OBJECTIVETo detect the gene expression profile in gastric cancer cell cycle and explain the mechanism of gastric cancer cell proliferation by a genomic study.

METHODSGastric cancer cells MKN45 were synchronized at G2/M and G1/S point by nocodazole-thymidine and double thymidine methods. The synchronizing degree of cells was monitored by flow cytometry. The gene expression profiles at G2/M point, M/G1 transition, G1 early phase, G1 late phase, G1/S point, S early phase, S late phase, G2 early phase and G2 late phase in MKN45 cell cycling were examined using cDNA microarray chips. Hierarchy analysis was conducted with a professional software package and the up-regulated genes at G1 late and G2 phase were analyzed according to gene database. Furthermore, the mRNA level of cyclin E, cyclin B, plk1 and STK15 in above mentioned nine points were measured by quatitative PCR.

RESULTS2001 genes were detected to be available at all 9 points via software processing, out of which 959 appeared up-regulated or down-regulated. 379 genes showed to be up-regulated at late G1 (147) or G2 phases (232), 40 at S and M phases (also up-regulated at G1 late and G2 phases). The 147 up-regulated genes at G1 late phase are involved in DNA metabolism, transcription and translation, protein transportation, ubiquitination and signal transduction, etc. The 232 up-regulated genes in G2 phase are involved in RNA synthesis and processing, intracellular protein transportation, cytoskeleton synthesis, signal transduction, apoptosis and anti-apoptosis, transcription regulation, ubiquitination, mitosis regulation and oncogene expression, etc. The mRNA level of 4 genes detected by quantitative PCR during cell cycle was in agreement with that detected by microarray.

CONCLUSIONDuring MKN45 cell cycling, the preparation for DNA synthesis and chromosome separation are conducted in G1 and G2, which are implicated in multiple genes, may be the main impetus of driving MKN45 cell cycle. Some of these genes may be related to tumor over-proliferation. The cDNA microarray technique has characteristic features such as reliability and can provide a great deal for future research on cell cycle related genes in gastric cancer.

Aurora Kinase A ; Aurora Kinases ; Cell Cycle ; genetics ; Cell Cycle Proteins ; genetics ; Cell Line, Tumor ; Cyclin B ; genetics ; Cyclin E ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Polymerase Chain Reaction ; methods ; Protein-Serine-Threonine Kinases ; genetics ; Proto-Oncogene Proteins ; genetics ; RNA, Messenger ; genetics ; metabolism ; Stomach Neoplasms ; genetics ; pathology

The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/AxC (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/AxC was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/AxC we firstly set up could be used to analyze cyclin E expression threshold quantitatively.
Caffeine/pharmacology ; Cell Cycle/*physiology ; Cell Line, Tumor ; Cyclin E/*analysis ; Cycloheximide/pharmacology ; DNA, Neoplasm/*analysis ; Flow Cytometry/methods ; Jurkat Cells ; Leukemia, Lymphoid/pathology

BACKGROUND/AIMS: Carcinogenesis is characterized by the abnormal regulation of cell cycle. The abnormal expression of the regulators of cell cycle may be related to the prognosis. Since the clinical significance of the expression of the three proteins in colorectal carcinomas is still controversial, we evaluated the prognostic value of the expression of cyclin E, p27 and mutant p53 in stage II colorectal cancer. METHODS: The expression levels of cyclin E, p27 and mutant p53 proteins in 41 patients with stage II colorectal carcinomas were analyzed by immunohistochemistry. RESULTS: In the univariate analysis, the level of CEA at diagnosis was associated with disease relapse. In the multivariate analysis, the clinicopathological variables such as age, gender, site of primary tumor, tumor size, state of tumor differentiation and preoperative plasma CEA level were not associated with disease relapse. When Kaplan-Meier survival curves were constructed to determine the prognosis, cyclin E, p27 and mutant p53 expressions did not predict poor prognosis. CONCLUSIONS: Our results suggested that the expression of cyclin E, p27 and mutant p53 proteins did not predict the clinical outcome in the stage II colorectal carcinomas.
Adenocarcinoma/chemistry/*diagnosis/mortality/pathology ; Adult ; Aged ; Aged, 80 and over ; Cell Cycle Proteins/*analysis ; Colorectal Neoplasms/chemistry/*diagnosis/mortality/pathology ; Cyclin E/*analysis ; Disease-Free Survival ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Mutation ; Prognosis ; Protein p53/*analysis/genetics ; Survival Rate ; Tumor Markers, Biological/analysis ; Tumor Suppressor Proteins/*analysis

Acetamides ; pharmacology ; Antineoplastic Agents ; pharmacology ; CD11b Antigen ; analysis ; Cell Differentiation ; drug effects ; Cyclin D ; Cyclin E ; analysis ; Cyclins ; analysis ; G1 Phase ; Genes, Retinoblastoma ; Genes, bcl-2 ; Genes, myc ; HL-60 Cells ; Humans ; Leukemia, Myeloid ; pathology ; Proliferating Cell Nuclear Antigen ; analysis ; RNA ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; U937 Cells

OBJECTIVETo investigate the effects of p57(kip2) and cyclinE proteins on the genesis and progression of human pancreatic cancer.

METHODSThe expression of p57(kip2) and cyclinE proteins in tumor tissues and adjacent tissues of pancreatic cancer in 32 patients was detected by SP immunohistochemical technique.

RESULTSThe p57(kip2) protein positive-expression rate in tumor tissues of pancreatic cancer was 46.9%, which was lower than that in adjacent pancreatic tissue (P < 0.05). The p57(kip2) protein positive-expression correlated significantly with tumor cell differentiation (P < 0.05) and did not correlate significantly with lymph node metastasis (P > 0.05). The cyclinE positive-expression rate in tumor tissues was 68.8%, which was higher than that in adjacent pancreatic tissues (P < 0.05). The cyclinE positive-expression also correlated significantly with tumor cell differentiation and lymph node metastasis (P < 0.05). The cyclinE protein positive-expression rate in the tumor tissues of the p57(kip2) protein positive-expression group was lower than that in the p57(kip2) protein negative-expression group, and there were no significant correlation between the two groups (r = -0.112, P > 0.05).

CONCLUSIONDecreased expression of the p57(kip2) protein and/or over-expression of the cyclinE protein may play an important role in the genesis and progression of human pancreatic cancer.

Adult ; Aged ; Cyclin E ; analysis ; Cyclin-Dependent Kinase Inhibitor p57 ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Nuclear Proteins ; analysis ; Pancreatic Neoplasms ; chemistry ; pathology

Hexamethylene bisacetamide (HMBA) is referred as a differentiation-inducer for the clinical treatment of acute myeloid leukemia and myelodysplastic syndrome. However, the molecular mechanism of the effects of HMBA on myeloid leukemic cells remains unknown. In this study, the effects of HMBA on cell cycle and expression of cell cycle regulatory proteins in HL-60 cell were investigated in order to explore its pharmacological mechanism. The altered distribution of cell cycle and expression of its regulatory proteins (cyclin D, cyclin E and p27) in HL-6 0 cell induced by HMBA were analyzed by flow cytometry. The effects on transcription for mRNA of CKI p15, p16 and p27 in HL-60 cell were further studied by RT-PCR. The results showed that HMBA could mainly commit HL-60 cell to G0/G1 arrest and the significantly decreased endocytic cyclin E protein and increased cyclin D/p27 protein after HMBA treatment were found. There was no expression of p15, p16 mRNA in untreated HL-60 cell and 3 mmol/L of HMBA could make them expressed after exposed for 24 h or 48 h respectively. The expression of p27 mRNA was positive and no obviously different in untreated HL-60 cells exposed for 24 h, 48 h and 72 h. These results suggested that one of the pharmacological mechanisms of HMBA was to elevate the expression of p27 and reduce the cyclin E expression as well as to activate the expression of p15, p16 gene mRNA, that arrested cell at G0/G1 and exerted its effects of anti-proliferation.
Acetamides ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; analysis ; genetics ; Cyclin D ; Cyclin E ; analysis ; Cyclin-Dependent Kinase Inhibitor p15 ; Cyclin-Dependent Kinase Inhibitor p27 ; Cyclins ; analysis ; Genes, p16 ; HL-60 Cells ; Humans ; RNA, Messenger ; analysis ; Tumor Suppressor Proteins ; analysis ; genetics

OBJECTIVETo investigate the expressions of cyclin E, cyclin dependent kinase 2 (CDK-2) and cyclin-dependent kinase inhibitor p57(KIP2) in human gastric cancer, and to evaluate the relationships between protein levels and clinicopathological parameters.

METHODSWestern blot was used to measure the expressions of cyclin E, CDK-2 and p57(KIP2) proteins in the surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 36 patients.

RESULTSCyclin E and CDK-2 protein levels were higher in gastric cancer tissues in comparison with normal tissues (P < 0.05). Overexpression of cyclin E was correlated with lymph node involvement, poor histological grade and serosa invasion (P < 0.05). Overexpression of CDK-2 was correlated with lymph nodes involvement (P < 0.05). No statistically significant difference between cyclin E and CDK-2 expression was found when samples were stratified according to tumor size (P > 0.05). Expression of cyclin E and CDK-2 showed a positive linear correlation (r = 0.451, P = 0.01). Protein levels of p57(KIP2) were lower in gastric cancer tissues than in the normal mucosa (P < 0.05). Decreased expression of p57(KIP2) was correlated with lymph node involvement (P < 0.05). No statistically significant difference in p57(KIP2) expression was found when sample were stratified according to tumor size, histological grade or serosa invasion (P > 0.05). In metastatic lymph nodes, expression of cyclin E was increased and the expression of p57(KIP2) decreased.

CONCLUSIONOverexpressions of cyclin E, CDK-2 and downregulated expression of p57(KIP2) may play important roles in tumorigenesis and metastatic potential of gastric cancer.

Blotting, Western ; CDC2-CDC28 Kinases ; Cyclin E ; analysis ; physiology ; Cyclin-Dependent Kinase 2 ; Cyclin-Dependent Kinase Inhibitor p57 ; Cyclin-Dependent Kinases ; analysis ; physiology ; Humans ; Lymphatic Metastasis ; Nuclear Proteins ; analysis ; physiology ; Protein-Serine-Threonine Kinases ; analysis ; physiology ; Stomach Neoplasms ; chemistry ; pathology

OBJECTIVETo investigate the expression of five kinds of cyclins in hepatocellular carcinoma (HCC) and their association with degree of tumor differentiation, metastasis and infection of hepatitis B virus (HBV).

METHODSThe HCC tissue microarrays were composed of those from 273 cases of HCC tissues, 144 surrounding-tumor liver tissues and 10 normal liver tissues obtained from autopsy. The diameter of each specimens on tissue microarrays was 2.0 mm. Immunohistochemistry was used to detect the expression of cyclin A, cyclin B, cyclin D1, cyclin D3 and cyclin E on HCC tissue microarrays. The association of the expression of these cyclins with the infection rate of HBV was also analyzed.

RESULTSThree paraffin-embedded HCC tissue microarrays were successfully constructed, including 136, 143 and 148 tissue spots, respectively. The positive rates of cyclins in 273 cases of HCC were cyclin A 52.7%, cyclin B 45.4%, cyclin D1 35.9%, cyclin D3 44.3% and cyclin E 23.1%; while the figures in 144 surrounding-tumor tissues were 8.3%, 5.6%, 4.9%, 6.3% and 1.4%, respectively. In 10 normal liver tissues these cyclins exhibited negative staining, with the exception that cyclin D1 was positive in one case of normal liver tissue. The positive rate of cyclins in HCC were significant higher than those in surrounding-tumor liver tissues (P < 0.01), in HCC tissues with histological grade II and III, the cyclins expression were stronger than that in grade I (P < 0.05). The positive rates of cyclins, except cyclin A in HCC with portal vein invasion were higher than those without portal vein invasion (P < 0.01). Infection of HBV did not have significant relationship with the expression of cyclins (P > 0.05).

CONCLUSIONCyclins in different cell cycles overexpressed at varied levels in hepatocellular carcinoma, and the increased expression of cyclins may shorten the tumor cell cycle phase, accelerate cell proliferation, and have a close relationship with HCC aggressiveness.

Carcinoma, Hepatocellular ; chemistry ; Cyclin A ; analysis ; Cyclin B ; analysis ; Cyclin D1 ; analysis ; Cyclin D3 ; Cyclin E ; analysis ; Cyclins ; analysis ; Hepatitis B ; metabolism ; Humans ; Immunohistochemistry ; Liver Neoplasms ; chemistry

BACKGROUND: Cyclin E plays a pivotal role in the regulation of G1-S transition and could consequently be a deregulated molecule in tumors. The activity of the cdk2-cyclin E complex is increased by degradation of cdk inhibitor p27kip1. Little is known about the expression and prognostic significance of cyclin E and p27 in non-small cell lung cancer(NSCLC). MATERIAL AND METHOD: The expression of cyclin E and p27 in eighty-one cases of resected stage I NSCLC tissues and its relation to major clinico-pathological factors, including histology, differentiation, size of tumor, pleural invasion and survival rate were studied and analyzed. Immunohistochemical analysis with monoclonal antibodies specific for cyclin E and p27 were performed by ABC method. RESULT: Expression rates of cyclin E and p27 in stage I NSCLC tissues were 29.6% and 28.4% respectively. Cyclin E was expressed higher in cases of pleural invasion(p=0.04), and p27 was expressed higher in diameter of tumor less than 3cm(p=0.015). The 5-years survival rate was lower in cases of positive expression of cyclin E than in cases of negative expression of cyclin E(44.4% vs 68.2%, p=0.015), and the 5-years survival rate was 72.2% in positive expression of p27 and 56.2% in negative expression of p27(p=0.09). The 5-years survival rate was higher in negative expression of cyclin E and positive expression of p27 than in cases of positive expression of cyclin E and negative expression of p27 (73.5% vs 36.3%, p=0.0029). In multivariate analysis, expression of cyclin E was an unfavorable prognostic factor(RR=3.578, p=0.006) and p27 was a favorable prognostic factor(RR=0.183, p=0.019) independently. CONCLUSION: Cyclin E and p27 may play a pivotal role for the biological behavior of stage I NSCLC, so that the expressions of cyclin E and p27 may be new prognostic markers.
Antibodies, Monoclonal ; Carcinoma, Non-Small-Cell Lung* ; Cyclin E* ; Cyclins* ; Lung ; Multivariate Analysis ; Survival Rate

PURPOSE: Cyclin E is a key regulatory protein in the G1-S transition during the cell cycle. The correlations between cyclin E protein and the clinical features of breast cancer were investigated in order to evaluate its clinical utility in invasive breast cancer. METHODS: An immunohistochemical assay for cyclin E was performed in 101 consecutive invasive breast cancers. The correlation between cyclin E expression and the clinicobiological parameters including patient survival was analyzed. RESULTS: Cyclin E expression was observed in 50 patients (49.5%). The scoring of the cyclin E expression level was divided into low (<25%) and high (>or=25%). In high nuclear grade tumors, cyclin E overexpression was much higher than that in low nuclear grade tumors (P=0.049). In the younger age group (<50 yrs), cyclin E expression was significantly higher than the older age group (P= 0.016). No significant correlation was observed between cyclin E and the tumor size, lymph node status, hormonal receptor status, histological grade, mitotic index and Ki67. In multivariate analysis, only the lymph node status was significantly associated with the patients' outcome (P= 0.002). CONCLUSION: Cyclin E overexpression did not have prognostic impact on the patients' survival rate in invasive breast cancer. In high nuclear grade tumors, the cyclin E expression level was much higher. The definite value of cyclin E as a clinicobiologic marker should be further investigated by prospective studies with other cell regulatory proteins.
Breast Neoplasms* ; Breast* ; Cell Cycle ; Cell Proliferation ; Cyclin E* ; Cyclins* ; Humans ; Lymph Nodes ; Mitotic Index ; Multivariate Analysis ; Prognosis ; Survival Rate