The effect of triamcinolone acetonide(TA) on the expression of Gl related genes was investigated the cultured keloid fibroblast. The addition of TA to the culture medium resulted in growth inhibition of keloid fibroblast. TA reduced the expression of cyclin A, B, E and cyclin dependent kinase(CDK) 2 mRNA, but unexpectedly, the expression of cyclin C, Dl and CDK4 mRAN was not affected significantly as compared with those of normal fibroblast. Expressions of p16, p21 and p27, the wellestabilished CDK-inhibitors, were also investigated. The level of p16 was not detected in both normal and keloid fibroblasts and the expression of p27 was significantly decreased in keloid fibroblast. The expression of p21 was dramatically increased in keloid fibroblast but not significantly changed in normal fibroblast. Also the expressions of p53 and pRb, the well known tumor suppressor genes, were increased by the addition of TA. These data suggested that the observed growth inhibitory effect of TA may be related to transcriptional inactivation of cyclin A, B, E and CDK2 and to the transcriptional activation of p21, but the mechanisms of unchanged expression of cyclin C, Dl and CDK4 mRNA remain to be elucidated.
Cell Cycle* ; Cyclin A ; Cyclin C ; Cyclins ; Fibroblasts* ; Genes, Tumor Suppressor ; Keloid* ; RNA, Messenger ; Transcriptional Activation ; Triamcinolone Acetonide* ; Triamcinolone*

PURPOSE: To evaluate changes in expression of cell cycle related genes during apoptosis induced in HL60 cells by X-irradiation to understand molecular biologic aspects in mechanism of radiation therapy. MATERIAL AND METHODS: HL-60 cell line (promyelocytic leukemia cell line) was grown in culture media and irradiated with 8 Gy by linear accelerator (6 MV X-ray). At various times after irradiation, ranging from 3 to 48 hours were analyzed apoptotic DNA fragmentation assay for apoptosis and by western blot analysis and semi-quantitative RT-PCR for expression of cell cycle related genes (cyclin A, cyclin B, cyclin C, cyclin D1, cyclin E, cdc2, CDK2, CDK4, p16INK4a, p21WAF1, p27KIP1, E2F, PCNA and Rb). RESULTS: X-irradiation (8 Gy) induced apoptosis in HL-60 cell line. Cycline A protein increased after reaching its peak 48 h after radiation delivery and cyclin E, E2F, CDK2 and RB protein increased then decreased after radiation. Radiation induced up-regulation of the expression of E2F is due to mostly increase of phosphorylated retinoblastoma proteins (ppRb). Cyclin D1, PCNA, CDC2, CDK4 and p16INK4a protein underwent no significant change at any times after irradiation. There was not detected p21WAF1 and p27KIP1 protein. Cyclin A, B, C mRNA decreased immediately after radiation and then increased at 12 h after radiation. Cyclin D1 mRNA increased immediately and then decreased at 48 h after radiation. After radiation, cyclin E mRNA decreased with the lapse of time. CDK2 mRNA decreased at 3 h and increased at 6h after radiation. CDK4 mRNA rapidly increased at 6 to 12 h after radiation. There was no change of expression of p16INK4a and not detected in expressin of p21WAF1 and p27KIP1 mRNA. CONCLUSION: We suggest that entry into S phase may contribute to apoptosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced apoptosis of HL60 cells and tosis of HL60 cells induced by irradiation. Increase of ppRb and decrease of pRb protein are related with radiation induced apoptosis of HL60 cells and this may be associated with induction of E2F and cyclinE/CDK2. These results support that p21WAF1 and p27KIP1 are not related with radiation induced-apoptosis.
Apoptosis* ; Blotting, Western ; Cell Cycle* ; Culture Media ; Cyclin A ; Cyclin B ; Cyclin C ; Cyclin D1 ; Cyclin E ; Cyclin-Dependent Kinase Inhibitor p16 ; Cyclin-Dependent Kinase Inhibitor p27 ; Cyclins ; DNA Fragmentation ; HL-60 Cells* ; Humans ; Leukemia ; Particle Accelerators ; Proliferating Cell Nuclear Antigen ; Retinoblastoma Protein ; RNA, Messenger ; S Phase ; Up-Regulation

OBJECTIVETo investigate the influence of aerosols on the expression of cyclin B(1), cyclin C and proliferating cell nuclear antigen (PCNA) in wound tissue healing of burned rat models.

METHODSSprague Dawley (SD) rats were inflicted as the deep partial thickness burn models. Rats were randomly divided into experimental group and control group. The experimental group were treated with aerosols. Samples were collected in 1 approximately 10 postburn days. Immunohistochemistry and image analysis methods were conducted to examine the expression of cyclin B(1), cyclin C and PCNA in both experimental and control groups.

RESULTSThe expression of cyclin C in experimental group was detected in nucleus of skin basal cell on the second postburn day, increased evidently at the fifth days and sustained at high expression level up to the tenth days after treatment. The expression of cyclin C in experimental group was significantly higher than control group (P < 0.05). The expression of PCNA was first observed in skin basal cell nucleus and hair follicle cell nucleus in both experimental and control group on the third postburn day. The expression of PCNA increased evidently at the fifth days in experimental after treatment and that increased evidently at the seventh days in control group, which showed there were lots of active proliferation cell. And the difference of the expression of PCNA between experimental and control group was significant (P < 0.01). The expression of cyclin B(1) was detected in nucleus and cytoplasm of skin basal cell in both groups on the third postburn day, and no difference between the experimental and control group (P > 0.05).

CONCLUSIONSAerosols can up-regulate the expression of cyclin C and PCNA in skin basal cell nucleus. Therefore the aerosols can accelerate wound tissue healing.

Aerosols ; Animals ; Burns ; metabolism ; therapy ; Cyclin B ; biosynthesis ; Cyclin B1 ; Cyclin C ; Cyclins ; biosynthesis ; Disease Models, Animal ; Electric Stimulation Therapy ; methods ; Female ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Wound Healing ; physiology

Eugenol is an essential oil found in cloves and cinnamon that is used widely in perfumes. However, the significant anesthetic and sedative effects of this compound have led to its use also in dental procedures. Recently, it was reported that eugenol induces apoptosis in several cancer cell types but the mechanism underlying this effect has remained unknown. In our current study, we examined whether the cytotoxic effects of eugenol upon human melanoma G361 cells are associated with cell cycle arrest and apoptosis using a range of methods including an XTT assay, Hoechst staining, immunocytochemistry, western blotting and flow cytometry. Eugenol treatment was found to decrease the viability of the G361 cells in both a time- and dose-dependent manner. The induction of apoptosis in eugenol-treated G361 cells was confirmed by the appearance of nuclear condensation, the release of both cytochrome c and AIF into the cytosol, the cleavage of PARP and DFF45, and the downregulation of procaspase-3 and -9. With regard to cell cycle arrest, a time-dependent decrease in cyclin A, cyclin D3, cyclin E, cdk2, cdk4, and cdc2 expression was observed in the cells after eugenol treatment. Flow cytometry using a FACScan further demonstrated that eugenol induces a cell cycle arrest at S phase. Our results thus suggest that the inhibition of G361 cell proliferation by eugenol is the result of an apoptotic response and an S phase arrest that is linked to the decreased expression of key cell cycle-related molecules.
Apoptosis ; Blotting, Western ; Caspase 3 ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Proliferation ; Cinnamomum zeylanicum ; Cyclin A ; Cyclin D3 ; Cyclin E ; Cyclins ; Cytochromes c ; Cytosol ; Down-Regulation ; Eugenol ; Flow Cytometry ; Humans ; Hypnotics and Sedatives ; Immunohistochemistry ; Melanoma ; S Phase ; Syzygium

Carcinoma of the uterine cervix is one of the most common malignancies among women worldwide. Human papillomaviruses (HPV) have been identified as the major etiological factor in cervical carcinogenesis. However, the time lag between HPV infection and the diagnosis of cancer indicates that multiple steps, as well as multiple factors, may be necessary for the development of cervical cancer. The development and progression of cervical carcinoma have been shown to be dependent on various genetic and epigenetic events, especially alterations in the cell cycle checkpoint machinery. In mammalian cells, control of the cell cycle is regulated by the activity of cyclin-dependent kinases (CDKs) and their essential activating coenzymes, the cyclins. Generally, CDKs, cyclins, and CDK inhibitors function within several pathways, including the p16INK4A-cyclin D1-CDK4/6-pRb-E2F, p21WAF1-p27KIP1-cyclinE-CDK2, and p14ARF-MDM2-p53 pathways. The results from several studies showed aberrant regulation of several cell cycle proteins, such as cyclin D, cyclin E, p16 INK4A, p21WAF1, and p27KIP1, as characteristic features of HPV- infected and HPV E6/E7 oncogene-expressing cervical carcinomas and their precursors. These data suggested further that interactions of viral proteins with host cellular proteins, particularly cell cycle proteins, are involved in the activation or repression of cell cycle progression in cervical carcinogenesis.
Uterine Cervical Neoplasms/*pathology ; Tumor Suppressor Protein p53/physiology ; Tumor Suppressor Protein p14ARF/physiology ; Retinoblastoma Protein/physiology ; Proto-Oncogene Proteins c-mdm2/physiology ; Humans ; Female ; E2F Transcription Factors/physiology ; Cyclin-Dependent Kinase Inhibitor p27/physiology ; Cyclin-Dependent Kinase Inhibitor p21/physiology ; Cyclin-Dependent Kinase Inhibitor p16/physiology ; Cyclin-Dependent Kinase 4/physiology ; Cyclin-Dependent Kinase 2/physiology ; Cyclin E/physiology ; Cyclin D1/physiology ; Cell Cycle/*physiology

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

This study was purposed to investigate the effects of TGF-beta1 and arsenic trioxide (As₂O₃) on cell apoptosis, cell cycle and changes of P27(Kip1), endogenous TGF-ß1, cyclin E and BCL-2 in HL-60 cells, and to explore the relationship between expression of P27(Kip1) and apoptosis induced by As₂O₃ and/or TGFß1. Cell apoptosis and cell cycle changes of HL-60 cells treated with As₂O₃ and/or TGFß1 were detected by cytomorphologic observation and flow cytometry, the protein expressions of P27(Kip1), TGF-ß1, cyclin E and BCL-2 were measured by immunohistochemistry. The results showed the effect of 5 μmol/L of As₂O₃ was the most strong among the different concentration of As₂O₃ ,and the effect on apoptosis at 48 hour was more strong than that at 24 hours (p < 0.05). The TGF-beta1 (5 ng/ml) induced arrest of cells in G₁ phase (p < 0.05) compared with As₂O₃ alone and As₂O₃ combined with TGF-ß1, while there was no difference with control. P27(Kip1) expression was up regulated (p < 0.05), cyclin E and BCL-2 expression was down-regulated (p < 0.05) in TGFß1-treated group. BCl-2 expression was down regulated, endogenesis TGFß1 expression was up regulated (p < 0.05), and the level of P27(kip1) and cyclin E were not changed in As₂O₃-treated group (p > 0.05). The down-regulating effect of TGF-ß1 combined with As₂O₃ on BCL-2 protein was more strong than that in single factor treated group (p < 0.05). It is concluded that TGFß1 induces cell cycle arrest and apoptosis in HL-60 cells, while the P27(kip1) expression is up regulated. P27 protein is the key effector of TGFß-induced cell cycle arrest. The effect of TGF-ß1 combined with As₂O₃ on apoptosis as well as the down-regulation of BCL-2 protein in HL-60 cells is more strong than that in single factor-treated groups, that indicates the passages linking up each other.
Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Oxides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo assess the ability of tetrandrine (Tet) to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice.

METHODSMCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry. Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B 1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo.

RESULTSClonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio (SER) of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells, whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis (M phase). Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice.

CONCLUSIONTetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.

Animals ; Benzylisoquinolines ; pharmacology ; CDC2-CDC28 Kinases ; metabolism ; Cell Line, Tumor ; Cyclin B ; metabolism ; Cyclin B1 ; Drug Screening Assays, Antitumor ; G2 Phase ; drug effects ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Radiation Tolerance

OBJECTIVETo explore the proliferation and invasive effects of inhibitors of kinase 4(INK4)(P15(ink4b) and P16(ink4a)/CDKN2) gene protein activation on RKO human colorectal cell in vivo and in vitro.

METHODSRKO human colorectal cell line was exposed to the specific DNA methyltransferase inhibitor 5-Aza-CdR and INK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression was detected by Western blotting. Soft agar cloning experiment and Transwell chamber assay were used to detect the proliferative and invasive ability in vitro. Tumorigenicity in nude mice was analyzed in vivo.

RESULTSINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression of RKO human colorectal cells after exposure to 1×10(-7), 5×10(-7) and 1×10(-6) mol/L 5-Aza-CdR concentrations(A, B, C groups) were 1.13, 1.38, 1.92 folds and 1.11, 1.45, 2.14 folds compared to positive control group respectively. Soft agar cloning experiment showed the number of cell colony significantly decreased from 36.8±5.1(positive control group) to 32.4±7.2, 21.3±5.4 and 19.5±6.4 (3 experiment groups, all P<0.05) respectively. Transwell chamber assay showed that migrated cell number in positive control group(67.4±7.2) was significantly higher than those of 3 experimental groups(35.3±4.6, 29.5±7.3 and 25.3±6.2, respectively). The tumor volume of metastasis model in nude mice was inhibited in experimental groups, but not significantly lower compared to control group (P>0.05). There were significant differences of tumor weight and inhibition rate between control group and 3 experimental groups in nude mice respectively(all P<0.01).

CONCLUSIONINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein activation can inhibit tumor proliferation, migration and suppress the tumor formation ability.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Metastasis ; Neoplasm Transplantation ; Transcriptional Activation

The aim of this study was to investigate the effect of Celastrol on induction of HL-60 cell apoptosis and its possible mechanism. The proliferative activity of HL-60 cells treated with 0.25 - 8.0 μmol/L of Celastrol for 24 - 72 hours was assayed by MTT method, the effects of Celastrol on apoptosis and cell cycle of HL-60 were detected by TUNEL staining and flow cytometry with Annexin V-FITC/PI double labeling, the expression of pAkt and cyclin D1 at protein and gene level in HL-60 cells treated with Celastrol were measured by Western blot and RT-PCR. The results showed that the Celastrol could obviously inhibit the proliferation of HL-60 cells in concentration-and time-dependent manners, the IC₅₀ value of Celastrol for 24 hours was 6.21 ± 0.242 μmol/L. The Celastrol concentration-dependently induced the apoptosis of HL-60 cells, accompanying with morphological changes of apoptotic cells, which may be related with arrest of cells in G₀/G₁ phase. The Celastrol suppressed the expression of pAkt and Cyclin D1 in HL-60 cells to a varying degree which showed obvious concentration-and time-dependent manners. It is concluded that the Celastrol inhibits the proliferation and induced the apoptosis of HL-60 cells. Its mechanism may be related with down-regulation of p-Act and cyclin D1 expressions.
Apoptosis ; drug effects ; Cyclin D1 ; metabolism ; Down-Regulation ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Proto-Oncogene Proteins c-akt ; metabolism ; Triterpenes ; pharmacology