OBJECTIVETo observe the effect of human cytomegalovirus infection on the host cellular DNA synthesis and expression of cyclones.

METHODSHCMV infected cell was established in vitro by incubating passage cultured HEL and HCMV AD169 strain with different titres. The cells were synchronized in the G0/G1 stage by contact inhibition and infected with strain AD169 of HCMV at an MOI of 5 PFU per cell. We harvested infected cell at different time 0 h, 3 h, 6 h, 24 h, 72 h and 96 h post infection. Then the cell cycle progress was measured. Meanwhile, the DNA content and expression of proteins of cycline E, cycline A and cycline D1 were determined with FCM and Western Blot respectively.

RESULTSWe found that the amount of S stage cell infected by HCMV had increased dramatically, and that of G2/M stage cell reduced during 24 h-96 h PI, and no G2/M stage cell was detected within 96 h PI. The content of 2N DNA maintained unchangeable for 24 h after infection and the content of total DNA in infected cells began to increase within 48 h PI, and the substantial cell with 2N DNA were observed 72 h after infection. However, DNA content was not altered in control group of normal HEL and HCMV PAA group. CyclinE protein was induced 12 h PI and peak induction occurred 24 h PI in contact-inhibited cells. CyclinA protein expression was not induced in HCMV infected density-arrested cells. The abundance of cyclinD1 decreased 24 h PI.

CONCLUSIONThe expression of cyclinE and activity of cyclinE/Cdk2 kinase are increased obviously in G0/G1 stage cells infected with HCMV, which may induce the cell cycle to overpass G1/S restriction point and make the cell cycle arrested in later G1 stage. HCMV can not activate cellular DNA synthesis, and increase of total DNA content in infected cells result from the viral DNA replication.

Cell Cycle ; Cells, Cultured ; Cyclins ; genetics ; metabolism ; Cytomegalovirus ; physiology ; Cytomegalovirus Infections ; genetics ; metabolism ; Gene Expression ; Humans

The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.
Anemia, Aplastic/*blood ; Antigens, CD34/*metabolism ; Cells, Cultured ; Colony-Forming Units Assay ; Cyclins/*biosynthesis ; Cyclins/genetics ; Fetal Blood/cytology ; Hematopoietic Stem Cells/*cytology ; Protein Isoforms/biosynthesis ; Protein Isoforms/genetics ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Serum

OBJECTIVETo construct cyclin D2 (CCND2) short hairpin RNA ( shRNA) plasmid for repressing the expression of CCND2 in human myeloma cell line LP-1,and to detect its effect on the proliferation and apoptosis of LP-1 cell.

METHODSA CCND2 shRNA model was constructed and cloned into plasmid pGensil-2, then the plasmid was transfected into LP-1 cell in vitro. The CCND2 expression cell proliferation, cell cycle and cell apoptosis of the transfected LP-1 cells were studied by RT-PCR, trypanosome staining, flow cytometry and annexin V assay.

RESULTSThe transfection efficiency of LP-1 cell was 34. 2%. In the transfected LP-1 cell CCND2 mRNA expression was reduced significantly, the cell growth was inhibited significantly and the cell cycle was partly arrested in G, phase. The apoptosis rate of the transfected LP-1 cell after 72 h was (25.7+/-4.8)%.

CONCLUSIONThe inhibition of CCND2 in LP-1 cells could inhibit the cell growth and induce cell apoptosis. CCND2 maybe a new therapeutic target.

Apoptosis ; Cell Proliferation ; Cyclin D2 ; Cyclins ; genetics ; Humans ; RNA Interference ; RNA, Double-Stranded ; RNA, Small Interfering ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo explore the relationship between human cyclin C (CCNC) gene and childhood acute lymphocytic leukemia (ALL).

METHODSThe total RNA isolated from myeloid tissues of normal children and of children with newly diagnosed ALL and from ALL cell line 6T-CEM was reversely transcribed into cDNA. Real-time fluorescence quantitative PCR method was used to detect CCNC gene expression.

RESULTSCCNC was expressed in myeloid tissues of normal children and of children with newly diagnosed ALL as well as 6T-CEM. The relative expression level of CCNC gene in children with newly diagnosed ALL was significantly lower than in normal controls (2.35 +/- 0.83 vs 13.5 +/- 0.30; P <0.05).

CONCLUSIONSCCNC gene shows lower expression in children with newly diagnosed ALL, suggesting that it may be a tumor suppressing gene in childhood ALL.

Child ; Cyclin C ; Cyclins ; genetics ; Female ; Fluorescence ; Humans ; Male ; Polymerase Chain Reaction ; methods ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism

OBJECTIVETo investigate the expression, localization and interrelationship of P27(kip1) and cyclin D3 in non-Hodgkin's lymphoma (NHL).

METHODSThe expressions of P27(kip1), cyclin D3 and index Ki-67 was detected in 100 NHL and 20 reactive lymph nodes by immunohistochemical technique. The expression and localization of P27(kip1) and cyclin D3 in 3 NHL cell lines were detected by Western blot, double immunolabelling and laser scanning confocal microscopy, respectively.

RESULTSIn general the expression of P27(kip1) in NHL was lower than in control group, and was negatively related to the tumor aggressiveness and proliferating activity; the expression of cyclin D3 in NHL was higher than in control group, and was positively related to the tumor aggressiveness and proliferating activity. There was a negative correlation between P27(kip1) and cyclin D3. Nevertheless, anomalous high P27(kip1) expression was found in a few NHL tissues with high expression of cyclin D3 and Ki-67. Overexpression and colocalization of P27(kip1) and cyclin D3 was found in Raji cell line.

CONCLUSIONSUnder expression of P27(kip1) and overexpression of cyclin D3 may play a role in the occurrence and development of NHL. Anomalous high P27(kip1) expression and its interaction with cyclin D3 may be another mechanism for tumor genesis of NHL.

Cell Line, Tumor ; Cyclin D3 ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Cyclins ; genetics ; metabolism ; Humans ; Lymphoma, Non-Hodgkin ; genetics ; metabolism ; pathology

Good progress has been made in the researches on correlating genes of breast cancer in recent years. Quite a few kinds of genes such as susceptibility gene, oncogene and tumor suppressor genes have been found with implications for diagnosis, therapy and prognosis. Abnormality of breast cancer susceptibility gene (BRCA) is of great significance, especially in the development of breast cancer.
BRCA1 Protein ; genetics ; BRCA2 Protein ; genetics ; Breast Neoplasms ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; Female ; Humans ; Mutation ; Neoplasm Proteins ; genetics ; Proto-Oncogene Proteins ; genetics ; Tumor Suppressor Protein p53 ; genetics

BACKGROUNDMutation and expression change of p21WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21WAF1/CIP1 gene in human osteosarcoma, p21WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.

METHODSp21WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry, respectively.

RESULTSThe occurrence of P21 protein in osteosarcoma was 17.78% (8/45), and p21WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma, there were 17 cases (47.22%) with C-->T at position 609; 10 normal blood samples' DNA sequence analysis yielded 8 cases (80.00%) with C-->T at the same position.

CONCLUSIONSAlong with the increase of malignancy, the expression of p21WAF1/CIP1mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research.

Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; Humans ; Osteosarcoma ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; RNA, Messenger ; analysis ; Sequence Analysis, DNA

OBJECTIVETo investigate c-myc and CCNE2 gene amplifications and their relationship in breast cancer.

METHODSSixty-six infiltrating ductal breast carcinomas with foci of ductal carcinoma in situ components collected from January 2005 to December 2007 were selected for tissue microarray and quantitative multi-gene FISH for c-myc and CCNE2 gene amplification, and the relationship with the clinicopathologic features was analyzed.

RESULTSOf the 66 cases, 18 (27.3%) showed c-myc amplification and 23 (34.8%) showed CCNE2 amplification. A strong correlation was found between c-myc and CCNE2 amplification (P < 0.01). The breast cancers showing c-myc and CCNE2 amplifications were all aneuploidy, and were HER2 positive (P < 0.05). Tumors with c-myc amplification also showed higher Ki-67 index (P < 0.05).

CONCLUSIONSC-myc and CCNE2 amplifications are common events in breast cancer, and they often coexist. C-myc and CCNE2 genes may play critical roles in the pathogenesis and development of breast cancer through unique and overlapping signaling pathways.

Aneuploidy ; Breast Neoplasms ; genetics ; Carcinoma in Situ ; genetics ; Carcinoma, Ductal, Breast ; genetics ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; Cyclins ; genetics ; Female ; Gene Amplification ; Genes, myc ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Tissue Array Analysis

OBJECTIVETo explore the role of F10 gene in regulating cell cycles of choriocarcinoma cells and the underlying mechanisms.

METHODSUsing untreated cells as the control, JAR cells with F10 gene silencing or stable F10 over-expression were examined for cell cycle changes by flow cytometry (FCM) and for expressions of cyclin and cyclin-dependent kinase (CDKs) with Western blotting and immunofluorescence technique.

RESULTSJAR cells over-expressing F10 gene showed reduced duration of cell cycle compared with untreated and with cells after F10 gene silencing. In F10-over-expressing cells, Western blotting revealed significantly up-regulated expressions of cyclin A2, B1, D1, E and CDK2, 6, and 7, but not CDK4, as compared with the control cells and cells with F10 gene silencing (P<0.05), and these results were consistent with those by immunofluorescence assay.

CONCLUSIONF10 gene may accelerate cell cycle progression and promote cell proliferation by up-regulating the expressions of cyclin A2, B1, D1, E and CDK 2, 4, 6, 7 in choriocarcinoma cells.

Cell Cycle ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Choriocarcinoma ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; Cyclins ; metabolism ; Factor X ; genetics ; Female ; Gene Silencing ; Humans ; Pregnancy

OBJECTIVETo investigate the relationship between cyclin D2 and P210(BCR/ABL) tyrosine kinase in chronic myelogenous leukemia (CML).

METHODSRT-PCR, Western blot and flow cytometry were performed to detect the expression of cyclin D2 in K562 cells and in K562-ib-eGFP cells which express intracellular single-chain antibody (sFv, intrabody) against ABL tyrosine kinase domain.

RESULTSCyclin D2 expression in K562-ib-eGFP cells was 18.90% which was lower than that of control K562 cells (48.10%), and the number of S-phase cells in K562-ib-eGFP was 40.40% which was much lower than that in K562 cells (64.34%).

CONCLUSIONCyclin D2 is a potential down-stream signal molecule of the p210(BCR/ABL) tyrosine kinase in CML. The altered expression of cyclin D2 may contribute to the over proliferation of CML cells.

Blotting, Western ; Cyclin D2 ; Cyclins ; analysis ; genetics ; Flow Cytometry ; Genes, abl ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction