OBJECTIVE: To investigate the expression profile and the clinical significance of CDK3 in the tissues of nasopharyngeal carcinoma. METHOD: Immunohistochemistry was utilized to detect the expression of CDK3 in 94 cases of NPC and 40 cases of nasopharyngeal inflammation. RESULT: CDK3 was highly expressed in NPC cell lines. Immunohistochemistry showed that CDK3 was mostly expressed in the cytoplasm. The positive expression rate of NPC was 67% and that of nasopharyngeal inflammation was only 12. 5%. The difference between these two groups was highly statistically significant. The CDK3 expression in NPC was related to the TNM clinical staging of NPC (P < 0.01). CONCLUSION The expression levels of CDK3 were obviously higher in NPC tissues. The CDK3 expression in NPC was related to the TNM clinical staging of NPC. It suggests that CDK3 expression may play an important role in the occurrence and development of nasopharyngeal carcinoma.
Adult ; Carcinoma ; Cyclin-Dependent Kinase 3 ; metabolism ; Female ; Humans ; Male ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Staging

PURPOSE: To explore the role of cell cycle and apoptosis regulators during hepatocarcinogenesis, the expression of cell cycle-related proteins (cyclin D1 and p27kip1) and apoptosis-related proteins (p53, survivin, caspase 3). METHODS: Sprague-Dawley rats were given 120 ppm diethylnitrosamine (DEN) as a carcinogen and sequentially sacrificed. The expression of cell cycle and apoptotic related proteins were examined by light microscopy and immunohistochemistry. RESULTS: During the DEN-induced hepatocarcinogenesis, sequential histologic changes from preneoplastic lesions (altered hepatic cellular foci, hyperplastic nodules, and hepatocellular adenomas) and ultimately overt hepatocellular carcinomas and metastatic lesions were noted. The cyclin D1 were progressively increased from preneoplastic lesions to hepatocellular carcinomas. However, the p27kip1 and the survivine proteins did not show any other difference with the increasing degree of carcinogenesis. The p53 and caspase 3 proteins were more significantly increased in hepatocellular carcinomas than preneoplastic lesions. The cyclin D1 protein expression did not show any correlation with the expression of p27Kip1 protein, but the p53 expression was related to the expression of survivin and caspase 3. CONCLUSION: From the above results, over-expression of cyclin D1 plays a role in the early and late stages of hepatocarcinogenesis. In addition p53 and caspase 3 might be useful markers for evaluating the risk of malignant transformation.
Animals ; Apoptosis ; Carcinoma, Hepatocellular ; Caspase 3 ; Cell Cycle ; Cyclin D1 ; Cyclin-Dependent Kinase Inhibitor p27 ; Diethylnitrosamine ; Light ; Microscopy ; Proteins ; Rats ; Rats, Sprague-Dawley

OBJECTIVESTo unravel the molecular mechanism of proliferation inhibition induced by transfection of pemt2-cDNA into rat CBRH-7919 hepatoma cells.

METHODSWe started with the highly expressed PEMT2 clone. Cell culture and Western blotting techniques were used to examine the expression of cyclinD1/CDK4, cyclinE/CDK2, phospho-Rb, caspase-3, c-jun and caveolins.

RESULTSOur results showed that CDK4, CDK2, phospho-Rb and c-jun were down regulated in the pemt2 highly expressed cell clone. The high expression clone of pemt2-transfected cells also showed over expression of caspase-3.

CONCLUSIONThe reductions of proliferation and apoptosis of pemt2 transfected cells could be related to the G1 phase arrest induced by down-regulation of the cell cycle-associated proteins.

Animals ; Apoptosis ; physiology ; Caspase 3 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase 2 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase 4 ; biosynthesis ; genetics ; Cyclin-Dependent Kinases ; biosynthesis ; genetics ; Down-Regulation ; Liver Neoplasms, Experimental ; genetics ; metabolism ; Phosphatidylethanolamine N-Methyltransferase ; genetics ; metabolism ; Rats ; Transfection ; Tumor Cells, Cultured

This study was aimed to investigate the effect of Honokiol (HNK) on proliferation and apoptosis of acute myeloid leukemia HL-60 cells and its potential mechanism. Inhibitory effect of HNK on the HL-60 cell proliferation was detected by MTT assay. Flow cytometry was used to detect the change of cell cycle and AnnexinV/PI staining was used to detect apoptosis. Western blot was applied to analyze the cell cycle protein (cyclins), cyclin-dependent kinase (CDK), P53, P21, P27, BCL-2, BCL-XL, Bax, caspase-3/9 and proteins for MAPK signal pathway. The results showed that HNK could inhibit the proliferation of HL-60 cells in time- and dose dependent ways. HNK arrested HL-60 cells in G0/G1 phase, and S phase cells decreased significantly (P < 0.05). The expression of cyclin D1, cyclin A, cyclin E and CDK2/4/6 were significantly down-regulated (P < 0.05), the expression of P53 and P21 was significantly upregulated after treating for 24 h with HNK (P < 0.05). After 24 h treatment with HNK, HL-60 cell apoptosis increased significantly with the upregulation of activated caspase-3, -9, BAX expression and the downregulation of BCL-2, BCL-XL expression. The MAPK subfamily, P38 and JNK were not significantly changed, but the expression of MEK1/2-ERK1/2 was significantly downregulated (P < 0.05). It is concluded that HNK arrestes the cells at G0/G1 phase and induces HL-60 cell apoptosis through the intervention of MEK1/2-ERK1/2 signaling pathway.
Apoptosis ; drug effects ; Biphenyl Compounds ; pharmacology ; Caspase 3 ; Cell Cycle ; Cell Proliferation ; drug effects ; Cyclin D1 ; Cyclin E ; Cyclin-Dependent Kinase 2 ; HL-60 Cells ; Humans ; Lignans ; pharmacology ; Oncogene Proteins ; Signal Transduction ; bcl-2-Associated X Protein

Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G(2)/M and G(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 micromol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/L) increased viable PC12 cells to 45.16%, (Z-DEVD-FMK) and 58.06%, (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 micromol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid a-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.
Animals ; Apoptosis ; drug effects ; physiology ; Caspase 3 ; physiology ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; PC12 Cells ; Protein Kinase Inhibitors ; pharmacology ; Purines ; pharmacology ; Rats

OBJECTIVETo investigate the regulatory effect of microRNA-221 (MIR221) on CDKN1C/p57 expression in colon carcinoma cells in vitro.

METHODSCaco2 cells were treated with or without anti-p57-siRNA prior to the addition of pre-MIR221 or anti-MIR221. The MIR221 expression pattern was detected by real-time RT-PCR, and the mRNA and protein levels of CDKN1C/p57 expression were detected using semi-quantitative RT-PCR and Western blotting. Caco2 cell proliferation following the treatment was detected with MTT assay. CDKN1C/p57 3'-UTR fragment was amplified by PCR from the genome DNA of human colon and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into Caco2 cells along with pre-MIR221 or anti-MIR221, and the luciferase activity in the transfected cells was detected.

RESULTSMIR221-specific inhibitor significantly up-regulated CDKN1C/p57 protein expression in Caco2 cells (P<0.01). Anti-MIR221 could markedly inhibit Caco2 cell proliferation, and the inhibitory effect was obviously abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57 (P<0.01). A significant increase of luciferase activity was detected in Caco2 cells co-transfected with the luciferase reporter plasmid construct and anti-MIR221 (P<0.01).

CONCLUSIONSMIR221 can interact with the target site on the 3'-UTR of CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote colon carcinoma cell proliferation, suggesting the value of MIR221 as a potential target for treatment of colon carcinoma.

3' Untranslated Regions ; Caco-2 Cells ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p57 ; genetics ; metabolism ; Down-Regulation ; Humans ; MicroRNAs ; pharmacology ; RNA, Messenger ; genetics ; metabolism

BACKGROUND/AIMS: Hypothyroidism has been reported in 36~85% of patients treated with sunitinib for renal cell carcinoma or gastrointestinal stromal tumor. However, the mechanism behind this hypothyroidism is unclear. This study evaluated the effects of sunitinib, a multi-target tyrosine kinase inhibitor, on the survival and proliferation of thyrocytes using FRTL-5 rat thyroid cells. METHODS: We examined the effect of sunitinib on cell proliferation in the presence and absence of thyroid stimulating hormone (TSH) in a colorimetric assay. Effects on the cell cycle were evaluated by flow cytometry, and on apoptosis using an annexin V apoptosis assay kit and by immunoblotting for caspase-3. Immunoblotting was also used to evaluate changes in the levels of intracellular proteins associated with the G1-S phase of the cell cycle. RESULTS: Sunitinib suppressed the proliferation of FRTL-5 cells in a dose- and time-dependent manner. This suppressive effect was enhanced by the presence of TSH (1 mU/mL). Sunitinib was subsequently shown, in flow cytometric analyses, to arrest the cell cycle at the G1-S phase. Furthermore, it induced apoptosis at a high concentration (15 micrometer) by activating caspase-3. G1-S phase arrest was associated with the induction of p27(kip1) and p21(cip1), whose expression is suppressed by TSH under control conditions. Sunitinib also decreased intracellular levels of cyclin D1 and cyclin-dependent kinase 2 in FRTL-5 cells. CONCLUSIONS: Sunitinib induced apoptosis in and suppressed the proliferation of FRTL-5 cells. Its suppression of proliferation was further enhanced by the presence of TSH. Sunitinib arrested the cell cycle in the G1-S phase by inducing the expression of p27(kip1)/p21(cip1), which are suppressed by TSH under normal conditions. Collectively, these findings suggest that sunitinib may interfere with TSH signaling pathways in normal thyrocytes.
Animals ; Annexin A5 ; Apoptosis ; Carcinoma, Renal Cell ; Caspase 3 ; Cell Cycle ; Cell Proliferation ; Cyclin D1 ; Cyclin-Dependent Kinase 2 ; Flow Cytometry ; Gastrointestinal Stromal Tumors ; Humans ; Hypothyroidism ; Immunoblotting ; Indoles ; Protein-Tyrosine Kinases ; Proteins ; Pyrroles ; Rats ; Thyroid Gland ; Thyrotropin

OBJECTIVECytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.

METHODSAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.

RESULTSCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.

CONCLUSIONSCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Antigens, Bacterial ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Gastric Mucosa ; cytology ; enzymology ; microbiology ; Helicobacter pylori ; metabolism ; pathogenicity ; physiology ; Humans ; Interleukin-8 ; secretion ; Intracellular Signaling Peptides and Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transcription, Genetic ; Virulence

OBJECTIVETo study the effects of LIF combined with bFGF on the proliferation, stemness and senescence of hUC-MSC.

METHODSExperiments were divided into 4 groups: control group, in which the cells were treated with complete medium (α-MEM containing 10% FBS); group LIF, in which the cells were treated with complete medium containing 10 ng/ml LIF; group bFGF, in which the cells were treated with complete medium containing 10 ng/ml bFGF; combination group, in which the cells were treated with complete medium containing 10 ng/ml LIF and 10 ng/ml bFGF. The growth curves of hUC-MSC at passage 4 in different groups were assayed by cell counting kit 8. Cellular morphologic changes were observed under inverted phase contrast microscope; hUC-MSC senescence in different groups was detected by β-galactosidase staining. The expression of PCNA, P16, P21, P53, OCT4 and NANOG genes was detected by RT-PCR.

RESULTSThe cell growth curves of each group were similar to the S-shape; the cell proliferation rate from high to low as follows: that in the combination group > group bFGF > group LIF > control group. Senescence and declining of proliferation were observed at hUC-MSC very early in control group; the cells in group LIF maintained good cellular morphology at early stage, but cell proliferation was slow and late senescence was observed; a few cells in group bFGF presented signs of senescence, but with quick proliferation; the cells in combination group grew quickly and maintained cellular morphology of hUC-MSC for long time. The LIF and bFGF up-regulated the expression of PCNA, OCT4 and NANOG, while they down-regulated the expression of P16, P21, P53, and their combinative effects were more significant.

CONCLUSIONLIF combined with bFGF not only can promote the proliferation and maintenance of stemness of hUC-MSC, but also can delay the senescence of hUC-MSC.

Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Genes, Homeobox ; Humans ; Leukemia Inhibitory Factor ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Octamer Transcription Factor-3 ; metabolism ; Organic Chemicals ; Proliferating Cell Nuclear Antigen ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Umbilical Cord ; cytology

OBJECTIVETo reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.

METHODSUsing a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association beta-galactosidase staining as well as senescence association CKIs, p16(INK4) and p21(Cip1) protein expressions were all measured in the low passages of 2BS cells.

RESULTSBoth 25 micro mol/L and 50 micro mol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G(1) phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association beta-galactosidase (P < 0.05). In addition, LY294002 could induce time-course expressions of p16(INK4) and p21(Cip1) in 2BS cell lines.

CONCLUSIONSPI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16(INK4) and p21(Cip1), might be involved in this senescence phenotype proceeding in 2BS cell lines.

Cells, Cultured ; Cellular Senescence ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; analysis ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fibroblasts ; drug effects ; physiology ; G1 Phase ; drug effects ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors