OBJECTIVE: To investigate the expression and clinicopathological significance of the cell cycle regulators cyclin E, cyclin D1, p21, p16 in laryngeal carcinogenesis tissus. METHOD: The expression of cell cycle regulators were detected by flow cytometry method in 23 cases of polyps of vocal cord, 69 cases of laryngeal precancerous change and 33 cases of laryngeal squamous cell carcinoma (LSCC), which tissue was paraffin embedded, sliced, dewaxed, and prepared into the cell suspension, then fluorescently labeled by cyclin E, cyclin D1, p21 and p16. RESULT: In polyps of vocal cord, laryngeal precancerous change and LSCC, The positive expression rate of cyclin E and cyclin D1 were respectively 13.04%, 20.29D, 42.420 and 26.09%, 43.48% and 93.94%. The positive expression rate of p16 and p21 were respectively 61.90%, 40.98%, 14.28% and 47.62%, 23.81%, 26.23%. Those showed the positive expression rate of cyclin D1, cyclin E gradually decreased from vocal cord polyps, laryngeal precancerous change to LSCC, (P < 0.05, P < 0.01), while the positive expression rate of p21 and p16 gradually decreased (P < 0.01). CONCLUSION The abnormal expression of cell cycle regulatory factors is the molecular events of laryngeal carcinoma. High expression of positive regulatory factors cyclin D1 and cyclin E, and low expression of negative regulatory factors p16 and p21, which showed the imbalance of multiple positive and negative regulatory factors related with cell cycle play an important role in the occurrence of laryngeal cancer.
Adult ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Female ; Flow Cytometry ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Oncogene Proteins ; metabolism

BACKGROUND/AIMS: The cyclin-dependent kinase inhibitors (CDKI) including p21, p27, and p57 of the kinase inhibitor protein (KIP) family are negative regulators of cell cycle progression and potentially act as tumor suppressor. Tumor behavior and growth are influenced by the extent of tumor cell proliferation. The aim of this study was to evaluate the expression of KIP family CDKI in gastric cancer tissue, and to examine the relationship between these expression and various clinicopathological parameters including tumor cell proliferation. METHODS: We conducted an immunohistochemical analysis of p21, p27, and p57 expression in 109 gastric cancer tissues. Tumor cell proliferation was assessed by immunohistochemistry with antibody against Ki-67. RESULTS: Negative expression of p21, p27, and p57 was demonstrated in 45.9%, 65.1%, and 57.8% of cancer tissues, respectively. Negative expression of p21 correlated with larger tumor size, poor differentiation, depth of invasion, lymph node metastasis and advanced TNM stage (p=0.048, 0.041, 0.001, 0.005, and 0.001 respectively). Negative expression of p21 correlated with poor survival (p=0.037). Tumors with negative p21 expression had higher Ki-67 expression than those with positive p21 expression (p=0.024). No significant correlation could be observed between status of p27 and p57 expression and various clinicopathological parameters including survival and tumor cell proliferation. CONCLUSIONS: These results suggest that negative expression of p21 may play an important role in carcinogenesis by stimulating tumor cell proliferation, and may help in predicting the prognosis of gastric cancer.
Adult ; Aged ; Cell Division ; Cyclin-Dependent Kinase Inhibitor Proteins/*metabolism ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cyclin-Dependent Kinase Inhibitor p57/metabolism ; English Abstract ; Female ; Humans ; Male ; Middle Aged ; Stomach Neoplasms/*metabolism/mortality/pathology ; Survival Rate

BACKGROUND/AIMS: Eukaryotic cell cycle is regulated by signal transduction pathways mediated by complexes of cyclin dependent kinases (CDKs) and their partner cyclins, or by interaction with CDK inhibitors. Thioacetamide (TA) is a weak hepatocarcinogen causing several types of liver damage in a dose dependent manner and ultimately producing malignant transformation. We investigated alterations of expression of cell cycle regulators in the rat liver, involved in G1 entry and progression during TA administration. METHODS: We studied expression patterns of cyclin D1, CDK4, CDK6, p21(CIP1) and p16(INK4a) during daily intraperitoneal injection of low dose TA (50 mg/kg) till 7 day. We used western blot and immunohistochemistry for detection. RESULTS: Expression of cyclin D1, CDK4, CDK6 and p21(CIP1) increased from 6 hour and peaked at 2, 3 day, then decreased next 2 days, and re-increased at 6 day. Cytoplasmo-nuclear translocation of cyclin D1 and p21(CIP1) was evident within 1 day and prominent at 2 and 7 day. Expression of p16(INK4a) increased immediately after TA treatment and remarkably increased from 3 day and progressed till 7 day, showing cytoplasmic location, suggestive of inactive form. Most of in situ immunoreactions occurred at the centrilobular hepatocytes. Concomitant nuclear translocation of p21(CIP1) and cyclin D1, different with p16(INK4a) suggests that p21(CIP1) might be a transporter for nuclear translocation rather than cell cycle inhibitor. CONCLUSIONS: Daily administration of low dose TA makes cell cycle open and G1 progress, possibly due to cyclin D1, CDK4 and CDK 6, their transporter p21(CIP1), and inactive p16(INK4a), which occur at quiescent hepatocytes, not stem cells.
Animals ; Cell Cycle Proteins/*metabolism ; Cyclin D1/metabolism ; Cyclin-Dependent Kinase 4/metabolism ; Cyclin-Dependent Kinase 6/metabolism ; Cyclin-Dependent Kinase Inhibitor p16/metabolism ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; G1 Phase ; Immunohistochemistry ; Liver/*drug effects/enzymology/metabolism ; Liver Diseases/chemically induced/metabolism/pathology ; Male ; Rats ; Rats, Sprague-Dawley ; Thioacetamide/*toxicity

This study is to examine the effects of NNIspm-mediated cellular senescence of HepG2 cells and elucidate its potential molecular mechanism. Cellular senescence was detected with senescence-associated beta-galactosidase staining. Cell cycle distribution, intracellular fluorescence intensity and accumulation of intracellular reactive oxygen species (ROS) were detected by high content screening (HCS). Protein expression was detected by Western blotting. Polyamines content was analyzed by high performance liquid chromatography (HPLC). The results demonstrated that NNIspm significantly induced HepG2 cells senescence. This effect was due to the decrease of intracellular polyamines, the arrest at G0/G1 phase and an increase of ROS level. The molecular senescence marker p21 increased significantly after NNIspm treatment. In contrast, the protein expressions of Cyclin E and CDK2 were obvious down-regulation. The results indicated that cellular senescence induced by NNIspm was one of its antitumor mechanisms.
Antineoplastic Agents ; metabolism ; pharmacology ; Cellular Senescence ; drug effects ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; G1 Phase ; Hep G2 Cells ; Humans ; Oncogene Proteins ; metabolism ; Polyamines ; metabolism ; pharmacology ; Reactive Oxygen Species ; metabolism

OBJECTIVETo verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid.

METHODSImmunofluorescence laser was used to confirm CASK and Id1 protein expression and localization in fibroblasts of the keloid and normal skin. RT-PCR and Western-blot were adopted to analysis the CASK and Id1 expression and differences between keloid and normal skin fibroblasts. The natural combination of CASK and Id1 protein of keloid fibroblasts was tested by immunoprecipitation.

RESULTSCASK and Id1 protein expression were both found in fibroblast cells of keloid and normal skin under normal circumstances. Most of CASK and Id1 were distributed in the cytoplasm and nucleus of fibroblasts. The results of RT-PCR showed that the expression of CASK mRNA in the keloid group was 0.658 +/- 0.024, which was lower than that in the normal control group (1.076 +/- 0.008, t = 11.159, P < 0.05). The expression of Id1 mRNA was 0.497 +/- 0.014, which was higher than that in the normal control group (0.307 +/- 0.017, t = 15.148, P < 0.05). The results of Western-blot showed that the expression level for CASK protein in the keloid group was 0.057 +/- 0.006, which was lower than that in the normal control group (0.168 +/- 0.012, t = 13.524, P < 0.05); the expression of Id1 protein was 0.812 +/- 0.035, which was higher than that in the normal control group (0.368 +/- 0.031, t = 16.356, P < 0.05). The results of immunoprecipitation showed that Id1 could be detected in the CASK precipitate, while CASK also could be detected in the Id1 precipitate. There was a natural binding of CASK and Id1 in keloid fibroblasts.

CONCLUSIONCASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts, which is related with the occurrence of keloid.

Cell Proliferation ; genetics ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; metabolism ; Fibroblasts ; metabolism ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; Keloid ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction

This study was aimed to investigate the effect of arsenic trioxide (As2O3) on proliferation and cell cycle of human Burkitt lymphoma cells and its related molecular mechanism, so as to provide experimental evidence for treatment of Burkitt lymphoma with As2O3. Human Burkitt lymphoma cell line Namalwa was used as the model, the effect of As2O3 on cell proliferation, cell cycle and apoptosis, as well as the expression of cell cycle modulation related genes, including mRNA and protein level, were detected by MTT method, flow cytometry, real-time quantitative PCR (RQ-PCR) and Western blot, respectively. The results showed that the As2O3 inhibited significantly the growth and proliferation of Namalwa cells in concentration-and time-dependent manner. The As2O3 arrested obviously cell cycle of Namalwa cells in G1 phase, and showed significant concentration-effect relationship. The As2O3 induced the apoptosis of Namalwa cells in concentration-and time-dependent manner, downregulated the expression of the important driving genes of cell cycle including Cyclin E and CDK2 in mRNA and protein level, upregulated the expression of the important inhibiting gene of cell cycle-P21 in mRNA and protein level in concentration-dependent manner. It is concluded that As2O3 inhibits significantly the growth and proliferation of Namalwa cells, and the effect was closely relates with its inducing the apoptosis and blocking the cell cycle of Namalwa. The action of blocking cell cycle is closely associated with its downregulating the expression of driving genes of cell cycle-Cyclin E and CDK2, upregulating the expression of the inhibiting gene of cell cycle-P21.
Apoptosis ; Arsenicals ; pharmacology ; Burkitt Lymphoma ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Humans ; Oncogene Proteins ; metabolism ; Oxides ; pharmacology

OBJECTIVETo clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.

METHODSsiRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.

RESULTSThe inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ± 0.10 vs. 0.73 ± 0.11 and 0.71 ± 0.07 respectively, P < 0.05). AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ± 0.35 vs. 3.47 ± 0.93 and 3.17 ± 0.78, P < 0.01). Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42)% vs. (70.58 ± 10.10)% and (69.83 ± 14.77)%, respectively, P < 0.01]. Cell cycle analysis by FACS showed that G(0)/G(1) cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ± 5.93)% vs. (57.05 ± 5.97)% and (52.99 ± 4.80)%, P < 0.05]. Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1(0.45 ± 0.21 vs. 1.25 ± 0.24 and 1.12 ± 0.15, respectively, P < 0.05)and CDK4 (0.55 ± 0.08 vs. 1.18 ± 0.32 and 1.10 ± 0.29, respectively, P < 0.05); the following genes were notably increased: p16(1.07 ± 0.23 vs. 0.36 ± 0.12 and 0.35 ± 0.13, respectively, P < 0.01)and p21(0.42 ± 0.12 vs. 0.17 ± 0.06 and 0.19 ± 0.08, respectively, P < 0.05). RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line. From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 and 0.58 ± 0.55, respectively, P < 0.05).

CONCLUSIONSRhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.

Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; rho GTP-Binding Proteins ; genetics ; metabolism ; rhoC GTP-Binding Protein

OBJECTIVETo investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.

METHODSEctopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.

RESULTSRIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.

CONCLUSIONSRIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

Cell Cycle ; genetics ; Cell Differentiation ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; U937 Cells

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Staging ; Phosphorylation ; S-Phase Kinase-Associated Proteins ; metabolism

OBJECTIVETo study the effect of lovastatin on the expression of IkappaBalpha and cell-cycle regulating proteins in MCF-7 cells.

METHODSMCF-7 cells were treated with 4, 8 and 16 micro mol/L lovastatin for 48 - 72 h. The distribution of cell cycles was assayed by flow cytometry (FCM). The protein expression of IkappaBalpha, CDK4, p16, pRb in cytoplasm and IkappaBalpha in the nucleus were detected by Western blot.

RESULTSLovastatin could arrest cellcycle in the G(0)/G(1) phase in a dose- and time-dependent manner, obviously lowering the expression of IkappaBalpha, CDK4 and pRb protein level in the cytoplasm and increasing IkappaBalpha in the nucleus, but not on p16 protein level.

CONCLUSIONLovastatin can induce the arrest of cell cycle in G(0)/G(1) phase by affecting the expression of IkappaBalpha and cell-cycle regulating protein in MCF-7 cells.

Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; Female ; Humans ; I-kappa B Proteins ; metabolism ; Lovastatin ; pharmacology ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; Proto-Oncogene Proteins ; metabolism ; Retinoblastoma Protein ; metabolism