OBJECTIVE: Cyclin is a family of regulatory proteins that play a key role in controlling the cell cycle. Abnormalities of cell cycle regulators, including cyclins and cyclin dependent kinases (CDKs), have been reported in malignant tumors. This study was undertaken to quantitatively detect cyclin B1 and D1 in cervical cancer. METHODS: A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were used to analyze the expression of cyclin B1, D1 mRNA and proteins, respectively, in fresh invasive cervical cancer (n=41) and normal cervix tissue (n=10). RESULTS: There was significantly greater cyclin B1 expression in invasive cervical cancer than in normal cervix tissue (p=0.019). However, cyclin D1 expression was not significantly different (p=0.967). A Western blot analysis yielded similar results. CONCLUSION: Our results were consistent with the concept that up-regulation of cyclin B1 expression occurred in cervical cancer and an aberrant expression of cyclin B1 might play an important role in cervical carcinogenesis.
Blotting, Western ; Carcinogenesis ; Cell Cycle ; Cervix Uteri ; Cyclin B1* ; Cyclin D1 ; Cyclin I ; Cyclin-Dependent Kinases ; Cyclins* ; Female ; Humans ; RNA, Messenger ; Up-Regulation ; Uterine Cervical Neoplasms*

In this study we investigated the effects of fucoidan on the proliferation of fibroblasts and the reconstruction of a skin equivalent (SE). Fucoidan significantly stimulated the proliferation of CCD-25Sk human fibroblasts and Western blot analysis demonstrated that fucoidan markedly increased the expression of cyclin D1 and decreased the expression of p27. Fucoidan was used to reconstruct SE. Immunohistochemical staining showed that the addition of fucoidan to dermal equivalents increased expression of proliferating cell nuclear antigen (PCNA) and p63. In addition, expression of alpha6-integrin was significantly increased by fucoidan, whereas expression of beta1-integrin, type 1 collagen, elastin, fibronectin did not markedly change. These results suggest that fucoidan has positive effects on epidermal reconstruction and will therefore be beneficial in the reconstruction of SE.
Blotting, Western ; Collagen Type I ; Cyclin D1 ; Elastin ; Fibroblasts ; Fibronectins ; Humans ; Proliferating Cell Nuclear Antigen ; Skin*

Serum used widely in mammalian cell culture is also a potential source of bacterial, mycoplasmal and viral contaminations. In addition, the complex biological components in serum make harder the subsequent product recovery process. High cost, high batch variation and potential source limitation are among the other shortcomings. So serum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO-dhfr- cells were genetically engineered to make them adapted to IMEM, a protein-free medium, and resistant to apoptosis. The genes in choice are insulin-like factor (Igf-1), Bcl-2 and cyclin E. Bcl-2 is a mitochondrial membrane-integrated protein. It can block the release of cytochrome c by maintaining the integrity of mitochondrial membrane, and thus inhibit apoptosis. Igf-1 is similar both in structure and function to insulin, a growth factor added to serum-free medium to promote cell growth and is the only protein component in many currently used serum-free media. cyclin E is a cell cycle protein expressed continuously in G1 phase. When cyclin E accumulates to certain amount, cell cycle was driven to S phase. So cyclin E is a proliferation-promoting protein. By co-express Igf-1/Bcl-2 or Bcl-2/ cyclin E in CHO-dhfr- cells with a dicistronic expression vector, we constructed two cell lines: CHO-IB and CHO-BC. The high expression of each protein was confirmed by Western blot and flow cytometry. Apoptosis was analyzed by flow cytometry and DNA ladder detection, and the two cell lines were both found much more resistant to apoptosis induced by withdrawal of serum or addition of actinomycin D than the CHO-dhfr- parent cell. Cell proliferation assay by MTT method showed that the two cell lines proliferated much faster than CHO-dhfr- in IMDM medium without serum. Continuously culture assay proved that the two cell lines grow very well in IMEM protein-free medium supplemented with fibronectin and vitronectin to ease adherence. When compared to CHO-dhfr-, the two cell lines exhibited much more viable cell numbers and faster growth rate.
Animals ; Apoptosis ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Culture Media ; Cyclin E ; genetics ; Genes, bcl-2 ; Insulin-Like Growth Factor I ; genetics

OBJECTIVETo study the effect of lovastatin on the expression of IkappaBalpha and cell-cycle regulating proteins in MCF-7 cells.

METHODSMCF-7 cells were treated with 4, 8 and 16 micro mol/L lovastatin for 48 - 72 h. The distribution of cell cycles was assayed by flow cytometry (FCM). The protein expression of IkappaBalpha, CDK4, p16, pRb in cytoplasm and IkappaBalpha in the nucleus were detected by Western blot.

RESULTSLovastatin could arrest cellcycle in the G(0)/G(1) phase in a dose- and time-dependent manner, obviously lowering the expression of IkappaBalpha, CDK4 and pRb protein level in the cytoplasm and increasing IkappaBalpha in the nucleus, but not on p16 protein level.

CONCLUSIONLovastatin can induce the arrest of cell cycle in G(0)/G(1) phase by affecting the expression of IkappaBalpha and cell-cycle regulating protein in MCF-7 cells.

Antineoplastic Agents ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; metabolism ; Cell Line, Tumor ; Cyclin-Dependent Kinase 4 ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinases ; metabolism ; Female ; Humans ; I-kappa B Proteins ; metabolism ; Lovastatin ; pharmacology ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; Proto-Oncogene Proteins ; metabolism ; Retinoblastoma Protein ; metabolism

OBJECTIVETo assess the value of rheumatoid factors IgM-RF, IgA-RF, IgG-RF, interleukin-1 receptor type I (IL-1RI) and cyclin-dependent kinase 2 (CDK2) in the diagnosis of rheumatoid arthritis (RA).

METHODSIgM-RF, IgA-RF, IgG-RF, IL-1RI and CDK2 were detected in 47 patients with active RA, 47 with inactive RA, 20 with active systemic lupus erythematosus (SLE), 20 with inactive SLE, 20 with acute upper respiratory tract infection (AURTI), and 20 healthy controls using enzyme-linked immunosorbent assay (IgM-RF, IgA-RF, IgG-RF, IL-1RI) and time-resolved fluoroimmunoassay (CDK2).

RESULTSPatients with active and inactive RA showed significant differences in peripheral serum CDK2 and IgA-RF levels (P<0.05). Between active and inactive RA patients, RA patients and SLE patients, RA patients and AURTI patients, and RA patients and the control subjects, the area under curve (AUC) of the receiver operating characteristic curve (ROC) was 0.561, 0.814, 0.799, and 0.888 for IgM-RF, 0.596, 0.678, 0.729, and 0.850 for IgA-RF, 0.614, 0.718, 0.692, and 0.791 for IgG-RF, 0.646, 0.691, 0.762, and 0.835 for IL-1RI, 0.803, 0.753, 0.741, and 0.840 for CDK2, respectively.

CONCLUSIONSIgM-RF may have high value in the diagnosis and differential diagnosis of RA, and CDK2 can be useful for differential diagnosis between active and inactive RA.

Adult ; Area Under Curve ; Arthritis, Rheumatoid ; blood ; diagnosis ; Cyclin-Dependent Kinase 2 ; blood ; Female ; Humans ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Male ; Middle Aged ; ROC Curve ; Receptors, Interleukin-1 Type I ; blood ; Rheumatoid Factor ; blood

PURPOSE: Chloride channel-3 (ClC-3) is a member of the chloride channel family and plays a critical role in a variety of cellular activities. The aim of the present study is to explore the molecular mechanisms underlying the antitumor effect of silencing ClC-3 in breast cancer. METHODS: Human breast cancer cell lines MDA-MB-231 and MCF-7 were used in the experiments. Messenger RNA and protein expression were examined by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was measured by the bromodeoxyuridine method, and the cell cycle was evaluated using fluorescence-activated cell sorting. Protein interaction in cells was analyzed by co-immunoprecipitation. Tumor tissues were stained with hematoxylin-eosin and tumor burden was measured using the Metamorph software. RESULTS: Breast cancer tissues collected from patients showed an increase in ClC-3 expression. Knockdown of ClC-3 inhibited the secretion of insulin-like growth factor (IGF)-1, cell proliferation, and G1/S transition in breast cancer cells. In the mouse xenograft model of human breast carcinoma, tumor growth was significantly slower in animals injected with ClC-3-deficient cells compared with the growth of normal human breast cancer cells. In addition, silencing of ClC-3 attenuated the expression of proliferating cell nuclear antigen, Ki-67, cyclin D1, and cyclin E, as well as the activation of extracellular signal-regulated protein kinases (ERK) 1/2, both in vitro and in vivo. CONCLUSION: Together, our data suggest that upregulation of ClC-3 by IGF-1 contributes to cell proliferation and tumor growth in breast cancer, and ClC-3 deficiency suppresses cell proliferation and tumor growth via the IGF/IGF receptor/ERK pathway.
Animals ; Blotting, Western ; Breast Neoplasms* ; Breast* ; Bromodeoxyuridine ; Cell Cycle ; Cell Line ; Cell Proliferation ; Chloride Channels ; Cyclin D1 ; Cyclin E ; Cyclins ; Flow Cytometry ; Heterografts ; Humans ; Immunoprecipitation ; In Vitro Techniques* ; Insulin-Like Growth Factor I ; Methods ; Mice ; Proliferating Cell Nuclear Antigen ; Protein Kinases ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; Tumor Burden ; Up-Regulation

To determine which genes are regulated by thyroid stimulating hormone (thyrotropin, TSH), insulin and insulin-like growth factor-1 (IGF-1) in the rat thyroid, we used the microarray technology and observed the changes in gene expression. The expressions of genes for bone morphogenetic protein 6, the glucagon receptor, and cyclin D1 were increased by both TSH and IGF-1; for cytochrome P450, 2c37, the expression was decreased by both. Genes for cholecystokinin, glucuronidase, beta, demethyl-Q 7, and cytochrome c oxidase, subunit VIIIa, were up-regulated; the genes for ribosomal protein L37 and ribosomal protein L4 were down-regulated by TSH and insulin. However, there was no gene observed to be regulated by all three: TSH, IGF-1, and insulin molecules studied. These findings suggest that TSH, IGF-1, and insulin stimulate different signal pathways, which can interact with one another to regulate the proliferation of thyrocytes, and thereby provide additional influence on the process of cellular proliferation.
Animals ; Bone Morphogenetic Protein 6 ; Bone Morphogenetic Proteins/biosynthesis ; Cell Line, Tumor ; Cyclin D1/biosynthesis ; *Gene Expression Profiling ; *Gene Expression Regulation ; Insulin/*biosynthesis/metabolism ; Insulin-Like Growth Factor I/*biosynthesis ; Models, Genetic ; *Oligonucleotide Array Sequence Analysis ; Rats ; Receptors, Glucagon/biosynthesis ; Thyroid Gland/*metabolism ; Thyrotropin/*biosynthesis/metabolism ; Time Factors

Squamous cell carcinoma(SCC) is a significant cause of cancer morbidity and mortality worldwide, with an incidence of up to 166 cases per 100 000 population. It arises in the skin, upper aerodigestive tract, lung, and cervix and affects more than 200 000 Americans each year. We report here that a microarray experiment comparing 41 SCC and 13 normal tissue specimens showed that Id2, a gene that controls the cell cycle, was significantly up-regulated in SCC. Enforced expression of Id2 in vitro stimulated the proliferation of SCC cells and up-regulated the transcription of nuclear factor kappa B (NF-κB) and cyclin D1. Enhancement of the NF-κB activity with p65 significantly increased the cell proliferation and the transcription of cyclin D1, whereas inhibition of the NF-κB activity with I kappa B alpha mutant (IκBαM) and pyrroline dithiocarbamate (PDTC) abrogated cell proliferation and transcription of cyclin D1. Furthermore, a mutated NF-κB binding site in the cyclin D1 promoter fully abrogated the Id2-induced transcription of cyclin D1. Taken together, these data indicate that Id2 induces SCC tumor growth and proliferation through the NF-κB/cyclin D1 pathway.
Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; I-kappa B Proteins ; metabolism ; Inhibitor of Differentiation Protein 2 ; genetics ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; Transcription Factor RelA ; metabolism ; Transcription, Genetic ; Up-Regulation

OBJECTIVETo investigate the way of nuclear factor kappa B (NF-kappaB) activation and the mechanism of NF-kappaB-promoted proliferation in estrogen receptor (ER)-negative breast cancer cells.

METHODSThe protein of IkappaB kinase alpha (IKKalpha) was measured by Western blot and the influence on cell-cycle was assayed by flow cytometry (FCM).

RESULTSThe IKKalpha was tested higher in three ER-negative breast cancer cell lines than in MCF-7. The influence caused by epidermal growth factor (EGF), tumor necrosis factor (TNF)-alpha and E(2) to tumor cells' proliferation was tested. EGF could remarkably enhance cyclin D(1) expression about 83% more in EGF group than that in control group and proliferation index from 0.22 to 0.31 (P < 0.01). On the other hand, TNF-alpha inhibited cyclin D(1) expression. Protein kinase C inhibitor, Go6976, could peculiarly prevent NF-kappaB over-expression caused by EGF. The cell-cycle was assayed by FCM in phase G(0)/G(1) 69.36% and in phase S 22.77% when adding EGF and in phase G(0)/G(1) 91.54% and in phase S 7.81% when adding EGF and Go6976. The proliferation index decreased from 0.31 to 0.09 (P < 0.01).

CONCLUSIONSEGF-EGFR pathway can provide ER-negative breast cancer cells the signal for the autonomous growth. This signal promoted tumor cells' proliferation is transmitted by activating NF-kappaB and expressing more cyclin D(1). In this pathway, NF-kappaB play an important role as signal transmitting. The strategy to NF-kappaB activating may provide new way to treat ER-negative breast cancers.

Breast Neoplasms ; metabolism ; pathology ; physiopathology ; Carbazoles ; pharmacology ; Cell Proliferation ; drug effects ; Cyclin D1 ; biosynthesis ; Epidermal Growth Factor ; pharmacology ; Estradiol ; pharmacology ; Female ; Humans ; I-kappa B Kinase ; metabolism ; Indoles ; pharmacology ; NF-kappa B ; metabolism ; physiology ; Receptors, Estrogen ; metabolism ; Signal Transduction ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; pharmacology

PURPOSE: Lactobacillus casei (L. casei) is known to exert anti-proliferation effects on many types of cancer cells. However, the effect of L. casei on liver cancer has not been reported. Accordingly, the aim of this study was to determine the anti-cancer effect of L. casei extract on Huh7 cells. MATERIALS AND METHODS: L. casei ATCC393 extract was prepared and purified. After the treatment of L. casei extract on Huh7 cells, cell viability, cell cycle arrest and cell death were analyzed by flow cytometry. The expression levels of tumor necrosis factor-alpha receptor 1 (TNFR1) and death receptor 3 (DR3) mRNA related with extrinsic apoptosis were assessed by reverse transcription polymerase chain reaction. Additionally, P21 and P27 cell cycle proteins as well as Caspase-3, -8, -9, phospho-Bad and Bcl-2 apoptosis proteins were analyzed by western blot analysis. To determine the effect of L. casei extract on cancer stem-like cells, we analyzed changes in side population fraction through flow cytometry. RESULTS: The cell viability of Huh7 cells treated with L. casei extract was decreased by 77%, potentially owing to increases in the rates of Huh7 cells arrested in the G2/M phase (3% increase) and that underwent apoptosis (6% increase). The expression levels of TNFR1 and DR3 mRNA, as well as P21 and P27 cell cycle proteins, were increased. Meanwhile, the expressions of caspase-8, -9, phospho-Bad and Bcl-2 proteins decreased. However, in the case of side population cells, no remarkable changes were observed. CONCLUSION: L. casei extract exerts a potent anti-tumor effect on the viability of liver cancer cells, although not on cancer stem-like cells.
Apoptosis/drug effects ; Carcinoma, Hepatocellular/*pathology ; Caspase 8/metabolism ; Caspase 9/metabolism ; Cell Cycle Checkpoints/drug effects ; Cell Extracts/*pharmacology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Cyclin-Dependent Kinase Inhibitor p27/metabolism ; Cytostatic Agents/*pharmacology ; Flow Cytometry ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Lactobacillus casei/*chemistry ; Liver Neoplasms/*pathology ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA, Messenger/metabolism ; Receptors, Tumor Necrosis Factor, Member 25/metabolism ; Receptors, Tumor Necrosis Factor, Type I/metabolism ; bcl-Associated Death Protein/metabolism