ABSTRACT
Polyploid giant cancer cells (PGCCs) are key contributors to cancer heterogeneity, and the formation of PGCCs is associated with changes in the expression of cell-cycle-related proteins. This study investigated the intracellular localization and expression level of multiple cell-cycle-related proteins in PGCCs derived from BT-549 and HEY cells. In addition, the formation of PGCCs and the clinicopathological significance of cell-cycle-related proteins in human breast and ovarian cancer were examined. The expression levels of cell-cycle-related proteins, including cyclin B1, CDC25B, CDC25C, and other cell cycle phosphoproteins, including Chk2, and Aurora-A kinase, were determined using immunostaining and western blotting both in vitro and in vivo. Migration, invasion, and proliferation in control cells, cyclin B1 knockdown cells and their PGCCs following CoCl2 treatment were compared. In addition, human breast and ovarian cancer samples were collected to determine the correlation of number of PGCCs, expression of cell-cycle-related proteins, and tumor pathologic grade and metastasis. Our results confirm that cyclin B1 was localized in the cytoplasm of PGCCs and in the nuclei of their budding daughter cells. The phosphorylated proteins Chk2 and Aurora-A kinase regulated the expression and subcellular localization of cyclin B1, CDC25B, and CDC25C. The rate of positive cytoplasmic staining of cyclin B1 and positive nuclear staining of both CDC25B and CDC25C increased with increase in tumor grade and lymph node metastasis. Cell-cycle-related proteins, including cyclin B1, CDC25B, and CDC25C play an important role in regulating the formation of PGCCs. The inhibition of cyclinB1 and CoCl2 treatment significantly promoted cell proliferation, invasion, and migration abilities. The subcellular localization of these cell-cycle-related proteins was regulated by other cell cycle phosphoproteins, and was associated with pathologic grade and metastasis of tumors in cases of human breast and ovarian cancer.
Subject(s)
Breast Neoplasms , Cell Cycle Proteins/metabolism , Cyclin B1/metabolism , Ovarian Neoplasms , cdc25 Phosphatases/metabolism , Breast Neoplasms/chemistry , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/physiopathology , Cell Cycle Proteins/analysis , Cell Line, Tumor , Cyclin B1/analysis , Female , Humans , Intracellular Space/metabolism , Neoplastic Processes , Ovarian Neoplasms/chemistry , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Ovarian Neoplasms/physiopathology , Polyploidy , cdc25 Phosphatases/analysisABSTRACT
PURPOSE: To investigate the expression of CDC25B, which is a member of the cyclin-dependent kinase activating phosphatase family, in diffuse astrocytoma (DA), anaplastic astrocytoma (AA), glioblastoma multiforme (GBM), pilocytic astrocytoma (PA) and reactive gliosis (RG). Also, to study the relationship of the expression level of CDC25B with clinical parameters and with p53 and Ki-67 proliferation index (PI). METHODS: Tissues were collected from 36 cases diagnosed with astrocytoma (10 DA, 6 AA, 20 GBM), 10 PA, 10 RG and 10 normal brain tissues for controlling purposes. The sections were immunohistochemically stained with CDC25B, Ki-67 and p53. For each marker, 1000 tumor cells were counted and the ratio of positive tumor cells was calculated. RESULTS: The average CDC2B staining index (CSI) was 0.6% in PA, 0.4% in DA , 7.7% in AA and 25.5% in GBM. The increase of CSI in parallel with the increase of WHO grade was significant (p=0.001). No expressions were identified in RG and normal brain. There was also significant relationship between the tumor size and CSI (p=0.027) and also between Ki-67 PI and CSI (p=0.001). Among the groups with low and high CSI in astrocytoma cases, the disease free survival (DFS) was significantly higher in the low CSI group (p=0.0001). CONCLUSIONS: Positive expression of CDC25B in astrocytoma affects the prognosis in an adverse manner. CSI can be used as a diagnostic method and CDC25B may be a possible target molecule for treatment.
Subject(s)
Astrocytoma/chemistry , Brain Neoplasms/chemistry , Brain/metabolism , Gliosis , Ki-67 Antigen/analysis , Tumor Suppressor Protein p53/analysis , cdc25 Phosphatases/analysis , Adolescent , Adult , Astrocytoma/pathology , Astrocytoma/therapy , Brain/pathology , Brain Neoplasms/pathology , Brain Neoplasms/therapy , Cell Proliferation , Chi-Square Distribution , Disease-Free Survival , Female , Glioblastoma/chemistry , Glioblastoma/pathology , Glioblastoma/therapy , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Middle Aged , Multivariate Analysis , Neoplasm Grading , Proportional Hazards Models , Risk Factors , Tumor BurdenABSTRACT
Flavonoids are polyphenolic compounds which display a vast array of biological activities and are among the most promising anti-cancer agents. The derivative of quercetin, 5,7,3'-trihydroxy-3,4'-dimethoxyflavone (THDF), is a natural flavonoid that inhibits cell proliferation and induces apoptosis in human leukemia cells. Here we show that THDF induces cell-cycle arrest in the M phase and inhibits tubulin polymerization. This was associated with the accumulation of cyclin B1 and p21(Cip1) , changes in the phosphorylation status of cyclin B1, Cdk1, Cdc25C, and MPM-2, and activation of the acidic sphingomyelinase (ASMase). Moreover, desipramine attenuated THDF-mediated cell death, indicating a crucial role of ASMase in the mechanism of cell death. In vivo studies on the athymic nude mouse xenograft model also confirmed that THDF inhibits growth of human leukemia cells and suggest that this compound may have therapeutic value.
Subject(s)
Flavones/pharmacology , Sphingomyelins/metabolism , Tubulin Modulators/pharmacology , Animals , Antineoplastic Agents/pharmacology , CDC2 Protein Kinase/analysis , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cyclin B1/metabolism , Desipramine/pharmacology , Female , HL-60 Cells , Humans , Leukemia/drug therapy , Mice , Mice, Nude , Phosphorylation , Polymerization/drug effects , Sphingomyelin Phosphodiesterase/analysis , Tubulin/metabolism , U937 Cells , Xenograft Model Antitumor Assays , cdc25 Phosphatases/analysisABSTRACT
Activation of cyclin-dependent kinase complexes (CDK) at key cell cycle transitions is dependent on their dephosphorylation by CDC25 dual-specificity phosphatases (CDC25A, B and C in human). The CDC25B phosphatase plays an essential role in controlling the activity of CDK1-cyclin B complexes at the entry into mitosis and together with polo-like kinase 1 (PLK1) in regulating the resumption of cell cycle progression after DNA damage-dependent checkpoint arrest in G2. In this study, we analysed the regulation of CDC25B-dependent mitosis entry by PLK1. We demonstrate that PLK1 activity is essential for the relocation of CDC25B from the cytoplasm to the nucleus. By gain and loss of function analyses, we show that PLK1 stimulates CDC25B-induced mitotic entry in both normal conditions and after DNA-damage induced G2/M arrest. Our results support a model in which the relocalisation of CDC25B to the nucleus at the G2-M transition by PLK1 regulates its mitotic inducing activity.
Subject(s)
Cell Cycle Proteins/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , cdc25 Phosphatases/metabolism , Cell Division , Cell Nucleus/metabolism , Cells, Cultured , Cytoplasm/metabolism , DNA Damage , Fluorescent Antibody Technique , G2 Phase , Humans , Transfection , cdc25 Phosphatases/analysis , Polo-Like Kinase 1ABSTRACT
Diets rich in n-3 polyunsaturated fatty acids (PUFAs) have been associated with a reduced risk of several types of cancer. Recent reports have suggested that these PUFAs enhance the cytotoxic effect of cancer chemoradiotherapy. The effect of docosahexaenoic acid (DHA) on key cell cycle regulators and target proteins of cancer therapy was investigated in the human malign colon cancer cell line SW620. Cell cycle check point proteins such as p21 and stratifin (14-3-3 sigma) increased at mRNA and protein level, whereas cell cycle progression proteins such as cell division cycle 25 homolog and cyclin-dependent kinase 1 decreased after DHA treatment. Protein levels of inhibitors of apoptosis family members associated with chemotherapy resistance and cancer malignancy, survivin and livin, decreased after the same treatment: likewise the expression of NF-kappaB. Levels of the proapoptotic proteins phosphorylated p38 MAPK and growth arrest-inducible and DNA damage-inducible gene 153/C/EBP-homologous protein (CHOP) increased. The results indicate that DHA treatment causes simultaneous cell cycle arrest in both the G1 and G2 phase. In conclusion, DHA affects several target proteins of chemotherapy in a favorable way. This may explain the observed enhanced chemosensitivity in cancer cells supplemented with n-3 PUFAs and encourage further studies investigating the role of n-3 PUFAs as adjuvant to chemotherapy and radiotherapy in vivo.
Subject(s)
Colonic Neoplasms/drug therapy , Docosahexaenoic Acids/pharmacology , Apoptosis , CDC2 Protein Kinase/analysis , Cell Line, Tumor , Colonic Neoplasms/chemistry , Colonic Neoplasms/pathology , Drug Resistance, Neoplasm , G1 Phase/drug effects , G2 Phase/drug effects , Humans , Transcription Factor RelA/analysis , cdc25 Phosphatases/analysis , p38 Mitogen-Activated Protein Kinases/analysisABSTRACT
CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.
Subject(s)
Meiosis , Oocytes/cytology , Oocytes/enzymology , cdc25 Phosphatases/metabolism , Animals , Cyclic AMP/metabolism , Female , Gene Expression , Mice , Oocytes/chemistry , cdc25 Phosphatases/analysisABSTRACT
AIM: The aim of this study was to investigate the mechanism of pseudolaric acid B (PLAB)-induced cell cycle arrest in human melanoma SK-28 cells. METHODS: Cell growth inhibition was detected by MTT assay, the cell cycle was analyzed by flow cytometry, and protein expression was examined by Western blot analysis. RESULTS: PLAB inhibited the growth of human melanoma cells and induced G(2)/M arrest in SK-28 cells, accompanied by an up-regulation of Cdc2 phosphorylation and a subsequent down-regulation of Cdc2 expression. Furthermore, PLAB decreased the expression of Cdc25C phosphatase and increased the expression of Wee1 kinase. Meanwhile, a reduction in Cdc2 activity was partly due to induction of the expression of p21(waf1/cip1) in a p53-dependent manner. In addition, PLAB activated the checkpoint kinase, Chk2, and increased the expression of p53, two major targets of ATM kinase. These effects were inhibited by caffeine, an ATM kinase inhibitor. We also found that PLAB significantly enhanced ATM kinase activity. CONCLUSION: Taken together, these results suggest that PLAB induced G(2)/M arrest in human melanoma cells via a mechanism involving the activation of ATM, and the effect of PLAB on Cdc2 activity was mediated via interactions with the Chk2-Cdc25C and p53 signalling pathways, two distinct downstream pathways of ATM. PLAB may be a promising chemopreventive agent for treating human melanoma.
Subject(s)
Cell Cycle Proteins/physiology , Cell Division/drug effects , DNA-Binding Proteins/physiology , Diterpenes/pharmacology , Drugs, Chinese Herbal/pharmacology , G2 Phase/drug effects , Protein Serine-Threonine Kinases/physiology , Signal Transduction/drug effects , Tumor Suppressor Proteins/physiology , Ataxia Telangiectasia Mutated Proteins , CDC2 Protein Kinase , Caffeine/pharmacology , Cell Cycle Proteins/analysis , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 2 , Cyclin B/analysis , Cyclin B1 , Cyclin-Dependent Kinase Inhibitor p21/analysis , Cyclin-Dependent Kinases , Humans , Melanoma/pathology , Nuclear Proteins/analysis , Protein-Tyrosine Kinases/analysis , Tumor Suppressor Protein p53/analysis , cdc25 Phosphatases/analysisABSTRACT
Cdc25 dual-specificity phosphatases coordinate entry into mitosis through activating dephosphorylation of the Mitosis-Promoting Factor, Cdk1-cyclin B1. Activation of Cdc25C at the G2/M transition, involves its dissociation from 14-3-3, together with its hyperphosphorylation on several sites within its regulatory N-terminal domain, mediated by cyclin-dependent kinases and Plk1. Growing evidence suggests that phosphorylation intermediates are likely to precede complete hyperphosphorylation of Cdc25C. To address whether such variants occur in mitotic cells, we raised antibodies directed against different mitotic phosphorylation sites of human Cdc25C, and characterized the phosphorylated species detectable in HeLa cells. In the present study, we provide first-time evidence for the existence of multiple species of Cdc25C in mitotic cell extracts, including full-length and splice variants with different phosphorylation patterns, thereby revealing an intricate network of Cdc25C phosphatases, likely to have distinct biological functions.
Subject(s)
Mitosis , cdc25 Phosphatases/metabolism , Alternative Splicing , Amino Acid Sequence , Antibodies, Phospho-Specific/immunology , Cell Extracts/chemistry , Electrophoresis , HeLa Cells , Humans , Interphase , Molecular Sequence Data , Phosphoproteins/analysis , Phosphorylation , cdc25 Phosphatases/analysis , cdc25 Phosphatases/geneticsABSTRACT
It is clear from a wide range of studies that the nuclear/cytoplasmic distribution of Cdc25C has important functional consequences for cell cycle control. It is now admitted that in somatic cells, the localization of Cdc25C in the cytoplasm is required to maintain the cell in an interphasic state and that Cdc25C has to translocate to the nucleus just before M-phase to induce mitotic events. We characterized the expression and localization of Cdc25C during oocyte maturation, the first embryo mitosis, and the first steps of somatic cell nuclear transfer (SCNT) in cattle. We demonstrated that Cdc25C was expressed throughout the maturation process and the early development. We clearly showed that Cdc25C was localized in the nucleus at the germinal vesicle stage and during the early development until the blastocyst stage. However, the signal change in blastocyst and Cdc25C became cytoplasmic as is the case in somatic cells. Thus, oocytes and early embryonic cells presented a specific nuclear Cdc25C localization different from the one observed in somatic cells, suggesting that Cdc25C could have a particular localization/regulation in undifferentiated cells. Following SCNT, Cdc25C became nuclear as soon as the nucleus swelled, and this localization persisted until the blastocyst stage, as is the case in in vitro fertilized embryos. The Cdc25C nuclear localization appeared to constitute a major change, which could be associated with the reorganization of the somatic nucleus upon nuclear transfer.
Subject(s)
Blastocyst/enzymology , Cell Nucleus/enzymology , Nuclear Transfer Techniques , Oocytes/enzymology , cdc25 Phosphatases/analysis , Animals , Blotting, Western , Cattle , Cytoplasm/enzymology , Female , Fertilization in Vitro , Immunohistochemistry , Microscopy, Fluorescence , PregnancyABSTRACT
BACKGROUND: Cell cycle progression is regulated by cyclin dependent kinases (cdk) and cdk inhibitors. Recent immunohistological studies suggested that dysregulation of cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), and p27(kip1) are of prognostic value in patients with breast cancer. Our study represents the first comprehensive immunohistochemical cell cycle marker analysis for cdc25A, cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), p27(kip1), and pRb in tumor tissue and adjacent benign breast tissue from 69 primarily untreated breast cancer patients. METHODS: Immunhistochemistry using primary monoclonal antibodies to detect cdc 25A, cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), p27(kip1), and pRb has been performed. RESULTS: Sixty-nine patients with untreated, invasive breast cancer (n = 69) were divided into a low/ intermediate and a high risk group according to the St. Gallen 2005 consensus conference. High risk patients (n = 22) had a significantly (p = 0.003) shorter mean and median survival (282.85 weeks; 383.0 weeks, respectively) than low/intermediate risk patients (375.41 weeks; not reached yet, respectively). A subgroup of high risk breast cancer patients characterized in addition by overexpression of cdc25A, cyclin A, cyclin E, p16(ink4a), and p27(kip1) experienced a shortened mean survival of 222.03, 235.71, 257.25, 239.18, and 261.94 weeks, respectively. Regarding benign breast tissue adjacent to breast cancer tissue, 59.4% of the patients investigated overexpressed cdc25A, 23.2% overexpressed pRb, and 63.2% exerted dysregulation of p27(kip1) while they proved to be negative for immunohistochemical staining regarding all other markers tested. CONCLUSION: The immunohistological analyses of cdc25A, cyclin A, cyclin E, p16(ink4a), and p27(kip1) have the potential for further refining the risk assessment in patients with untreated breast cancer who belong to the high risk category defined according to the St. Gallen 2005 consensus conference. These cell cycle markers define a subgroup of high risk patients with even higher risk of metastazation and shortened survival. For confirmation a prospective study using standardized laboratory procedures in a larger population is needed.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/chemistry , Breast Neoplasms/mortality , Cell Cycle Proteins/analysis , Cell Cycle , Cell Proliferation , Adult , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cyclin A/analysis , Cyclin D , Cyclin E/analysis , Cyclin-Dependent Kinase Inhibitor p16/analysis , Cyclin-Dependent Kinase Inhibitor p21/analysis , Cyclin-Dependent Kinase Inhibitor p27 , Cyclins/analysis , Disease-Free Survival , Female , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/analysis , Kaplan-Meier Estimate , Neoplasm Invasiveness , Proportional Hazards Models , Retinoblastoma Protein/analysis , Retrospective Studies , Risk Assessment , Treatment Outcome , cdc25 Phosphatases/analysisABSTRACT
Withaferin A (WA) is derived from the medicinal plant Withania somnifera that has been safely used for centuries in the Indian Ayurvedic medicine for treatment of various ailments. We now demonstrate that WA treatment causes G2 and mitotic arrest in human breast cancer cells. Treatment of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) cell lines with WA resulted in a concentration- and time-dependent increase in G2-M fraction, which correlated with a decrease in levels of cyclin-dependent kinase 1 (Cdk1), cell division cycle 25C (Cdc25C) and/or Cdc25B proteins, leading to accumulation of Tyrosine15 phosphorylated (inactive) Cdk1. Ectopic expression of Cdc25C conferred partial yet significant protection against WA-mediated G2-M phase cell cycle arrest in MDA-MB-231 cells. The WA-treated MDA-MB-231 and MCF-7 cells were also arrested in mitosis as judged by fluorescence microscopy and analysis of Ser10 phosphorylated histone H3. Mitotic arrest resulting from exposure to WA was accompanied by an increase in the protein level of anaphase promoting complex/cyclosome substrate securin. In conclusion, the results of this study suggest that G2-M phase cell cycle arrest may be an important mechanism in antiproliferative effect of WA against human breast cancer cells.
Subject(s)
Breast Neoplasms/drug therapy , Cell Division/drug effects , Ergosterol/analogs & derivatives , G2 Phase/drug effects , Medicine, Ayurvedic , Breast Neoplasms/pathology , Cell Line, Tumor , Ergosterol/pharmacology , Female , Histones/metabolism , Humans , Mitosis/drug effects , Phosphorylation , Reactive Oxygen Species/metabolism , Withanolides , cdc25 Phosphatases/analysis , cdc25 Phosphatases/physiologyABSTRACT
Dual-specific phosphatases Cdc25 play a critical role in the cell cycle regulation by activating kinases of Cdk/cyclin complexes. Three Cdc25 isoforms (A, B and C) have been identified in mammalians. Cdc25A and B display oncogenic properties and are over-expressed in different tumors. Cdc25 phosphatases are therefore attractive targets for therapeutic strategies. Novel maleic anhydride derivatives bearing a fatty acid chain of variable size have been synthesized and tested for their Cdc25 inhibitory potential using an in vitro assay. We report biological activity of ineffective, moderate, and efficient inhibitors on breast cancer cells (MCF7) and its counterpart resistant to vincristine (Vcr-R). The most potent compounds induced Cdk2 inhibition and accumulation in G0/G1 phase of the cell cycle. Moreover, apoptosis was triggered within 48-h treatment, without oxidative burst and modulation of the Bax to Bcl-2 ratio. When used as pre-treatments, these derivatives were also able to potentiate adriamycin and cisplatin toxicity in both cell lines. Thus, maleic anhydride derivatives may mediate apoptosis through a cell cycle blockage via inhibition of Cdc25. This class of inhibitors may present potential interest in therapeutic strategies against cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Enzyme Inhibitors/pharmacology , Maleic Anhydrides/pharmacology , cdc25 Phosphatases/antagonists & inhibitors , Apoptosis/drug effects , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Line, Tumor , Cisplatin/pharmacology , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Drug Synergism , Drug Therapy, Combination , Enzyme Inhibitors/chemistry , Female , G1 Phase/drug effects , Humans , Maleic Anhydrides/chemistry , Quantitative Structure-Activity Relationship , Resting Phase, Cell Cycle/drug effects , Vincristine/pharmacology , cdc25 Phosphatases/analysisABSTRACT
PURPOSE: The purpose of this study is to systematically study the cell-cycle alterations of glioblastoma stem-like cells (GSLCs) after irradiation, possibly enriching the mechanisms of radioresistance of GSLCs. MATERIALS AND METHODS: GSLCs were enriched and identified, and then the radioresistance of GSLCs was validated by analyzing cell survival, cell proliferation, and radiation-induced apoptosis. The discrepancy of the cell-cycle distribution and expression of cell-cycle-related proteins between GSLCs and glioblastoma differentiated cells (GDCs) after irradiation was completely analyzed. RESULTS: The survival fractions and the cell viabilities of GSLCs were significantly higher than those of GDCs after irradiation. Radiation-induced apoptosis was less prominent in GSLCs than in GDCs. After irradiation with high-dose X-rays, the percentages of GDCs in G2/M phase was evidently increased. However, radiation-induced G2/M arrest occurred less frequently in GSLCs, but S-phase arrest occurred in GSLCs after irradiation with 8 Gy. Further mechanistic studies showed that the expressions levels of Cdc25c, Cdc2, and CyclinB1 in GSLCs were not apparently changed after irradiation, while those of p-ATM and p-Chk1 were sharply increased after irradiation in GSLCs. The basal level of Cdc25c expression in GSLCs was much higher than that in GDCs. CONCLUSIONS: We explored the cell-cycle alterations and cell-cycle-related proteins expression levels in GSLCs after irradiation, providing a novel mechanism of radioresistance of GSLCs.
Subject(s)
G2 Phase Cell Cycle Checkpoints/radiation effects , Glioblastoma/radiotherapy , M Phase Cell Cycle Checkpoints/radiation effects , Neoplastic Stem Cells/radiation effects , Apoptosis/radiation effects , Ataxia Telangiectasia Mutated Proteins/physiology , Cell Line, Tumor , Checkpoint Kinase 1/physiology , Glioblastoma/pathology , Humans , Radiation Tolerance , Spheroids, Cellular , cdc25 Phosphatases/analysisABSTRACT
Cells expressing high-risk human papillomavirus (HPV) E7 protein display impaired checkpoint control after DNA damage and exhibit elevated rates of mutagenesis. Repression of HPV E7 expression results in the subsequent accumulation of hypophosphorylated retinoblastoma protein and repression of the Cdc25A genes. No study has been conducted to elucidate the role of Cdc25A in the development and progression of human oral carcinomas. To confirm Cdc25A protein expression together with HPV, immunohistochemistry, Western blotting, polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR were performed using various histological subtypes of oral carcinomas. Cdc25A protein was localized predominantly in the cell nuclei in carcinomas, and high expression was found in 54% of primary tumors. HPV-16 E7 was not found in non-neoplastic oral tissues, whereas it was observed in eight (36%) of 22 oral carcinomas. We found a significant correlation between Cdc25A over-expression and HPV-16 E7 positive carcinomas. There was a strong positive correlation between Cdc25A over-expression and tumor size and TNM stage. This study suggests that Cdc25A is likely to be an important mediator in the progression of oral tumors, and HPV-16 E7 may be a sensitive indicator of the involvement of viral oncogenes in oral carcinogenesis.
Subject(s)
Carcinoma, Squamous Cell/virology , Mouth Neoplasms/virology , Oncogene Proteins, Viral/genetics , cdc25 Phosphatases/analysis , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/chemistry , Carcinoma, Squamous Cell/pathology , DNA, Viral/analysis , Female , Humans , Immunohistochemistry , Male , Middle Aged , Mouth Neoplasms/chemistry , Mouth Neoplasms/pathology , Papillomavirus E7 Proteins , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , RiskABSTRACT
To clarify the significance of cdc25B, which plays an important physiologic role in regulation of the G2/M check point, in progression of gastric cancer, 125 samples of paraffin-embedded gastric cancers were investigated by immunohistochemistry. In addition, 3 human gastric cancer cell lines were studied to determine the cellular localization of cdc25B by immunohistochemistry and cell fractionation followed by Western blotting. In the cell lines cdc25B was found to be present in both nuclei and cytoplasm, but predominantly in nuclei. High labeling indices of cdc25B in invasion front of gastric cancer was observed in 31 out of 125 cases (24.8%), linked to an advanced depth of cancer invasion (P=0.02), high rates of lymphatic invasion (P=0.03), and lymph node metastasis (P<0.01). Furthermore, the Kaplan-Meier method demonstrated a poor prognosis for cdc25B high labeling indices cases (P=0.02), although multivariate analysis revealed it not to be an independent factor. In conclusion, it seems likely that cdc25B is located predominantly in nuclei when overexpressed and this has some linkage with progression of gastric cancer.
Subject(s)
Cell Cycle Proteins/analysis , Stomach Neoplasms/diagnosis , Stomach Neoplasms/mortality , cdc25 Phosphatases/analysis , Aged , Blotting, Western , Cell Fractionation , Cell Line, Tumor , Disease Progression , Female , Humans , Immunohistochemistry , Male , Multivariate Analysis , Prognosis , Stomach Neoplasms/pathology , Survival AnalysisABSTRACT
Cdc25 activates maturation promoting factor (MPF) and promotes mitosis by removing the inhibitory phosphate from the Tyr-15 of Cdc2 in human cells. In this study, we searched the interacting protein(s) of human Cdc25C using the yeast two-hybrid screen and identified proliferating cell nuclear antigen (PCNA) as an interacting partner of Cdc25C. The interaction between Cdc25C and PCNA was confirmed in vitro and in vivo. Co-immunoprecipitation analyses using human T cell line, Jurkat, further revealed that Cdc25C interacted with PCNA transiently when cells began to enter mitosis. Immunofluorescence analysis also showed that Cdc25C and PCNA were transiently co-localized in the nucleus at the beginning of M phase. Together with the previous observations of the interaction between various cdc/cyclin and PCNA, our findings strongly suggested a potential role of PCNA at the G2 to M phase transition of cell cycle.
Subject(s)
Cell Cycle Proteins/physiology , G2 Phase , Mitosis , Proliferating Cell Nuclear Antigen/physiology , cdc25 Phosphatases/physiology , Cell Cycle Proteins/analysis , Humans , Jurkat Cells , Proliferating Cell Nuclear Antigen/analysis , Two-Hybrid System Techniques , cdc25 Phosphatases/analysisABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths. Deregulation of cell-cycle control is thought to be a crucial event in malignant transformation, and CDC25 phosphatases are a family of cyclin-dependent kinase activators, which act at different points of the cell cycle, including G1-S and G2-M transition. Here, we investigated the expression and functional significance of CDC25s in PDAC. CDC25B mRNA expression levels in human pancreatic tissue samples were analysed by cDNA array, quantitative PCR and Northern blotting. Immunohistochemistry was carried out to localize and quantify CDC25B expression. Two specific CDC25B inhibitors were utilized to determine the functional relevance of CDC25B. By quantitative RT-PCR, CDC25B mRNA was overexpressed in pancreatic cancer (7.5-fold) in comparison to the normal pancreas. Strong nuclear CDC25B immunoreactivity was present in both pancreatic and metastatic cancer samples, and there was a marked increase of the percentage of positive cells in primary cancer (48.6+/-16.3%) and metastatic tissues (71.7+/-3.1%) compared to normal samples (8.3+/-1.8%). Two CDC25B inhibitors reduced the growth of pancreatic cancer cell lines, resulting in the accumulation of phosphorylated CDC2 and G2/M arrest. These findings demonstrate an important role of CDC25B in cell-cycle progression, raising the possibility that inhibition of CDC25B may have therapeutic potential in pancreatic cancer.
Subject(s)
Adenocarcinoma/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Cell Cycle Proteins/genetics , Pancreatic Neoplasms/metabolism , cdc25 Phosphatases/genetics , Adenocarcinoma/pathology , Adult , Aged , Aged, 80 and over , Carcinoma, Pancreatic Ductal/pathology , Cell Cycle Proteins/analysis , Cell Cycle Proteins/physiology , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Male , Middle Aged , Pancreatic Neoplasms/pathology , RNA, Messenger/analysis , cdc25 Phosphatases/analysis , cdc25 Phosphatases/physiologyABSTRACT
Most cyclin-dependent kinases are negatively regulated by phosphorylation of two residues, a threonine at residue 14 and a tyrosine at residue 15. These residues are dephosphorylated by the cdc25 family of dual-specificity phosphatases leading to cell cycle progression. These phosphatases are inactivated by cellular checkpoint pathways in response to DNA damage leading to cell cycle arrest. Checkpoint pathways regulate the function of these phosphatases by regulating their stability, localization, association with substrate, and their activity. Hence, determining these properties for the cdc25 family of phosphatases becomes crucial for understanding how checkpoint pathways regulate the function of the cdc25 family members and, hence, cell cycle progression. This chapter describes methods to determine the activity, levels, phosphorylation status, and localization of both endogenous and overexpressed cdc25 proteins.
Subject(s)
cdc25 Phosphatases/analysis , Binding Sites , Cell Cycle/physiology , Cell Fractionation , Cell Line , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/metabolism , Escherichia coli/genetics , Fluorescent Antibody Technique , HeLa Cells , Humans , Immunoprecipitation , Isoenzymes/analysis , Isoenzymes/genetics , Isoenzymes/metabolism , Phosphorylation , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , cdc25 Phosphatases/genetics , cdc25 Phosphatases/metabolismABSTRACT
PURPOSE: This study aims to identify differentially expressed genes in esophageal squamous cell carcinoma (ESCC) through the use of a membrane-based cDNA array. EXPERIMENTAL DESIGN: Two newly established human ESCC cell lines (HKESC-1 and HKESC-2) and one corresponding to a morphologically normal, esophageal epithelium tissue specimen, prospectively collected from the HKESC-2-related patient, were screened in parallel using a cDNA expression array containing gene-specific fragments for 588 human genes spotted onto nylon membranes. RESULTS: The results of cDNA expression array showed that 53 genes were up-regulated 2-fold or higher and 8 genes were down-regulated 2-fold or higher in both ESCC cell lines at the mRNA level. Semiquantitative RT-PCR analysis of a subset of these differentially expressed genes gave results consistent with cDNA array findings. Four of the differentially expressed genes that belong to the categories of oncogenes/tumor suppressor genes (Fra-1 and Neogenin) and cell cycle-related genes (Id-1 and CDC25B) were studied more extensively for their protein expression by immunohistochemistry. The two ESCC cell lines and their corresponding primary tissues, 61 primary ESCC resected specimens and 16 matching, morphologically normal, esophageal epithelium tissues were analyzed. The immunostaining results showed that Fra-1, Neogenin, Id-1, and CDC25B were overexpressed in both ESCC cell lines and their corresponding primary tumors at the protein level, validating the microarray findings. The results of the clinical specimens showed that the Fra-1 gene was overexpressed in ESCC compared with normal esophageal epithelium in 53 of 61 cases (87%), Neogenin in 57 of 61 cases (93%), Id-1 in 57 of 61 cases (93%), and CDC25B in 48 of 61 cases (79%). Furthermore, the expression of Fra-1, Neogenin, and Id-1 in ESCC correlated with tumor differentiation. CONCLUSIONS: Overall, this study demonstrates that multiple genes are differentially expressed in ESCC and provides the first evidence that oncogenes Fra-1 and Neogenin and cell cycle-related genes Id-1 and CDC25B are overexpressed in ESCC.
Subject(s)
Carcinoma, Squamous Cell/genetics , DNA, Complementary/genetics , Esophageal Neoplasms/genetics , Gene Expression Profiling , Repressor Proteins , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Cycle Proteins/analysis , Cell Cycle Proteins/genetics , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Esophageal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Inhibitor of Differentiation Protein 1 , Membrane Proteins/analysis , Membrane Proteins/genetics , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-fos/analysis , Proto-Oncogene Proteins c-fos/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/analysis , Transcription Factors/genetics , Tumor Cells, Cultured , cdc25 Phosphatases/analysis , cdc25 Phosphatases/geneticsABSTRACT
Microtubule-disruption (MTD) is often thought to arrest the mammalian cell cycle only during mitosis. However, MTD has also been demonstrated to arrest cells during interphase at a G(1)-phase point we call G(1)MTA. Microtubule integrity is now shown to be required for progression past G(1)MTA and the mammalian restriction-point. Neither p21(waf1) nor p27(kip1) are required for MTD-induced G(1)-arrest. Only p21(waf1) is crucial for normal G(1)MTA passage. The p21(waf1)-Chk1-cdc25C-cdc2-checkpoint-pathway is implicated in monitoring this passage. P21(waf1) deletion deregulates G(1)MTA transition and decreases MTD-G(1) arrest, possibly via Chk1 disregulation. Oncogene-induced overexpression of p21(waf1) produced opposite effects on the Chk1-cdc25C-cdc2 pathway and enhanced MTD-G(1) arrest. G(1)MTA thus represents a novel facet of mammalian G(1)/S checkpoint.