CDK2 and CDK4 known promoter of cell cycling catalyze phosphorylation of RB protein. Enzyme specificity between two CDKs that work at a different cell cycle phase is not clearly understood. In order to define kinase properties of CDK2 and CDK4 in complex with cycline A or cycline D1 in relation to their respective role in cell cycling regulation, we examined enzymatic properties of both CDK4/cycline D1 and CDK2/cycline A in vitro. Association constant, Km for ATP in CDK4/cyclin D1 was found as 418 micrometer, a value unusually high whereas CDK2/cyclin A was 23 micrometer, a value close to most of other regulatory protein kinases. Turnover value for both CDK4/cyclin D1 and CDK2/cyclin A were estimated as 3.4 and 3.9 min(-1)respectively. Kinetic efficiency estimation indicates far over one order magnitude less efficiency for CDK4/cyclin D1 than the value of CDK2/cycline A (9.3 pM(-1)min(-1)and 170 pM(-1)min(-1)respectively). In addition, inhibition of cellular CDK4 caused increase of cellular levels of ATP, even though inhibition of CDK2 did not change it noticeably. These data suggest cellular CDK4/cyclin D1 activity is tightly associated with cellular ATP concentration. Also, analysis of phosphorylated serine/threonine sites on RB catalyzed by CDK4/cyclin D1 and CDK2/cyclin A showed significant differences in their preference of phosphorylation sites in RB C-terminal domain. Since RB is known to regulate various cellular proteins by binding and this binding is controlled by its phosphorylation, these data shown here clearly indicate significant difference in their biochemical properties between CDK4/cyclin D1 and CDK2/cyclin A affecting regulation of cellular RB function.
Adenosine Triphosphate/metabolism ; Amino Acid Sequence ; Baculoviridae/genetics ; CDC2-CDC28 Kinases/genetics/isolation&purification/*metabolism ; Cyclin A/genetics/isolation&purification/*metabolism ; Cyclin D1/genetics/isolation&purification/*metabolism ; Cyclin-Dependent Kinases/antagonists&inhibitors/genetics/isolation&purification/*metabolism ; Human ; Kinetics ; Molecular Sequence Data ; Phosphorylation ; Protein Conformation ; Protein p16/metabolism ; Recombinant Proteins/genetics/isolation&purification/metabolism

OBJECTIVETo design DNA microarray and investigate the molecular anti-tumor mechanism of herbs of traditional Chinese medicine.

METHODcDNA microarrays consisting of 56 probes representing 24 human cell cycle genes were constructed, Four anti-hepatocarcinoma herbs including Radix Linderae, Hebra Artemisiae Annuae, Radix Amebiae, Radix Astragli, were chosen. Effects of herbs on SMMC-7721 cell cycle were observed by flow cytometry assay. Effects of herbs on cell cycle gene expression in SMMC-7721 cells were analyzed by comparing hybridization of Dig-Labeled cDNAs from herb-treated cells and cDNAs from untreated cells.

RESULTExpressions of cell cycle geneswere changed in different degrees after herbs treated. Some genes were down-regulated and some genes were up-regulated. The changes in gene expression agreed with the results of flow cytometry assay.

CONCLUSIONThe results suggest that these herbs may have effects on cell cycle and DNA damage checkpoint genes which may be the mechanism of the herbs, and DNA microarray can be used to investigate the biological function of extracts of traditional Chinese medicine.

Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Artemisia ; chemistry ; Astragalus membranaceus ; chemistry ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Cyclin-Dependent Kinases ; genetics ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Gene Amplification ; Gene Expression Profiling ; Genes, cdc ; drug effects ; Humans ; Lindera ; chemistry ; Lithospermum ; chemistry ; Liver Neoplasms ; metabolism ; pathology ; Oligonucleotide Array Sequence Analysis ; methods ; Plants, Medicinal ; chemistry ; Proliferating Cell Nuclear Antigen ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; cdc25 Phosphatases ; genetics ; metabolism

OBJECTIVETo investigate the effects of ginsenoside Rh2 (GS-Rh2) on growth inhibition and cell cycle of Eca-109 esophageal carcinoma cell line in culture.

METHODThe effects of GS-Rh2 on cell growth inhibition was detected by MTT assay. Cell cycle was analyzed by flow cytometry (FCM). Cell morphology was observed by a light microscope after HE staining. The protein expression of cell cycle components (cyclinE, CDK2, p21WAF1) were examined by immunocytochemistry and Western blot. The mRNA expression were examined by semiquantitative RT-PCR.

RESULTGS-Rh2 inhibited the proliferation of Eca-109 cells in dose and time-dependent manners. The inhibition rate was about 50% after 1-day treatment with 20 microg x mL(-1) GS-Rh2 x 20 microg x mL(-1) GS-Rh2 induced the mature differentiation and morphological reversion. With increasing dose of GS-Rh2 treatment, the cell number of G0/G1 phase was increased, whereas it decreased at S and G2/M phase. There was significant difference between 10, 20 microg x mL(-1) GS-Rh2 groups and the corresponding group without GS-Rh2 treatement. After treating cells by 20 microg x mL(-1) GS-Rh2 for 1, 2, 3 days individually, the protein and mRNA expression of both cyclinE and CDK2 reduced, while the expression of p21WAF1 enhanced gradually.

CONCLUSIONGS-Rh2 could arrest Eca-109 cells at G0/G1 phase and induce cell differentiation tending to normal. Furthermore, GS-Rh2 had an effect on expression of cell cycle components (cyclinE, CDK2 and p21WAF1) to inhibit Eca-109 cell proliferation.

Carcinoma, Squamous Cell ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin E ; biosynthesis ; genetics ; Cyclin-Dependent Kinase 2 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Esophageal Neoplasms ; metabolism ; pathology ; Ginsenosides ; administration & dosage ; isolation & purification ; pharmacology ; Humans ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Time Factors

OBJECTIVETo evaluate the overexpression of cyclin G1 in cervical intraepithelial neoplasia (CIN) and cervical carcinoma, and the correlation between cyclin G1 and high-risk human papilloma virus (HPV) infection.

METHODSAll of the specimens were obtained from the Department of Pathology of China-Japan Friendship Hospital from January 2000 to August 2004. We detected the expression of cyclin G1 with immunohistochemistry, HPV16/18 infection with in situ hybridization, and high-risk HPV infection with Hybrid capture system II (HC-II) in normal group (25 cases), CIN I (48 cases), CIN II (56 cases), CIN III (54 cases), and invasive cervical squamous-cell carcinoma (SCC, 31 cases).

RESULTSThe positive rates of cyclin G1 expression in CIN (77.85%) and SCC cervical tissues (87.10%) were significantly higher than normal (8.00%, P < 0.01), and the intensities of cyclin G1 expression in CIN (40.60%) and SCC cervical tissues (61.51%) were significantly higher than normal (2.72%, P < 0.05). The positive rates and intensities of cyclin G1 expression increased gradually with the grade of cervical lesions. High-risk HPV infection rates were higher in CIN and SCC than normal groups (P < 0.05). There was a positive correlation between cyclin G1 expression and high-risk HPV infection detected with HC-II (Kendall's tau-b = 0.316, 0.269, 0.352, and 0.474 in CIN I, CINII, CIN III, and SCC, respectively, P < 0.05).

CONCLUSIONSCyclin G1 is overexpressed in CIN and SCC. Cyclin G1 may be a biomarker for detecting CIN and SCC. Cyclin G1 may play an important role in the oncogenesis of CIN and SCC by high-risk HPV infection.

Carcinoma, Squamous Cell ; metabolism ; virology ; Case-Control Studies ; Cervical Intraepithelial Neoplasia ; metabolism ; virology ; Cyclin G ; Cyclin G1 ; Cyclins ; metabolism ; Female ; Human papillomavirus 16 ; genetics ; isolation & purification ; Human papillomavirus 18 ; genetics ; isolation & purification ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Papillomavirus Infections ; metabolism ; virology ; Uterine Cervical Neoplasms ; metabolism ; virology

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.
Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 9 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; drug effects ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Nucleosomes ; drug effects ; metabolism ; pathology ; Plant Extracts ; chemistry ; Prostate ; drug effects ; metabolism ; pathology ; Reishi ; chemistry ; Signal Transduction ; Triterpenes ; isolation & purification ; pharmacology

OBJECTIVETo investigate the clinicopathological significance of p16(INK4A) expression and DNA ploidy status in HPV-negative uterine cervical cancers and their precursors.

METHODSHPV-negative cervical lesions, including 20 cases of cervicitis, 20 cases of cervical intraepithelial neoplasm (CIN), 3 cases of cervical glandular intraepithelial neoplasm (CGIN), 38 cases of invasive squamous cell carcinoma (SCCs) and 15 cases of invasive adenocarcinoma were selected and subject to screening for HPV infection by PCR method. The p16(INK4A) protein expression and DNA ploidy status were studied by immunohistochemistry and flow cytometry respectively.

RESULTSSpecific expression of p16(INK4A) was seen in both the nucleus and cytoplasm of the dysplastic and malignant cells of CIN, CGIN, cervical SCC and adenocarcinoma. In contrast, no expression was present in normal and inflammatory squamous or glandular epithelium. DNA aneuploidy was significantly more frequent in invasive SCCs and adenocarcinomas than in CIN (P < 0.01). Aneuploid was also more frequent in the lymph node positive group than lymph node negative group, although no statistic significance was found. Among the 8 cases of p16(INK4A) negative SCCs, two showed DNA aneuploidy.

CONCLUSIONSImmunohistochemical detection for p16(INK4A) can be an early diagnostic marker for HPV-negative cervical SCC and adenocarcinoma. DNA ploidy analysis may further assist the diagnosis of cervical malignancies.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Aneuploidy ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; DNA, Neoplasm ; genetics ; Female ; Flow Cytometry ; Humans ; Immunohistochemistry ; Papillomaviridae ; genetics ; isolation & purification ; Polymerase Chain Reaction ; Uterine Cervical Neoplasms ; genetics ; metabolism ; pathology ; Uterine Cervicitis ; genetics ; metabolism ; pathology

The aim of this study was to investigate the effect of evodiamine on the proliferation and the immune function of thymocytes and splenocyte of mice from three germlines, which were 8 weeks old masculinity BALB/c, C57BL/6 and F1 hybridization mice. Cells of thymus and spleen were harvested and prepared as unicellular suspension. Cell proliferation was detected by MTT method, while the concentration of IL-2 was detected by ELISA, mRNA levels of bcl-2 and cdk2 in cells treated with evodiamine were detected by RT-PCR, the apoptosis rate and intracellular reactive oxygen species (ROS) concentration were analyzed by FCM, and the protein levels of BCL-2, CDK2 and BAX were determined by fluorescence microscope. The results indicated that at 0.5, 0.75 and 1 micromol/L, evodiamine inhibited the proliferation and externalization of thymocytes and splenocytes stimulated with ConA (p < 0.05). At 0.75 micromol/L, evodiamine inhibited the secretion of IL-2, decreased the mRNA level of bcl-2 and cdk2, and induced apoptosis of thymocytes and splenocytes (p < 0.05). Intracellular ROS concentration increased significantly after treatment with evodiamine for 12 hours (p < 0.05). The death rate increased at a prolong period of time. After treatment with evodiamine for 24 and 48 hours, the cells were divided into two groups, one of which was negatively stained by 2 7-dichlorofluorescein (DCF), which indicated that ROS level decreased significantly in the dying cells. It is concluded that evodiamine inhibits proliferation and induces apoptosis of thymocytes and splenocytes from different germline mice, and at the same time decreases secretion of IL-2 through down-regulating bcl-2 and cdk2 levels.
Adjuvants, Immunologic ; pharmacology ; Animals ; Cyclin-Dependent Kinase 2 ; genetics ; metabolism ; Drugs, Chinese Herbal ; chemistry ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Plant Extracts ; isolation & purification ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Quinazolines ; isolation & purification ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Species Specificity ; Spleen ; cytology ; immunology ; Thymus Gland ; cytology ; immunology

Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.
Carcinoma, Squamous Cell/genetics/*pathology/virology ; Comparative Study ; Cyclin-Dependent Kinase Inhibitor p16/analysis/*genetics ; *DNA Methylation ; DNA, Viral/genetics/isolation & purification ; Epstein-Barr Virus Infections/genetics/*pathology/virology ; Epstein-Barr Virus Nuclear Antigens/genetics ; Female ; Herpesvirus 4, Human/genetics ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Polymerase Chain Reaction ; Precancerous Conditions/genetics/*pathology/virology ; RNA, Viral/genetics ; Research Support, Non-U.S. Gov't ; Uterine Cervical Neoplasms/genetics/*pathology/virology

OBJECTIVETo investigate the effect of curcumin (Cur) on histone deacetylase (HDAC1) and P21(WAF1/CIP1), a cyclin dependent kinase inhibitor, in HepG2 cells for exploring the mechanism of Cur in anti-cancer.

METHODThe HDAC1, P21(WAF1/CIP1) proteins and P21(WAF1/CIP1) mRNA were extracted from human hepatoma cells treated with or without Cur of different concentrations at different time points. Western blot analysis was performed to determine the levels of HDAC1 and P21(WAF1/CIP1) proteins, respectively. RT-PCR was performed to detect the level of P21(WAF1/CIP1) mRNA.

RESULTThe IC50 of concentration treated by Cur was 25 micromol x L(1) on HepG2 cell. The level of HDAC1 was obviously inhibited by Cur, and decreased at 4 hours at IC, and lasted for 48 h in a time-dependent manner. The inhibition of HDAC1 was significant at the Cur concentration of 12.5 micromol x L(-1) but there was no difference between 50 and 100 micromol x L(-1). The levels of P21(WAF1/CIP1) mRNA and protein were up-regulated by Cur in dose and time-dependent manner, and the change of mRNA and protein was detected at 8 hours and lasted for 48 hours.

CONCLUSIONCur can inhibit the level of HDAC1 and enhance the expression of P21(WAF1/CIP1) protein and mRNA, and the results suggest that inhibiting HDAC1 and increasing P21(WAF1/CIP1) may be one of the possible mechanisms of anti-cancer by Cur.

Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Curcuma ; chemistry ; Curcumin ; administration & dosage ; isolation & purification ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Neoplastic ; drug effects ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

OBJECTIVETo verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo.

METHODSChromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein.

RESULTSThe p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples.

CONCLUSIONSp53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.

Binding Sites ; Chromatin ; genetics ; isolation & purification ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; metabolism ; Gene Expression Regulation ; HSP90 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Polymerase Chain Reaction ; methods ; Precipitin Tests ; methods ; Promoter Regions, Genetic ; Protein Binding ; Transcription, Genetic ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; physiology