OBJECTIVETo study the effects of arsenic trioxide (As(2)O(3)) on cell cycle and expression of cyclin dependent kinase inhibitors (CDKIs) in multiple myeloma (MM) cells, and explore its pharmacological mechanism.

METHODSThe DNA content of MM cells line HS-Sultan was analyzed by flow cytometry after exposure to As(2)O(3), the effects on expression of CDKI P15, P16 AND P21 were studied by reverse transcriptase PCR.

RESULTSDNA flow cytometric analysis showed that As(2)O(3) induced most of HS-Sultan cells, arrest at G(0)/G(1) phase and a small fraction at G(2)/M phase and apoptosis occurred mainly in S phase. There was no expression of P15 and P16 mRNA in untreated HS-Sultan cells and 1.0 micromol/L As(2)O(3) could make them expressed after exposed 24 or 48 hours respectively. Expression of P12 mRNA was obviously elevated by As(2)O(3) comparing with that of control.

CONCLUSIONOne of the pharmacological mechanisms of As(2)O(3) is to activate the expression of CDKI P15, P16 and P21, and consequently affect cell proliferation cycle.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; physiology ; Cyclin-Dependent Kinase Inhibitor p15 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; RNA, Messenger ; genetics ; Tumor Cells, Cultured

This paper was designed to investigate the expression of p53, p21 of A549 cell strains under hypoxic condition and the effect of trichostatin A (TSA), the inhibitor of histone deacetylasel (HDAC1) on their expression. The authors designed 1 normoxia group (control group) and 6 hypoxia groups (experimental group): hypoxia 6 h group (A), TSA+hypoxia 6 h (B), hypoxia 12 h group (C), hypoxia 24 h group (D), TSA+hypoxia 24 h (E), hypoxia 48 h group (F). The expression of HDAC1 in A549 cells was examined by using Western blot and the expression of p53, p21 in A549 cells and the effect of TSA on them were determined by using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The A value expressed by HDAC1 in A549 cell strains was 138+/-11 in the control group, 78+/-4, 86+/-5, 124+/-3, 120+/-9 in experimental groups A, C, D, F, respectively. The A value of the expression of the protein and mRNA of p53 in A549 cell strains were 0.12+/-0.02, 0.62+/-0.02 in the control group, 0.10+/-0.03, 0.32 +/-0.03; 0.11+/-0.01, 0.33+/-0.02; 0.13+/-0.03, 0.58+/-0.01; 0.12+/-0.02, 0.56+/-0.02 in experimental group A, B, D, E, respectively. The A value of the expression of the protein and mRNA of p21 in A549 cell strains were 0.17+/-0.03, 0.62+/-0.03 in the control group, 0.16+/-0.02, 0.50+/-0.02; 0.14+/-0.02, 0.36+/-0.02; 0.15+/-0.03, 0.49+/-0.03; 0.13+/-0.02, 0.33+/-0.02 in experimental groups A, B, D, E, respectively. These results indicate that the expression of HDAC1 is regulated by hypoxia and the effect of TSA is closely related to the expression of P21 under hypoxia condition.
Adenocarcinoma ; metabolism ; pathology ; Cell Hypoxia ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; biosynthesis ; genetics ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; Lung Neoplasms ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo study the effect of allicin on cell cycle of human gastric cancer cell lines, MGC-803 and SGC-7901, and its possible mechanisms.

METHODSThe gastric cancer cell lines MGC-803 and SGC-7901 were treated with allicin. Proliferation inhibitory rate was detected by trypan-blue exclusion. Morphologic changes were observed by electron microscopy. The cell cycle was examined by flow cytometry and Giemsa staining. Expression of p21WAF1, p16INK4 protein and mRNA was detected by immunohistochemistry and RT-PCR.

RESULTSThe gastric cancer cells were inhibited after exposure to allicin for 24 hr, The IC50 was 6.4 microg/ml in MGC-803 cells and 7.3 microg/ml in SGC-7901cells. After exposure to allicin of 12 microg/ml for 24 hr, it caused the cytotoxic effect on the cells, including cellular membrane breakagy. After exposure to allicin of 3 microg/ml, 6 microg/ml and 9 microg/ml for 24 hr, compared with the control group, the proportion of cells in the G0/G1 phase was decreased and that in the G2/M phase was increased significantly (P < 0.01). After exposure to allicin of 6 microg/ml for 24 hr, compared with the control group, cell division index was much higher, suggesting that allicin could induce cell arrest in M phase. The expression levels of p21WAF1 and p16INK4 protein and mRNA in MGC-803 cells and p21WAF1 protein and mRNA in SGC-7901 cells were markedly up-regulated.

CONCLUSIONAllicin induce cell arrest of gastric cancer in M phase, which may be related to the up-regulated expression of p21WAF1 and p16INK4 genes.

Adenocarcinoma, Mucinous ; metabolism ; pathology ; ultrastructure ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Division ; drug effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Neoplasms ; metabolism ; pathology ; ultrastructure ; Sulfinic Acids ; pharmacology

OBJECTIVETo investigate the expression of p21(WAF1) gene in human osteosarcoma and their relationships with clinicopathological parameters and prognostic value.

METHODSThe p21(WAF1) gene mRNA and p21 protein expression in 45 osteosarcoma and 10 fibrous dysplasia of bone specimens were analyzed by in situ hybridization and immunohistochemistry respectively.

RESULTS(1) The positive rate of p21 protein expression in osteosarcoma was 17.7% (8/45). (2) The expression rate in high proliferation (4/10) significantly higher than that in low proliferation (4/35) osteosarcoma (chi2 = 4.34, P < 0.05). (3) The positive rate of p21(WAF1) mRNA expression in osteosarcoma was 42.2% (19/45). The expression rate in high proliferation (6/10) significantly higher than that in low proliferation (13/35) osteosarcoma (chi2 = 20.6, P < 0.01). (4) The survival time after operation of the patients with p21(WAF1) mRNA expression were higher than that of the patients with p21(WAF1) mRNA negative expression (P < 0.05).

CONCLUSIONS(1) With the increase in degree of malignancy, the expression of p21(WAF1) mRNA and p21 protein in osteosarcoma tend decrease. (2) The expression of p21(WAF1) mRNA has a definite value in judging prognosis in osteosarcoma.

Adolescent ; Adult ; Bone Neoplasms ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Female ; Follow-Up Studies ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; secondary ; Male ; Neoplasm Staging ; Osteosarcoma ; metabolism ; pathology ; Prognosis ; RNA, Messenger ; biosynthesis ; genetics ; Survival Rate

OBJECTIVETo study the growth inhibitory effects of p21WAF1 and p53 overexpression in human lung adenocarcinoma cell line.

METHODSThe p21WAF1 and p53 gene were transfected respectively into a human lung adenocarcinoma cell line, GLC-82. Flow cytometry (FLC), transmission electron microscopy (EM) and TUNEL technique were used to evaluate cell growth and identify apoptosis.

RESULTSThe GLC-82 transfected by p21 plasmid showed increased cell number in G1 phase of cell cycle, decreased proliferation potential and decreased cloning efficiency. Apoptosis have not been detected neither on EM nor by TUNEL technique, whereas the GLC-82 infected by Ad-p53 showed significantly decreased proliferation potential and some of them even died, in addition apoptosis was confirmed by TUNEL technique.

CONCLUSIONThe results indicate that p21WAF1 and p53 can inhibit proliferation; p53 also can induce apoptosis of lung adenocarcinoma cell. Therefore, these two genes should have a wide application in gene therapy of tumors in future.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Apoptosis ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; biosynthesis ; genetics ; Gene Expression ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

To investigate the effect of Protein kinase B on the expression and location of p21 in mouse early development. Immunopreciptation technology was used to detect the localization of p21 and Western blotting was used to analyze the expression of p21 after microinjecting mRNA of WT-PKB, myt-PKB and PKB-KD to mouse eggs. There was no obvious difference between the three kinds of mRNA in the expression of p21. But the cell localization altered. The p21 retain in cytoplasm after microjecting myt-PKB. In mouse fertilized egg PKB/Akt controls the cell cycle by changing the cell localization of p21.
Animals ; Cell Nucleus ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cytoplasm ; metabolism ; Female ; Fluorescent Antibody Technique ; Male ; Mice ; Microinjections ; Mutation ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; administration & dosage ; genetics ; metabolism ; Time Factors ; Zygote ; cytology ; growth & development ; metabolism

OBJECTIVETo explore the effect of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell proliferation and cell cycle of gastric carcinoma cells, and its possible molecular mechanism.

METHODSControl siRNA and AEG-1 siRNA were transfected into gastric carcinoma SGC-7901 cells. 48 h after transfection, the cells were divided into 3 groups including untransfected, siRNA control and AEG-1 siRNA transfection groups. Expressions of AEG-1 mRNA and protein in the 3 group cells were detected by real-time quantitative PCR and Western blot. The changes of cell proliferation were examined using CCK-8 kit, and the cell cycle distribution was detected by flow cytometry. Finally, expressions of cell proliferation and cell cycle related proteins were detected by Western blot.

RESULTSReal-time quantitative PCR and Western blot demonstrated that compared with the untransfected and siRNA control groups, expressions of AEG-1 mRNA and protein were significantly down-regulated in the AEG-1 siRNA transfection group (P < 0.05), but there was no significant difference between the untransfected and siRNA control groups (P > 0.05). Furthermore, in vivo experiment confirmed a significant down-regulation of AEG-1 protein in the AEG-1 siRNA transfection group (P < 0.05). In addition, AEG-1 siRNA obviously inhibited the proliferation of SGC-7901 cells at different time points after transfection with AEG-1 siRNA. The percentage of cells in G0/G1 phase in the AEG-1 siRNA transfection group [(61.26 ± 1.25)%] was significantly higher than those in the untransfected group [(46.17 ± 1.91)%] and siRNA control group [(46.46 ± 1.96)%], and there was a significant difference between them (all P < 0.001). Furthermore, the result of Western blotting revealed that down-regulation of AEG-1 expression evoked the down-regulation of cdk2 and cyclin D1 expressions and elevation of p21 expression in vitro and in vivo.

CONCLUSIONSThe inhibition of cell proliferation and cell cycle arrest mediated by down-regulation of AEG-1 expression may be closely associated with the changes of expression of cell cycle related proteins including cdk2, cyclin D1 and p21.

Animals ; Cell Adhesion Molecules ; biosynthesis ; genetics ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Down-Regulation ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Stomach Neoplasms ; metabolism ; pathology ; Transfection

OBJECTIVECytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.

METHODSAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.

RESULTSCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.

CONCLUSIONSCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Antigens, Bacterial ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Gastric Mucosa ; cytology ; enzymology ; microbiology ; Helicobacter pylori ; metabolism ; pathogenicity ; physiology ; Humans ; Interleukin-8 ; secretion ; Intracellular Signaling Peptides and Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transcription, Genetic ; Virulence

OBJECTIVETo study effects of the expression of transforming growth factor (TGF)-beta1 on the growth of Smad4-null pancreatic cancer cells.

METHODSTGF-beta1 eukaryotic expression vector was transfected into pancreatic cancer cell line BxPC3. Effects of the expressison of TGF-beta1 was studied by growth curve analysis and flow cytometry. Cell motility was monitored by wound-healing assay. Western blot was used to estimate the expression level of p21(WAF/CLIP1), a cyclin-dependent kinase inhibitor.

RESULTSTransfection of TGF-beta1 changed the morphology of BxPC3 into spindle shaped cells. The growth rate of BxPC3 began to decrease after the fourth day of TGF-beta1 transfection, compared with the control groups. Flow cytometry showed that the percentages of cells in the S phase were (27.53 +/- 0.02)%, (26.32 +/- 0.01)% and (17.01 +/- 0.03)% in naïve BxPC3, vector-control group and TGF-beta1 transfection group respectively. Lesser cells entered the S phase after TGF-beta1 transfection (P < 0.01), but no difference was seen between the BxPC3 and vector groups (P > 0.05). The expression of p21(WAF/CLIP1) increased upon the expression of TGF-beta1.

CONCLUSIONThe Smad4-independent pathway of TGF-beta1 not only induces epithelial-mesenchymal transition in pancreatic cancer BxPC3, but also inhibits its growth through the up-regulation of p21(WAF/CLIP1).

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Deletion ; Genetic Vectors ; Humans ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; S Phase ; Smad4 Protein ; genetics ; Transfection ; Transforming Growth Factor beta1 ; biosynthesis ; genetics ; physiology ; Up-Regulation

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.
Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Enzyme Inhibitors ; pharmacology ; Flow Cytometry ; Histone Deacetylase Inhibitors ; Humans ; Hydroxamic Acids ; pharmacology ; Leukemia, Myeloid ; metabolism ; pathology