PURPOSE: In the present study the effects of amiloride on the growth of human gastric adenocarcinoma cell line, AGS cells were examined with or without the addition of 5-fluorouracil (5-FU) in vitro. MATERIALS AND METHODS: The growth of AGS cells was examined by counting number of cells on two and four days post-treatment with 50 micrometer, 100 micrometer, 200 micrometer, 400 micrometer, 800 micrometer, amiloride, and 0.1 microgram/ml, 0.3 microgram/ml 5-FU, after plating AGS cells into 6 well plates at a density of 10 x 10(4) cells/well. The reversibility of the effects of amiloride was examined on two to eight days post-treatment with 400 micrometer amiloride after seeding 2 x 10(4) cells/dish. Cell cycle analysis was performed after four day-treatment with 400 micrometer amiloride. RESULTS: Amiloride (50~800 micrometer) significantly inhibited the growth of AGS in a dose-dependent fashion (p<0.05). The inhibitory effect of amiloride on growth of AGS was reversible since removal of amiloride after 24 hours treatment led to resumption of rapid growth up to control levels. Amiloride combined with 5-FU markedly inhibited the growth of AGS in a dose-dependent fashion compared to that of amiloride or 5-FU alone (p<0.05). The fraction of S phase, G0-G1 phase and G2-M phase was 19.3%, 55.7%, 18.8%, in the amioride group (400 micrometer) and 43.9%, 37.4%, 25.1% in the control group, respectively, showing significantly higher G1 fraction in amiloride group compared to control. CONCLUSION: This is the first paper which reported that amiloride inhibited in vitro growth of human gastric adenocarcinoma cells and that its effect of growth inhibition may be synergistic with 5-FU. Amiloride given with or without 5-FU may be useful agent in the treatment of gastric carcinomas. The inhibitory effects of amiloride on the growth of AGS may be mediated in part by blocking G1-S transition of cell cycle.
Adenocarcinoma ; Amiloride* ; Cell Cycle ; Cell Line ; Fluorouracil ; Humans* ; S Phase

In this study, a new parameter, S phase cell percentage (S fraction) normalized BrdU (SFN-BrdU) incorporation rate, was introduced to detect S arrest. The results showed a positive linear correlation between the BrdU incorporation rate and the S fraction in unperturbed 16HBE cells. Theoretical analysis indicated that only S arrest could result in a decrease in the SFN-BrdU incorporation rate. Additionally, the decrease in SFN-BrdU incorporation rate and the activation of DNA damage checkpoints further demonstrated that S arrest was induced by diethyl sulfate treatment of 16HBE cells. In conclusion, SFN-BrdU incorporation rate can be used to detecting S arrest.
Bromodeoxyuridine ; pharmacokinetics ; Cell Proliferation ; DNA Damage ; Epithelial Cells ; cytology ; Humans ; S Phase ; S Phase Cell Cycle Checkpoints

BACKGROUND: S Phase Kinase Associated Protein 2 (Skp2), an F-box protein necessary for DNA replication, has recently been demonstrated to be an oncogene. The purpose of this study was to examine the Skp2 expression and to investigate its association with expressions of estrogen receptor (ER), androgen receptor (AR) and HER-2, as well as clinicopathological variables including tumor recurrence. METHODS: The expressions of Skp2, ER and AR were examined by immunohistochemistry and HER-2 amplification by chromogenic in situ hybridization (CISH) in 117 cases of breast carcinoma. RESULTS: Skp2 was expressed in 26 patients (22.2%) and was significantly correlated with tumor type (p=0.031), tumor grade (p=0.017) and ER expression (p=0.038). Twenty four (20.5%) of 117 patients had a tumor recurrence, and 6 patients (5.1%) died of multifocal metastases. Tumor recurrence was significantly correlated with histological grade (p=0.041) and lymph node status (p<0.001). CONCLUSIONS: Although Skp2 expression was statistically insignificant in association with tumor recurrence, it might be useful as a biologic predictor in breast cancer. The simple and reliable immunohistochemical assay presented in this study can be a routine part of breast cancer evaluation and may influence patient management.
Breast Neoplasms* ; Breast* ; DNA Replication ; Estrogens ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Lymph Nodes ; Neoplasm Metastasis ; Oncogenes ; Receptors, Androgen ; Recurrence ; S Phase* ; S-Phase Kinase-Associated Proteins*

BACKGROUND: The function of the p53 protein is known to regulate cell proliferation by inhibiting cells entering S phase, so DNA damaged cell proliferation is inhibited by apoptosis. p21 is a cyclin dependent kinase inhibitor induced by wild type p53, not mutant p53. Thus p21 is thought to mediate the signal of p53 induced by DNA damaged agents to arrest the cell cycle in G1 phase. p53 and p21 are expressed in many malignant tumors, and its role in oncogenesis, tumor progression and prognosis are important. OBJECTIVE: The purpose of this study was to analyze immunohistochemical expression of mutant p53 and p21 protein in melanocytic lesions. METHOD: 11 cases of intradermal nevus, 7 cases of junctional nevus and 6 cases of malignant melanoma were immunohistochemically stained with p53 and p21 monoclonal antibodies. RESULTS: 1. In intradermal nevus, the p53 was negative in 100% and the p21 was negative in 98%. These findings suggest that the composing cells of intradermal nevus is completely mature cell. 2. The positive rates of p53 and p21 in junctional nevus were 43% and 43%, respectively. The positive rates of p53 and p21 in malignant melanoma were 82% and 67%, respectively. CONCLUSION: If the expression of p21 is induced by p53 independent pathway, the cell cycle can be arrested in G1 phase, so the tumor cell proliferation is inhibited. But if the expressed p21 is mutated as p53, it means that the natural function of p21 disappears. More research is necessary about the nature of p21 which is expressed with mutant p53.
Antibodies, Monoclonal ; Apoptosis ; Carcinogenesis ; Cell Cycle ; Cell Proliferation ; Cyclins ; DNA ; G1 Phase ; Melanoma* ; Nevus* ; Nevus, Intradermal* ; Phosphotransferases ; Prognosis ; S Phase

OBJECTIVE: We compared the cell responsiveness of activated lymphocytes to rapamycin, which blocks the G1/S transition, between patients with Alzheimer's disease (AD) and normal controls to assess the early phase control defect in cell cycle. METHODS: Blood samples of 26 patients with AD and 28 normal controls were collected to separate peripheral lymphocytes. We measured the proportion of each cell cycle phase in activated lymphocytes using flow cytometry and evaluated the responsiveness of these lymphocytes to rapamycin. RESULTS: The patients with AD were older than the normal controls (AD 74.03+/-7.90 yr vs. control 68.28+/-6.21 yr, p=0.004). The proportion of G1 phase cells in the AD group was significantly lower than that in the control group (70.29+/-6.32% vs. 76.03+/-9.05%, p=0.01), and the proportion of S phase cells in the AD group was higher than that in control group (12.45+/-6.09% vs. 6.03+/-5.11%, p=0.001). Activated lymphocytes in patients with AD were not arrested in the G1 phase and they progressed to the late phase of the cell cycle despite rapamycin treatment, in contrast to those of normal subjects. CONCLUSION: The patients with AD probably have a control defect of early phase cell cycle in peripheral lymphocytes that may be associated with the underlying pathology of neuronal death.
Alzheimer Disease ; Cell Cycle ; Cell Cycle Checkpoints ; Flow Cytometry ; G1 Phase ; Humans ; Lymphocytes ; Neurons ; S Phase ; Sirolimus

The cell cycle is the set of events that is responsible for the duplication of the cells. Recent studies indicate that cell cycle regulatory proteins, mainly the cyclins and cyclin-related genes, can be critical targets during oncogenesis. The genes and gene products normally control specific events in the cell cycles, particularly during the late G1 and early S phase and G2/M phase. A large body of date implicates cyclins in oncogenesis. The first evidence came from human cyclin A in oncogenesis. Cyclin A is expressed from the late G1 phase through the M-phase of the cell cycle. Cyclin A is known as positive regulator of cell cycle and participates in the tumorigenesis. Overexpression of cyclin A has been reported in several cancers. Ki-67 is a nuclear protein expressed during the cell cycle except in Go. The labeling index of Ki-67 in the tumor cell nuclei has been used as a good prognostic factor. In this study, we compared labeling index of cyclin A and Ki-67 to assess the feasibility between them with 30 cases of cervical intraepithelial neoplasia(CIN) and 20 cases of invasive squamous cell carcinoma(SCC)by immunohistochemistry. The results were as follow; 1. Cyclin A expressed in normal parabasal cells and their labeling index was 0.8+/-0.4%, while in CIN and invasive SCC 65.5+/-9.4% and 86.5+/-12.3% respectively. Ki-67 expressed in normal parabasal cells as 1.3+/-0.7% while in CIN and invasive SCC as 77.8+/-12.9% and 92.2+/-17.6% respectively. 2. In CIN, the expression of cyclin A increased according to the grades of the CIN as 32.5+/-5.7%, 75.8+/-9.0%, and 83.2+/-13.4% in CIN I, II and III respectively. The expression of the Ki-67 also increased according to the grades of the CIN as 51.8+/-9.8%, 87.9+/-11.3%, and 93.6+/-17.5% respectively in CIN I, II and III. 3. There was no differences of cyclin A and Ki-67 expressions according to the histologic types of invasive SCC. Above results suggests that the cyclin A labeling index could be used as a marker of tumor progression in the uterine cervical carcinoma as Ki-67.
Carcinogenesis ; Cell Cycle ; Cell Cycle Proteins ; Cell Nucleus ; Cyclin A* ; Cyclins* ; G1 Phase ; Humans ; Immunohistochemistry ; Nuclear Proteins ; S Phase

OBJECTIVETo explore the changes of liver regeneration after partial hepatectomy on rats with nonalcoholic fatty liver disease (NAFLD) caused by a high fat diet.

METHODSOne hundred Wistar rats were randomly divided into a control group (group C, n = 45), fed with normal diet, and a NAFLD group, fed with fat-rich diet (group F, n = 55). All rats had a 70% partial hepatectomy at the end of the 12th week. They were sacrificed at postoperative 0, 1, 12, 24, or 36 hours and the percentages of their regenerated liver masses were calculated. The mitosis index was measured microscopically and the changes of cell ultrastructure were observed under an electron microscope. The expression of proliferating cell nuclear antigen (PCNA) was detected using immunohistochemistry. The expression of mRNA of cyclin D1 was measured by RT-PCR.

RESULTSThe light and electron microscopy showed that the hepatic sinusoids expanded at an early period after the partial hepatectomy. The mitochondria and rough endoplasmic reticulum (RER) expanded and their number increased. The mitosis index was increased. The sinusoids of the livers in group F were narrow and irregular. The nuclei were smaller and the necrotic cells increased. As compared with the control group, the mitosis index was significantly decreased (P < 0.01), and the regenerative liver weight ratio in group F was lower at postoperative 12 h, 24 h, 36 h (P < 0.01). PCNA labeling index in group F also was lower than that in group C. The peak of the PCNA in group F was later than that of the control group (P < 0.01). In group C, the mRNA of cyclin D1 peaked at the 24th hour after the partial hepatectomy, and then decreased afterwards. In group F, it was lower than that of group C at the same time (P < 0.01).

CONCLUSIONAfter NAFLD rats had partial hepatectomies, the capacity of their liver regeneration decreased and the peak of DNA synthesis was delayed, and at the same time the morbidity of the rats increased.

Animals ; Fatty Liver ; pathology ; physiopathology ; G1 Phase ; Hepatectomy ; Liver Regeneration ; Male ; Postoperative Period ; Rats ; Rats, Wistar ; S Phase

OBJECTIVE: To show that Stat3 played a key role in the G1 to S phase transition in laryngocarcinoma cells. METHOD: Human laryngocarcinoma cell lines Hep-2 were transfected with Stat3 antisense oligonucleotide mediated by liposome, MTT assay was used to measure the proliferation, flow cytometry was applied to analyze the cell cycle, and the expressions of Stat3, phosphorylation specific Stat3 (tyrosine705), CyclinD1, Cyclin E, CDK2, CDK4, CDK6, p21 and p27 were detected by western blot. RESULT: Hep-2 laryngocarcinoma cell lines expressed constitutively activated Stat3. Antisense oligonucleotide which directed blocked up the translation site resulted in growth inhibition, downregulation of Stat3, p-Stat3, Cyclins and CDKs, and upregulation of p21 and p27. CONCLUSION Our findings suggested that Stat3 played an important role in the G1 to S phase transition in laryngocarcinoma cells, Stat3 orchestrated cell cycle by regulating the balance between CDK/Cyclin complex and CKI.
Cell Line, Tumor ; G1 Phase ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; S Phase ; STAT3 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; Transfection

BACKGROUND: Flow cytometric measurement of DNA can reveal G0/G1, S, G2/M phases of cell cycle, and BrdU labeling can determine the percentage of cells in active DNA synthesis. A monoclonal antibody (MoAb), Ki-67, recognizes a protein that is present only in the nucleus of cycling cells but absent in resting cells. We analyzed whether the resting and the proliferating fraction could be differentiated by double staining with Ki-67 MoAb and propidium iodide (PI), and observed the effects of GM-CSF on cell cycle in acute myelogenous leukemia (AML) cells by Ki-67 MoAb. METHODS: Blast cells were prepared from 9 AML patients. The cells were incubated for 48 hours with or without GM-CSF. Cells were stained with BrdU/PI and Ki-67/PI. Cell cycle was analyzed by flow cytometry. RESULTS: The average fraction of G0/G1, S, and G2/M phases was 84.6%, 10.9%, and 4.5 % by BrdU/PI and 87.8%, 8.6%, and 3.7% by Ki-67/PI, respectively. Ki-67/PI staining dis-criminated between G0 and G1 phases and the average was 71.5% and 16.3%, respectively. In cells incubated with GM-CSF, BrdU/ PI method showed that the average S phase fraction (SPF) significantly increased from 10.9 to 16.2% (P=0.01) and the fraction of G0/G1 phase decreased from 84.6% to 78.4% (P= .02). Ki-67/PI method showed that the median SPF significantly increased from 8.6% to 13.7% (P=0.05) and G0 fraction decreased from 71.5% to 58.1% (P=0.02) but G1 fraction increased from 16.3% to 22.3% (P=0.01). CONCLUSION: Cell cycle analysis by Ki-67 MoAb and PI in AML is rapid and simple. It is especially useful to determine the growth fraction and G0 fraction compared to BrdU/PI staining.
Bromodeoxyuridine ; Cell Cycle* ; DNA ; Flow Cytometry ; G0 Phase ; G1 Phase ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Ki-67 Antigen ; Leukemia, Myeloid, Acute* ; Propidium ; S Phase

BACKGROUND: Renin is secreted from the juxtaglomerular (JG) cells in response to a wide variety of extracellular stimuli. To study the underlying mechanism of regulation of renin secretion at molecular level, pure JG cell lines (As 4.1) cloned from renal JG tumor was used. In this study, to explore the feasibility of As 4.1 cells as an in vitro model for renin secretion, the changes of renin secretion from As 4.1 in culture during cell cycle were characterized. METHODS: To address this issue, As 4.1s were synchronized in G0, G1, S, G2, early M and late M phase during experiment. RESULTS: The rate of renin secretion was above 1 ng AI/well/hr in G0, G2/M and early mitotic phase and 0.5 ng AI/well/hr in G1, G1/S, S and late mitotic phase. ML-7 (6x10(-5) M), an inhibitor of MLCK which is known to stimulate renin secretion, increased the rate of renin secretion much greater in G1, G1/S, S and late M phase than the other phases; in particular, in early mitotic phase it had no stimulation. On the other hand, the rate of renin secretion was not influenced through out cell cycles by calyculin A, an inhibitor of type 1 protein phosphatase. Forskolin, an activator of adenlyate cyclase resulting in an elevation of intracellular cyclic AMP, stimulated renin secretion only in S phase in a concentration dependent manner. CONCLUSION: The present study demonstrated that As 4.1 cells in culture secrete active renin in much the similar manner to JG cells in situ but its rate varies during each phase of the cell cycle. Thus As 4.1 cells can be utilized as an in vitro model for renin secretion. But, changes in the rate of renin secretion and the secretory responses to stimulators or inhibitors during cell cycle must be considered in conducting experiments to elucidate the cellular and molecular mechanism of the renin secretion.
Cell Cycle* ; Cell Division ; Cell Line ; Clone Cells ; Colforsin ; Cyclic AMP ; Hand ; Renin* ; S Phase