescence-activated cell sorter analysis after 70% ethanol fixation. Cofilin phosphorylation was measured by isoelectric focusing (IEF) with 5 x 105 cells. The young and old HDF cells were washed with ice-cold PBS; the nuclei were broken by sonication (Sonic Dismembrator 550; Fisher Scientific, Hampton, NH) in IEF lysis buffer [0.02 M Tris-HCl (pH 7.5), 2 x 10-3 M MgCl2, 0.07 M KCl, 1 x 10-3 M Na3VO4, 0.3 x 10-6 M okadaic acid, 2 µg/ml leupeptin, 1 x 10-3 M phenylmethylsulfonyl fluoride, and 0.5% Triton X-100]; and the suspension was centrifuged at 4400 x g for 10 min. IEF of the supernatant was performed with IEF gel (pH 3–10) according to the manufacturer’s instruction. Immunoblot analyses were performed with anticofilin antibody. IEF criterion gel (pH 3–10) and cathode buffer were obtained from Bio-Rad (Hercules, CA).

[3H]-thymidine Incorporation Assay.
Old HDF cells were plated, and the monolayer was stabilized for 48 h until 70% confluent, refed with a fresh medium, and incubated for another 24 h. HDF young cells also were plated on a six-well plate and incubated for 24 h until 70% confluent. TPA (50 ng/ml), epidermal growth factor (EGF; 10 ng/ml), TPA inhibitor (Go6976; 0.4 x 10-6 M, 0.8 x 10-6 M), or DMSO as a vehicle was added individually to the culture medium, and the cells were harvested at 4, 8, 20, and 32 h of incubation. Before harvest, the cells were treated with 2 µCi/ml [3H]thymidine for 4 h; the harvested cells were lysed with 200 µl of 0.04 M Tris-HCl buffer (pH 8.0) containing 0.12 M NaCl and 0.5% NP-40 by vortex mixing at 4°C for 20 min; and the lysate was rinsed thoroughly with PBS. Radioactivity of the lysate (100 µl) was counted by a liquid scintillation counter (Microbeta Counter 1450; Perkin Elmer, Wellesley, MA) using 1 ml scintillation mixture solution. Data presented are mean ± SD of quadruplicate wells from more than three experiments. Multiple linear regression analyses were applied for the data analyses obtained from [3H]thymidine incorporation assay by calculating B value in the equation composed of the affecting factors and using P = 0.05 as cutoff for significance.

LMB Treatment and Mutant Cofilin Transfection to HDF Cells.
To inhibit nuclear export of actin, HDF young cells (1.3 x 105 cell/6 well) were treated with leptomycin B (LMB; 10 nM or 20 nM; Sigma Chemical Co.) for 24 h after 18 h of cell seeding, and the cells were treated additionally with TPA (50 ng/ml) for 32 h and then subjected to cell growth analysis and [3H]-thymidine incorporation. To prove nuclear accumulation of actin caused by dephosphorylated cofilin, S3A cofilin (pKEX2-A3 Flag) was transfected to HDF young cells (1 x 106 cells/100 mm) using 20 µl of lipofectamine (Invitrogen). The control vector, pKEX2-Flag, was prepared by ligating the double-stranded flag oligonucleotides into the XbaI-XhoI site of the pKEX2. Sense and antisense flag oligonucleotides, which were synthesized in our laboratory, were denatured (100 pmol each) at 100°C and annealed slowly at room temperature for 30 min before ligating into pKEX-XR plasmid. After transfection with 4 µg of pKEX2-A3 Flag or the control vector, hygromycin (100 µg/ml) -resistant HDF cells were selected and used for additional experiments.

Preparation of Recombinant Cofilin Protein.
Wild-type cofilin clone (6-His tagged) was cultured in Luria-broth medium with ampicillin (100 µg/ml; Sigma Chemical Co.) at 37°C until absorbance at 600 nm reached 0.6 and incubated additionally with 0.4 x 10-3 M isopropyl ß-D-thiogalactoside (Sigma Chemical Co.) overnight at 28°C. Bacterial pellet was centrifuged at 1500 x g for 15 min at 4°C and washed with extraction/wash buffer [50 x 10-3 M sodium phosphate and 300 x 10-3 M NaCl (pH 7.0)]. Additional procedures were carried out basically according to the manufacturer’s recommendation (BD Biosciences, Palo Alto, CA). Hexa-histidine tagged cofilin protein was purified with TALON resin (50% slurry; BD Biosciences), and cofilin was eluted three times from the resin with 300 µl of elution buffer [50 x 10-3 M sodium phosphate, 300 x 10-3 M NaCl, and 150 x 10-3 M imidazole (pH 7.0)].

Immunoprecipitation of LIMK-1 and Its Activity Assay.
Cells were washed twice with ice-cold PBS and broken by sonication in LIMK assay buffer [50 x 10-3 M HEPES (pH 7.4), 150 x 10-3 M NaCl, 1% NP-40, 5% glycerol, 1 x 10-3 M DTT, 1 x 10-3 M MgCl2, 1 x 10-3 M MnCl2, 10 x 10-3 M NaF, 1 x 10-3 M NaVO4, 1 x 10-3 M phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin]. Whole cell lysates (500 µg) were incubated with anti-LIMK-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 8 h at 4°C. Using protein G-agarose (Invitrogen), LIMK-1 immune complex was harvested at 4°C for 4 h and then washed vigorously three times with 500 µl of kinase buffer. LIMK-1 assay was performed with 5 µg cofilin as a substrate, 50 x 10-6 M ATP, and 5 µCi of [{gamma}-32P]ATP in 30 µl of kinase buffer at 30°C for 45 min. The reaction was terminated by boiling for 5 min, and 32P-labeled cofilin was analyzed by autoradiography in 15% (w/v) SDS-PAGE.

Evaluation of Senescence Marker Expressions.
To monitor degree of cellular senescence, expressions of phosphorylated RB, p21WAF1, p-Erk1/2, and caveolin-1 and caveolin-2 in the old cells were examined by immunoblot analyses before and after TPA treatment. For quantitation, the number of the cells with nuclear actin accumulated was counted under a confocal microscope; however, the cells in mitosis were excluded from the total counting, which included >1000 cells. Assay of SA-ß-gal activity followed basically the published method (47) . Cells were fixed to plates with 3% formaldehyde for 5 min after washing with PBS and then were incubated overnight in freshly prepared staining solution [0.04 M citric acid/sodium phosphate (pH 6.0), 1 mg/ml of X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), 5 x 10-3 M potassium ferrocyanide, 5 x 10-3 M potassium ferricyanide, 0.15 M NaCl, and 2 x 10-3 M MgCl2]. Stain was visible after 12 h of incubation at 37°C. By counting the number of the cells with blue color and the total cells per field (0.5 x 0.5 cm) under an inverted microscope, the percentage of the SA-ß-gal-positive blue-stained cells was calculated. More than 1000 cells were counted from five fields and presented as mean ± SD.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Nuclear Accumulation of G-Actin and Cofilin during HDF Senescence.
To investigate a mechanism of actin accumulation in the nucleus, subcellular localization of actin and cofilin was examined by immunocytochemistry, and markedly increased actin and cofilin expressions were found in the nucleus of the old, but not the young, cells (Fig. 1A)Citation . When treated with rhodamine-conjugated phalloidin for 1 h, F-actin was found spread over the cytoplasm; however, the F-actin was not concentrated in nuclei of young and old cells (Fig. 1B)Citation . To eliminate the possibilities that the phalloidin-binding sites were not accessible to the potential binding site in the nucleus of fixed cell and that long actin filaments in the nucleus might have been covered with a protein that obstructs the phalloidin-binding pocket, HDF cells were treated briefly with Triton X-100 and DNase I. This treatment revealed no actin accumulation in the nucleus (Fig. 1C)Citation , suggesting that the accumulated actin in the nucleus (Fig. 1A)Citation would be G-actin. When the nuclear matrix was extracted, nuclear actin was seen as a matrix scaffold, which is located inside nuclear membrane. Furthermore, IEF revealed dephosphorylated cofilin exclusively in the old HDF (Fig. 2A)Citation , whereas young HDF and NIH3T3 cells contained phosphorylated and dephosphorylated forms of cofilin. To elucidate possible dependency of changes of cofilin activity on the cell division cycle, IEF analysis of cofilin also was performed with HDF cells synchronized (Fig. 2B)Citation by double thymidine block, nocodazole treatment, or serum deprivation and refeeding. When harvested by shake-off after nocodazole treatment, fluorescence-activated cell sorter analysis showed 100% mitotic cells (N0). On analyses of the synchronized cells by fluorescence-activated cell sorter and IEF, completely phosphorylated cofilin was found exclusively in the mitotic cells (Fig. 2C)Citation . These data could explain why the dephosphorylated cofilin was present in the old HDF cells, which were arrested in late G1 phase, potentially dissociating G-actin from F-actin.



View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Nuclear accumulation of globular actin (G-actin) and cofilin during human diploid fibroblast (HDF) senescence. A, young and old cells up to 50% confluent were incubated with antiactin and anticofilin antibodies (1:200) at 4°C in a chamber overnight and treated additionally with FITC or Texas red conjugated secondary antibody and Hoechst 33258 (0.1 µg/ml) for 1 h. Actin and cofilin were accumulated in the nucleus of old cells; confocal microscope (x600 Fluoview; Olympus, Tokyo, Japan). B, distribution of fibrous actin (F-actin) in HDF cells was examined by immunofluorescence microscope (x400 Axiophot; Zeiss, Esslingen, Germany) before chromatin removal. Much more F-actin expression was found in the old cells than in the young cells over the cytoplasm. C, no evidence of F-actin accumulation in the nucleus of HDF cells, except for G-actin in the nuclear matrix. Nuclear matrix was prepared after removal of soluble proteins by 1.5 min of Triton X-100 and DNase I (100 units/ml) treatment. The cells were permeabilized to stain F-actin and G-actin in the nuclear matrix. Rhodamine-phalloidin was applied for 1 h at room temperature, and actin was visualized with FITC-conjugated secondary antibody.

 


View larger version (48K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Presence of dephosphorylated cofilin in the old human diploid fibroblast (HDF) cells, except the G2/M phase cells. A, phosphorylation of cofilin was evaluated by isoelectric focusing (IEF) in the young and old HDF cells and also NIH3T3 fibroblasts (control). Note dephosphorylated cofilin exclusively in the old HDF but the mixture in the young HDF and NIH3T3 cells. B, to investigate cell cycle-dependent cofilin phosphorylation, young HDF cells were synchronized with double thymidine block, nocodazole treatment, or serum starvation, and the cells then were harvested 4 and 6 h after thymidine release (Th4 and Th6), 0 and 3 h after nocodazole release (N0 and N3), or 7, 16, and 20 h after serum addition (Sr7, Sr16, and Sr20). The phase of the harvested cells was determined by fluorescence-activated cell sorter analysis. C, phosphorylation status of cofilin was determined by IEF. Note p-cofilin exclusively in mitotic cells (Lane 3, N0); however, phosphorylated and dephosphorylated forms of cofilin appeared rapidly within 3 h after nocodazole release (Lane 4, N3).

 
Nuclear Export of G-Actin Significantly Increased DNA Synthesis of Senescent Cells.
To investigate the consequence of nuclear export of G-actin, young and old HDF cells were treated with either EGF or TPA. As shown in Fig. 3Citation , TPA induced export of actin from the nucleus to cytoplasm 8 h after the treatment, followed by morphologic changes of large and flat old cells to small and cylindrical young cell-like ones in 20 h. However, EGF failed to stimulate actin export from nuclei and to change the old cell morphology. To elucidate the effect of TPA-induced nuclear export of actin on G1 arrest of senescent cells, [3H]-thymidine incorporation assay was performed, and statistically significant induction of DNA synthesis was found only in the old cells in 20 h (Fig. 4B)Citation as compared with no response in the young cells (Fig. 4A)Citation . When measured by IEF, TPA also induced cofilin phosphorylation (Fig. 4C)Citation . Furthermore, when added to old HDF cells, the protein kinase C (PKC) inhibitor, Go6976, inhibited significantly TPA-induced [3H]-thymidine incorporation (Fig. 4D)Citation , indicating an important role of PKC in the nuclear export of G-actin and induction of DNA synthesis only in the senescent cells. To investigate whether the nuclear actin accumulation was responsible for the decreased DNA synthesis in the senescent cells, young HDF cells were treated with LMB with or without TPA. LMB induced clearly actin accumulation in the nucleus (Fig. 5A)Citation and large and flat old cell-like phenotypes (Fig. 5B)Citation ; however, subsequent treatment of the cells with TPA reversed old cell morphology to the young cell-like phenotypes (Fig. 5C)Citation . Moreover, LMB inhibited significantly cell proliferation (Fig. 5D)Citation . When treated subsequently with TPA, however, significantly increased [3H]-thymidine incorporation was found as compared with that in the LMB-treated cells (Fig. 5E)Citation . These data indicate that failure of nuclear export of actin duplicates senescence phenotypes and the nuclear export of G-actin, accompanied by re-entry of G1-arrested senescent cells into the cell division cycle.



View larger version (51K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Immunofluorescence findings showing the export of nuclear actin by 12-O-tetradecanoylphorbol-13-acetate (TPA) but not by epidermal growth factor (EGF). Old human diploid fibroblast cells plated at ~40% confluence were treated with TPA, EGF, or DMSO as a vehicle. The cells were harvested at 4, 8, and 20 h and then subjected to actin immunocytochemistry. Note the young cell-like morphology and nuclear export of actin in 20 h only after TPA treatment (x400). White arrows indicate the periphery of each cell to reveal the morphologic changes after TPA treatment.

 


View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. 12-O-tetradecanoylphorbol-13-acetate (TPA) induced DNA synthesis only in the old, but not in the young, human diploid fibroblast cells. A, young cells were treated with epidermal growth factor (EGF), TPA, or DMSO when the cell density reached 70% and were incubated with [3H]-thymidine (2 µCi/ml) for 4 h before harvest at the indicated times. B, mid-old (doubling time, 10 days) cells with 70% cell density were stabilized for 48 h before being subjected to [3H]-thymidine incorporation assay. Note the significantly increased DNA synthesis after 20 h of TPA treatment (*P = 0.024 versus TPA 8 h; #P < 0.01 versus all other samples). C, to evaluate whether cofilin phosphorylation occurred after TPA treatment, the mid-old cells were subjected to isoelectric focusing, and cofilin phosphorylation induced by TPA treatment was determined. D, to evaluate the role of protein kinase C (PKC), Go6976 (0.4 and 0.8 µM) was added to mid-old cells, which were stabilized for 48 h, and the concentration-dependent inhibition of DNA synthesis was measured by the method described earlier. *P < 0.001 versus all other samples; **P < 0.005 versus all other samples.

 


View larger version (77K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Leptomycin B (LMB)-induced nuclear actin accumulation in young human diploid fibroblast (HDF) cells could duplicate old cell phenotypes morphologically and biochemically; however, subsequent 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment reversed to young cell-like phenotypes. Young HDF cells (1.3 x 105 cells/6 well) were seeded for 18 h; LMB (10 nM, 20 nM) then was added for 24 h and treated additionally with either TPA or DMSO for 32 h. A, confocal finding (x900) of the LMB-induced nuclear accumulation of actin in 24 h. B, the cell appeared flat and large under inverted microscope. C, however, TPA reversed LMB-induced morphologic change. D, the viable cell numbers were counted after trypan blue stain. E, the rate of DNA synthesis was measured by [3H]-thymidine incorporation assay. LMB treatment inhibited significantly the growth rate (*P < 0.01 versus control; #P = 0.024 versus 10 nM), whereas TPA increased significantly DNA synthesis of the LMB-induced slow-growing cells (*P = 0.001 versus control).

 
Biochemical Evidence of Reversal of Cellular Senescence.
In addition to [3H]-thymidine incorporation, TPA-induced actin export also was accompanied by reversed expression of several other senescence markers, including RB phosphorylation (Fig. 6B)Citation , inhibition of p21WAF1 and p-Erk1/2 expressions (Fig. 6C)Citation , and reduced caveolin-1 and caveolin-2 expressions in 20 h of TPA treatment (Fig. 6D)Citation . RB protein in the senescent HDF was mostly dephosphorylated as compared with that in the young cells (Fig. 6A)Citation . These results are in accordance with previous findings that phosphorylated and under-phosphorylated forms of RB proteins are found in young cells, whereas only under-phosphorylated RB protein is found in senescent cells (48) and in the long-term confluent cultures (49) . These findings indicate strongly that TPA induced export of nuclear actin in old cells and changed not only the old morphologic phenotypes but also biochemical markers.



View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Biochemical evidence for reversal of cellular senescence. Young and old human diploid fibroblast (HDF) cells at exponential phase were harvested, and the known cell cycle factors regulating at G1-S phase were investigated by immunoblot analyses. A, RB protein was phosphorylated in the young HDF cells as opposed to dephosphorylated in the old HDF cells. When the old cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), induction of RB protein phosphorylation (B), decreased expression of p21WAF1 and p-extracellular signal-regulated kinase (Erk) 1/2 (C), and reduced expression of caveolin-1 and caveolin-2 (D) occurred in 20 h.

 
Regulation of LIMK-1 Activity in the Old and H2O2-treated Young HDF Cells.
To investigate whether the dephosphorylated cofilin is responsible for nuclear actin accumulation in the senescent cells, S3A cofilin-transfected HDF cells were used for cofilin and actin immunocytochemistry. S3A cofilin transfection revealed nuclear accumulation of cofilin (Fig. 7A)Citation and actin (Fig. 7B)Citation compared with that in the vector-transfected cells. The regulation of the accompanying LIMK-1 activity during cellular senescence then was investigated by in vitro LIMK-1 assay with recombinant cofilin and LIMK-1 immunoprecipitate. As shown in Fig. 8ACitation , the activity was lower significantly in the old cells than in the young cells; however, TPA treatment could recover the LIMK-1 activity up to the level of young cells. Interestingly, by treating young cells with H2O2 for 7 days, LIMK activity also was inhibited, and the inhibition was eliminated by TPA treatment, reaching the control level (Fig. 8B)Citation . Phosphorylation of pRb and induction of p21WAF1 expression were changed concomitantly in the young cells by repeated treatment with H2O2 (Fig. 8C)Citation .



View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Nuclear accumulation of actin occurred in the serine-3 mutant (S3A) cofilin-transfected cells but not in the vector-transfected cells. Young human diploid fibroblast cells were (1 x 106 cells/100 mm) transfected with 4 µg of S3A cofilin or vector plasmids and selected with hygromycin (100 µg/ml) treatment. Anticofilin (A) and antiactin (B) antibodies were used for immunocytochemistry, and Hoechst 33258 was applied for nuclear staining in the same field. Note actin accumulation in the nucleus of the S3A-transfected cells, whereas there is no definite change of actin accumulation in the vector-transfected cells. Confocal microscopic finding (x900).

 


View larger version (33K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. In vitro LIM kinase (LIMK) activity assay in the young and old human diploid fibroblast (HDF) cells. A, LIMK activity was decreased significantly in old (O) HDF cells compared with that in the young (Y) cells. However, treatment of the old cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 50 ng/ml) for 20 h (O+T) recovered LIMK activity up to the young cell level. Upper, autoradiography of the 32p-labeled cofilin used as a substrate for LIMK assay. Lower, immunoblot analyses against {alpha}-tubulin as loading control. B, changes of LIMK activity in the induced senescence by H2O2 treatment. HDF cells were treated with 0.2 x 10-3 M H2O2 for 7 days (H2O2), and the cells then were treated additionally with TPA for 20 h (H+T). Control cells indicate DMSO-treated cells. Note the reduced LIMK activity by H2O2 treatments; however, TPA can recover the activity up to the young cell level. C, immunoblot analyses reveal pRb and p21WAF1 changes in the young (Y) HDF cells and H2O2-induced premature senescent cells (Y+H2O2). Note the decrease of hyperphosphorylated Rb and the increase of p21WAF1 proteins in the H2O2-treated cells.

 
Nuclear Actin Accumulation Is Highly Sensitive and an Earlier Event during Cellular Senescence.
To evaluate the possibility of nuclear actin accumulation as a new marker of cellular senescence, various senescence models were examined, including premature senescence of HDF primary culture by H-ras double mutant virus infection (Fig. 9A)Citation , induced senescence of HDF cells by treatment with H2O2 (0.2 x 10-3 M), induced senescence of Mv1Lu epithelial cells by treatment with hydroxyurea (1 x 10-3 M), and replicative senescence of human chondrocyte primary cultures (Fig. 9B)Citation in addition to replicative senescence of HDF cells. The results obtained indicate strongly that nuclear actin accumulation is a universal phenomenon in cellular senescence. When compared with SA-ß-gal activity, nuclear actin accumulation was much more sensitive and an earlier event. Nuclear actin accumulation was observed in ~40% of the cells in 2 days and in >50% in 4 days after H2O2 treatment; however, there were no SA-ß-gal-positive cells until 4 days under the same condition (Table 1)Citation , although morphology of the cells changed to large and flat at this time point.1



View larger version (65K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. Nuclear accumulation of actin is a universal event in the replicative senescence and the variously induced senescent cells. A, premature senescence of human diploid fibroblast (HDF) cells was induced by H-ras double mutant (V12C40, V12G37, or V12S35) virus infection to HDF cells, and the virus-infected HDF cells revealed its doubling time over 3 days (V12C40) and 6 days (V12G37 and V12S35). Actin immunocytochemistry was performed with FITC-conjugated secondary antibody with Hoechst 33258 stain (x400). B, induced senescence also was reproduced by treating HDF primary culture with 0.2 x 10-3 M H2O2 for 4 days (confocal x900) and by treating Mv1Lu epithelial cells with hydroxyurea (1 x 10-3 M) for 4 days (confocal x1200). Chondrocyte primary cultures obtained from female knee were maintained by culture from passage 2 (control cell) to passage 6 (treated cell) and evaluated by rhodamine-conjugated actin immunocytochemistry (confocal x1200). All of the flat cells revealed nuclear actin accumulation, regardless of the origin of cells and the methods of senescence induction used.

 

View this table:
[in this window]
[in a new window]

  		Table 1 Comparison of nuclear actin accumulation and SA-ß-galactosidase activity in the H2O2-induced senescent HDF cells

 
Actin Accumulation in the Nucleus of Senescent Cancer Cells.
To investigate whether the nuclear accumulation of actin molecule is the universal phenomenon applicable even to the cancer cells, Huh7 human hepatoma cells were treated with either deferoxamine (0.5 x 10-3 M) once or H2O2 (0.2 x 10-3 M) for 3 days. Induction of senescence phenotype was monitored by SA-ß-gal assay (Fig. 10A)Citation and the actin immunocytochemistry with nuclear stain with Hoechst 33258 (Fig. 10B)Citation . Deferoxamine and H2O2 treatments induced senescence phenotypes of Huh7 cells in morphology and expression of senescence markers such as SA-ß-gal and actin accumulation. This finding suggests strongly that nuclear actin accumulation may be used as a sensitive and universal marker of senescence in normal and cancer cells.



View larger version (50K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 10. Actin accumulation in the nucleus of the Huh7 cancer cells treated with either deferoxamine (DFO) or H2O2. A, inverted microscopic findings show senescence-associated ß-galactosidase (SA-ß-gal) expression in the Huh7 hepatocellular carcinoma cells by treatment with either DFO once or H2O2 once a day for 3 days (x100). SA-ß-gal was positive in 3 days after DFO and H2O2 treatment. We have reported previously that DFO induced premature senescence of Huh7 cells in the culture system (44 , 45) . B, actin immunocytochemistry of Huh7 cells treated with either DFO once or H2O2 every day. Note the cells became large and flat with actin accumulation in the nucleus by DFO and H2O2 treatments as compared with the control. Lower, site of the nucleus in each cell.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been reported that actin contains two putative leucine-rich nuclear export signals (50) ; therefore, it is expected to be localized in cytoplasm as an important cytoskeleton protein. Thus, a question of why G-actin is accumulated in the nucleus during cellular senescence arises. In our study, because actin accumulated in the nucleus contemporaneously with colocalization of dephosphorylated cofilin in the senescent cells (Fig. 1A)Citation , there is a strong possibility of generating actin monomers or short oligomers by the active cofilin. IEF analysis revealed that the old cells contained only dephosphorylated (active) cofilin as opposed to the mixture of phosphorylated and dephosphorylated forms in young HDF and NIH3T3 cells (Fig. 2A)Citation . Furthermore, maximum level of p-cofilin was found at G2/M phase (N0; Fig. 2CCitation ). However, once the cells entered into G1 phase, p-cofilin was dephosphorylated within 3 h after the release from the mitotic arrest (N3; Fig. 2CCitation ). This supports the previous report that LIMK phosphorylates cofilin (31) and that LIMK is most active during mitosis (51) . To investigate whether the LIMK activity can be regulated in the replicative and induced senescent cells, LIMK-1 activity was measured in vitro in the young and old cells treated with either H2O2 or TPA (Fig. 8)Citation , and the activity was found to be regulated by the treatment in the replicative and induced senescence, supporting the potential role of dephosphorylated cofilin for actin accumulation in nuclei of the senescent cells. Furthermore, the assay of S3A mutant cofilin transfected into young HDF cells (Fig. 7)Citation supported the cooperative function of cofilin phosphorylation by LIMK-1 and G-actin accumulation during cellular senescence. Because senescent cells are arrested at G1 phase, it is evident that the dephosphorylated cofilin may have severed G-actin from F-actin. Cofilin and gelsolin, one of the actin-solubilizing factors, were accumulated in the nucleus of old HDF (52) .1 Contemporaneous accumulation of active cofilin and gelsolin in the nucleus also could explain continuous severing of actin and accumulation of G-actin during cellular senescence.

A few questions then arise as to whether nuclear export of actin could reverse the senescence phenomenon and what signal could induce actin export and stimulate the cell division cycle of the senescent cells. It has been shown recently that, when activated by TPA, PKC in vascular smooth muscle cell line A7r5 disassembles actin stress fiber to membranous ruffles through Src- and Rho-dependent pathways (53) , suggesting strongly that the PKC isozymes are involved in the nuclear actin accumulation during the senescence process. In addition, phospholipase D and diacylglycerol pathways are defective in senescent WI38 human fetal lung fibroblast (54) , indicating significantly reduced PKC activity in the senescent cells. Furthermore, stimulation of PKC-{alpha} translocation by serum is possible only in the young cells but not in the old cells; however, exogenous PKC activator, TPA, strongly induces PKC-{alpha} translocation in the young and old cells. Conversely, depletion of G-actin pool could stimulate serum response factor activity in neuronal cell lines (55) . Therefore, our observations that G-actin accumulated in the nucleus of senescent cells and that TPA induced G-actin export in the old cells (Fig. 3)Citation accompanied by the induction of DNA synthesis (Fig. 4B)Citation imply strongly that the G1 arrest during cellular senescence may be caused by defect of the PKC pathway. Involvement of PKC was confirmed additionally by the use of Go6976, a PKC inhibitor, which inhibited TPA-induced DNA synthesis in the old cells (Fig. 4D)Citation , indicating a significant role of PKC in nuclear actin export and the reversal of senescence phenotypes. The failure of PKC activation by EGF may be explained by recent reports that up-regulation of caveolin and down-regulation of amphiphysin-1 attenuate EGF signaling in the senescent HDF cells through the receptor-mediated endocytosis (56, 57, 58) . These findings are strengthened additionally by the recent observation that reduction of caveolin expression reverses the senescent phenotype in HDF cells (43) . However, we are unable to explain the failure of PKC activation by EGF even in the young cells. Detailed signal transduction mechanism of actin nuclear export through a specific PKC isozyme remains to be explored further.

The earlier report that RB protein phosphorylation occurred 10–20 h after serum stimulation, accompanied by induction of DNA synthesis, is in good accord with our present observation that TPA started to reverse senescent cell morphology to young cell-like phenotypes in 8 h and that the reversal became evident in 20 h (Fig. 3Citation ; Ref. 48 ). TPA treatment also reversed the well-known biochemical markers of cellular senescence in 20 h, including RB protein phosphorylation, down-regulation of p21WAF1 and p-Erk, and caveolin-1 and caveolin-2 expressions (Fig. 6)Citation .

CRM1 has been known as an essential mediator of the signal-dependent nuclear export of proteins and as an essential nuclear protein for proliferation and chromosome region maintenance in eukaryotic cells (59) . Therefore, it also is called an export receptor for nuclear export signals and RanGTP (60 , 61) . Human CRM1 has been cloned, and its preferential localization is the nuclear envelope. Therefore, to examine whether the accumulated nuclear actin was responsible for induction of senescence phenotype, LMB, a specific inhibitor of CRM1 (59 , 61) , was applied to young HDF cells, and as shown in Fig. 5Citation , nuclear actin accumulation, changing cellular morphology, and significant growth arrest were duplicated. These phenomena give additional credence to the function of the accumulated nuclear actin to induce senescent phenotypes. Therefore, we suggest strongly that failure of nuclear export of G-actin is involved directly in the process of cellular senescence.

Finally, we examined the feasibility of nuclear accumulation of actin as a senescence marker, not only in the replicative but also in the induced senescence, by using H2O2-treated HDF cells, hydroxyurea-induced senescence of Mv1Lu, H-ras virus infection of HDF, culture passage-induced senescence of human chondrocyte primary cultures (Fig. 9)Citation , and Huh7 cancer cell senescence by treatment with either deferoxamine or H2O2 (Fig. 10)Citation . To our delight, actin accumulation in the nucleus was found to be a much earlier and universal event for the various cellular senescence than for SA-ß-gal activity (Table 1)Citation . Nuclear actin accumulation was highly sensitive to H2O2 exposure; ~40% of the cells exposed to H2O2 for 2 days revealed the typical nuclear actin accumulation, whereas it took 10 days for SA-ß-gal expression. Therefore, we suggest strongly the nuclear actin accumulation as a sensitive and new marker for cellular senescence.


   ACKNOWLEDGMENTS
 
We thank Professors Woon Ki Paik at Hanyang University and Ronald A. DePinho at Harvard Medical School for their encouragement and careful discussion. pKEX2-A3 Flag was donated by Prof. Dr. Yvonne Samstag at Ruprecht-Karls-University, Heidelberg, Germany. 6-His tagged wild-type mouse cofilin clone was donated by Dr. Kensaku Mizuno at Tohoku University, Japan, and V12S35, V12G37, and V12C40 in pWZL constructs were from Dr. Scott W. Lowe at Cold Spring Harbor Laboratory.


   FOOTNOTES
 
Grant Support: Grant R11-2002-097-01002-0 to Aging & Apoptosis Research Center, Grant R05-2002-000-00054-0 to I. K. L. from Korea Science & Engineering Foundation, and a grant from Health Fellowship Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: In Kyoung Lim, Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, 443-721, Korea. Phone: 82-31-219-5051; Fax: 82-31-219-5059; E-mail: iklim{at}ajou.ac.kr

1 Unpublished observations. Back

Received 6/24/03. Revised 11/ 4/03. Accepted 11/10/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hayflick L. Intracellular determinants of cell aging. Mech. Ageing Dev., 28: 177-185, 1984.[CrossRef][Medline]
  2. Goldstein S. Replicative senescence: the human fibroblast comes of age. Science, 249: 1129-1133, 1990.[Abstract/Free Full Text]
  3. Kirkland J. L. The biochemistry of mammalian senescence. Clin. Biochem., 25: 61-75, 1992.[CrossRef][Medline]
  4. Macieira-Coelho A., Loria E., Berumen L. Relationship between cell kinetic changes and metabolic events during cell senescence in vitro. Adv. Exp. Med. Biol., 53: 51-65, 1975.[Medline]
  5. Start R. D., Loomes R. S., Shortland J. R. The relationship between donor age and the growth characteristics of human smooth muscle cultures of aorta and stomach. Int. J. Exp. Pathol., 72: 647-654, 1991.[Medline]
  6. Ponten J. The relationship between in vitro transformation and tumor formation in vivo. Biochim. Biophys. Acta, 458: 397-422, 1976.[Medline]
  7. Sager R. Senescence as a mode of tumor suppression. Environ. Health Perspect., 93: 59-62, 1991.[Medline]
  8. Kim N. W., Piatyszek M. A., Prowse K. R., Harley C. B., West M. D., Ho P. L., Coviello G. M., Wright W. E., Weinrich S. L., Shay J. W. Specific association of human telomerase activity with immortal cells and cancer. Science (Wash. DC), 266: 2011-2015, 1994.[Abstract/Free Full Text]
  9. Brenner A. J., Stampfer M. R., Aldaz C. M. Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation. Oncogene, 17: 199-205, 1998.[CrossRef][Medline]
  10. Shay J. W., Wright W. E., Werbin H. Defining the molecular mechanisms of human cell immortalization. Biochim. Biophys. Acta, 1072: 1-7, 1991.[Medline]
  11. Wright W. E., Pereira-Smith O. M., Shay J. W. Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts. Mol. Cell. Biol., 9: 3088-3092, 1989.[Abstract/Free Full Text]
  12. Shay J. W., Pereira-Smith O. M., Wright W. E. A role for both RB and p53 in the regulation of human cellular senescence. Exp. Cell Res., 196: 33-39, 1991.[CrossRef][Medline]
  13. Beausejour C. M., Krtolica A., Galimi F., Narita M., Lowe S. W., Yaswen P., Campisi J. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J., 22: 4212-4222, 2003.[CrossRef][Medline]
  14. Sharpless N. E., DePinho R. A. p53: good cop/bad cop. Cell, 110: 9-12, 2002.[CrossRef][Medline]
  15. Harley C. B., Kim N. W., Prowse K. R., Weinrich S. L., Hirsch K. S., West M. D., Bacchetti S., Hirte H. W., Counter C. M., Greider C. W., et al Telomerase, cell immortality, and cancer. Cold Spring Harb. Symp. Quant. Biol., 59: 307-315, 1994.[Abstract/Free Full Text]
  16. Blasco M. A., Lee H. W., Hande M. P., Samper E., Lansdorp P. M., DePinho R. A., Greider C. W. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell, 91: 25-34, 1997.[CrossRef][Medline]
  17. Rudolph K. L., Chang S., Lee H. W., Blasco M., Gottlieb G. J., Greider C., DePinho R. A. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell, 96: 701-712, 1999.[CrossRef][Medline]
  18. Hastie N. D., Dempster M., Dunlop M. G., Thompson A. M., Green D. K., Allshire R. C. Telomere reduction in human colorectal carcinoma and with ageing. Nature (Lond.), 346: 866-868, 1990.[CrossRef][Medline]
  19. Lin A. W., Barradas M., Stone J. C., van Aelst L., Serrano M., Lowe S. W. Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling. Genes Dev., 12: 3008-3019, 1998.[Abstract/Free Full Text]
  20. Zohn I. M., Campbell S. L., Khosravi-Far R., Rossman K. L., Der C. J. Rho family proteins and Ras transformation: the RHOad less traveled gets congested. Oncogene, 17: 1415-1438, 1998.[CrossRef][Medline]
  21. Palmero I., Serrano M. Induction of senescence by oncogenic Ras. Methods Enzymol., 333: 247-256, 2001.[CrossRef][Medline]
  22. Bar-Sagi D., Hall A. Ras and Rho GTPases: a family reunion. Cell, 103: 227-238, 2001.
  23. Mackay D. J., Hall A. Rho GTPases. J. Biol. Chem., 273: 20685-20688, 1998.[Free Full Text]
  24. Pomorski P., Grebecka L. Is actin involved in the nuclear division in Amoeba proteus?. Cell Biol. Int., 17: 521-524, 1993.[CrossRef][Medline]
  25. Satterwhite L. L., Pollard T. D. Cytokinesis. Curr. Opin. Cell Biol., 4: 43-52, 1992.[CrossRef][Medline]
  26. Carlsson L., Nystrom L. E., Sundkvist I., Markey F., Lindberg U. Actin polymerizability is influenced by profilin, a low molecular weight protein in non-muscle cells. J. Mol. Biol., 115: 465-483, 1977.[CrossRef][Medline]
  27. Pring M., Weber A., Bubb M. R. Profilin-actin complexes directly elongate actin filaments at the barbed end. Biochemistry, 31: 1827-1836, 1992.[CrossRef][Medline]
  28. Nishida E., Maekawa S., Sakai H. Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin. Biochemistry, 23: 5307-5313, 1984.[CrossRef][Medline]
  29. Iida K., Moriyama K., Matsumoto S., Kawasaki H., Nishida E., Yahara I. Isolation of a yeast essential gene, COF1, that encodes a homologue of mammalian cofilin, a low-M(r) actin-binding and depolymerizing protein. Gene, 124: 115-120, 1993.[CrossRef][Medline]
  30. Edwards K. A., Montague R. A., Shepard S., Edgar B. A., Erikson R. L., Kiehart D. P. Identification of Drosophila cytoskeletal proteins by induction of abnormal cell shape in fission yeast. Proc. Natl. Acad. Sci. USA, 91: 4589-4593, 1994.[Abstract/Free Full Text]
  31. Yang N., Higuchi O., Ohashi K., Nagata K., Wada A., Kangawa K., Nishida E., Mizuno K. Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization. Nature (Lond.), 393: 809-812, 1998.[CrossRef][Medline]
  32. Rando O. J., Zhao K., Janmey P., Crabtree G. R. Phosphatidylinositol-dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex. Proc. Natl. Acad. Sci. USA, 99: 2824-2829, 2002.[Abstract/Free Full Text]
  33. Zhao K., Wang W., Rando O. J., Xue Y., Swiderek K., Kuo A., Crabtree G. R. Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell, 95: 625-636, 1998.[CrossRef][Medline]
  34. Rando O. J., Zhao K., Crabtree G. R. Searching for a function for nuclear actin. Trends Cell Biol., 10: 92-97, 2000.[CrossRef][Medline]
  35. Iida K., Matsumoto S., Yahara I. The KKRKK sequence is involved in heat shock-induced nuclear translocation of the 18-kDa actin-binding protein, cofilin. Cell Struct. Funct., 17: 39-46, 1992.[Medline]
  36. Abe H., Nagaoka R., Obinata T. Cytoplasmic localization and nuclear transport of cofilin in cultured myotubes. Exp. Cell Res., 206: 1-10, 1993.[CrossRef][Medline]
  37. Samstag Y., Eckerskorn C., Wesselborg S., Henning S., Wallich R., Meuer S. C. Costimulatory signals for human T-cell activation induce nuclear translocation of pp19/cofilin. Proc. Natl. Acad. Sci. USA, 91: 4494-4498, 1994.[Abstract/Free Full Text]
  38. Nebl G., Meuer S. C., Samstag Y. Dephosphorylation of serine 3 regulates nuclear translocation of cofilin. J. Biol. Chem., 271: 26276-26280, 1996.[Abstract/Free Full Text]
  39. Sotiropoulos A., Gineitis D., Copeland J., Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell, 98: 159-169, 1999.[CrossRef][Medline]
  40. Lim I. K., Hong K. W., Kwak I. H., Yoon G., Park S. C. Cytoplasmic retention of p-Erk1/2 and nuclear accumulation of actin proteins during cellular senescence in human diploid fibroblasts. Mech. Ageing Dev., 119: 113-130, 2000.[CrossRef][Medline]
  41. Kim H. S., Song M. C., Kwak I. H., Park T. J., Lim I. K. Constitutive induction of p-Erk1/2 accompanied with reduced activities of protein phosphatases 1 and 2A and MKP3 due to reactive oxygen species during cellular senescence. J. Biol. Chem., 278: 37497-37510, 2003.[Abstract/Free Full Text]
  42. Kawamura S., Miyamoto S., Brown J. H. Initiation and transduction of stretch-induced RhoA and Rac1 activation through caveolae. J. Biol. Chem., 278: 31111-31117, 2003.[Abstract/Free Full Text]
  43. Cho K. A., Ryu S. J., Park J. S., Jang I. S., Ahn J. S., Kim K. T., Park S. C. Senescent phenotype can be reversed by reduction of caveolin status. J. Biol. Chem., 278: 27789-27795, 2003.[Abstract/Free Full Text]
  44. Yoon G., Kim H. J., Yoon Y. S., Cho H., Lim I. K., Lee J. H. Iron chelation-induced senescence-like growth arrest in hepatocyte cell lines: association of transforming growth factor â1 (TGF-â1)-mediated p27Kip1 expression. Biochem. J., 366: 613-621, 2002.[CrossRef][Medline]
  45. Chenoufi N., Baffet G., Drenou B., Cariou S., Desille M., Clement B., Brissot P., Lescoat G., Loreal O. DFO arrests in vitro the proliferation of porcine hepatocyte in G1 phase of the cell cycle. Liver, 18: 60-66, 1998.[Medline]
  46. Clubb B. H., Locke M. Peripheral nuclear matrix actin forms perinuclear shells. J. Cell Biochem., 70: 240-251, 1998.[CrossRef][Medline]
  47. Dimri G. P., Lee X., Basile G., Acosta M., Scott G., Roskelley C., Medrano E. E., Linskens M., Rubelj I., Pereira-Smith O., Peacocke M., Campisi J. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA, 92: 9363-9367, 1995.[Abstract/Free Full Text]
  48. Futreal P. A., Barrett J. C. Failure of senescent cells to phosphorylate the RB protein. Oncogene, 6: 1109-1113, 1991.[Medline]
  49. Munro J., Steeghs K., Morrison V., Ireland H., Parkinson E. K. Human fibroblast replicative senescence can occur in the absence of extensive cell division and short telomeres. Oncogene, 20: 3541-3552, 2001.[CrossRef][Medline]
  50. Wada A., Fukuda M., Mishima M., Nishida E. Nuclear export of actin: a novel mechanism regulating the subcellular localization of a major cytoskeletal protein. EMBO J., 17: 1635-1641, 1998.[CrossRef][Medline]
  51. Sumi T., Matsumoto K., Nakamura T. Mitosis-dependent phosphorylation and activation of LIM-kinase1. Biochem. Biophy. Res. Commun., 290: 1315-1320, 2002.[CrossRef][Medline]
  52. Chen P., Murphy-Ullrich J. E., Wells A. A role for gelsolin in actuating epidermal growth factor receptor-mediated cell motility. J. Cell Biol., 134: 689-698, 1996.[Abstract/Free Full Text]
  53. Brandt D., Gimona M., Hillmann M., Haller H., Mischak H. Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway. J. Biol. Chem., 277: 20903-20910, 2002.[Abstract/Free Full Text]
  54. Venable M. E., Blobe G. C., Obeid L. M. Identification of a defect in the phospholipase D/diacylglycerol pathway in cellular senescence. J. Biol. Chem., 269: 26040-26044, 1994.[Abstract/Free Full Text]
  55. Geneste O., Copeland J. W., Treisman R. LIM kinase and diaphanous cooperate to regulate serum response factor and actin dynamics. J. Cell Biol., 157: 831-838, 2002.[Abstract/Free Full Text]
  56. Park S. C., Park J. S., Park W. Y., Cho K. A., Ahn J. S., Jang I. S. Down-regulation of receptor-mediated endocytosis is responsible for senescence-associated hyporesponsiveness. Ann. N. Y. Acad. Sci., 959: 45-49, 2002.[Medline]
  57. Park J. S., Park W. Y., Cho K. A., Kim D. I., Jhun B. H., Kim S. R., Park S. C. Down-regulation of amphiphysin-1 is responsible for reduced receptor-mediated endocytosis in the senescent cells. FASEB J., 15: 1625-1627, 2001.[Abstract/Free Full Text]
  58. Park W. Y., Park J. S., Cho K. A., Kim D. I., Ko Y. G., Seo J. S., Park S. C. Up-regulation of caveolin attenuates epidermal growth factor signaling in senescent cells. J. Biol. Chem., 275: 20847-20852, 2000.[Abstract/Free Full Text]
  59. Fukuda M., Asano S., Nakamura T., Adachi M., Yoshida M., Yanagida M., Nishida E. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature, 390: 308-311, 1997.[CrossRef][Medline]
  60. Fornerod M., Ohno M., Yoshida M., Mattaj I. W. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell, 90: 1051-1060, 1997.[CrossRef][Medline]
  61. Ossareh-Nazari B., Bachelerie F., Dargemont C. Evidence for a role of CRM1 in signal-mediated nuclear protein export. Science (Wash. DC), 278: 141-144, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
H. S. Kim and I. K. Lim
Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1
J. Biol. Chem., June 5, 2009; 284(23): 15475 - 15486.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Rastogi, B. Joshi, P. Dasgupta, M. Morris, K. Wright, and S. Chellappan
Prohibitin Facilitates Cellular Senescence by Recruiting Specific Corepressors To Inhibit E2F Target Genes.
Mol. Cell. Biol., June 1, 2006; 26(11): 4161 - 4171.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. Matsunobu, K. Tanaka, T. Nakamura, F. Nakatani, R. Sakimura, M. Hanada, X. Li, T. Okada, Y. Oda, M. Tsuneyoshi, et al.
The Possible Role of EWS-Fli1 in Evasion of Senescence in Ewing Family Tumors
Cancer Res., January 15, 2006; 66(2): 803 - 811.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Barker, M. Weinfeld, J. Zheng, L. Li, and D. Murray
Identification of Mammalian Proteins Cross-linked to DNA by Ionizing Radiation
J. Biol. Chem., October 7, 2005; 280(40): 33826 - 33838.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kwak, I. H.
Right arrow Articles by Lim, I. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kwak, I. H.
Right arrow Articles by Lim, I. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online