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1.  Regulated overexpression of the survival factor bcl-2 in CHO cells increases viable cell density in batch culture and decreases DNA release in extended fixed-bed cultivation 
Cytotechnology  2000;32(1):45-61.
Using multicistronic expression technology we generated a stable Chinese hamster ovary (CHO) cell line (MG12) expressing a model secreted heterologous glycoprotein, the secreted form of the human placental alkaline phosphatase (SEAP), and bcl-2, best known as an apoptosis inhibitor, in a tetracycline-repressible dicistronic configuration. In batch cultivations in serum-containing medium, MG12 cells reached twice the final viable cell density when Bcl-2 was overexpressed (in the absence oftetracycline) compared to MG12 populations culturedunder tetracycline-containing conditions (bcl-2repressed). However, bcl-2-expressing MG12 cellsshowed no significant retardation of the decline phasecompared to batch cultures in which the dicistronicexpression unit was repressed.Genetic linkage of bcl-2 expression with the reporter protein SEAP in our multicistronic construct allowed online monitoring of Bcl-2 expression over an extended, multistage fixed-bed bioreactor cultivation. The cloned multicistronic expression unit proved to be stable over a 100 day bioreactor run. CHO MG12 cells in the fixed-bed reactor showed a drastic decrease in the release of DNA into the culture supernatant under conditions of reduced tetracycline (and hencederepressed SEAP and bcl-2 overexpression). This observation indicated enhanced robustness associated with bcl-2 overexpression, similar to recent findings for constitutive Bcl-2-overexpressing hybridoma cells under the same bioprocess conditions. These findings indicate, in these serum-containing CHO cell cultures, that overexpression of Bcl-2 results in desirable modifications in culture physiology.
doi:10.1023/A:1008168522385
PMCID: PMC3449443  PMID: 19002966
apoptosis; Bcl-2; fixed-bed reactor; regulated gene expression
2.  Regulation of expression by promoters versus internal ribosome entry site in the 5′-untranslated sequence of the human cyclin-dependent kinase inhibitor p27kip1 
Nucleic Acids Research  2005;33(12):3763-3771.
p27kip1 regulates cell proliferation by binding to and inhibiting the activity of cyclin-dependent kinases and its expression oscillates with cell cycle. Recently, it has been suggested from studies using the traditional dicistronic DNA assay that the expression of p27kip1 is regulated by internal ribosome entry site (IRES)-mediated translation initiation, and several RNA-binding protein factors were thought to play some role in this regulation. Considering the inevitable drawbacks of the dicistronic DNA assay, which could mislead a promoter activity or alternative splicing to IRES as previously demonstrated, we decided to reanalyze the 5′-untranslated region (5′-UTR) sequence of p27kip1 and test whether it contains an IRES element or a promoter using more stringent methods, such as dicistronic RNA and promoterless dicistronic and monocistronic DNA assays. We found that the 5′-UTR sequence of human p27kip1 does not have any significant IRES activity. The previously observed IRES activities are likely generated from the promoter activities present in the 5′-UTR sequences of p27kip1. The findings in this study indicate that transcriptional regulation likely plays an important role in p27kip1 expression, and the mechanism of regulation of p27 expression by RNA-binding factors needs to be re-examined. The findings in this study also further enforce the importance that more stringent studies, such as promoterless dicistronic and monocistronic DNA and dicistronic RNA tests, are required to safeguard any future claims of cellular IRES.
doi:10.1093/nar/gki680
PMCID: PMC1174905  PMID: 16006622
3.  Hes1 Directly Controls Cell Proliferation through the Transcriptional Repression of p27Kip1 
Molecular and Cellular Biology  2005;25(10):4262-4271.
A transcriptional regulator, Hes1, plays crucial roles in the control of differentiation and proliferation of neuronal, endocrine, and T-lymphocyte progenitors during development. Mechanisms for the regulation of cell proliferation by Hes1, however, remain to be verified. In embryonic carcinoma cells, endogenous Hes1 expression was repressed by retinoic acid in concord with enhanced p27Kip1 expression and cell cycle arrest. Conversely, conditional expression of a moderate but not maximal level of Hes1 in HeLa cells by a tetracycline-inducible system resulted in reduced p27Kip1 expression, which was attributed to decreased basal transcript rather than enhanced proteasomal degradation, with concomitant increases in the growth rate and saturation density. Hes1 induction repressed the promoter activity of a 5′ flanking basal enhancer region of p27Kip1 gene in a manner dependent on Hes1 expression levels, and this was mediated by its binding to class C sites in the promoter region. Finally, hypoplastic fetal thymi, as well as livers and brains of Hes1-deficient mice, showed significantly increased p27Kip1 transcripts compared with those of control littermates. These results have suggested that Hes1 directly contributes to the promotion of progenitor cell proliferation through transcriptional repression of a cyclin-dependent kinase inhibitor, p27Kip1.
doi:10.1128/MCB.25.10.4262-4271.2005
PMCID: PMC1087711  PMID: 15870295
4.  p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes. 
Molecular Biology of the Cell  1997;8(9):1815-1827.
p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.
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PMCID: PMC305739  PMID: 9307976
5.  Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment 
Cell Stress & Chaperones  2008;14(1):49-60.
Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (qp), NaBu enhanced the qp by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the qp by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca2+ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca2+ ([Ca2+]c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the qp of cells treated with NaBu, but it also sensitized cells to apoptosis.
doi:10.1007/s12192-008-0054-0
PMCID: PMC2673906  PMID: 18663604
Calnexin; Chinese hamster ovary (CHO) cells; Apoptosis
6.  Efficient and inducible production of human interleukin 6 in Chinese hamster ovary cells using a novel expression system 
Cytotechnology  1997;25(1-3):53-60.
High level and inducible production of human interleukin 6 (hIL-6) was achieved using a novel expression system in Chinese hamster ovary (CHO) cells. In this system, the transcription of hIL-6 gene under the control of PhCMV*-1 promoter composed of tetracycline operator sequences and a minimal promoter is activated by a chimeric transactivator (tTA) composed of tetracycline repressor and transactivating domain of VP16 protein of herpes simplex virus. The transcription of tTA gene, which is also under the control of PhCMV*-1 promoter, is activated by itself via a positive feedback cycle. The expression of both genes is further enhanced by potentiating the VP16 transactivating domain of tTA transactivator with pX protein of hepatitis B virus. In the presence of tetracycline, the tTA transactivators can not bind to PhCMV*-1 promoter, therefore, the expression of hIL-6 and tTA gene is suppressed, and the pX will not activate basal transcription. In the absence of tetracycline, tTA transactivators bind to PhCMV*-1 promoter and activate efficient transcription of hIL-6 and tTA gene, and the transcription is further enhanced by pX via VP16 transactivating domain. Using this strategy, we isolated a clone (UX1) producing hIL-6 at a rate about 1425 ng/106 cells/day. Furthermore, the hIL-6 production is stringently regulated by tetracycline. This results suggested a novel strategy to establish highly efficient, inducible and cell type independent recombinant protein production system by using an artificial promoter to recruit transactivators and coactivators which can synergistically activate transcription.
doi:10.1023/A:1007972002180
PMCID: PMC3466749  PMID: 22358879
CHO cells; coactivator; recombinant protein production system; transactivator
7.  Cell Density-Dependent Inhibition of Epidermal Growth Factor Receptor Signaling by p38α Mitogen-Activated Protein Kinase via Sprouty2 Downregulation▿  
Molecular and Cellular Biology  2009;29(12):3332-3343.
Contact inhibition is a fundamental process in multicellular organisms aimed at inhibiting proliferation at high cellular densities through poorly characterized intracellular signals, despite availability of growth factors. We have previously identified the protein kinase p38α as a novel regulator of contact inhibition, as p38α is activated upon cell-cell contacts and p38α-deficient cells are impaired in both confluence-induced proliferation arrest and p27Kip1 accumulation. Here, we establish that p27Kip1 plays a key role downstream of p38α to arrest proliferation at high cellular densities. Surprisingly, p38α does not directly regulate p27Kip1 expression levels but leads indirectly to confluent upregulation of p27Kip1 and cell cycle arrest via the inhibition of mitogenic signals originating from the epidermal growth factor receptor (EGFR). Hence, confluent activation of p38α uncouples cell proliferation from mitogenic stimulation by inducing EGFR degradation through downregulation of the EGFR-stabilizing protein Sprouty2 (Spry2). Accordingly, confluent p38α-deficient cells fail to downregulate Spry2, providing them in turn with sustained EGFR signaling that facilitates cell overgrowth and oncogenic transformation. Our results provide novel mechanistic insight into the role of p38α as a sensor of cell density, which induces confluent cell cycle arrest via the Spry2-EGFR-p27Kip1 network.
doi:10.1128/MCB.01955-08
PMCID: PMC2698726  PMID: 19364817
8.  CDK inhibitor p57Kip2 is downregulated by Akt during HER2-mediated tumorigenicity 
Cell Cycle  2013;12(6):935-943.
HER2/neu oncogene is frequently deregulated in cancers, and the (PI3K)-Akt signaling is one of the major pathways in mediating HER2/neu oncogenic signal. p57Kip2, an inhibitor of cyclin-depependent kinases, is pivotal in regulating cell cycle progression, but its upstream regulators remain unclear. Here we show that the HER2-Akt axis is linked to p57Kip2 regulation, and that Akt is a negative regulator of p57Kip2. Ectopic expression of Akt can decrease the expression of p57Kip2, while Akt inhibition leads to p57Kip2 stabilization. Mechanistic studies show that Akt interacts with p57Kip2 and causes cytoplasmic localization of p57Kip2. Akt phosphorylates p57 on Ser 282 or Thr310. Akt activity results in destabilization of p57 by accelerating turnover rate of p57 and enhancing p57 ubiquitination. Importantly, the negative impact of HER2/Akt on p57 stability contributes to HER2-mediated cell proliferation, transformational activity and tumorigenicity. p57 restoration can attenuate these defects caused by HER2. Significantly, Kaplan-Meier analysis of tumor samples demonstrate that in tumors where HER2 expression was observed, high expression levels of p57Kip2 were associated with better overall survival. These data suggest that HER2/Akt is an important negative regulator of p57Kip2, and that p57 restoration in HER2-overexpressing cells can reduce breast tumor growth. Our findings indicate the applicability of employing p57 regulation as a therapeutic intervention in HER2-overexpressing cancers.
doi:10.4161/cc.23883
PMCID: PMC3637352  PMID: 23421998
Akt; cell cycle; HER2; p57 Kip2; phosphorylation; subcellular localizaiton
9.  Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells ▿ †  
Molecular and Cellular Biology  2010;30(21):5057-5070.
The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
doi:10.1128/MCB.00249-10
PMCID: PMC2953065  PMID: 20805359
10.  Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells 
Cytotechnology  1997;24(1):55-64.
Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.
doi:10.1023/A:1007969502256
PMCID: PMC3449609  PMID: 22358597
CHO; IGF-I; serum-free; autocrine growth; cell culture
11.  The Sp1 Family of Transcription Factors Is Involved in p27Kip1-Mediated Activation of Myelin Basic Protein Gene Expression 
Molecular and Cellular Biology  2003;23(12):4035-4045.
p27Kip1 levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27Kip1 levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27Kip1 enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27Kip1. Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27Kip1 can activate gene transcription by using the MBP gene in oligodendrocytes as a model. We show that the enhancement of MBP promoter activity by p27Kip1 is mediated by a proximal region of the MBP promoter that contains a conserved GC box binding sequence. This sequence binds transcription factors Sp1 and Sp3. Increased expression of p27Kip1 increases the level of Sp1 promoter binding to the GC box but does not change the level of Sp3 binding. The binding of Sp1 to this element activates the MBP promoter. p27Kip1 leads to increased Sp1 binding through a decrease in Sp1 protein turnover. Enhancement of MBP promoter activity by an increase in the level of p27Kip1 involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1.
doi:10.1128/MCB.23.12.4035-4045.2003
PMCID: PMC156141  PMID: 12773549
12.  3,3′-Diindolylmethane Induces a G1 Arrest in Human Prostate Cancer Cells Irrespective of Androgen Receptor and p53 Status 
Biochemical pharmacology  2009;78(5):469-476.
3,3′-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study we characterized the effect of DIM on cell cycle regulation in both androgen dependent LNCaP and androgen receptor negative-p53 mutant DU145 human prostate cancer cells. DIM had an antiproliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM mediated G1 cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased expression of the cell cycle inhibitor p27Kip1 protein in LNCaP and DU145 cells. Promoter deletion studies with p27Kip1 reporter gene constructs showed that this DIM-mediated increase in p27Kip1 was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27Kip1 and subsequent G1 arrest by DIM involves activation of the p38 MAPK pathway in the DU145 cells. Taken together, our results indicate that DIM is able to stop the cell cycle progression of human prostate cancer cells regardless of their androgen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle regulatory effect in DU145 cells.
doi:10.1016/j.bcp.2009.05.008
PMCID: PMC2706920  PMID: 19433067
3; 3′-Diinolylmethane; Prostate cancer; Cell cycle arrest; p27Kip1; p38 MAPK; Cancer
13.  CSIG Inhibits PTEN Translation in Replicative Senescence▿  
Molecular and Cellular Biology  2008;28(20):6290-6301.
Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) that was abundant in young human diploid fibroblast cells but declined upon replicative senescence. Overexpression or knockdown of CSIG did not influence p21Cip1 and p16INK4a expressions. Instead, CSIG negatively regulated PTEN and p27Kip1 expressions, in turn promoting cell proliferation. In PTEN-silenced HEK 293 cells and PTEN-deficient human glioblastoma U87MG cells, the effect of CSIG on p27Kip1 expression and cell division was abolished, suggesting that PTEN was required for the role of CSIG on p27Kip1 regulation and cell cycle progression. Investigation into the underlying mechanism revealed that the regulation of PTEN by CSIG was achieved through a translational suppression mechanism. Further study showed that CSIG interacted with PTEN mRNA in the 5′ untranslated region (UTR) and that knockdown of CSIG led to increased luciferase activity of a PTEN 5′ UTR-luciferase reporter. Moreover, overexpression of CSIG significantly delayed the progression of replicative senescence, while knockdown of CSIG expression accelerated replicative senescence. Knockdown of PTEN diminished the effect of CSIG on cellular senescence. Our findings indicate that CSIG acts as a novel regulatory component of replicative senescence, which requires PTEN as a mediator and involves in a translational regulatory mechanism.
doi:10.1128/MCB.00142-08
PMCID: PMC2577433  PMID: 18678645
14.  p57Kip2 Stabilizes the MyoD Protein by Inhibiting Cyclin E-Cdk2 Kinase Activity in Growing Myoblasts 
Molecular and Cellular Biology  1999;19(11):7621-7629.
We show that expression of p57Kip2, a potent tight-binding inhibitor of several G1 cyclin–cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation. We examined the effect of p57Kip2 on the activity of the transcription factor MyoD. In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57Kip2, p21Cip1, and p27Kip1 but not p16Ink4a induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57Kip2. Forced expression of p57Kip2 correlated with hypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mutant arrested cells at the G1 phase transition and induced hypophosphorylation of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57Kip2. In addition, the NH2 domain of p57Kip2 necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts. Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57Kip2 could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.
PMCID: PMC84790  PMID: 10523650
15.  Shp-1 mediates the anti-proliferative activity of TIMP-2 in human microvascular endothelial cells 
The Journal of biological chemistry  2005;281(6):3711-3721.
The tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase (MMP) activity required for cell migration/invasion associated with cancer progression and angiogenesis. TIMPs also modulate cell proliferation in vitro and angiogenesis in vivo independent of their MMP-inhibitory activity. Here, we show that TIMP-2 mediates G1 growth arrest in human endothelial cells through de novo synthesis of the cyclin-dependent kinase inhibitor p27Kip1. TIMP-2-mediated inhibition of Cdk4 and Cdk2 activity is associated with increased binding of p27Kip1 to these complexes in vivo. Protein tyrosine phosphatase inhibitors or expression of a dominant negative Shp-1 mutant ablates TIMP-2 induction of p27Kip1. Finally, angiogenic responses to FGF-2 and VEGF-A in ‘motheaten viable’ Shp-1 deficient mice are resistant to TIMP-2 inhibition, demonstrating that Shp-1 is an important negative regulator of angiogenesis in vivo.
doi:10.1074/jbc.M509932200
PMCID: PMC1361361  PMID: 16326706
TIMP-2, tissue inhibitor of metalloproteinase-2; MMP, matrix metalloproteinase; Shp-1, SH2-containing protein tyrosine phosphatase-1; PTP, protein tyrosine phosphatase; hMVECs, human microvascular endothelial cells; Cdks, cyclin-dependent kinases; ECM, extracellular matrix; FGF-2, fibroblast growth factor; PDGF, platelet derived growth factor; EGF, epidermal growth factor; VEGF-A, vascular endothelial growth factor-A; INK4, inhibitors of Cdk4; PBS, phosphate-buffered saline; pRb, retinoblastoma protein
16.  TRIP6 Regulates p27KIP1 To Promote Tumorigenesis 
Molecular and Cellular Biology  2013;33(7):1394-1409.
TRIP6 is an adaptor protein that regulates cell motility and antiapoptotic signaling. Although it has been implicated in tumorigenesis, the underlying mechanism remains largely unknown. Here we provide evidence that TRIP6 promotes tumorigenesis by serving as a bridge to promote the recruitment of p27KIP1 to AKT in the cytosol. TRIP6 regulates the membrane translocation and activation of AKT and facilitates AKT-mediated recognition and phosphorylation of p27KIP1 specifically at T157, thereby promoting the cytosolic mislocalization of p27KIP1. This is required for p27KIP1 to enhance lysophosphatidic acid (LPA)-induced ovarian cancer cell migration. TRIP6 also promotes serum-induced reduction of nuclear p27KIP1 expression levels through Skp2-dependent and -independent mechanisms. Consequently, knockdown of TRIP6 in glioblastoma or ovarian cancer xenografts restores nuclear p27KIP1 expression and impairs tumor proliferation. As TRIP6 is upregulated in gliomas and its levels correlate with poor clinical outcomes in a dose-dependent manner, it may represent a novel prognostic marker and therapeutic target in gliomas.
doi:10.1128/MCB.01149-12
PMCID: PMC3624266  PMID: 23339869
17.  Phosphorylation of the Cyclin-Dependent Kinase Inhibitor p21Cip1 on Serine 130 Is Essential for Viral Cyclin-Mediated Bypass of a p21Cip1-Imposed G1 Arrest 
Molecular and Cellular Biology  2006;26(6):2430-2440.
K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.
doi:10.1128/MCB.26.6.2430-2440.2006
PMCID: PMC1430279  PMID: 16508017
18.  p57KIP2 control of actin cytoskeleton dynamics is responsible for its mitochondrial pro-apoptotic effect 
Cell Death & Disease  2012;3(5):e311-.
p57 (Kip2, cyclin-dependent kinase inhibitor 1C), often found downregulated in cancer, is reported to hold tumor suppressor properties. Originally described as a cyclin-dependent kinase (cdk) inhibitor, p57KIP2 has since been shown to influence other cellular processes, beyond cell cycle regulation, including cell death and cell migration. Inhibition of cell migration by p57KIP2 is attributed to the stabilization of the actin cytoskeleton through the activation of LIM domain kinase-1 (LIMK-1). Furthermore, p57KIP2 is able to enhance mitochondrial-mediated apoptosis. Here, we report that the cell death promoting effect of p57KIP2 is linked to its effect on the actin cytoskeleton. Indeed, whereas Jasplakinolide, an actin cytoskeleton-stabilizing agent, mimicked p57KIP2's pro-apoptotic effect, destabilizing the actin cytoskeleton with cytochalsin D reversed p57KIP2's pro-apoptotic function. Conversely, LIMK-1, the enzyme mediating p57KIP2's effect on the actin cytoskeleton, was required for p57KIP2's death promoting effect. Finally, p57KIP2-mediated stabilization of the actin cytoskeleton was associated with the displacement of hexokinase-1, an inhibitor of the mitochondrial voltage-dependent anion channel, from the mitochondria, providing a possible mechanism for the promotion of the mitochondrial apoptotic cell death pathway. Altogether, our findings link together two tumor suppressor properties of p57KIP2, by showing that the promotion of cell death by p57KIP2 requires its actin cytoskeleton stabilization function.
doi:10.1038/cddis.2012.51
PMCID: PMC3366085  PMID: 22592318
p57KIP2; cell migration; cancer; cytoskeleton
19.  Decreased proliferation of human melanoma cell lines caused by antisense RNA against translation factor eIF-4A1 
British Journal of Cancer  2002;86(12):1957-1962.
Control of translation initiation was recognised as a critical checkpoint for cell proliferation and tumorigenesis. In human melanoma cells, we have previously reported consistent overexpression of translation initiation factor eIF-4A1. Here, we investigated by transfection of antisense constructs its significance for the control of melanoma cell growth. The tetracycline-inducible expression system was established in melanoma cells, and three fragments of the 5′-, central-, and 3′-portion of the eIF-4A1 cDNA were subcloned in antisense and in sense orientation after a tetracycline inducible promoter. Significant proliferation decrease was obtained after transient transfection and induction of antisense RNA directed against the 5′- and the central portion (up to 10%), whereas, no effects were seen after induction of the 3′-fragment and the sense controls. Cell clones stably transfected with the central antisense fragment revealed after doxycycline induction reduced expression of endogeneous eIF-4A1 mRNA correlated with decreased proliferation rates (up to 6%). These data demonstrate the applicability of antisense strategies against translation factors in melanoma cells. Translation initiation factor eIF-4A1 contributes to the control of melanoma cell proliferation and may be taken into consideration when scheduling new therapeutic approaches targeting the translational control.
British Journal of Cancer (2002) 86, 1957–1962. doi:10.1038/sj.bjc.6600351 www.bjcancer.com
© 2002 Cancer Research UK
doi:10.1038/sj.bjc.6600351
PMCID: PMC2375438  PMID: 12085193
melanoma; translation control; antisense; proliferation; apoptosis
20.  MyoD regulates p57kip2 expression by interacting with a distant cis-element and modifying a higher order chromatin structure 
Nucleic Acids Research  2012;40(17):8266-8275.
The bHLH transcription factor MyoD, the prototypical master regulator of differentiation, directs a complex program of gene expression during skeletal myogenesis. The up-regulation of the cdk inhibitor p57kip2 plays a critical role in coordinating differentiation and growth arrest during muscle development, as well as in other tissues. p57kip2 displays a highly specific expression pattern and is subject to a complex epigenetic control driving the imprinting of the paternal allele. However, the regulatory mechanisms governing its expression during development are still poorly understood. We have identified an unexpected mechanism by which MyoD regulates p57kip2 transcription in differentiating muscle cells. We show that the induction of p57kip2 requires MyoD binding to a long-distance element located within the imprinting control region KvDMR1 and the consequent release of a chromatin loop involving p57kip2 promoter. We also show that differentiation-dependent regulation of p57kip2, while involving a region implicated in the imprinting process, is distinct and hierarchically subordinated to the imprinting control. These findings highlight a novel mechanism, involving the modification of higher order chromatin structures, by which MyoD regulates gene expression. Our results also suggest that chromatin folding mediated by KvDMR1 could account for the highly restricted expression of p57kip2 during development and, possibly, for its aberrant silencing in some pathologies.
doi:10.1093/nar/gks619
PMCID: PMC3458561  PMID: 22740650
21.  Increased Expression of the Integral Membrane Protein ErbB2 in Chinese Hamster Ovary Cells Expressing the Anti-apoptotic Gene Bcl-xL 
Receptor tyrosine kinases (RTKs) are the second largest family of membrane receptors and play a key role in the regulation of vital cellular processes, such as control of cell growth, differentiation, metabolism, and migration. The production of whole-length RTKs in large quantities for biophysical or structural characterization, however, is a challenge. In this study, a cell engineering strategy using the anti-apoptotic Bcl-2 family protein, Bcl-xL, was tested as a potential method for increasing stable expression levels of a recombinant RTK membrane protein in Chinese hamster ovary (CHO) cells. Wild type and CHO cells stably overexpressing heterologous Bcl-xL were transformed with the gene for a model RTK membrane protein, ErbB2, on a plasmid also containing the Zeocin resistance gene. While CHO cells exhibited a gradual decrease in expression with passaging, CHO-bcl-xL cells offered an increased and sustained level of ErbB2 expression following continuous passaging over more than 33 days in culture. The increased ErbB2 expression in CHO-bcl-xL cells was evident both in stable transfected pools and in clonal isolates, and demonstrated both in western blot analysis and flow cytometry. Furthermore, the sustained high-level protein expression in CHO-bcl-xL cells does not alter the correct membrane localization of the ErbB2 protein. Our results demonstrate that cellular engineering, specifically anti-apoptosis engineering, can provide increased and stable ErbB2 membrane protein expression in mammalian cells. This approach may also be useful for other membrane proteins in which large quantities are needed for biophysical and structural studies.
doi:10.1016/j.pep.2009.04.007
PMCID: PMC2688385  PMID: 19376231
membrane protein; epidermal growth factor receptors; ErbB2; Neu; Zeocin; Bcl-xL; anti-apoptosis engineering; CHO; mammalian culture; metabolic engineering
22.  A novel role of proteasomal β1 subunit in tumorigenesis 
Bioscience Reports  2013;33(4):e00050.
p27Kip1 is a key cell-cycle regulator whose level is primarily regulated by the ubiquitin–proteasome degradation pathway. Its β1 subunit is one of seven β subunits that form the β-ring of the 20S proteasome, which is responsible for degradation of ubiquitinated proteins. We report here that the β1 subunit is up-regulated in oesophageal cancer tissues and some ovarian cancer cell lines. It promotes cell growth and migration, as well as colony formation. β1 binds and degrades p27Kip1directly. Interestingly, the lack of phosphorylation at Ser158 of the β1 subunit promotes degradation of p27Kip1. We therefore propose that the β1 subunit plays a novel role in tumorigenesis by degrading p27Kip1.
doi:10.1042/BSR20130013
PMCID: PMC3712487  PMID: 23725357
degradation; p27Kip1; phosphorylation; tumorigenesis; β1 subunit; CBB, Coomassie Brilliant Blue; CDK, cyclin-dependent kinase; GST, glutathione transferase; HCC, hepatocellular carcinoma; HEK-293T, HEK-293 cells expressing the large T-antigen of SV40 (simian virus 40); PKA, protein kinase A; Rb, retinoblastoma; Rfp, red fluorescent protein; RP, regulatory particle; shRNA, small hairpin RNA
23.  Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8 
Molecular cell  2011;43(5):751-763.
SUMMARY
The kinesin-8 family of microtubule motors plays a critical role in microtubule length control in cells. These motors have complex effects on microtubule dynamics: they destabilize growing microtubules yet stabilize shrinking microtubules. The budding yeast kinesin-8, Kip3, accumulates on plus ends of growing but not shrinking microtubules. Here we identify an essential role of the tail domain of Kip3 in mediating both its destabilizing and stabilizing activities. The Kip3-tail promotes Kip’s accumulation at the plus ends and facilitates the destabilizing effect of Kip3. However, the Kip3-tail also inhibits microtubule shrinkage and is required for promoting microtubule rescue by Kip3. These effects of the tail domain are likely to be mediated by the tubulin- and microtubule-binding activities that we describe. We propose a concentration-dependent model for the coordination of the destabilizing and stabilizing activities of Kip3 and discuss its relevance to cellular microtubule organization.
doi:10.1016/j.molcel.2011.06.027
PMCID: PMC3181003  PMID: 21884976
24.  Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors. 
Molecular Biology of the Cell  1997;8(6):1025-1033.
Two Saccharomyces cerevisiae kinesin-related motors, Cin8p and Kip1p, perform an essential role in the separation of spindle poles during spindle assembly and a major role in spindle elongation. Cin8p and Kip1p are also required to prevent an inward spindle collapse prior to anaphase. A third kinesin-related motor, Kar3p, may act antagonistically to Cin8p and Kip1p since loss of Kar3p partially suppresses the spindle collapse in cin8 kip1 mutants. We have tested the relationship between Cin8p and Kar3p by overexpressing both motors using the inducible GAL1 promoter. Overexpression of KAR3 results in a shrinkage of spindle size and a temperature-dependent inhibition of the growth of wild-type cells. Excess Kar3p has a stronger inhibitory effect on the growth of cin8 kip1 mutants and can completely block anaphase spindle elongation in these cells. In contrast, overexpression of CIN8 leads to premature spindle elongation in all cells tested. This is the first direct demonstration of antagonistic motors acting on the intact spindle and suggests that spindle length is determined by the relative activity of Kar3p-like and Cin8p/Kip1p-like motors.
Images
PMCID: PMC305711  PMID: 9201713
25.  Krüppel-like Factor 4 Induces p27Kip1 Expression in and Suppresses the Growth and Metastasis of Human Pancreatic Cancer Cells 
Cancer research  2008;68(12):4631-4639.
The zinc finger transcription factor Krüppel-like factor 4 (KLF4) has been implicated in both tumor suppression and progression. However, its function in pancreatic cancer has not been well characterized. Here, we show that pancreatic cancer cell lines expressed various levels of KLF4 RNA and protein. Ectopic expression of KLF4 by FG and BxPC-3 pancreatic cancer cells resulted in cell cycle arrest and marked inhibition of cell growth in vitro and attenuation of tumor growth and metastasis in an orthotopic mouse model. Overexpression of KLF4 also led to significant induction of p27Kip1 expression, at both the RNA and protein levels, in a dose- and time-dependent manner, indicating that KLF4 transcriptionally regulates the expression of p27Kip1. Chromatin immunoprecipitation assays consistently showed that KLF4 protein physically interacts with the p27Kip1 promoter. Promoter deletion and point mutation analyses indicated that a region between nucleotides −435 and −60 of the p27Kip1 promoter and intact of the three KLF4-binding sites within that region were required for the full induction of p27Kip1 promoter activity by KLF4. Our findings suggest that KLF4 transactivates p27Kip1 expression and inhibits the growth and metastasis of human pancreatic cancer.
doi:10.1158/0008-5472.CAN-07-5953
PMCID: PMC2481517  PMID: 18559508

Results 1-25 (511939)