KARAKTERISTIK SEL KANKER ORAL BARU (Sp-C1) DAN UJI HAMBATAN PERTUMBUHAN SEL Sp-C1 MENGGUNAKAN TERAPI GEN pcDNA3.1 -p27Kip1mutant type in vitro

2014 ◽  
Vol 18 (2) ◽  
pp. 120-125
Author(s):  
Supriatno
Keyword(s):  
Cyclin E ◽  
Cyclin Dependent Kinase ◽  
Empty Vector

Sel primer kanker oral (sel Sp-C1) mempunyai beberapa karakteristik pertumbuhan sel dan ekspresi protein antiapoptosis maupun protein regulator positif siklus sel. Karakteristik sel Sp-C1 penting diketahui untuk memudahkanpeneliti menggunakan model in vitro sel kanker oral. Tujuan penelitian adalah memperkenalkan jenis sel kanker oral baru(sel Sp-C1) dengan karakteristiknya, serta menguji hambatan pertumbuhan sel Sp-C1 mengunakan terapi gen p27Kip1mt secara in vitro. Isolasi sel Sp-C1 berasal dari jaringan limfonodi servikal yang termetastasis kanker lidah. Proses isolasididapatkan fibroblas dan sel primer kanker oral yang diinkubasi pada refrigerator -800C. Hambatan pertumbuhan sel SpC1 diperlakukan dengan terapi gen pcDNA3.1 -p27Kip1 mt dan pcDNA3.1-neo (empty vector) menggunakan uji MTT.Hasil penelitian menunjukkan karakteristik pertumbuhan sel Sp-C1 yang relatif cepat dan ekspresi protein p45Skp2, αtubulin, cyclin-dependent kinase-2 (CDK-2), cyclin-E, metastatic associated protein-1 (MTA-1) dan protein antiapoptosis maspin. Selanjutnya, sel transfektan Sp-C1-pcDNA3.1-p27Kip1 mt mempunyai potensi hambatanpertumbuhan sel yang signifikan dibandingkan pcDNA3.1-neo (P< 0.05). Kesimpulan, sel Sp-C1 mempunyaikarakteristik tertentu dan dapat digunakan sebagai model penelitian in vitro kanker mulut. pcDNA3.1-p27Kip1 mtmempunyai potensi hambatan pertumbuhan sel Sp-C1 yang kuat. Metode transfer gen secara in vitro merupakan proseduryang sederhana dan suatu strategi baru terapi gen terhadap sel kanker oral.

An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3985-3987 ◽  
Author(s):  
Mu-Shui Dai ◽  
Charlie R. Mantel ◽  
Zhen-Biao Xia ◽  
Hal E. Broxmeyer ◽  
Li Lu
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
S Phase ◽  
Cyclin D3 ◽  
G1 Phase ◽  
Cyclin Dependent Kinase ◽  
Terminal Erythroid Differentiation

The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34+cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)–derived erythroblasts from a 7-day culture of CD34+ cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.

scholarly journals Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E–cyclin-dependent kinase 2 complex in vitro

1999 ◽  
Vol 340 (3) ◽  
pp. 775-782 ◽  
Author(s):  
Stéphane MANENTI ◽  
Emiko YAMAUCHI ◽  
Odile SOROKINE ◽  
Martine KNIBIEHLER ◽  
Alain VAN DORSSELAER ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclin E ◽  
Cyclin Dependent Kinase ◽  
Kinase Substrate ◽  
Cyclin Dependent Kinases ◽  
Kinase C ◽  
Cdk2 Complex

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.

scholarly journals Noncatalytic Requirement for Cyclin A-cdk2 in p27 Turnover

2004 ◽  
Vol 24 (13) ◽  
pp. 6058-6066 ◽  
Author(s):  
Xin-Hua Zhu ◽  
Hoang Nguyen ◽  
H. Dorota Halicka ◽  
Frank Traganos ◽  
Andrew Koff
Keyword(s):  
Ubiquitin Ligase ◽  
Cyclin E ◽  
Flow Cytometric Analysis ◽  
E3 Ubiquitin Ligase ◽  
Cyclin A ◽  
Cyclin B1 ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Flow Cytometric

ABSTRACT Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27kip1 is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G1 cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCFskp2 complex.

scholarly journals Modulation of β-Catenin Phosphorylation/Degradation by Cyclin-dependent Kinase 2

2004 ◽  
Vol 279 (19) ◽  
pp. 19592-19599 ◽  
Author(s):  
Chun Shik Park ◽  
Sung Il Kim ◽  
Mi Su Lee ◽  
Cho-ya Youn ◽  
Dae Joong Kim ◽  
...  
Keyword(s):  
Cyclin E ◽  
Signal Transduction Pathway ◽  
Transduction Pathway ◽  
Cyclin Dependent Kinase ◽  
Rapid Degradation ◽  
Subsequent Degradation ◽  
Proteasome Pathway ◽  
Fine Tune

β-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway, and inappropriate control of cytosolic β-catenin is a crucial step in the genesis of several human cancers. Here we demonstrate that cyclin-dependent kinase 2 (CDK2) in association with cyclin A or cyclin E directly binds to β-catenin.In vivoandin vitrokinase assays with cyclin-CDK2 demonstrate β-catenin phosphorylation on residues Ser33, Ser37, Thr41, and Ser45. This phosphorylation promotes rapid degradation of cytosolic β-catenin via the β-TrCP-mediated proteasome pathway. Moreover, cyclin E-CDK2 contributes to rapid degradation of cytosolic β-catenin levels during G1phase by regulating β-catenin phosphorylation and subsequent degradation. In this way, CDK2 may “fine tune” β-catenin levels over the course of the cell cycle.

scholarly journals Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E‒cyclin-dependent kinase 2 complex in vitro

1999 ◽  
Vol 340 (3) ◽  
pp. 775 ◽  
Author(s):  
Stéphane MANENTI ◽  
Emiko YAMAUCHI ◽  
Odile SOROKINE ◽  
Martine KNIBIEHLER ◽  
Alain VAN DORSSELAER ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclin E ◽  
Cyclin Dependent Kinase ◽  
Kinase C

scholarly journals Maturation of Human Cyclin E Requires the Function of Eukaryotic Chaperonin CCT

1998 ◽  
Vol 18 (12) ◽  
pp. 7584-7589 ◽  
Author(s):  
Kwang-Ai Won ◽  
Robert J. Schumacher ◽  
George W. Farr ◽  
Arthur L. Horwich ◽  
Steven I. Reed
Keyword(s):  
Cyclin E ◽  
Positive Control ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Native State ◽  
Human Proteins ◽  
Reticulocyte Lysate ◽  
Dependent Processes

ABSTRACT Cyclin E, a partner of the cyclin-dependent kinase Cdk2, has been implicated in positive control of the G1/S phase transition. Whereas degradation of cyclin E has been shown to be exquisitely regulated by ubiquitination and proteasomal action, little is known about posttranscriptional aspects of its biogenesis. In a yeast-based screen designed to identify human proteins that interact with human cyclin E, we identified components of the eukaryotic cytosolic chaperonin CCT. We found that the endogenous CCT complex in yeast was essential for the maturation of cyclin E in vivo. Under conditions of impaired CCT function, cyclin E failed to accumulate. Furthermore, newly translated cyclin E, both in vitro in reticulocyte lysate and in vivo in human cells in culture, is efficiently bound and processed by the CCT. In vitro, in the presence of ATP, the bound protein is folded and released in order to become associated with Cdk2. Thus, both the acquisition of the native state and turnover of cyclin E involve ATP-dependent processes mediated by large oligomeric assemblies.

An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3985-3987 ◽  
Author(s):  
Mu-Shui Dai ◽  
Charlie R. Mantel ◽  
Zhen-Biao Xia ◽  
Hal E. Broxmeyer ◽  
Li Lu
Keyword(s):  
Cell Cycle ◽  
Cord Blood ◽  
Cyclin E ◽  
Erythroid Differentiation ◽  
S Phase ◽  
Cyclin D3 ◽  
G1 Phase ◽  
Cyclin Dependent Kinase ◽  
Terminal Erythroid Differentiation

Abstract The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34+cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)–derived erythroblasts from a 7-day culture of CD34+ cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.

scholarly journals p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.

1997 ◽  
Vol 17 (7) ◽  
pp. 3566-3579 ◽  
Author(s):  
M S Woo ◽  
I Sánchez ◽  
B D Dynlacht
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Kinase Activity ◽  
Cyclin E ◽  
Critical Role ◽  
Cyclin A ◽  
Terminal Region ◽  
Cyclin Dependent Kinase ◽  
Amino Terminal

The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases.

scholarly journals Inhibition of Cyclin-Dependent Kinase Activity and Induction of Apoptosis by Preussin in Human Tumor Cells

2000 ◽  
Vol 44 (10) ◽  
pp. 2794-2801 ◽  
Author(s):  
Tatjana V. Achenbach ◽  
Emily P. Slater ◽  
Harm Brummerhop ◽  
Thorsten Bach ◽  
Rolf Müller
Keyword(s):  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Human Cancer ◽  
Cyclin E ◽  
Human Tumor ◽  
Chemotherapeutic Agents ◽  
Cyclin Dependent Kinase ◽  
Human Cancer Cells ◽  
Induction Of Apoptosis

ABSTRACT In this paper, we report that (+)-preussin, a pyrrolidinol alkaloid originally identified as an antifungal agent, has growth-inhibitory and cytotoxic effects on human cancer cells. Preussin was found to be a potent inhibitor of cyclin E kinase (CDK2-cyclin E) in vitro (50% inhibitory concentration; ∼500 nM) and to inhibit cell cycle progression into S phase. In agreement with these findings, the level of the cyclin-dependent kinase inhibitor p27KIP-1 is increased in response to preussin treatment while the expression of both cyclin A and the transcription factor E2F-1 is down-regulated. Preussin also induces programmed cell death (apoptosis), which requires caspase activation and involves the release of cytochrome cfrom mitochondria. This induction of apoptosis is not blocked by high levels of Bcl-2, which usually confers resistance to chemotherapeutic agents. Taken together, our data indicate that preussin could be a promising lead compound for the development of a new class of potent antitumor drugs.

scholarly journals Combined Luteolin and Indole-3-Carbinol Synergistically Constrains ERα-Positive Breast Cancer by Dual Inhibiting Estrogen Receptor Alpha and Cyclin-Dependent Kinase 4/6 Pathway in Cultured Cells and Xenograft Mice

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2116
Author(s):  
Xiaoyong Wang ◽  
Lijuan Zhang ◽  
Qi Dai ◽  
Hongzong Si ◽  
Longyun Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
Cyclin Dependent Kinase ◽  
Xenograft Mice ◽  
The Individual

The high concentrations of individual phytochemicals in vitro studies cannot be physiologically achieved in humans. Our solution for this concentration gap between in vitro and human studies is to combine two or more phytochemicals. We screened 12 phytochemicals by pairwise combining two compounds at a low level to select combinations exerting the synergistic inhibitory effect of breast cancer cell proliferation. A novel combination of luteolin at 30 μM (LUT30) and indole-3-carbinol 40 μM (I3C40) identified that this combination (L30I40) synergistically constrains ERα+ breast cancer cell (MCF7 and T47D) proliferation only, but not triple-negative breast cancer cells. At the same time, the individual LUT30 and I3C40 do not have this anti-proliferative effect in ERα+ breast cancer cells. Moreover, this combination L30I40 does not have toxicity on endothelial cells compared to the current commercial drugs. Similarly, the combination of LUT and I3C (LUT10 mg + I3C10 mg/kg/day) (IP injection) synergistically suppresses tumor growth in MCF7 cells-derived xenograft mice, but the individual LUT (10 mg/kg/day) and I3C (20 mg/kg/day) do not show an inhibitory effect. This combination synergistically downregulates two major therapeutic targets ERα and cyclin dependent kinase (CDK) 4/6/retinoblastoma (Rb) pathway, both in cultured cells and xenograft tumors. These results provide a solid foundation that a combination of LUT and I3C may be a practical approach to treat ERα+ breast cancer cells after clinical trials.

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