Identification of Molecular Signatures in Mild Intrinsic Atopic Dermatitis by Bioinformatics Analysis

BACKGROUND: Atopic dermatitis (AD) is recognized as a common inflammatory skin disease and frequently occurred in Asian and Black individuals.OBJECTIVE: Since the limitation of dataset associated with human severe AD, this study aimed to screen potential novel biomarkers involved in mild AD.METHODS: Expression profile data (GSE75890) were obtained from the database of Gene Expression Omnibus. Using limma package, the differentially expressed genes (DEGs) between samples from AD and healthy control were selected. Furthermore, function analysis was conducted. Meanwhile, the protein-protein interaction (PPI) network and transcription factor (TF)-miRNA-target regulatory network were constructed. And quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the expressions patterns of key genes.RESULTS: In total, 285 DEGs including 214 upregulated and 71 downregulated genes were identified between samples from two groups. The upregulated DEGs were mainly involved in nine pathways, such as hematopoietic cell lineage, pertussis, p53 signaling pathway, staphylococcus aureus infection, and cell cycle, while tight junction was the only pathway enriched by the downregulated DEGs. Cyclin B (CCNB)1, CCNB2, cyclin A (CCNA)2, C-X-C motif chemokine ligand (CXCL)10, and CXCL9 were key nodes in PPI network. The TF-miRNA-target gene regulatory network focused on miRNAs such as miR-106b, miR-106a, and miR-17, TFs such as nuclear factor kappa B subunit 1, RELA proto-oncogene, Sp1 transcription factor, and genes such as matrix metallopeptidase 9, peroxisome proliferator activated receptor gamma , and serpin family E member 1. Moreover, the upregulation of these genes, including CCNB1, CCNB2, CCNA2, CXCL10, and CXCL9 were confirmed by qRT-PCR.CONCLUSION: CCNB1, CCNB2, CCNA2, and CXCL9 might be novel markers of mild AD. miR-106b and miR-17 may involve in regulation of immune response in AD patients.

RNA-sequencing Profiles of Cell Cycle–Related Genes Upregulated during the G2-Phase in Giardia lamblia

To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.

Development of a Novel Nonradioisotopic Assay and Cdc25B Overexpression Cell Lines for Use in Screening for Cdc25B Inhibitors

PURPOSE: The cyclin-dependent kinase 1 (Cdk1) and cyclin B complex performs important roles in the transition from the G2 to M phase in the cell cycle through removal of inhibitory phosphates on Cdk1, and Cdc25B, which is a dual-specific phosphatase, mediates these dephosphorylation events. However, measuring Cdc25B activity by existing methods is hampered by inadequate nonspecific substrates and the need to use a radiolabeled isotope. The present study aimed to develop an improved method with which to properly measure Cdc25B activity using a novel nonradioisotopic assay and Cdc25B overexpression cell lines. MATERIALS AND METHODS: A nonradioisotopic Cdk1 kinase assay, based on Western blotting for retinoblastoma protein and histone H1, was used to analyze Cdc25B activity. Also, stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines were constructed using the tetracycline-regulated expression system and were applied as a tool for screening for inhibitors of Cdc25B. RESULTS: The present study developed and optimized a nonradioisotopic assay method to properly measure Cdc25B activity. Furthermore, we constructed stable Cdc25B2 and Cdc25B3 overexpression HeLa cell lines for the establishment of a strong assay system with which to evaluate the specificity of Cdc25B inhibitors under conditions similar to the intracellular environment. These methods were confirmed as useful tools for measuring Cdc25B activity. CONCLUSION: The nonradioisotopic Cdk1 kinase assay and Cdc25B overexpression cell lines developed in this study can be conveniently used as tools for screening inhibitors of Cdc25B phosphatase as anticancer drugs.

Overexpression of Cell Cycle Proteins of Peripheral Lymphocytes in Patients with Alzheimer's Disease

OBJECTIVE: Biological markers for Alzheimer's disease (AD) will help clinicians make objective diagnoses early during the course of dementia. Previous studies have suggested that cell cycle dysregulation begins earlier than the onset of clinical manifestations in AD. METHODS: We examined the lymphocyte expression of cell cycle proteins in AD patients, dementia controls (DC), and normal controls (NC). One-hundred seventeen subjects (36 AD, 31 DC, and 50 NC) were recruited. The cell cycle proteins CDK2, CDK4, CDK6, cyclin B, and cyclin D were measured in peripheral lymphocytes. Cell cycle protein expression in the three groups was compared after adjusting for age and sex. RESULTS: The levels of cell cycle proteins CDK2, CDK4, CDK6, cyclin B, and cyclin D were significantly higher in AD patients than in the NC subjects. The DC group manifested intermediate levels of cell cycle proteins compared with the AD patients and the NC subjects. The present study indicates that cell cycle proteins are upregulated in the peripheral lymphocytes of AD patients. CONCLUSION: Cell cycle dysregulation in peripheral lymphocytes may present a promising starting point for identifying peripheral biomarkers of AD.

Sorbus rufopilosa Extract Exhibits Antioxidant and Anticancer Activities by Inducing Cell Cycle Arrest and Apoptosis in Human Colon Adenocarcinoma HT29 Cells

BACKGROUND: Sorbus rufopilosa, a tsema rowan, is a species of the small ornamental trees in the genus Sorbus and the family Rosaceae found in East Asia. The bioactivities of S. rufopilosa have not yet been fully determined. The objective of this study is to evaluate the antioxidant and anticancer effects of ethanol extract of S. rufopilosa (EESR) and to determine the molecular mechanism of its anticancer activity in human colon carcinoma HT29 cells. METHODS: To examine the antioxidant activity of EESR, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay was performed. Inhibitory effect of EESR on cancer cell growth and proliferation was determined by water-soluble tetrazolium salt assay. To investigate the mechanism of EESR-mediated cytotoxicity, HT29 cells were treated with various concentrations of EESR and the induction of cell cycle arrest and apoptosis was analyzed by flow cytometry, 4,6-diamidino-2-phenylindole staining, and Western blot analysis. RESULTS: EESR showed significant antioxidant activity and inhibitory effect on HT29 cell growth in a dose-dependent manner. EESR induced cell cycle arrest at G2/M phase in a dose-dependent manner by modulating cyclin B, cyclin-dependent kinase 1 (CDK1), and CDK inhibitor p21 expression. EESR-induced apoptosis was associated with the upregulation of p53, a death receptor Fas, and a pro-apoptotic protein Bax and the activation of caspase 3, 8, and 9, resulting in the degradation of PARP. CONCLUSIONS: EESR possessing antioxidant activity efficiently inhibits proliferation of HT29 cells by inducing both cell cycle arrest and apoptosis. EESR may be a possible candidate for the anticancer drug development.

Effects of suppressed autophagy on mitochondrial dynamics and cell cycle of N2a cells / 华中科技大学学报（医学）（英德文版）

Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.

Simultaneously expressed miR-424 and miR-381 synergistically suppress the proliferation and survival of renal cancer cells---Cdc2 activity is up-regulated by targeting WEE1

OBJECTIVES: MiRNAs are intrinsic RNAs that interfere with protein translation. Few studies on the synergistic effects of miRNAs have been reported. Both miR-424 and miR-381 have been individually reported to be involved in carcinogenesis. They share a common putative target, WEE1, which is described as an inhibitor of G2/M progression. Here, we studied the synergistic effects of miR-424 and miR-381 on renal cancer cells. METHODS: The viability of 786-O cells was analyzed after transfection with either a combination of miR-424 and miR-381 or each miRNA alone. We investigated cell cycle progression and apoptosis with flow cytometry. To confirm apoptosis and the abrogation of G2/M arrest, we determined the level of pHH3, which is an indicator of mitosis, and caspase-3/7 activity. The expression levels of WEE1, Cdc25, γH2AX, and Cdc2 were manipulated to investigate the roles of these proteins in the miRNA-induced anti-tumor effects. To verify that WEE1 was a direct target of both miR-424 and miR-381, we performed a dual luciferase reporter assay. RESULTS: We showed that the combination of these miRNAs synergistically inhibited proliferation, abrogated G2/M arrest, and induced apoptosis. This combination led to Cdc2 activation through WEE1 inhibition. This regulation was more effective when cells were treated with both miRNAs than with either miRNA alone, indicating synergy between these miRNAs. WEE1 was verified to be a direct target of each miRNA according to the luciferase reporter assay. CONCLUSIONS: These data clearly demonstrate that these two miRNAs might synergistically act as novel modulators of tumorigenesis by down-regulating WEE1 expression in renal cell cancer cells. .

Preferential Cytotoxic Effect of Genistein on G361 Melanoma Cells Via Inhibition of the Expression of Focal Adhesion Kinase

Resistance to the induction of apoptosis is a possible mechanism by which tumor cells can survive anti-neoplastic treatments. Melanoma is notoriously resistant to anti-neoplastic therapy. Previous studies have demonstrated focal adhesion kinase (FAK) overexpression in melanoma cell lines. Given its probable role in mediating resistance to apoptosis, many researchers have sought to determine whether the downregulation of FAK in melanoma cells would confer a greater sensitivity to anti-neoplastic agents. Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting the PTK-mediated signaling pathway. This present study was undertaken to investigate the effect of genistein on the expression of FAK and cell cycle related proteins in the G361 melanoma cell line. Genistein was found to have a preferential cytotoxic effect on G361 melanoma cells over HaCaT normal keratinocytes. Genistein decreased the expression of 125 kDa phosphotyrosine kinase and the FAK protein in particular. Genistein treatment did not affect the expression of p53 in G361 cells in which p21 is upregulated. The expression of cyclin B and cdc2 was downregulated by genistein treatment. Taken together, our data indicate that genistein induces the decreased proliferation of G361 melanoma cells via the inhibition of FAK expression and regulation of cell cycle genes. This suggests that the use of genistein may be a viable approach to future melanoma treatments.

Essential role of TRPC6 in the proliferation of gastric cancer and its mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the essential role and mechanism of TRPC6 gene in the development of gastric cancer.</p><p><b>METHODS</b>The expression of TRPC6 protein was assessed in gastric cancer tissues and normal tissues adjacent to the cancer from 30 patients with gastric cancer. The inhibiting effect of TRPC6 activity on cell growth, cell cycle of a human gastric cancer cell line AGS cells, tumor progression and development of xenografted human gastric cancer in a mouse model was tested using dominant-negative mutant TRPC6 (DNC6). The survival of mice bearing xenografted tumors in the GFP and DNC6 was compared using Kaplan-Meier analysis. All statistical tests were two-sided.</p><p><b>RESULTS</b>The TRPC6 protein in the tumor tissues and para-tumor tissues was (21.60 ± 8.32)% versus (7.14 ± 2.24)%. After transfection of DNC6 virus for 24 hours, 48 hours, 72 hours and 96 hours, the growth inhibition rates of gastric cancer cells were (36.90 ± 1.13)%, (44.06 ± 2.17)%, (52.12 ± 2.76)% and (50.89 ± 1.97)%, respectively. The clone formation rates of control group and DNC6 group were (14.70 ± 3.00)% versus (43.80 ± 7.00)%. After transfection with DNC6 virus for 0, 24, 36 and 48 hours, the G(2)/M phase arrest was (20.34 ± 1.98)%, (24.31 ± 2.37)%, (27.70 ± 2.36)%, (35.10 ± 3.0)% in the DNC6 group and (18.40 ± 2.01)%, (18.0% ± 1.72)%, (17.50 ± 1.74)%, (16.80 ± 1.71)% in the control group, respectively. Inhibition of TRPC6 activity also reduced the subcutaneous tumor volume in the mouse models with xenografted human tumors (P < 0.05).</p><p><b>CONCLUSION</b>In the preclinical models tested, TRPC6 channels are essential for gastric cancer development via regulation of G(2)/M phase transition.</p>

Crystal structure of human Gadd45γ corrected reveals an active dimer

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45γ [corrected], revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45γ [corrected] identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45γ [corrected] is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.

Overexpressions of Cyclin B1, cdc2, p16 and p53 in Human Breast Cancer: The Clinicopathologic Correlations and Prognostic Implications

PURPOSE: The molecular mechanisms that are responsible for the initiation and progression of breast cancers are largely unknown. This study was to analyze the cyclin B1, cdc2, p53 and p16 tumor suppressor genes in human breast cancer. MATERIALS AND METHODS: To investigate the role of cyclin B1, cdc2, p53 and p16 in the pathogenesis and progression of breast carcinomas, 98 cases of breast cancers were examined by immunohistochemical method. The correlations of cyclin B1, cdc2, p53 and p16 expression with various clinico-pathologic findings were analysed. RESULTS: In the normal breast tissues, cyclin B1, cdc2 and p16 were weakly expressed, while p53 was not expressed. On the other hand, cyclin B1, cdc2, p53 and p16 were overexpressed in breast cancer, showing correlation between the expression of cyclin B1 and cdc2 and breast cancers (p=0.00). The overexpressions of cdc2 and p16 were correlated with an infiltrative tumor border pattern and this was statistically significant (p<0.05). In addition, the overexpression of cdc2 was correlated with histologic high grade carcinomas (p=0.00). CONCLUSION: Cyclin B1 and cdc2 appeared to be involved in the genesis or progression of breast cancers. In addition, the overexpressions of p16 and p53 may play important roles in more aggressive tumor and the overexpression of cdc2 is associated with progression of tumor to a higher grade of breast carcinomas. The deranged overexpressions of cyclin B1, cdc2, p16 and p53 may play an important role in human breast carcinogenesis.

Pseudomonas aeruginosa Exotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells

Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4',5-trimethoxy-3'-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.

The Expression of Hypoxia Inducible Factor-1alpha and Its Correlation with the Expressions of Cyclin A1 and Cyclin B1 and the Clinicopathologic Factors of Uterine Cervical Carcinoma

BACKGROUND: Hypoxia inducible factor-1alpha(HIF-1alpha) is a transcription factor for various target genes that are involved in adapting cells to hypoxia. It promotes cell proliferation and survival via modulation of such cell cycle regulators such as cyclin A1 and cyclin B1 in response to hypoxia. This is associated with local failure of radiotherapy, which renders a poor prognosis for cervical carcinoma. METHODS: Using the tissue histologic sections and a tissue microarray of the archived biopsy and surgical specimens of uterine cervical carcinoma from 57 patients who were treated with radiation therapy alone, we performed immunohistochemical staining for HIF-1alpha and cyclin A1 and B1 to evaluate the correlations between the expressions of these proteins in tumors and the clinicopathologic parameters associated with the prognosis. RESULTS: The large tumor cell nests and invasive front margins of the tumors showed comparatively intense immunoreactivity of HIF-1alpha. There was no significant correlation between the HIF-1alpha, cyclin A1 and cyclin B1 expressions and the clinicopathologic factors. CONCLUSIONS: The HIF-1alpha expression showed marked intra-tumoral heterogeneity. The HIF-1alpha expression is neither a powerful predictor of resistance to radiotherapy nor is it a poor prognostic marker in cervical carcinoma patients who are treated with radiotherapy. The expressions of cyclin A1 and cyclin B1 are neither independently associated with the response of radiation therapy nor are they associated with the prognostic parameters of uterine cervical carcinoma.

Long-term stable expression of antisense cDNA of cyclin B1 profoundly inhibits the proliferation of tumor cells and suppresses tumorigenicity in implanted mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cyclin B1 (CLB1) is necessary for mitotic initiation in mammalian cells and plays important roles in cancer development. Therefore, a potential strategy in cancer therapy is to suppress the activity of CLB1 by delivering antisense constructs of CLB1 into tumor cells. In previous CLB1 studies, antisense constructs with a short half life were often used and these constructs might not persistently inhibit CLB1.</p><p><b>METHODS</b>We successfully created a recombinant plasmid encoding the full-length antisense cDNA of mouse cyclin B1 (AS-mCLB1) and transfected this construct to the murine Lewis lung carcinoma (LL/2) and CT-26 colon carcinoma (CT-26) cells. We isolated clones of LL/2 and CT-26 transfectants with stable expression of AS-mCLB1. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression of the mRNA and protein levels of CLB1. To further test the efficacy of this strategy in vivo, AS-mCLB1-expressing LL/2 and CT-26 transfectants were implanted into mice.</p><p><b>RESULTS</b>We found the expression of the mRNA and protein levels of CLB1 decrease in these transfectants. The inhibition of CLB1 caused prominent G1 arrest, abnormal morphology, retarded cell growth and an increase in apoptosis. In AS-mCLB1-expressing LL/2 and CT-26 transfectants implanted mice, tumorigenicity was effectively suppressed compared with the controls. In addition, the expression of AS-mCLB1 also significantly increases the survival duration of implanted animals.</p><p><b>CONCLUSION</b>AS-mCLB1 is likely to be useful in future cancer therapy, which may be associated with its ability to down-regulate the expression of CLB1 and then induce G1arrest and apoptosis in tumor cells.</p>

Induction of G2 /M phase arrest and apoptosis of MCF-7 cells by novel benzofuran lignan via suppressing cell cycle proteins / 药学学报

In the present study, a newly synthesized benzofuran lignan 4-formyl-2-(4-hydroxy-3methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo [b] furan (ERJT-12) was tested for its antiproliferative activity on human tumor cells. The related mechanisms were also investigated. In vitro growth inhibitory effects of ERJT-12 on various cancer cell lines were determined by MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The integrity of DNA was assessed by agarose gel electrophoresis. Activation of Caspase-3/7 and Caspase-6 was measured by colorimetric assay. The expressions of cell cycle proteins cell divide cycle 25c (Cdc25c), cyclin dependent kinase 1 (CDK1), CyclinB1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blotting. MTT assay showed that ERJT-12 inhibited the proliferation of several cancer cell lines including multidrug resistant cells. MCF-7 cells were markedly arrested at gap2/mitosis (G2/M) phase after treatment with ERJT-12 and progressed into apoptosis. The increased activities of Caspase-3/7 and Caspase-6 in MCF-7 cells were observed. The expression of CyclinB1 was down-regulated. The activities of Cdc25c and CDK1 protein were suppressed and Bcl-2 protein was phosphorylated. ERJT-12 displays potent antiproliferative activity towards cancer cells through suppressing cell cycle proteins, arresting cell cycle at G2/M phase and inducing apoptosis. It might be a novel candidate for cancer therapy.

Tetrandrine: a potent abrogator of G2 checkpoint function in tumor cells and its mechanism / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>MCF-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.</p><p><b>RESULTS</b>Clonogenic 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.</p><p><b>CONCLUSION</b>Tetrandrine 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.</p>

Prognostic Implications of Cyclin B1, p34cdc2, p27(Kip1) and p53 Expression in Gastric Cancer

PURPOSE: Cell cycle progression is regulated by interactions of specific cyclins and cyclin dependent kinases (CDKs) at the G1-S and G2-M checkpoints and cell cycle deregulation plays a major role in carcinogenesis of human cancers. PATIENTS AND METHODS: To investigate the role of cell cycle regulators in the pathogenesis and progression of human gastric cancers, 23 cases of gastric carcinomas were examined for the expression of cyclin B1, p34cdc2, p27(Kip1) and p53 by immunohistochemical methods, and gene expression was correlated with various clinicopathologic findings. RESULTS: Out of 23 cases studied, cyclin B1 was diffusely expressed in 20 cases (87.0%), p34cdc2 in 14 cases (60.9%) and p53 in 12 cases (52.2%), whereas in normal gastric tissues, cyclin B1 and p34cdc2 were weakly expressed and p53 was not expressed. In contrast, p27(Kip1) was expressed in only 8.7% of gastric carcinomas compared with 78.3% of normal gastric tissues. There was correlation between the expression of cyclin B1 and expression of p34cdc2 (p=0.002), between the expression of cyclin B1 and loss of p27(Kip1) (p=0.025), and between the expression of p34cdc2 and loss of p27(Kip1) (p=0.065). In addition, expression of cyclin B1 was correlated with regional lymph node metastasis (p=0.032). CONCLUSION: Our results indicate that cyclin B1 and p34cdc2 are involved in the genesis or progression of gastric cancers. Furthermore, overexpression of cyclin B1 may play an important role in lymph node metastatic potential of gastric cancer. Thus, abnormal expression of cyclin B1 and CDKs, overexpression of p53 and loss of p27(Kip1) expression may play important roles in human gastric carcinogenesis.

Effect of arsenic trioxide on the expression of apoptosis-related genes in NB4 cells / 中国实验血液学杂志

The aim of this study was to investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with arsenic trioxide (As2O3) by using cDNA microarray. cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without arsenic trioxide. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 201 different human genes, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in the gene expression profile. The results showed that after the treatment of arsenic trioxide (2 micromol/L), 6 genes were up-regulated, and 12 genes related to apoptosis and signal transduction were down-regulated. The p21, survivin, cdc2 and Wee1Hu genes may be related to the differentiation and/or apoptosis of NB4 cells induced by As2O3. It is concluded that p21, survivin, cdc2 and Wee1Hu may play an important role in the mechanism underling arsenic trioxide-mediated NB4 cell apoptosis.

Analysis of the genetic interactions between Cyclin A1, Atm and p53 during spermatogenesis / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To analyze the functional interactions of Cyclin with p53 and Atm in spermatogenesis and DNA double-strand break repair.</p><p><b>METHODS</b>Two lines of double knockout mice were generated. Spermatogenesis and double strand break repair mechanisms were analyzed in Cyclin A1 (Ccna1); p53- and Ccna1; Atm-double knockout mice.</p><p><b>RESULTS</b>The block in spermatogenesis observed in Cyclin A1-/- (Ccna1-/-) testes at the mid-diplotene stage is associated with polynucleated giant cells. We found that Ccna1-deficient testes and especially the giant cells accumulate unrepaired DNA double-strand breaks, as detected by immunohistochemistry for phosphorylated H2AX. In addition, the giant cells escape from apoptosis. The development of giant cells occurred in meiotic prophase I, because testes lacking ATM, which are known to develop spermatogenic arrest earlier than prophase I, do not develop giant cells in the absence of cyclin A1. Cyclin A1 interacted with p53 and phosphorylated p53 in complex with CDK2. Interestingly, p53-deficiency significantly increased the number of giant cells in Ccna1-deficient testes. Gene expression analyses of a panel of DNA repair genes in the mutant testes revealed that none of the genes examined were consistently misregulated in the absence of cyclin A1.</p><p><b>CONCLUSION</b>Ccna1-deficiency in spermatogenesis is associated with defects in DNA double-strand break repair, which is enhanced by loss of p53.</p>