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 cell cycle is a complex process that involves DNA replication, protein expression, and cell division. Dysregulation of the cell cycle is associated with various diseases. Cyclin-dependent kinases (CDKs) and their corresponding cyclins are major proteins that regulate the cell cycle. In contrast to inhibition, a new approach called proteolysis-targeting chimeras (PROTACs) and molecular glues can eliminate both enzymatic and scaffold functions of CDKs and cyclins, achieving targeted degradation. The field of PROTACs and molecular glues has developed rapidly in recent years. In this article, we aim to summarize the latest developments of CDKs and cyclin protein degraders. The selectivity, application, validation and the current state of each CDK degrader will be overviewed. Additionally, possible methods are discussed for the development of degraders for CDK members that still lack them. Overall, this article provides a comprehensive summary of the latest advancements in CDK and cyclin protein degraders, which will be helpful for researchers working on this topic.
Humans ; Cell Cycle/physiology* ; Cell Division ; Cyclin-Dependent Kinases/metabolism* ; Cyclins/metabolism*

OBJECTIVETo explore a method which can remove the gastric mucus in order to prepare mucous membrane single cell suspension for the research of cytomics.

METHODSEnzymology was used to remove the mucus gel and to separate mucous layer from the normal fresh gastric tissue. The mucous layer was broken to prepare single cell suspension with machine method. Expression of major cyclins in mucous layer cells was examined by cytoimmunochemistry, flow cytometry(FCM) and confocal microscopy.

RESULTSThe 0.1% pepsin could dissolve the mucus gel and 1.2-2.4 U/L dispase could separate the mucous layer completely. The single mucous cell suspension was prepared successfully. FCM results from mucous single cell suspension revealed that expression of cyclin D(3), B(1) was obvious, that of cyclin D(2) was weak and that of cyclin D(1), A, E was the least. Similar results were found with confocal microscopy.

CONCLUSIONSSingle cell suspension from mucous layer can be easily prepared by pepsin and dispase. Cyclins schedule expression in vivo is different from cyclins schedule expression in vitro.

Cell Line ; Cell Proliferation ; Cyclins ; metabolism ; Flow Cytometry ; Gastric Mucins ; metabolism ; Gastric Mucosa ; cytology ; metabolism ; Humans ; Mucous Membrane ; cytology ; metabolism

OBJECTIVETo investigate the effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit.

METHODSBilateral mandibular osteotomy was performed in 45 New-Zeland rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/day for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups. 2 microg (0.1 microg/microl) of pIRES-hVEGF165-hBMP2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES and the same volume of normal saline (NS) was injected into the distraction area in each group, respectively. After injection, electroporation was performed in every group. Three animals in each group were sacrificed at 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations. The expression of cyclins A, D1 ,E in positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with the single factor analysis of variance and q test.

RESULTSCyclins A, D1, E staining was mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues around the new bone. The expression reached to the peak at 7th day of consolidation, and decreased at 14th day, and weak at 28th day. Image analysis results showed that, at 7th day, the expression absorbance A in group C (0.59 +/- 0.14) was the strongest, compared to group A (0.41 +/- 0.13), B (0.38 +/- 0.14), D (0.34 +/- 0.12) and E (0.31 +/- 0.10), showing a significant difference (P < 0.05, P < 0.01). There was no significance difference between group A and B (P > 0.05), but the difference between group A/B and group D/E (P < 0.05). At 14th and 28th day, there was no significant difference among group A (0.39 +/- 0.11), B (0.34 +/- 0.10) and C (0.33 +/- 0.09) (P > 0.05), but there was significant difference between group A/B/C and group D (0.19 +/- 0.12) or E (0.14 +/- 0.04) (P < 0.05 or P < 0.01).

CONCLUSIONSElectroporation-mediated gene transfection can promote cyclins A, D1, E expression effectively, which may promote cell differentiation and proliferation, stimulate extracellular matrix synthesis and new bone formation in distraction gap.

Animals ; Cyclins ; metabolism ; Electroporation ; Genetic Therapy ; Mandible ; surgery ; Osteogenesis, Distraction ; methods ; Plasmids ; Rabbits ; Transfection

OBJECTIVEThe effects of lentivirus-mediated suppression of Cyclin Y (CCNY) expression on the proliferation of laryngeal cancer cells were investigated in vitro.

METHODSThe lentivirus vectors containing a small hairpin RNA (shRNA) to target CCNY were constructed.Hep-2 cells were divided into the following two experimental groups:the negative control group (control lentivirus infected cells) and CCNY knockdown group (CCNY shRNA-expressing lentivirus infected cells). After Hep-2 cells were infected, Real-time PCR was used to measure CCNY expression. The influence of CCNY on the proliferation of laryngeal cancer cells were assessed using MTT and colony formation experiments.Each experiment was performed in triplicate and repeated three times.

RESULTSLentiviruses expressing shRNA against CCNY were constructed and Hep-2 cells were infected with above mentioned lentivirus at MOI (Multiplicity of infection) of 120.Real-time PCR analysis showed that the mRNA expression of CCNY in Hep-2 cells in the knockdown group was significantly decreased (P < 0.05); the mRNA level of CCNY was 75.3% lower in the si-CCNY group than in the si-CTRL group. After 5 days of lentiviral infection, the cell viability was significantly lower in cells infected with the CCNY-shRNA lentivirus compared to cells infected with the control lentivirus following a 6-day incubation. The colony number was decreased by 60% in Hep-2 cells infected with the CCNY-shRNA-lentivirus infected cells following a 10-day incubation.

CONCLUSIONSThe results suggested that lentivirus-mediated downregulation of CCNY expression decreased the proliferation and growth potency of laryngeal cancer cells.Lentiviruses delivering shRNA against CCNY may be a promising tool for laryngeal cancer therapy.

Cell Line, Tumor ; Cell Proliferation ; Cyclins ; Humans ; Laryngeal Neoplasms ; metabolism ; Lentivirus ; genetics ; RNA, Small Interfering ; genetics

Partial hepatectomy (PH) endorses quiescent hepatocytes to reenter the cell cycle. The regenerating liver returns to its preresection weight after 7 days, following one or two cell division and maintains nearly its original volume after then. We focused on the inhibition of further hepatocyte proliferation, hypothesizing possible involvement of cell cycle upregulators and inhibitors. We studied protein levels in expression of cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs), and their in situ hepatic lobular distributions in partial hepatectomized rat liver. Cyclin E was expressed in the same levels in normal liver and after PH. Expression of cyclin A, not detected in normal liver, increased in following times after PH and reached a maximum at 7 day. CDK2 and 4 showed increased expression toward terminal period. Contradictory findings of cyclin A and these CDKs might play an important role in the inhibition of further cell division, although still unclear. Constitutively expressed CDK6 decreased after 1 day. p18 showed peak expression within 1 day, and p16, p21, p27 and p57 were stronger at terminal periods. During the expected period of their activity, intranuclear translocations were observed in cyclin E, p18 and p16. There was no evidence of regional distribution in hepatic lobular architecture, instead, diffuse in situ expression, corroborating synchronous event, was found.
Animal ; Cell Cycle/physiology* ; Cyclin-Dependent Kinases/metabolism ; Cyclin-Dependent Kinases/antagonists & inhibitors ; Cyclins/metabolism* ; Cyclins/immunology ; Flow Cytometry ; Hepatectomy ; Immunoblotting ; Immunohistochemistry ; Interphase/physiology ; Liver/metabolism* ; Liver Regeneration/physiology* ; Male ; Rats ; Rats, Sprague-Dawley ; S Phase/physiology

Cyclin/cdc complexes are known to function in cell-cycle regulation. Cyclin D1/cdk4 and -6 complexes, which functions as a G1-S checkpoint and cyclin B1/cdc2 complexes, a G2-M checkpoint are essential for DNA synthesis and mitosis, respectively. Thus, dysregulated overexpression of cyclins appears to be involved in uncontrollable cell proliferation and early tumor development. We investigated the expression and proliferative index of cyclin D1 (PIcyclin D1), cyclin B1 (PIcyclin B1) and Ki-67 (PIKi-67) using immunohistochemical staining on 15 cases of ductal hyperplasia (DH), 26 cases of atypical ductal hyperplasia (ADH) and 43 cases of ductal carcinoma in situ (DCIS) of the breast in order to evaluate whether these cyclins are associated with abnormal cell proliferation and play a role in tumor development from ADH to carcinoma. Furthermore, we investigated whether the expression and proliferative index of the cyclins and Ki-67 are correlated with the histologic grade according to the Van Nuys classification and with the histologic subtype according to traditional classification. Finally, we estimated the correlation coefficient among PIcyclin D1, PIcyclin B1, PIKi-67 and estrogen receptor in ADH and DCIS. The expression of cyclin D1 was detected in 39.5% of DCIS and 7.7% of ADH cases. In the DH cases, expression of cyclin D1 was not found. Expression of cyclin B1 was also detected in 69.7% of DCIS, 50.0% of ADH and 93.3% of the DH cases. The PIcyclin D1 was significantly different among these three groups. Moreover, the PIcyclin D1 and PIKi-67 were differed significantly between the low grade DCIS and ADH cases. However, PIcyclin B1 only appeared to be significantly different between the total DCIS and ADH. Results of the correlation coefficient among PIcyclin D1, PIcyclin B1 and PIKi-67 were positively correlated with each other. No significant correlation was found between the expression of ER and cyclin D1 in ADH and DCIS. In summary, our results support the hypothesis that a cyclin D1 and cyclin B1 protein aberration, along with Ki-67, may act as a relatively early event in the tumor development from ADH to carcinoma.
Breast/pathology* ; Breast/metabolism* ; Breast Neoplasms/pathology* ; Breast Neoplasms/metabolism* ; Carcinoma, Infiltrating Duct/pathology* ; Carcinoma, Infiltrating Duct/metabolism* ; Cyclins/metabolism* ; Female ; Human ; Hyperplasia ; Ki-67 Antigen/metabolism

OBJECTIVETo study the expression of A-kinase anchor protein 95 (AKAP95), cyclin E(2), and connexin 43 (Cx43) in lung cancer tissue, the clinical significance of their expression, and the expression correlation among the three proteins.

METHODSFifty-one samples of lung cancer tissue were examined by immunohistochemistry to measure the expression of AKAP95, cyclin E2, and Cx43.

RESULTSThe positive rate of AKAP95 expression in lung cancer tissue was significantly higher than that in paracancerous tissue (82.35% vs 33.33%, P < 0.05); AKAP95 expression was associated with the cell differentiation and histopathological type of lung cancer (P < 0.05). The positive rate of cyclin E(2) expression in lung cancer tissue was significantly higher than that in paracancerous tissue (43.14% vs 13.33%, P < 0.05); cyclin E(2) expression was associated with the lymph node metastasis and histopathological type of lung cancer (P < 0.05). The positive rate of Cx43 expression in lung cancer tissue was lower than that in paracancerous tissue (60.78% vs 80.00%); Cx43 expression was associated with the cell differentiation, lymph node metastasis, and histopathological type of lung cancer (P < 0.05). There was correlation between each two of AKAP95 expression, cyclin E(2) expression, and Cx43 expression in lung cancer tissue.

CONCLUSIONHigh expression of AKAP95 and cyclin E(2) plays an important role in the occurrence and development of lung cancer. AKAP95 expression is associated with the cell differentiation and histopathological type of lung cancer, and cyclin E2 expression is associated with lymph node metastasis and histopathological type. There is correlation between each two of AKAP95 expression, cyclin E(2) expression, and Cx43 expression in lung cancer tissue.

A Kinase Anchor Proteins ; metabolism ; Adult ; Aged ; Connexin 43 ; metabolism ; Cyclins ; metabolism ; Female ; Humans ; Lung ; metabolism ; pathology ; Lung Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged

This study investigated the potential role of ERK1/2-cyclinE1 signaling pathway in rat pulmonary artery smooth muscle cells (rPASMCs) proliferation and pulmonary vascular remodeling induced by cigarette smoke exposure. A total of 24 male Wistar rats were randomly divided into 4 groups: control group (C group), S-1M, S-3M and S-6M groups (animals in the groups were exposed to smoke for 1, 3, and 6 months, respectively). HE staining and anti-α-smooth muscle actin antibody staining were performed to observe the degree of pulmonary vascular remodeling. Immunohistochemistry and Western blotting were performed to evaluate ERK1/2 and cyclinE1 expression in pulmonary vessels. Primary cultured rat pulmonary artery smooth muscle cells (rPASMCs) were exposed to cigarette smoke extract (CSE). ERK inhibitor (PD98059) and cyclinE1 siRNA were used to verify the role of ERK1/2 and cyclinE1 in CSE-induced rPASMCs proliferation. Cell proliferation was assessed by cell counting and 5-bromo-2-deoxyuridine (BrdU) incorporation. Our results showed that abnormal pulmonary vascular remodeling was found in cigarette smoked rats. Compared to C group, activated ERK1/2 and cyclinE1 expression was significantly increased in smoke-exposure groups. This up-regulated expression was positively correlated with the severity of pulmonary vascular remodeling, and there was positive correlation between the expression of ERK1/2 and cyclinE1. PD98059 and cyclinE1 siRNA inhibited the proliferation of rPASMCs. The expression of cyclinE1 could be down-regulated by PD98059. Our data demonstrated that increased expression of ERK1/2 and cyclinE1 might be involved in the pathogenesis of abnormal rPASMCs proliferation and rat pulmonary vascular remodelling induced by cigarette smoke exposure.
Animals ; Cells, Cultured ; Cyclins ; metabolism ; MAP Kinase Signaling System ; Male ; Myocytes, Smooth Muscle ; metabolism ; pathology ; Pulmonary Artery ; metabolism ; pathology ; Rats ; Rats, Wistar ; Smoking ; metabolism ; pathology ; Up-Regulation

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