Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 37
Filter
Add more filters










Publication year range
1.
Mol Med Rep ; 16(5): 6340-6345, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28849059

ABSTRACT

The present study aimed to evaluate the inhibitory effects of an irinotecan derivative, ZBH­1208, on brain tumors, and to explore the underlying molecular mechanisms. To determine the effects of ZBH­1208, a brain tumor mouse model was established by transplanting B22 cells. Subsequently, the visceral indices of immune organs and white blood cell counts were determined, and the effects of ZBH­1208 on the expression levels of cell cycle­related proteins were assessed by western blotting. The tumor inhibition rates of 20 and 40 mg/kg ZBH­1208 were 11.7 and 54.1%, respectively. Compared with the negative control group, ZBH­1208 barely affected visceral indices or white blood cell count. Furthermore, the expression levels of p53, p21, cyclin­dependent kinase 7 (CDK7), Wee1, phosphorylated (p)­cell division cycle 2 (CDC2) (Tyr15), p­CDC2 (Thr161) and cyclin B1 proteins were upregulated, whereas the expression levels of cyclin E were downregulated, and those of CDC2, CDK2 and CDC25C were barely altered. In conclusion, the present study demonstrated that ZBH­1208 suppressed the growth of B22 mouse brain tumor xenografts, but did not affect their visceral indices or white blood cell counts. It was suggested that ZBH­1208 exerted its effects by regulating the expression of p53, p21, Wee1, p­CDC2 (Tyr15) and cyclin E proteins.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Brain Neoplasms/drug therapy , Camptothecin/analogs & derivatives , Gene Expression Regulation, Neoplastic , Immunity, Innate/drug effects , Animals , Brain Neoplasms/genetics , Brain Neoplasms/immunology , Brain Neoplasms/pathology , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/immunology , Camptothecin/pharmacology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/immunology , Cell Line, Tumor , Cyclin B1/genetics , Cyclin B1/immunology , Cyclin E/genetics , Cyclin E/immunology , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/immunology , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/immunology , Dose-Response Relationship, Drug , Drug Administration Schedule , Germ-Free Life , Irinotecan , Male , Mice , Mice, Inbred ICR , Neoplasm Transplantation , Nuclear Proteins/genetics , Nuclear Proteins/immunology , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/immunology , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/immunology , Cyclin-Dependent Kinase-Activating Kinase
2.
Methods Mol Biol ; 1342: 337-48, 2016.
Article in English | MEDLINE | ID: mdl-26254935

ABSTRACT

Quantitative measurement of enzyme activity is a valuable approach to study how cells function. We present a method to measure the activity of the enzyme Cdk1/cyclin B. This enzyme is required by all eukaryotic cells to enter mitosis. Therefore, a biochemical assay to measure Cdk1/cyclin B activity can be used to identify cell populations that are in mitosis or to detect inhibitors of Cdk1/Cyclin B in vitro. A key distinction of the method presented here, compared to others, is that it uses a recombinant protein, a specific antibody, and a western blot apparatus, which makes the technique available to cell and molecular biology laboratories who do not wish to use radioisotopes, which are commonly required for other protein kinase assays.


Subject(s)
Antibody Specificity , Blotting, Western/methods , CDC2 Protein Kinase/immunology , CDC2 Protein Kinase/metabolism , Cyclin B/immunology , Cyclin B/metabolism , Enzyme Assays/methods , CDC2 Protein Kinase/antagonists & inhibitors , Cell Cycle Checkpoints/drug effects , Electrophoresis, Polyacrylamide Gel , Humans , Mitosis/drug effects , Protein Kinase Inhibitors/pharmacology
3.
Sci Rep ; 5: 11046, 2015 Jun 16.
Article in English | MEDLINE | ID: mdl-26078204

ABSTRACT

Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.


Subject(s)
Cell Cycle Checkpoints/genetics , Cell Cycle Proteins/genetics , Genome , Helicobacter pylori/immunology , Macrophages/metabolism , Animals , Aurora Kinase B/genetics , Aurora Kinase B/immunology , Bacterial Load , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/immunology , Cell Cycle Checkpoints/immunology , Cell Cycle Proteins/immunology , Cell Line, Transformed , Cell Proliferation , DNA Replication/genetics , DNA Replication/immunology , Gene Expression Profiling , Gene Expression Regulation , Helicobacter pylori/pathogenicity , Immune Evasion , Macrophages/microbiology , Mice , Oligonucleotide Array Sequence Analysis , Phosphorylation
5.
J Biol Chem ; 287(2): 1261-8, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22110135

ABSTRACT

The maintenance of T cell memory is critical for the development of rapid recall responses to pathogens, but may also have the undesired side effect of clonal expansion of T effector memory (T(EM)) cells in chronic autoimmune diseases. The mechanisms by which lineage differentiation of T cells is controlled have been investigated, but are not completely understood. Our previous work demonstrated a role of the voltage-gated potassium channel Kv1.3 in effector T cell function in autoimmune disease. In the present study, we have identified a mechanism by which Kv1.3 regulates the conversion of T central memory cells (T(CM)) into T(EM). Using a lentiviral-dominant negative approach, we show that loss of function of Kv1.3 mediates reversion of T(EM) into T(CM), via a delay in cell cycle progression at the G2/M stage. The inhibition of Kv1.3 signaling caused an up-regulation of SMAD3 phosphorylation and induction of nuclear p21(cip1) with resulting suppression of Cdk1 and cyclin B1. These data highlight a novel role for Kv1.3 in T cell differentiation and memory responses, and provide further support for the therapeutic potential of Kv1.3 specific channel blockers in T(EM)-mediated autoimmune diseases.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cyclin-Dependent Kinase Inhibitor p21/immunology , Immunologic Memory , Kv1.3 Potassium Channel/immunology , Signal Transduction/immunology , Smad3 Protein/immunology , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Autoimmune Diseases/metabolism , CD4-Positive T-Lymphocytes/metabolism , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/immunology , CDC2 Protein Kinase/metabolism , Cell Cycle Checkpoints/genetics , Cell Cycle Checkpoints/immunology , Cell Division/genetics , Cell Division/immunology , Cells, Cultured , Cyclin B1/genetics , Cyclin B1/immunology , Cyclin B1/metabolism , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , G2 Phase/genetics , G2 Phase/immunology , Humans , Kv1.3 Potassium Channel/genetics , Kv1.3 Potassium Channel/metabolism , Phosphorylation/genetics , Phosphorylation/immunology , Signal Transduction/genetics , Smad3 Protein/genetics , Smad3 Protein/metabolism
6.
Immunol Lett ; 116(1): 55-63, 2008 Feb 15.
Article in English | MEDLINE | ID: mdl-18192028

ABSTRACT

OBJECTIVE: The expression of cytokine-associated genes in dendritic cells (DCs) derived from umbilical cord blood (UCB) and adult peripheral blood (APB) was comprehensively compared in order to elucidate the difference in DC function between newborns and adults. STUDY DESIGN: Immature DCs were obtained from UCB and APB of healthy human donors. Several cytokines were added to generate mature DCs. Gene expression was compared using cDNA microarray containing 553 cytokine-associated genes. Eleven genes with differential expression were selected and determined their expression levels in DCs by quantitative real-time RT-PCR. RESULTS: The expression of the Th1 response-related genes (IL-12B and IL-18) and chemokine genes (CXCL9, CXCL13, CCL18 and CCL24) was significantly lower in UCB-DCs than in APB-DC in both maturation states. On the other hand, calgranulins A and B, which are speculated to induce immune tolerance, showed higher expression in UCB-DCs. The expression of cell cycle-related genes (CDC2 and cyclin B1) was significantly higher in UCB-DCs than in APB-DCs, and immature UCB-DCs proliferated more rapidly than immature APB-DCs. CONCLUSION: The expression of genes related to immune responses was significantly different between UCB- and APB-DCs, which may cause a decreased DC-mediated immunity and an increased susceptibility to infection in newborns.


Subject(s)
Dendritic Cells , Fetal Blood , Adult , Apoptosis/genetics , Apoptosis/immunology , Baculoviral IAP Repeat-Containing 3 Protein , Biomarkers/blood , Biomarkers/metabolism , Blood Cells/cytology , Blood Cells/immunology , Blood Cells/metabolism , CDC2 Protein Kinase/blood , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/immunology , Cell Culture Techniques , Cell Cycle/genetics , Cell Cycle/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cyclin B/blood , Cyclin B/genetics , Cyclin B1 , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Fetal Blood/cytology , Fetal Blood/immunology , Fetal Blood/metabolism , Gene Expression Profiling , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Gene Regulatory Networks , Humans , Infant, Newborn , Inflammation/genetics , Inflammation/immunology , Inhibitor of Apoptosis Proteins/blood , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/immunology , Leukocyte L1 Antigen Complex/blood , Leukocyte L1 Antigen Complex/genetics , Leukocyte L1 Antigen Complex/immunology , Oligonucleotide Array Sequence Analysis , Ubiquitin-Protein Ligases
7.
J Immunol ; 179(10): 6485-93, 2007 Nov 15.
Article in English | MEDLINE | ID: mdl-17982037

ABSTRACT

CCR7 and its ligands, CCL19 and CCL21, are responsible for directing the migration of T cells and dendritic cells into lymph nodes, where these cells play an important role in the initiation of the immune response. Recently, we have shown that systemic application of CCL19-IgG is able to inhibit the colocalization of T cells and dendritic cells within secondary lymphoid organs, resulting in pronounced immunosuppression with reduced allograft rejection after organ transplantation. In this study, we demonstrate that the application of sustained high concentrations of either soluble or immobilized CCL19 and CCL21 elicits an inhibitory program in T cells. We show that these ligands specifically interfere with cell proliferation and IL-2 secretion of CCR7(+) cells. This could be demonstrated for human and murine T cells and was valid for both CD4(+) and CD8(+) T cells. In contrast, CCL19 had no inhibitory effect on T cells from CCR7 knockout mice, but CCR7(-/-) T cells showed a proliferative response upon TCR-stimulation similar to that of CCL19-treated wild-type cells. Furthermore, the inhibition of proliferation is associated with delayed degradation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) and the down-regulation of CDK1. This shows that CCR7 signaling is linked to cell cycle control and that sustained engagement of CCR7, either by high concentrations of soluble ligands or by high density of immobilized ligands, is capable of inducing cell cycle arrest in TCR-stimulated cells. Thus, CCR7, a chemokine receptor that has been demonstrated to play an essential role during activation of the immune response, is also competent to directly inhibit T cell proliferation.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Cycle/immunology , Cell Proliferation , Chemokine CCL19/immunology , Chemokine CCL21/immunology , Receptors, CCR7/immunology , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , CDC2 Protein Kinase/immunology , CDC2 Protein Kinase/metabolism , Cell Cycle/drug effects , Cell Movement/drug effects , Cell Movement/genetics , Cell Movement/immunology , Cell Proliferation/drug effects , Chemokine CCL19/metabolism , Chemokine CCL19/pharmacology , Chemokine CCL21/metabolism , Chemokine CCL21/pharmacology , Cyclin-Dependent Kinase Inhibitor p27/immunology , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Dose-Response Relationship, Drug , Dose-Response Relationship, Immunologic , Down-Regulation/drug effects , Down-Regulation/genetics , Down-Regulation/immunology , Graft Rejection/immunology , Graft Rejection/metabolism , Immune Tolerance/drug effects , Immune Tolerance/genetics , Immunoglobulin G/immunology , Immunoglobulin G/pharmacology , Interleukin-2/immunology , Interleukin-2/metabolism , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Organ Transplantation , Receptors, CCR7/genetics , Receptors, CCR7/metabolism , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacology , Transplantation, Homologous
8.
Int J Cancer ; 119(12): 2784-94, 2006 Dec 15.
Article in English | MEDLINE | ID: mdl-17019715

ABSTRACT

The majority of cancer therapeutics induces DNA damage to kill cells. Normal proliferating cells undergo cell cycle arrest in response to DNA damage, thus allowing DNA repair to protect the genome. DNA damage induced cell cycle arrest depends on an evolutionarily conserved signal transduction network in which the Chk1 kinase plays a critical role. In mammalian cells, the p53 and RB pathways further augment the cell cycle arrest response to prevent catastrophic cell death. Given the fact that most tumor cells suffer defects in the p53 and RB pathways, it is likely that tumor cells would depend more on the Chk1 kinase to maintain cell cycle arrest than would normal cells. Therefore Chk1 inhibition could be used to specifically sensitize tumor cells to DNA-damaging agents. We have previously shown that siRNA-mediated Chk1 knockdown abrogates DNA damage-induced checkpoints and potentiates the cytotoxicity of several DNA-damaging agents in p53-deficient cell lines. In this study, we have developed 2 potent and selective Chk1 inhibitors, A-690002 and A-641397, and shown that these compounds abrogate cell cycle checkpoints and potentiate the cytotoxicity of topoisomerase inhibitors and gamma-radiation in p53-deficient but not in p53-proficient cells of different tissue origins. These results indicate that it is feasible to achieve a therapeutic window with 1 or more Chk1 inhibitors in potentiation of cancer therapy based on the status of the p53 pathway in a wide spectrum of tumor types.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Tumor Suppressor Protein p53/deficiency , Urea/analogs & derivatives , Antibodies/pharmacology , Blotting, Western , CDC2 Protein Kinase/immunology , CDC2 Protein Kinase/metabolism , Camptothecin/pharmacology , Caspases/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cell Survival/drug effects , Cell Survival/radiation effects , Checkpoint Kinase 1 , DNA Damage , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Drug Synergism , HeLa Cells , Humans , Molecular Structure , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinases/genetics , Protein Kinases/immunology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/genetics , Time Factors , Tumor Suppressor Protein p53/genetics , Urea/chemistry , Urea/pharmacology , cdc25 Phosphatases/genetics , cdc25 Phosphatases/metabolism
9.
Biol Reprod ; 71(3): 894-900, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15151934

ABSTRACT

The G2 to M phase transition in perch oocytes is regulated by maturation promoting factor (MPF), a complex of Cdc2 and cyclin B. In Anabas testudineus, a fresh water perch, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, the maturation inducing hormone (MIH), induced complete germinal vesicle breakdown (GVBD) of oocytes at 21 h. An unusual cyclin, p30 cyclin B, has been identified in oocyte extract using both monoclonal and polyclonal antibodies. Surprisingly, Cdc2 could not be identified, although a Northern blot with Cdc2 cDNA demonstrated expression of the gene. Purification of MPF through an immunoaffinity column followed by SDS-PAGE showed three proteins, Cdc2, cyclin B, and a 20 kDa fragment, indicating earlier failure in immunodetection may be due to the interference by this fragment. In uninduced oocytes, p30 cyclin B was present, and its expression was increased by MIH. MIH increased p30 cyclin B accumulation at 3 h, a high level which was maintained between 9 and 21 h, but an effective increase in GVBD and H1 kinase activation could only be observed between 15 and 21 h. This delay in active MPF formation was found to be related to the activation of Cdc25, phosphorylation of which was detected at 12 h, and a substantial increase occurred during 15-18 h. Sodium orthovanadate, a tyrosine phosphatase inhibitor, inhibited H1 kinase activity and GVBD, suggesting the requirement of Cdc25 activity in MPF activation. Our results show occurrence of pre-MPF in uninduced oocytes and its conversion to active MPF requires dephosphorylation by Cdc25, the existence of which has not yet been shown in fish.


Subject(s)
CDC2 Protein Kinase/metabolism , Cyclin B/metabolism , Oocytes/enzymology , Perches/physiology , cdc25 Phosphatases/metabolism , Animals , Antibodies, Monoclonal , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/immunology , Cell Division/physiology , Female , G2 Phase/physiology , Maturation-Promoting Factor/genetics , Maturation-Promoting Factor/metabolism , Oocytes/cytology , Phosphorylation , Protein Kinases/metabolism
10.
Plant Cell Rep ; 22(12): 878-84, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15042411

ABSTRACT

We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.


Subject(s)
Antibodies/metabolism , Calreticulin/immunology , Plants/immunology , Protoplasts/immunology , Spectrin/immunology , Animals , Antibodies/pharmacology , Arabidopsis/immunology , Biological Transport , CDC2 Protein Kinase/immunology , Cell Line , Cells, Cultured , Chickens , Helianthus/physiology , Humans , Protoplasts/drug effects , Nicotiana/immunology , Tubulin/immunology
12.
Eur J Biochem ; 267(23): 6775-83, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11082187

ABSTRACT

In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single-chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N-terminally fused to a His6-tag (His-CDC2aAt) and the other was made out of an anti-PSTAIRE hybridoma cell line. Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western blots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen-binding activity of these scFv fragments in a reducing environment were assessed. No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two-hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen-binding activity.


Subject(s)
Antibodies, Monoclonal/chemistry , Arabidopsis Proteins , Arabidopsis/chemistry , Arabidopsis/immunology , CDC2 Protein Kinase/immunology , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/immunology , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/isolation & purification , Amino Acid Sequence , Animals , Blotting, Western , CDC2 Protein Kinase/chemistry , Codon, Terminator , Enzyme-Linked Immunosorbent Assay , Epitopes , Escherichia coli/metabolism , Hybridomas , Mice , Molecular Sequence Data , Peptide Library , Peptides/chemistry , Protein Binding , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Tumor Cells, Cultured , Two-Hybrid System Techniques
13.
Indian J Biochem Biophys ; 35(4): 193-9, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9854898

ABSTRACT

A key regulator of the cell cycle is a highly conserved protein kinase whose catalytic subunit, p34cdc2, is encoded by cdc2 gene. Immunoblotting with a polyclonal antibody raised against PSTAIRE sequence (found in the N-terminal region of all cdc2 and cdc2 related proteins throughout the phylogenetic scale including higher plants), was used to study the presence of p34cdc2 in onion scale leaves and root tip cells. p34cdc2 homologues are beyond the detection level in scale leaves. PSTAIRE antibody was used to estimate p34cdc2 kinase protein levels during cell cycle in highly synchronous population of Allium cepa L. root meristem cells. p34cdc2 kinase protein showed gradual increase in their levels from S phase to G2 phase boundary. Immunoprecipitation followed by in vitro histone H1 kinase assays also depicted that its kinase activity increased parallel to the increase in p34cdc2 level.


Subject(s)
CDC2 Protein Kinase/metabolism , Onions/cytology , Onions/metabolism , Amino Acid Sequence , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/immunology , Cell Cycle , Epitopes/genetics , HeLa Cells , Humans , Meristem/cytology , Meristem/metabolism , Molecular Sequence Data
14.
J Neurosci ; 17(10): 3588-98, 1997 May 15.
Article in English | MEDLINE | ID: mdl-9133382

ABSTRACT

We have shown previously that M-phase phospho-epitopes accumulate in neuronal tau proteins incorporated into the hallmark neurofibrillary tangles (NFT) of Alzheimer's disease (AD). In M phase, the epitopes are produced by cdc2/cyclin B1 kinase by a highly conserved mechanism believed to be quiescent in terminally differentiated neurons of adult brain. To determine whether an M-phase mechanism is possible in AD neurons, we first investigated the presence of cdc2 and cyclin B1 in AD. Both proteins were enriched in neurons with NFT and in neurons susceptible to NFT. An antibody specific for catalytically active cdc2 stained numerous NFT-containing neurons in AD but did not react with normal neurons. Double-labeling studies showed that active cdc2 and cyclin B1 coexist in AD neurons and co-localize with AD-specific mitotic phospho-epitopes. Mitotic kinase purified from AD and normal brain, using the yeast p13suc1 protein as affinity ligand, showed higher histone H1 phosphorylation activity in AD. Accordingly, the levels of cdc2 and cyclin B1 in p13suc1 fractions from AD were higher than normal. Consistent with a physiological relationship between NFT and mitotic kinase, NFT proteins co-purified with and became phosphorylated by the p13suc1-bound kinase in vitro. Furthermore, cdc2/cyclin B1 is the only one of several proline-directed kinases that created the TG/MC mitotic phospho-epitopes in recombinant tau in vitro. These findings suggest that aberrantly reexpressed cdc2/cyclin B1 in NFT-bearing neurons in AD brain contributes to the generation of M-phase phospho-epitopes in NFT.


Subject(s)
Alzheimer Disease/metabolism , CDC2 Protein Kinase/metabolism , Cyclin B , Cyclin-Dependent Kinases , Cyclins/metabolism , Neurons/enzymology , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal , CDC2 Protein Kinase/biosynthesis , CDC2 Protein Kinase/immunology , Cross Reactions , Cyclin B1 , Cyclin-Dependent Kinase 5 , Cyclins/biosynthesis , Cyclins/immunology , Epitopes/analysis , Epitopes/metabolism , Humans , Middle Aged , Mitosis/physiology , Nerve Degeneration/physiology , Neurofibrillary Tangles/chemistry , Neurofibrillary Tangles/metabolism , Neurons/pathology , Phosphorylation , Protein Serine-Threonine Kinases/immunology , Recombinant Proteins/analysis , Recombinant Proteins/immunology , tau Proteins/immunology , tau Proteins/metabolism
15.
Chromosoma ; 105(6): 349-59, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9087377

ABSTRACT

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex.


Subject(s)
CDC2-CDC28 Kinases , Cell Cycle/genetics , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DNA Replication/physiology , Protein Serine-Threonine Kinases/metabolism , Simian virus 40/genetics , Animals , Antigens, Viral, Tumor/genetics , Antigens, Viral, Tumor/metabolism , CDC2 Protein Kinase/genetics , CDC2 Protein Kinase/immunology , CDC2 Protein Kinase/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinases/genetics , Cyclins/genetics , Cyclins/immunology , Cyclins/isolation & purification , DNA Polymerase II/genetics , DNA Polymerase II/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , G1 Phase/genetics , Precipitin Tests , Protein Serine-Threonine Kinases/genetics , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Replication Origin , Replication Protein A , S Phase/genetics , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication
16.
J Cell Physiol ; 169(1): 149-58, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8841431

ABSTRACT

Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these activated extracts, we previously showed that purified, bacterially expressed oncogenic human RasH protein arrests cell cycle progression. Because oncogenic Ras activates many serine/threonine protein kinases in Xenopus oocytes and egg extracts, it is possible that induction of cell cycle arrest involves the action of oncogenic Ras-activated kinases. Thus, the identification of the physiological substrates for oncogenic Ras-activated kinases is important for elucidating the molecular mechanism underlying oncogenic Ras-induced cell cycle arrest. We used 32P-orthophosphate as a label to identify the potential substrates. Our results demonstrated that the 32P-labeling of both a 32 and a 33 kDa protein were greatly enhanced by oncogenic Ras during the incubation of activated Xenopus egg extracts. The enhanced labeling correlated with the induced cell cycle arrest and was contributed by serine phosphorylation. Moreover, the 33 kDa protein was detected only in the presence of oncogenic Ras and was a serine-hyperphosphorylated form of the 32 kDa protein. Furthermore, new protein synthesis was not required for the enhanced labeling, consistent with the concept that the enhanced serine phosphorylation of the 32 kDa protein is by oncogenic Ras-activated protein kinases. In addition to serine phosphorylation, our results also suggested that an as yet unidentified modification of the 32 kDa protein might also be induced by oncogenic Ras. Our results suggest that the 32 kDa protein is a potential physiological substrate for oncogenic Ras-activated protein kinases.


Subject(s)
Ovum/chemistry , Protein Kinases/physiology , Tissue Extracts/analysis , Tyrosine 3-Monooxygenase , ras Proteins/pharmacology , 14-3-3 Proteins , Animals , Antibodies/immunology , CDC2 Protein Kinase/immunology , Cell Cycle/drug effects , Humans , Isomerism , Molecular Weight , Phosphorylation/drug effects , Proteins/chemistry , Proteins/immunology , Proteins/metabolism , Serine/metabolism , Substrate Specificity , Xenopus
17.
Cancer Biother Radiopharm ; 11(4): 259-66, 1996 Aug.
Article in English | MEDLINE | ID: mdl-10851546

ABSTRACT

We here describe an alternative way to microinjection by which cellular transport of immunoglobulins through surface membranes can be achieved after binding to specific surface receptors either induced or constutively present, or via Fc receptors (Ig-mediated). In this report, the internalisation of two antibodies in two different cellular systems is analysed: the anti-p21ras monoclonal antibody (MoAb) after surface Ig binding on murine placental cells and anti-cdc2 MoAb that binds directly to its surface receptor expressed on the human promyelocytic leukemia cell line HL-60. In both cases, binding and internalisation is followed by Electron Microscopy (EM) and function is assessed by different assays. The first involves abrogation of class II antigen expression induced by Interferon-gamma (IFN-gamma) and 5-Azacytidine (5-AzaC) known to be mediated by activation of the ras pathway. The second involves growth cessation of HL-60 cells after antibody adsorption when a G1-S-specific culture supernatant containing anti-cdc2 activity is employed, whereas no growth hindrance is observed when a G2-M-specific anti-cdc2 MoAb is used. Thus, the antibodies do not follow the lysosyme pathway and do not lose their functional activity. This method may be applied in the future in order to achieve biological or clinical therapies.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Endocytosis , Adsorption , Animals , Azacitidine/pharmacology , CDC2 Protein Kinase/immunology , Female , HL-60 Cells , Histocompatibility Antigens Class II/metabolism , Humans , Interferon-gamma/pharmacology , Male , Mice , Mice, Inbred BALB C , Microinjections , Proto-Oncogene Proteins p21(ras)/immunology
18.
J Cell Sci ; 109 ( Pt 6): 1565-73, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8799843

ABSTRACT

An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phosphorylation of NF-H by the cdc2-like activity was not affected by 3 microM olomoucine but was inhibited by 10 microM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.


Subject(s)
Brain/enzymology , CDC2 Protein Kinase/isolation & purification , Cyclin-Dependent Kinases , Neurofilament Proteins/isolation & purification , Protein Serine-Threonine Kinases/isolation & purification , Animals , CDC2 Protein Kinase/chemistry , CDC2 Protein Kinase/immunology , Cells, Cultured , Cyclin-Dependent Kinase 5 , Ganglia, Spinal/enzymology , Immunoblotting , Immunochemistry , Intermediate Filaments/chemistry , Intermediate Filaments/enzymology , Mice , Molecular Weight , Phosphorylation , Precipitin Tests , Rats
19.
Int J Hematol ; 63(3): 235-9, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8936337

ABSTRACT

Since malignant cell proliferation occurs at the expense of cell differentiation, we examined the possibility of differentiating malignant cells from mature cells by altering the regulation of the cell cycle. Using the human promyelocytic leukemia cell line HL-60, which expresses p34cdc2, the product of the cdc2 gene that controls the cell cycle, we showed that guided internalization of an anti-cdc2 monoclonal antibody could initiate differentiation not yet described for other cells of myeloid malignant origin. Experimental cellular manipulation may be employed as a method of inducing in vitro differentiation for transplantation purposes.


Subject(s)
Leukemia, Promyelocytic, Acute/pathology , Antibodies, Monoclonal/pharmacology , CDC2 Protein Kinase/immunology , Cell Cycle/drug effects , Cell Differentiation , HL-60 Cells/enzymology , HL-60 Cells/pathology , Humans , Leukemia, Promyelocytic, Acute/enzymology
20.
J Immunol ; 155(12): 5479-82, 1995 Dec 15.
Article in English | MEDLINE | ID: mdl-7499827

ABSTRACT

Intercellular adhesion molecules (ICAM)-1 and -3 coexist on T lymphocytes and are counter-receptors for the integrin LFA-1. Signaling through ICAM-3 stimulates a number of T cell functions and involves phosphorylation of Fyn, Lck, CD45, and other proteins. In contrast, this type of specific signaling event has not been described for signaling through ICAM-1. Here, tyrosine phosphorylation of cellular proteins was examined after cross-linking of ICAM-1. Tyrosine phosphorylation of the 34-kDa cdc2 protein kinase was induced transiently after stimulation of the leukemic T cell line, Molt-3, or peripheral blood T cells. Stimulation through ICAM-1 had no effect on constitutive presence of cdc2 or phosphorylation of cdc2 on threonine. cdc2 kinase activity was constitutive in peripheral blood T cells, and transient inhibition of kinase activity after ICAM-1 stimulation correlated kinetically with phosphorylation of cdc2 on tyrosine.


Subject(s)
Antigens, CD , Antigens, Differentiation , CDC2 Protein Kinase/antagonists & inhibitors , Intercellular Adhesion Molecule-1/metabolism , Protein-Tyrosine Kinases/metabolism , T-Lymphocytes/enzymology , Antibodies, Monoclonal/immunology , CDC2 Protein Kinase/immunology , CDC2 Protein Kinase/metabolism , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/pharmacology , Cell Line , Humans , Intercellular Adhesion Molecule-1/pharmacology , Phosphorylation/drug effects , Protein-Tyrosine Kinases/immunology , T-Lymphocytes/immunology
SELECTION OF CITATIONS
SEARCH DETAIL
...