In an oxygen-depleted environment, endothelial cells initiate an adaptive pattern of synthesis, which may enable them to survive hypoxic crises. Using high-resolution two-dimensional gel electrophoresis in conjunction with mass spectroscopy, we obtained a 24 differential display of proteins in the pancreatic endothelial cell line, MS-1, at four time points following induction of hypoxia. The induction of Wee1 under hypoxia was confirmed both at the mRNA and protein levels. The phosphorylation of cell division cycle 2, which is downstream of Wee1, was also increased after hypoxic exposure. In addition, pre-exposure to hypoxia attenuated a decrease in hydrogen peroxide-induced cell number. The induction of bax (a pro-apoptotic protein) and reduction of bcl (an anti-apoptotic protein) after hypoxia stimulus were also attenuated by hypoxic pre-exposure. Moreover, hydrogen peroxide-induced morphologic damage did not appear in the wild-type Wee1-expressing cells. Taken together, our results suggest that Wee1 may have important role in hypoxia-induced pathophysiological situations in endothelial cells.
Animals ; CDC2 Protein Kinase/metabolism ; Cell Cycle Proteins/*genetics/metabolism ; Cell Hypoxia ; Cell Line ; Cell Survival ; Endothelial Cells/cytology/*metabolism ; *Gene Expression Regulation ; Hydrogen Peroxide/*metabolism ; Mice ; Nuclear Proteins/*genetics/metabolism ; Pancreas/cytology ; Phosphorylation ; Protein-Tyrosine Kinases/*genetics/metabolism

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45γ [corrected], revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45γ [corrected] identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45γ [corrected] is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.
Amino Acid Motifs ; Animals ; Apoptosis ; radiation effects ; CDC2 Protein Kinase ; Cell Cycle ; Cell Survival ; Crystallography, X-Ray ; Cyclin B ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinases ; HeLa Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Mice ; Mutagenesis, Site-Directed ; Mutation, Missense ; Proliferating Cell Nuclear Antigen ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Protein Structure, Quaternary ; Ultraviolet Rays

Acute Lung Injury ; chemically induced ; metabolism ; pathology ; Animals ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle ; drug effects ; Checkpoint Kinase 1 ; Female ; Lung ; metabolism ; pathology ; ultrastructure ; Male ; Malondialdehyde ; metabolism ; Microscopy, Electron, Transmission ; Organometallic Compounds ; toxicity ; Oxidative Stress ; Protein Kinases ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVEOur previous cDNA array data have shown that expression level of CDK1 increased along with the malignant progression of ganglioglioma, and decreased with the differentiation process of neural stem cells. The purpose of this study was to investigate the CDK1 expression levels in gliomas and the effects of CDK1 knockdown on phenotype of glioma cells.

METHODSGlioma tissue array was constructed, which was composed of surgical specimens of gliomas with different malignancy grades, glioma xenografts in nude mice, cellular spheroids of brain tumor stem cells, normal neural stem cells and glioma cell line. CDK1 expression was detected in glioma tissue array with immunohistochemical techniques. CDK1 expression in human brain glioma cell line and relevant xenogeneic graft tumor was inhibited by retroviral vectors expressing short hairpin RNAs (shRNAs). Both in vitro and in vivo changes of biological characteristics were further observed.

RESULTSThe expression level of CDK1 increased along with the malignancy progression of glioma in clinical specimens. The positive expression rates of CDK1 in human brain glioma tissues were 22.2% (grade I), 40.0% (grade II), 69.6% (grade III) and 78.6% (grade IV), P = 0.01, respectively. The positive expression rate of CDK1 in glioma cell line and implanted xenografts was similar as the clinical tumors with high malignancy, and higher than those in neural stem cells and brain tumor stem cells (P = 0.0014). Expression of CDK1 was high in human fetal brain tissues and bone marrows of nude mice, but low in normal adult human brain tissues. Downregulation of CDK1 inhibited the proliferation activities notably both in SHG-44 cells in vitro and relevant xenogeneic graft tumors, and induced apoptosis of tumor cells prominantly as well.

CONCLUSIONOverexpression of CDK1 may promote oncogenesis and progression of human gliomas. Downregulation of CDK1 expression can inhibit the proliferation activities of human malignant gliomas.

Animals ; Apoptosis ; drug effects ; Astrocytoma ; genetics ; metabolism ; pathology ; Brain Neoplasms ; genetics ; metabolism ; pathology ; Brain Stem Neoplasms ; metabolism ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Ganglioglioma ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Glioma ; genetics ; metabolism ; pathology ; Humans ; Mice ; Mice, Nude ; Neoplasm Staging ; Neoplasm Transplantation ; RNA, Messenger ; metabolism

The acquisition of metastasis potential is a critical point for malignant tumors. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a potential tumor suppress gene and frequently down-regulated in malignant tumors. It has been implicated that overexpression of MDA-7 led to proliferation inhibition in many types of human tumor. Invasion is an important process which is potential to promote tumor metastasis. However, the role and potential molecular mechanism of mda-7/IL-24 to inhibit the invasion of human melanoma cancer is not fully clear. In this report, we identified a solid role for mda-7/IL-24 in invasion inhibition of human melanoma cancer LiBr cells, including decreasing of adhesion and invasion in vitro, blocking cell cycle, down-regulating the expression of ICAM-1, MMP-2/9, CDK1, the phosphorylation of ERK and Akt, NF-kappaB and AP-1 transcription activity. Meanwhile, there was an increased expression of PTEN in mda-7/IL-24 over-expression LiBr cells. Our results demonstrated that mda-7/IL-24 is a potential invasion suppress gene, which inhibits the invasion of LiBr cells by the down-regulation of ICAM-1, MMP-2/9, PTEN, and CDK1 expression. The molecular pathways involved were the MAPK/ERK, PI3K-Akt, NF-kappaB, and AP-1. These findings suggest that mda-7/IL-24 may be used as a possible therapeutic strategy for human melanoma cancer.
CDC2 Protein Kinase/genetics/metabolism ; Cell Line, Tumor ; Cell Movement ; Down-Regulation ; G2 Phase Cell Cycle Checkpoints ; Humans ; Intercellular Adhesion Molecule-1/genetics/metabolism ; Interleukins/genetics/*metabolism ; M Phase Cell Cycle Checkpoints ; Matrix Metalloproteinase 2/genetics/metabolism ; Matrix Metalloproteinase 9/genetics/metabolism ; Melanoma/metabolism/pathology ; NF-kappa B/genetics/metabolism ; PTEN Phosphohydrolase/genetics/metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt/genetics/metabolism ; Transcription Factor AP-1/genetics/metabolism ; Up-Regulation

OBJECTIVETo explore the molecular elements responsible for the enhanced apoptotic susceptibility in U937 cells mediated by silencing atactic telangiectasis mutation (ATM) gene.

METHODSTwo U937 cell mutants, U937-ASPI3K (ATM gene negative) and U937-pZEOSV2 (+) (ATM gene positive) were used as a cell model system. Apoptosis was examined by measuring free nucleosome concentrations in U937 cells. Western blotting was employed to measure nuclear protein abundances of cdc25A, cdc25B, cdc25C, total p34cdc2, p34cdc2 (Thr161) or p34cdc2 (Thr14, Tyr15). RT-PCR was used to estimate cdc25 transcript levels.

RESULTSU937-ASPI3K exhibited an enhanced apoptotic susceptibility to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine-theronine phosphatase inhibitor or cyclin-dependent kinase (CDK) inhibitors could abolish the enhancement. Upon irradiation, p34cdc2 in U937-pZEOSV2 (+) was in an inactive state owing to phosphorylation of Thr 14 and Tyr15, which was associated with a dramatic decrease of nuclear cdc25A, cdc25B and cdc25C poteins. In contrast, p34cdc2 in U937-ASPI3K was in an active state owing to the low phosphorylation of Thr14 and Tyr15, which was associated with constant nuclear cdc25A, cdc25B and cdc25C protein abundance before and after irradiation. The responsive decrease of nuclear cdc25 proteins occurred at the post-transcription level.

CONCLUSIONSilencing ATM gene blocks the irradiation induced responsive decrease of nuclear cdc25 proteins, resulting in an abnormal activation p34cdc2 is the critical molecular mechanism for the enhanced apoptotic responses.

Apoptosis ; drug effects ; genetics ; radiation effects ; Ataxia Telangiectasia Mutated Proteins ; Blotting, Western ; CDC2 Protein Kinase ; genetics ; metabolism ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin-Dependent Kinases ; genetics ; metabolism ; Cycloheximide ; pharmacology ; DNA-Binding Proteins ; Enzyme Inhibitors ; pharmacology ; Gene Silencing ; drug effects ; radiation effects ; Humans ; Nuclear Proteins ; genetics ; metabolism ; Oxazoles ; pharmacology ; Phosphoprotein Phosphatases ; antagonists & inhibitors ; Protein Synthesis Inhibitors ; pharmacology ; Protein-Serine-Threonine Kinases ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Proteins ; U937 Cells ; cdc25 Phosphatases ; genetics ; metabolism

Silencing ATM gene gave rise to enhanced apoptotic response to irradiation and irradiation-like chemotherapy agents, this paper explored the crucial identities of the molecular elements responsible for the enhanced apoptotic response in U937 cells mediated by silencing ATM gene. Two U937 cell mutants named U937-ASPI3K (ATM, negative) and U937-pZeosv2(+) (ATM, wild-type) were used as a cell model system to identify the critical molecule(s) responsible for the varied apoptotic response in the absence or presence of ATM gene. Apoptosis was examined by measuring concentrations of free nucleosome in U937 cells. Western blot was employed to measure nuclear protein abundance of CDC25A, CDC25B, CDC25C, total p34cdc2, p34cdc2, (Thr 161) or p34cdc2 (Thr 14, Tyr 15). RT-PCR was used to estimate CDC25 transcript levels. U937-ASPI3K exhibited an enhanced apoptotic response to lower dosage of irradiation, which could not be blocked by protein synthesis inhibitor. Protein serine-threonine phosphatase inhibitor or cyclin-dependent kinase (CDK) inhibitors, on the other hand, abolished the enhancement indicated that protein phosphorylation/dephosphorylation modification and CDK activity are required for the enhanced apoptotic response in the absence of ATM gene. Upon irradiation, p34cdc2 in U937-pZeosv2(+) was maintained in an inactive state by phosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with a dramatic decrease of nuclear CDC25A, CDC25B and CDC25C proteins. In contrast, p34cdc2 in U937-ASPI3K maintained in an active state by dephosphorylation on threonine 14 (Thr 14) and tyrosine 15 (Tyr 15), which was associated with constant nuclear CDC25A, CDC25B and CDC25C protein abundance before and after irradiation. The responsive decrease of nuclear CDC25 proteins occurred at the post-transcription level. Silencing ATM gene blocks the responsive decrease of nuclear CDC25 proteins, which is responsible for failure to inactivate p34cdc2 after irradiation. Active p34cdc2 and CDK2, in turn, acts as the death executors to trigger apoptosis. In summary, aberrantly activated CDK activity is the critical molecular mechanism central to enhanced apoptotic responses in the absence of ATM gene.
Apoptosis ; drug effects ; genetics ; radiation effects ; Ataxia Telangiectasia Mutated Proteins ; CDC2 Protein Kinase ; metabolism ; Cell Cycle Proteins ; Cyclin-Dependent Kinases ; metabolism ; DNA-Binding Proteins ; Gene Silencing ; drug effects ; radiation effects ; Humans ; Protein-Serine-Threonine Kinases ; genetics ; Tumor Suppressor Proteins ; U937 Cells ; cdc25 Phosphatases ; metabolism

The aim of this study was to investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with arsenic trioxide (As2O3) by using cDNA microarray. cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without arsenic trioxide. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 201 different human genes, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in the gene expression profile. The results showed that after the treatment of arsenic trioxide (2 micromol/L), 6 genes were up-regulated, and 12 genes related to apoptosis and signal transduction were down-regulated. The p21, survivin, cdc2 and Wee1Hu genes may be related to the differentiation and/or apoptosis of NB4 cells induced by As2O3. It is concluded that p21, survivin, cdc2 and Wee1Hu may play an important role in the mechanism underling arsenic trioxide-mediated NB4 cell apoptosis.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; genetics ; Arsenicals ; pharmacology ; CDC2 Protein Kinase ; Cell Line, Tumor ; Cyclin B ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinases ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Promyelocytic, Acute ; pathology ; Microtubule-Associated Proteins ; metabolism ; Oxides ; pharmacology

OBJECTIVETo investigate the essential role and mechanism of TRPC6 gene in the development of gastric cancer.

METHODSThe expression of TRPC6 protein was assessed in gastric cancer tissues and normal tissues adjacent to the cancer from 30 patients with gastric cancer. The inhibiting effect of TRPC6 activity on cell growth, cell cycle of a human gastric cancer cell line AGS cells, tumor progression and development of xenografted human gastric cancer in a mouse model was tested using dominant-negative mutant TRPC6 (DNC6). The survival of mice bearing xenografted tumors in the GFP and DNC6 was compared using Kaplan-Meier analysis. All statistical tests were two-sided.

RESULTSThe TRPC6 protein in the tumor tissues and para-tumor tissues was (21.60 ± 8.32)% versus (7.14 ± 2.24)%. After transfection of DNC6 virus for 24 hours, 48 hours, 72 hours and 96 hours, the growth inhibition rates of gastric cancer cells were (36.90 ± 1.13)%, (44.06 ± 2.17)%, (52.12 ± 2.76)% and (50.89 ± 1.97)%, respectively. The clone formation rates of control group and DNC6 group were (14.70 ± 3.00)% versus (43.80 ± 7.00)%. After transfection with DNC6 virus for 0, 24, 36 and 48 hours, the G(2)/M phase arrest was (20.34 ± 1.98)%, (24.31 ± 2.37)%, (27.70 ± 2.36)%, (35.10 ± 3.0)% in the DNC6 group and (18.40 ± 2.01)%, (18.0% ± 1.72)%, (17.50 ± 1.74)%, (16.80 ± 1.71)% in the control group, respectively. Inhibition of TRPC6 activity also reduced the subcutaneous tumor volume in the mouse models with xenografted human tumors (P < 0.05).

CONCLUSIONIn the preclinical models tested, TRPC6 channels are essential for gastric cancer development via regulation of G(2)/M phase transition.

Adenoviridae ; genetics ; Animals ; CDC2 Protein Kinase ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin B ; metabolism ; Cyclin-Dependent Kinases ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Recombinant Proteins ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; TRPC Cation Channels ; metabolism ; TRPC6 Cation Channel ; Transfection ; Tumor Burden

OBJECTIVETo investigate the inhibitory effect of recombinant polypeptide CH50 of fibronectin on invasion and angiogenesis of tumors, and analyze the possible molecular mechanism of the therapeutic effect of polypeptide CH50 on tumors.

METHODSThe tumor model was established by inoculation of H22 hepatocarcinoma cells in mice. The tumor gene therapy was performed by in vivo gene transfection with a method based on hydrodynamics to express polypeptide CH50. After treatment, the inhibitory effect on tumor invasion and angiogenesis was observed by histotology with HE staining of tumor tissues. The expresison of MMP-9 mRNA and protein at the edge of tumor tissue was evaluated by RT-PCR and gelatin zymography, respectively. RT-PCR was used to detect the expression of the related genes in H22 cells treated with polypeptide CH50. Cell adhesion assay was used to analyze the influence of polypeptide CH50 on the binding of cells to fibrinogen.

RESULTS(1) Eukaryotic expression plasmid pCH510 was expressed in vivo in a non-targeting manner and produced a significant inhibitory effect on tumor growth. The therapy with polypeptide CH50 resulted in pronounced necrosis of tumor cells in pCH510 group, compared with that in control groups at histological level. (2) Polypeptide CH50 could inhibit the growth, invasion and angiogenesis of the tumor, and interfere the formation of new collateral circulation in the tumor. (3) The expression level of MMP-9 protein at the edge of tumor tissue was significantly decreased after treatment, especially the activation of pro-MMP-9 was inhibited significantly, whereas the expression level of MMP-9 mRNA was not influenced. (4) The expression of alphav, 33 and cdc2 mRNAs in H22 cells treated with polypeptide CH50 was down-regulated. (5) Cell adhesion assay manifested that polypeptide CH50 can affect the adhesion ability of H22 cells.

CONCLUSIONPolypeptide CH50 can inhibit tumor growth and angiogenesis by suppressing the functions of MMP-9 and integrin alphavbeta3.

Animals ; CDC2 Protein Kinase ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; metabolism ; pathology ; therapy ; Cell Adhesion ; genetics ; physiology ; Cell Line, Tumor ; Fibronectins ; biosynthesis ; genetics ; physiology ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; methods ; Humans ; Integrin alphaVbeta3 ; biosynthesis ; genetics ; Liver Neoplasms, Experimental ; metabolism ; pathology ; therapy ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Neovascularization, Pathologic ; genetics ; metabolism ; therapy ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction