Upregulation of miR-223 in the rat liver inhibits proliferation of hepatocytes under simulated microgravity

Long-term spaceflight affects numerous organ systems in the body, including metabolic dysfunction. Recently, ample evidence has demonstrated that the liver is a vulnerable organ during spaceflight. However, the changes in hepatocyte proliferation and cell cycle control under microgravity remain largely unexplored. In the present study, we first confirmed that the serum levels of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase, biochemical markers of liver function, were altered in rats under tail suspension (TS) conditions to simulate microgravity, as shown in previous reports. Next, we demonstrated that the cell proliferation activity, determined by Ki67, PCNA and PH3, was significantly decreased at the different TS time points (TS for 14, 28 and 42 days) compared with that in the control group. Consistently, the positive cell cycle regulators Ccna2, Ccnd1, Cdk1, Cdk2 and cyclin D3 were also significantly decreased in the TS groups as shown by quantitative real-time PCR and western blotting analysis. Subsequent analysis revealed that the aberrant hepatocyte proliferation inhibition under simulated microgravity was associated with the upregulation of miR-223 in the liver. We further found that miR-223 inhibited the proliferation of Hepa1-6 cells and identified CDK2 and CUL1 as its direct targets. In addition, the decreased expression of CDK2 and CUL1 was negatively correlated with the level of p27 in vitro and in vivo, which may have been responsible for retarding hepatocyte proliferation. Collectively, these data indicate that upregulation of miR-223 was associated with the inhibition of liver cell growth and reveal the role of miR-223 in rat hepatocyte proliferation disorders and the pathophysiological process under simulated microgravity.

Effects of eukaryotic translation initiation factor 5A2 down-regulation by small interfering RNA on aggressiveness of MKN28 human / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effects of eukaryotic translation initiation factor 5A2 (EIF5A2) down-regulation by small interfering RNA (siRNA) on aggressiveness of human gastric cancer cell and its potential mechanisms.</p><p><b>METHODS</b>The expressions of EIF5A2 in human gastric cancer cell lines (MKN28 and HGC27) and immortalized gastric mucosal epithelial cells (GES-1) were measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. EIF5A2 gene in MKN28 cells was silenced by RNA interference and the inhibitory effect was evaluated by both qRT-PCR and Western blotting. Cell proliferation was assessed by CCK-8 assay. Cell migration and invasion were assessed by Transwell assay. The possible downstream targets of EIF5A2, such as CyclinD1, CyclinD3, matrix metallopeptidase-9 (MMP-9), E-cadherin, vimintin, C-myc, and metastasis-associated protein 1 (MTA1) expression levels, were examined by Western blotting.</p><p><b>RESULTS</b>High expressions of EIF5A2 were found in MKN28 cells and human gastric adenocarcinoma tissues. Both EIF5A2 mRNA and protein expression in MKN28 cells were significantly down-regulated by siRNA#1 and siRNA#2, especially siRNA#1. Knockdown of EIF5A2 caused an apparent suppression of MKN28 cell proliferation (all P<0.01), migration (P<0.001), and invasion (P<0.001). After the knockdown of EIF5A2 in MKN28 cells, E-cadherin levels were upregulated, whereas vimentin, Cyclin D1, Cyclin D3, C-myc and MTA1 levels were downregulated.</p><p><b>CONCLUSION</b>Knockdown of EIF5A2 may inhibit MKN28 cell proliferation by downregulating the CyclinD1 and CyclinD3 and suppressing the cell migration and invasion by inhibiting MTA1, C-myc and epithelial-mesenchymal transition.</p>

Prognostic value of clinical characteristics and immunophenotypic biomarkers in 115 patients with primary central nervous system lymphoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Clinical outcome in patients with primary central nervous lymphoma (PCNSL) is variable and poorly predictable. This study investigated the association of clinical features and immune markers with prognosis of patients with PCNSL.</p><p><b>METHODS</b>One hundred and fifteen newly diagnosed PCNSL patients at the study institution were considered eligible for this study. Clinical characteristics and biochemical assay data were collected. Immunohistochemical staining of Cyclin D3, Cyclin E, Foxp1, and LMO2 were performed. All cases were followed-up regularly.</p><p><b>RESULTS</b>The common sites of involvement were frontal lobe (54.8%) and thalamus (16.5%). Diffuse large B-cell lymphoma composed of 96.5% of the cases. The median overall survival was 22 (4 - 41) months, and the 5-year survival rate was 22.8%. Age > 65 years, serum globulin > 40 g/L, large size of tumor, lymphocyte count ≥ 1 × 10(9)/L, and expression of Cyclin D3 and Cyclin E were associated with poor prognosis of PCNSL. Expressions of Foxp1, LMO2, and CD44 were not related to the survival. Expression of Cyclin E, large tumor size, and high serum globulin were independent prognostic factors for PCNSL.</p><p><b>CONCLUSIONS</b>PCNSL prognosis is relatively poor. Age, high tumor burden, higher lymphocyte count, expression of Cyclin D3, and Cyclin E are inferior prognostic factors for PCNSL.</p>

Expression of Cyclin A, B1, D1, D3, and E in Non-Small Lung Cancers

PURPOSE: Cyclins, and their associated cyclin dependent kinases, regulate progression of the cell cycle through the G1 phase and into the S-phase during the DNA replication process. Cyclin E regulation is an important event in cell proliferation. Despite its importance, abnormalities of these genes and their protein products have yet to be found in lits asoociation with lung cancer. MATERIALS AND METHODS: The relationships between expression of cyclin A, cyclin B1, cyclin D1, cyclin D3, and cyclin E and clinicopathologic factors were investigated in 103 cases with non-small cell carcinomas, using immunohistochemical analysis. RESULTS: The positive immunoreactivity was observed in 51 cases (50%) for cyclin A, 33 cases (32%) for cyclin B1, 83 cases (81%) for cyclin D1, 19 cases (18%) for cyclin D3, and 11 cases (11%) for cyclin E. Expression of cyclin E was significant for lymph node metastasis (p=0.004, Chi-square test). There was no relationship between cyclin A, B1, D1, and E and histological typing, tumor size, lymph node metastasis, or pathological tumor, node and metastasis staging. CONCLUSION: These findings suggest that the expression of cyclin E played a role, to some degree, in the lymph node metastasis.

S Phase Cell Cycle Arrest and Apoptosis is Induced by Eugenol in G361 Human Melanoma Cells

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.

Significance of myeloid antigen expression in precursor T lymphoblastic lymphoma / 癌症

<p><b>BACKGROUND AND OBJECTIVE</b>Precursor T lymphoblastic lymphoma (T-LBL) is a highly aggressive lymphoma. Myeloid antigen expression was found in some of the patients, and its clinical significance is worth studying. This study was to compare the clinical features, short-term efficacy and survival of T-LBL patients with or without myeloid antigen expression so as to evaluate its prognostic significance.</p><p><b>METHODS</b>Forty-five T-LBL patients, with a median age of 14 years, were treated at Sun Yet-sen University Cancer Center between January 2000 and July 2008. These patients were divided into myeloid antigen-positive group (My(+) group) and myeloid antigen-negative group (My(-) group) based on the flow cytometric (FCM) analysis in bone marrow or pleural fluid. Myeloid antigen expression and its correlation with the short-term efficacy and overall survival were assessed in the two groups.</p><p><b>RESULTS</b>There were 18 patients (40.0%) in the My(+) group and 27 (60.0%) in the My(-) group. The myeloid antigen expression was negatively correlated with the initial level of lactate dehydrogenase (LDH), but not with other clinical features. The remission rate was lower in the My(+) group than in the My(-) group (38.8% vs. 70.3%, P = 0.028). The 2-year overall survival rate was lower in the My(+) group than in the My(-) group (51.9% vs. 78.7%, P = 0.036). By age subgroup analysis, there were no differences in response and survival rate among children and adolescents with or without myeloid antigen expression. But the remission rate and the 2-year overall survival rate were significantly lower in adult patients with myeloid antigen expression than in patients without it. Univariate and multivariate analysis demonstrated that age and myeloid antigen expression were adverse prognostic factors.</p><p><b>CONCLUSION</b>Myeloid antigen expression is a predictor of a poor response to chemotherapy, and adverse prognostic factor in adult T-LBL, but not in children with T-LBL.</p>

Clinicopathologic significance of bcl-6 gene rearrangement and expression in three molecular subgroups of diffuse large B-cell lymphoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the role of bcl-6 gene rearrangement and bcl-6 expression in three molecular subgroups of diffuse large B-cell lymphoma (DLBCL) and its clinicopathological significance.</p><p><b>METHODS</b>Tissue microarray including 163 newly diagnosed DLBCL was constructed. Fluorescence in situ hybridization (FISH) was performed to detect the bcl-6 gene rearrangement and immunohistochemistry (EnVision method) was used to evaluate the expression of bcl-6, Ki-67, cyclin D3, Geminin and P27(Kip1) proteins in DLBCL. The association with clinicopathological features was analyzed.</p><p><b>RESULTS</b>One hundred and forty nine of 163 cases were further classified into three molecular subgroups: 40 cases of germinal center B-cell-like (GCB) type, 75 cases of activated non-germinal center B-cell-like (ABC) type, 34 cases of Type 3. Of these 149 cases, FISH for bcl-6 gene rearrangement was successful in 118 cases. bcl-6 gene rearrangement was observed in 33 of 118 (28.0%) cases. The bcl-6 gene rearrangement was more frequently seen in the ABC subgroup (22/62, 35.5%) than in GCB (6/31, 19.4%) and Type 3 subgroups (5/25, 20.0%, P=0.16). The correlation of bcl-6 gene rearrangement and expression of its encoded protein was further analyzed. Most of DLBCL (26/33, 78.8%) with bcl-6 gene rearrangement presented with overexpression of its encoded protein, which was higher than those without bcl-6 gene rearrangement (53/84, 62.4%, P=0.088). DLBCL with bcl-6 gene rearrangement (24/33, 72.7%) more frequently expressed cyclin D3, and had a higher proliferative activity than those without bcl-6 gene rearrangement (37/81, 45.7% , P=0.009). Twenty-nine of 33 (87.9%) cases of DLBCL with bcl-6 gene rearrangement presented with advanced stage (Ann Arbor stage III/IV), which was higher than those without bcl-6 gene rearrangement (65/85, 76.5% , P=0.167). Univariate Cox proportional hazards regression analysis showed that bcl-6 gene rearrangement was associated with an increased relative risk (at 1.842) of death in DLBCL cases compared with those without bcl-6 gene rearrangement.</p><p><b>CONCLUSION</b>Overexpression of bcl-6 protein caused by bcl-6 gene rearrangement may play some important roles in the development and/or progression of a subset of DLBCL.</p>

Effect of betulinic acid on proliferation and apoptosis in Jurkat cells and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the anticancer effects of betulinic acid (BA) on Jurkat cells in vitro and its molecular mechanism.</p><p><b>METHODS</b>The effects of betulinic acid on the growth of Jurkat cells were studied by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was assessed by Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effect of betulinic acid on the cell cycle of Jurkat cells was studied by propidium iodide staining. RT-PCR and Western blot were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid.</p><p><b>RESULTS</b>The proliferation of Jurkat cells was decreased in betulinic acid-treated group at a 24 h IC50 value of 70.0 micromol/L. The effect of betulinic acid to induce apoptosis in Jurkat cells was in a time- and dose-dependent manner. Jurkat cells treated with betulinic acid showed an increase of G0/G1 phase and decrease of S phase. The Jurkat cells treated with 0, 20, 60, 100 micromol/L betulinic acid for 24 h, showed an increased G0/G1 phase from 31.0% to 58.8%, whereas decreased S phase from 61.5% to 35.8%, respectively. PBMC was less sensitive to the cytotoxic effect of betulinic acid than Jurkat cells. The expression of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid.</p><p><b>CONCLUSION</b>Betulinic acid can inhibit the proliferation of Jurkat cells by regulating the cell cycle that arrests cells at G0/G1 phase and induces apoptosis in Jurkat cells. The antitumor effects of betulinic acid may be related to down-regulation of the expression of cyclin D3 and bcl-xl.</p>

Chios Gum Mastic Induces Cell Cycle Arrest and Apoptosis in YD9 Human Oral Squamous Carcinoma Cells / 체질인류학회지

Chios gum mastic (CGM) is obtained from the stem and leaves of Pistacia lentiscus trees and has been extensively used for centuries in Mediterranean and Middle Eastern countries, both as a dietary supplement and herbal remedy. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying modulation of cell cycle and induction of apoptosis in YD9 human oral squamous carcinoma cell line treated with CGM. The viability of YD9 cells and human normal keratinocyes (HaCaT cells), and the growth inhibition of YD9 cells were assessed by the MTT assay and clonogenic assay respectively. The hoechst staining and DNA electrophoresis were conducted to observe the YD9 cells undergoing apoptosis. YD9 cells were treated with CGM, and Western blotting, immunocytochemistry, confocal microscopy and FACScan flow cytometry were conducted. Mitochondrial membrane potential change and proteasome activity were measured. CGM treatment on YD9 cells resulted in a does-dependent inhibition of cell growth and induced apoptotic cell death. And tested YD9 cells showed several lines of apoptotic manifestation. Flow cytometric analysis revealed that CGM resulted in G1 arrest in cell cycle progression which was associated with decrease in the protein expression of cyclin D1, cyclin D3, Cdk2 and Cdk4, and increase in the protein expression of p21(WAF1/CIP1) and p53. These results demonstrate that CGM induces G1 the cell cycle arrest via the modulation of cell cycle-related proteins, and apoptosis via mitochondria and caspase pathway in YD9 cells, suggesting that CGM can be considered as a novel therapeutic strategy for human oral squamous cell carcinoma from its strong cell cycle arrest and apoptosis-inducing activity.

Effects of betulinic acid on proliferation and apoptosis in Jurkat cells and its in vitro mechanism / 华中科技大学学报（医学）（英德文版）

The anti-cancer effects of betulinic acid (BA) on Jurkat cells and its in vitro mechanism were examined by using MTT assay. Apoptosis was detected by using Hoechst33258 staining and annexin-V/PI double-labeled cytometry. The effects of betulinic acid on the cell cycle of Jurkat cells were studied by propidium iodide method. RT-PCR and Western blotting were used to analyze the changes of cyclin D3, bcl-xl mRNA and protein levels in Jurkat cells after treatment with betulinic acid. Our results showed the proliferation of Jurkat cells was decreased in betulinic acid-treated group with a 24-h IC50 value being 70.00 mumol/L. Betulinic acid induced apoptosis of Jurkat cells in a time- and dose-dependent manner. The number of Jurkat cells treated with betulinic acid showed an increase in G(0)/G(1) phase and decrease in S phase. After treatment with 0, 20, 60, 100 mumol/L betulinic acid for 24 h, the number of Jurkat cells was increased from (31.00+/-1.25)% to (58.84+/-0.32)% in G(0)/G(1) phase, whereas it was decreased from (61.45+/-1.04)% to (35.82+/-1.95)% in S phase. PBMCs were less sensitive to the cytotoxicity of betulinic acid than Jurkat cells. The expressions of cyclin D3, bcl-xl mRNA and protein were decreased sharply in Jurkat cells treated with betulinic acid. It is concluded that betulinic acid is able to inhibit the proliferation of Jurkat cells by regulating the cell cycle, arrest cells at G(0)/G(1) phase and induce the cell apoptosis. The anti-tumor effects of betulinic acid are related to the down-regulated expression of cyclin D3 and bcl-xl.

Peroxisome proliferator-activated receptor gamma activator inhibits cell growth of MDA-MB-231 breast cancer cells through induction of apoptosis / 한국유방암학회지

PURPOSE: Peroxisome proliferator-activated receptor gamma (PPARgamma) has become a potential target for the prevention and treatment of human cancers. PPARgamma ligands inhibit cell proliferation of estrogen receptoralpha(ERalpha)-positive breast cancer cells. However, it has recently been shown that ERalpha-negatively inhibits PPARgamma signaling in breast cancer cells, indicating that PPARgamma ligand may be more useful for treating ERalpha-negative breast cancer cells compared to ERalpha-positive breast cancer cells. In this study, we attempted to elucidate the role of PPARg in ERalpha-negative breast cancer cells. METHODS: The effect of PPARgamma ligand on the growth of MDA-MB-231 cells was measured by MTT assay and flow cytometric analysis. TUNEL staining and Hoechst 33342 fluorescent staining were used to observe the effects of PPARgamma ligand on cell apoptosis. The regulatory proteins of the cell cycle were measured by Western blot. RESULTS: The treatment of MDA-MB-231 human breast cancer cells with the PPARgamma ligand, trgoglitazone, was shown to induce inhibition of cell growth in a dose-dependent manner. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. The apoptotic effect by troglitazone demonstrated that apoptotic cells were elevated from 2.5-fold of the control level at 10 mM, to 3.1-fold at 50micrometer and to 3.5-fold at 75 mM of troglitazone. Moreover, troglitazone treatment dose-dependently caused a marked decrease in the pRb, cyclin D1, cyclin D2, cyclin D3, cdk2, Cdk4 and Cdk6 expressions and there was a significant increase in the p21 and p27 expressions. CONCLUSION: These results indicate that trgoglitazone induces cell-cycle G1 arrest and apoptosis in ERalpha-negative MDA-MB-231 breast cancer cells. Collectively, this paper shows that PPARgamma ligand is an important player as a member of the chemotherapeutic candidates for treating ERalpha-negative breast cancer.

Expression, localization and interrelationship of P27kip1 and cyclin D3 in non-Hodgkin's lymphoma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression, localization and interrelationship of P27(kip1) and cyclin D3 in non-Hodgkin's lymphoma (NHL).</p><p><b>METHODS</b>The expressions of P27(kip1), cyclin D3 and index Ki-67 was detected in 100 NHL and 20 reactive lymph nodes by immunohistochemical technique. The expression and localization of P27(kip1) and cyclin D3 in 3 NHL cell lines were detected by Western blot, double immunolabelling and laser scanning confocal microscopy, respectively.</p><p><b>RESULTS</b>In general the expression of P27(kip1) in NHL was lower than in control group, and was negatively related to the tumor aggressiveness and proliferating activity; the expression of cyclin D3 in NHL was higher than in control group, and was positively related to the tumor aggressiveness and proliferating activity. There was a negative correlation between P27(kip1) and cyclin D3. Nevertheless, anomalous high P27(kip1) expression was found in a few NHL tissues with high expression of cyclin D3 and Ki-67. Overexpression and colocalization of P27(kip1) and cyclin D3 was found in Raji cell line.</p><p><b>CONCLUSIONS</b>Under expression of P27(kip1) and overexpression of cyclin D3 may play a role in the occurrence and development of NHL. Anomalous high P27(kip1) expression and its interaction with cyclin D3 may be another mechanism for tumor genesis of NHL.</p>

Effects of serum from aplastic anemia patients on the expression of cyclin D3 isoform in umbilical cord blood CD34+ cells / 华中科技大学学报（医学）（英德文版）

The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.

Immunohistochemical demonstration of cyclins A, B, D1, D3 and E in hepatocellular carcinomas using tissue microarrays / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of five kinds of cyclins in hepatocellular carcinoma (HCC) and their association with degree of tumor differentiation, metastasis and infection of hepatitis B virus (HBV).</p><p><b>METHODS</b>The HCC tissue microarrays were composed of those from 273 cases of HCC tissues, 144 surrounding-tumor liver tissues and 10 normal liver tissues obtained from autopsy. The diameter of each specimens on tissue microarrays was 2.0 mm. Immunohistochemistry was used to detect the expression of cyclin A, cyclin B, cyclin D1, cyclin D3 and cyclin E on HCC tissue microarrays. The association of the expression of these cyclins with the infection rate of HBV was also analyzed.</p><p><b>RESULTS</b>Three paraffin-embedded HCC tissue microarrays were successfully constructed, including 136, 143 and 148 tissue spots, respectively. The positive rates of cyclins in 273 cases of HCC were cyclin A 52.7%, cyclin B 45.4%, cyclin D1 35.9%, cyclin D3 44.3% and cyclin E 23.1%; while the figures in 144 surrounding-tumor tissues were 8.3%, 5.6%, 4.9%, 6.3% and 1.4%, respectively. In 10 normal liver tissues these cyclins exhibited negative staining, with the exception that cyclin D1 was positive in one case of normal liver tissue. The positive rate of cyclins in HCC were significant higher than those in surrounding-tumor liver tissues (P < 0.01), in HCC tissues with histological grade II and III, the cyclins expression were stronger than that in grade I (P < 0.05). The positive rates of cyclins, except cyclin A in HCC with portal vein invasion were higher than those without portal vein invasion (P < 0.01). Infection of HBV did not have significant relationship with the expression of cyclins (P > 0.05).</p><p><b>CONCLUSION</b>Cyclins in different cell cycles overexpressed at varied levels in hepatocellular carcinoma, and the increased expression of cyclins may shorten the tumor cell cycle phase, accelerate cell proliferation, and have a close relationship with HCC aggressiveness.</p>

Expression of cyclin genes in human gastric cancer and in first degree relatives / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To clarify the role of these cyclins in human gastric cancer.</p><p><b>METHODS</b>38 gastric cancer patients, 29 first degree relatives of gastric cancer patients, as well as 18 healthy subjects were included. The mRNA expression of cyclins D1, D2, D3 and E in gastric biopsies was evaluated by RT-PCR analysis using specific primers. Histomorphological features such as intestinal metaplasia, atrophy, H. pylori infection and severity of gastritis were determined by the updated Sydney System.</p><p><b>RESULTS</b>Significant mRNA overexpression was found for cyclins D2, D3 and E compared with healthy normal specimen, but cyclin D1 expression was not different between tumor and normal tissues. In addition, cyclin D2 and D3 overexpression was significantly more frequent in first degree relatives than in healthy controls (P < 0.05). Among the various pathological findings, the overexpression of cyclins D2 and E was associated with intestinal metaplasia, and the overexpression of cyclin D3 was associated with intestinal metaplasia as well as atrophy. The overexpression of cyclins D2 and D3 was significantly correlated with H. pylori infection. No correlation was observed between the overexpression of cyclin D1 and any pathological variables.</p><p><b>CONCLUSION</b>The overexpression of cyclins D2, D3 and E is a frequent event in patients with gastric cancer and their first degree relatives and may be an early event in gastric carcinogenesis.</p>

The Cell Cycle Regulatory Effects of High Dose 5-fluorouracil on Breast Cancer Cell Line

BACKGROUND: Chemotherapy with 5-fluorouracil (5-FU) has been one of the mainstay in breast cancer treatment. The effects of high dose 5-FU on cell cycle regulation were studied in breast caner cells. METHODS: A breast cancer cell line MCF-7 was used. Protein expressions of G1/S cyclins, p21(Waf1/Cip1), cdk2, E2F1 and retinoblastoma were tested by western blot analysis. Immunoprecipitation and immune complex kinase assay were done for the assessment of E2F1/RB interacton and the activity of cdk2 respectively. RESULTS: p21(Waf1/Cip1) expression was barely detectable in control cells. With addition of 5-FU level of p21(Waf1/Cip1) were induced and cyclin D3 level was decreased as cell growth decreases. In accordance with increased expression of p21(Waf1/Cip1), cyclin E-associated cdk2 kinase activity was reduced. Retinoblastoma protein (RB) became dephosphorylated and E2F-1 binding activity with RB was increased. CONCLUSION: In this situation of high concentration of 5-FU breast cancer cells tend to be G1/S cell cycle arrested. Overexpression of p21(Waf1/Cip1) and dephosphorylation of RB may mediate the effectss of 5-FU by inhibiting E2F-1 activity, which contributes to G1/S cell cycle arrest. These results could be an indicating landmark for further study of high dose chemotherapy with 5-FU.

Cell Cylce Regulatory Effects of Cyclic AMP in Cancer Cells Which Lack Wild-type p53 / 대한암학회지

PURPOSE: The activator of protein kinase A, cyclic AMP, has been a recognized growth inhibitor of certain cell types. The present study aimed to investigate the effects of dibutyryl cAMP on the growth of cancer cells which lack wild-type p53 and to determine the mechanism of growth inhibition. MATERIALS AND METHODS: Prostate and breast cancer cells were treated with dibutyryl cAMP and compared with untreated cells. Growth patterns of cells were assessed by trypan blue-excluding method and western blot was done to determine protein levels of cell cycle regulatory proteins which govern G1 and G1/S phase. Northern blot and immunoprecipitation were done to determine the level of mRNA of p21 and the association between cell cycle regulatory proteins. In vitro immune complex kinase assay was done to assess the activity of cdk2. RESULTS: cAMP reduced cell growth by 48 h. Cyclin D3 level was downregulated and RB protein level was decreased and mostly unphosphorylated forms remained. The association of RB with E2F1 was increased. While cdk2 levels remained constant throughout cAMP treatment, the activity of cdk2/cyclin E complex, which is responsible for entry into S phase, was downregulated. Cdk inhibitors, p27 and p21 were induced with cAMP treatment. CONCLUSION: These observation suggest that the growth inhibitory effects of dibutyryl cAMP on prostate and breast cancer cells were mediated by induction of cdk inhibitors such as p21 and p27 and RB activation in accordance with downregulation of cdk2.

CHANGES OF CYCLINS, CYCLIN DEPENDENT KINASES, CYCLIN DEPENDENT KINASE INHIBITORS DURING GLOSSAL DEVELOPMENT IN THE RATS

The molecular mechanisms that regulate glossal muscle cell cycle and terminal differentiation remain largely unknown. To determine which cyclins, cyclin dependent kinases (CDKs), cyclin dependent kinase inhibitors (CKIs) are important for glossal cell proliferation, we have examined expression of cyclins CDKs, CKIs during normal glossal muscle development in the rat. All cyclins, CDKs, and KIP/CIP family of CKIs were highly expressed during fetal glossal muscle development, then they decreased at different rates after birth. While the mRNAs of cyclin Dl, D3, E , A, and B decreased gradually in glossal muscle during all stages of development, the protein levels of these cyclins decreased differently in tongue during pre- and postnatal development. While the functionally active formed of cyclin Dl, cyclin D3 and E proteins were observed until 7 days after birth, cyclin A and B proteins were decreased more slowly. While the CDK4, CDK6, CDK2, cdc2, and proliferating cell nuclear antigen (PCNA) proteins were higllly present during fetal glossal muscle development and gradually decreased during postnatal development. Particularly, cdc2 levels decreased markedly after birth. Immunohistochemical data for PCNA was consistent with Western blotting data for PCNA temporally and spatially. The mRNA and protein levels of p21, p27, and p57 were high, then their levels changed differently during glossal development. While the mRNA levels of p21 and p57 decreased gradually, the mRNA level of p27 did not change during glossal development. While the protein levels of p21 and p57 in tongue decreased markedly after birth, the protein levels of p27 increased slightly after birth, then decreased at adulthood. These findings suggest that the all cyclins and CDKs observed are involved in glossal muscle cell cycle, and reduction of cyclins and CDKs and induction of p21 are associated with the withdrawal of glossal muscle cell cycle after birth.