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.
Adenine ; administration & dosage ; analogs & derivatives ; Apoptosis ; drug effects ; Autophagy ; drug effects ; genetics ; CDC2 Protein Kinase ; Cell Cycle ; drug effects ; genetics ; Cell Division ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin B ; biosynthesis ; Cyclin-Dependent Kinases ; Gene Expression Regulation ; drug effects ; Humans ; Membrane Proteins ; biosynthesis ; Microtubule-Associated Proteins ; biosynthesis ; Mitochondrial Dynamics ; drug effects ; genetics ; Mitochondrial Proteins ; biosynthesis ; Neuroblastoma ; Signal Transduction ; drug effects

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cyclin D1 ; biosynthesis ; Hepatitis B virus ; Humans ; Liver ; pathology ; Liver Cirrhosis ; metabolism ; pathology ; NF-kappa B ; biosynthesis ; Trans-Activators ; biosynthesis

To investigate the relationship between the expression of DNMT and clinical prognosis in adult patients with acute leukemia (AL), the mRNA expressions of DNMT, p15(INK4B), mdr1 were measured in 72 AL patients and 20 normal controls by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR); the ratio of p15 CpG land methylation was measured in 56 AL patients and 14 normal controls by methylation-specific PCR (MSP-PCR). The results showed that all three DNMT mRNA expressions in AL patients were significantly higher than that in normal controls (P < 0.01). When the internal control was changed into PCNA, a kind of cell proliferation marker gene, the difference still showed a statistic significance. All three DNMT genes were significantly expressed and positively correlated with AL patients, showing high synergistic expression, and there was a negative correlation between the levels of p15, mdr1 gene expression and DNMT. The complete remission (CR) rate in AL patients with the positive expression of all DNMT genes was significantly higher than that of AL patients with partially positive or negative expression (P < 0.01) of DNMT genes. In 56 AL patients, the P15I(NK4B) was completely methylated in 55.4% (31 of 56), partly methylated in 21.4% (12 of 56) and all 14 cases of normal controls were not methylated. It is concluded that DNMT genes are abnormally high expressed in adult AL patients, which lead to methylation-silence of tumor suppressor genes by CpG land hypermethylation, the AL patients with high expression of DNMT are more sensitive to chemotherapy, which may be a good prognostic factor for AL patients.
ATP Binding Cassette Transporter, Sub-Family B ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; Acute Disease ; Adult ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia ; genetics ; pathology ; Male ; Prognosis ; Proliferating Cell Nuclear Antigen ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

<b>OBJECTIVEb>To investigate the influence of aerosols on the expression of cyclin B(1), cyclin C and proliferating cell nuclear antigen (PCNA) in wound tissue healing of burned rat models.

<b>METHODSb>Sprague Dawley (SD) rats were inflicted as the deep partial thickness burn models. Rats were randomly divided into experimental group and control group. The experimental group were treated with aerosols. Samples were collected in 1 approximately 10 postburn days. Immunohistochemistry and image analysis methods were conducted to examine the expression of cyclin B(1), cyclin C and PCNA in both experimental and control groups.

<b>RESULTSb>The expression of cyclin C in experimental group was detected in nucleus of skin basal cell on the second postburn day, increased evidently at the fifth days and sustained at high expression level up to the tenth days after treatment. The expression of cyclin C in experimental group was significantly higher than control group (P < 0.05). The expression of PCNA was first observed in skin basal cell nucleus and hair follicle cell nucleus in both experimental and control group on the third postburn day. The expression of PCNA increased evidently at the fifth days in experimental after treatment and that increased evidently at the seventh days in control group, which showed there were lots of active proliferation cell. And the difference of the expression of PCNA between experimental and control group was significant (P < 0.01). The expression of cyclin B(1) was detected in nucleus and cytoplasm of skin basal cell in both groups on the third postburn day, and no difference between the experimental and control group (P > 0.05).

<b>CONCLUSIONSb>Aerosols can up-regulate the expression of cyclin C and PCNA in skin basal cell nucleus. Therefore the aerosols can accelerate wound tissue healing.

Aerosols ; Animals ; Burns ; metabolism ; therapy ; Cyclin B ; biosynthesis ; Cyclin B1 ; Cyclin C ; Cyclins ; biosynthesis ; Disease Models, Animal ; Electric Stimulation Therapy ; methods ; Female ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Wound Healing ; physiology

<b>OBJECTIVEb>To 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.

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

<b>RESULTSb>The 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).

<b>CONCLUSIONSb>EGF-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

<b>OBJECTIVEb>To investigate the feasibility of detecting cyclin D1 mRNA in paraffin-embedded tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and competitive RT-PCR and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL).

<b>METHODSb>Paraffin-embedded samples of 36 cases of MCL, 71 cases of other small B-cell lymphomas and 20 cases of lymphoid reactive hyperplasia as control group were retrieved from archival materials. Cyclin D1 protein and its mRNA was detected by EnVision and RT-PCR and competitive RT-PCR in all samples. House-keeping gene PGK was choosen as internal control.

<b>RESULTSb>(1) Cyclin D1 protein was expressed in 27 of the 38 MCL (71.1%). No cyclin D1 expression was found in the control group. (2) PGK was detected in 103 of the 116 cases (88.8%) and also detected in 34 of 36 MCL cases (94.7%). (3) cyclin D1 mRNA was detected in 34 nodal mantle cell lymphoma cases by RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA was 94.4% in mantle cell lymphomas after exclusion of the 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia, except 1 case of B-SLL. Sequencing analysis showed that sequences were identical to cyclin D1. (4) Cyclin D1 mRNA overexpression was detected in 27 cases of nodal mantle cell lymphoma by competitive RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA overexpression was 75.0% in mantle cell lymphomas after exclusion of 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA overexpression was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia.

<b>CONCLUSIONb>RT-PCR and competitive RT-PCR detection of cyclin D1 mRNA overexpression could be used for the diagnosis and differential diagnosis of mantle cell lymphoma in paraffin-embedded blocks.

Cyclin D1 ; biosynthesis ; genetics ; Diagnosis, Differential ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; pathology ; Lymphoma, Follicular ; genetics ; metabolism ; Lymphoma, Mantle-Cell ; genetics ; metabolism ; pathology ; Paraffin Embedding ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Cyclin B1, a positive regulator, controls mitosis occurrence, plays an important role in cell proliferation. To investigate the clinical significance of cyclin B1, the expression of cyclin B1 in acute leukemia (AL) patients was measured; the expression of cyclin B1 and p21(cipl), and their cell cycle distribution were assayed by flow cytometry in 136 adult patients with newly diagnosed AL, 10 continuous complete remission (CCR) AL and 17 normal controls; the mRNA of cyclin B1 and p21(cipl), and the proliferation cell nuclear antigen (PCNA) in patients and normal controls were detected with semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The results showed that the expression of cyclin B1 in newly diagnosed AL patients was significantly higher than that in normal controls. For the relapsed AL patients, the cyclin B1 expression was also higher than that in normal controls, but lower than that in newly diagnosed cases, there was no significant difference between the remission cases and normal controls, nor difference between CCR AL patients and normal controls. All patients with high cyclin B1 expression had an unscheduled expression manner, that cyclin B1 protein appeared in G(1) phase, and in some case it even higher than that of G(2) phase. The response rate (partial remission + complete remission) and survival rate in the cyclin B1 high expressed patients were higher than that of cyclin B1 low expressed patients. The relapse rate in cyclin B1 high expressed patients was higher than that in cyclin B1 normally expressed patients. The survival rate in cyclin B1 high expressed patients was higher than that in cyclin B1 low expression patients. A negative correlation between the expression of cyclin B1 and p21(cipl) was observed. Additionally, cyclin B1 protein expression was generally correlated with proliferation index (PI) and proliferation cell nuclear antigen (PCNA). It is concluded that this study demonstrates for the first time cyclin B1 overexpression and abnormally distribution in cell cyclin of newly diagnosed AL patients. It was considered that cyclin B1 may play an important role in leukemic pathogeneses and can be one of the factors influencing the prognosis of AL patients.
Acute Disease ; Adolescent ; Adult ; Aged ; Cell Proliferation ; Cyclin B ; genetics ; Cyclin B1 ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Female ; HL-60 Cells ; Humans ; Kaplan-Meier Estimate ; Leukemia ; drug therapy ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Prognosis ; Proliferating Cell Nuclear Antigen ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

In order to investigate the relationship between the expression of cyclin A and drug resistance in adult patients with acute leukemia (AL), the mRNA expression of cyclin A, mdr1, Top II alpha, bcl-2 was detected in 64 adult patients with AL and 20 normal controls by semi-reverse transcription polymerase chain reaction (semi-RT-PCR). It was found that the cyclin A and Top II alpha mRNA expression levels in drug resistant group were significantly lower than in sensitive group (P < 0.01). Under the same experimental condition no cyclin A mRNA expression was detectable in all normal controls. The mdr1 and bcl-2 mRNA expression levels in resistant group were significantly higher than in sensitive group (P < 0.01), cyclin A and Top II alpha gene expression levels were closely correlated (rs = +0.794, P = 0.000, n = 64) in all AL patients, but cyclin A was not correlated with mdr1 and bcl-2 gene expression levels. In drug resistant group there was a negative correlation between the gene expression levels of cyclin A and mdr1 (rs = -0.337, P = 0.029). The 10 AL patients with positive lower expression of both cyclin A and Top II alpha were all resistant to drugs. Logistic regression of Binary analysis showed the correlation between the lower expression of cyclin A and drug resistance. It was concluded that lower expression of cyclin A gene might be an unfavorable prognostic factor for patients with AL, and detection of both cyclin A and Top II alpha gene expression would predict drug resistance in AL patients.
ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; Adolescent ; Adult ; Aged ; Cyclin A ; biosynthesis ; genetics ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Female ; Genes, MDR ; genetics ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Leukemia, Promyelocytic, Acute ; genetics ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; RNA, Messenger ; biosynthesis ; genetics

<b>OBJECTIVEb>To investigate the relation between p16 gene expression and the carcinogenesis and progress of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC).

<b>METHODSb>In 35 specimens of HCC tissue and the adjacent liver tissue, the integration of HBV X gene was detected by polymerase chain reaction (PCR) and Southern blot. The point mutation of exon-1alpha, 2 and 3 of p16 gene were detected by PCR-single strand conformation polymorphism (SSCP). The expression of p16 mRNA and p16 protein was detected by RT-PCR and Western blot.

<b>RESULTSb>The integration of X gene correlated with the expression loss of p16 mRNA and p16 protein in HCC (P < 0.05). The expression loss rates of p16 protein in HCC and adjacent tissues were 62.9% (22/35) and 40.0% (14/35) with significant difference (P < 0.05). The expression loss of p16 protein in HCC correlated with the differentiation degrees of HCC and the infiltration of tumor cells (P < 0.05).

<b>CONCLUSIONb>The integration of X gene correlates with the expression loss of p16 protein. The alteration of p16 gene, playing an important role in all stages of hepatocarcinogenesis, correlates with the progress and invasion of hepatocellular carcinoma.

Carcinoma, Hepatocellular ; genetics ; metabolism ; virology ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Exons ; Female ; Genes, p16 ; Hepatitis B Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; genetics ; metabolism ; virology ; Male ; Middle Aged ; Point Mutation ; RNA, Messenger ; biosynthesis ; genetics ; Trans-Activators ; genetics ; Virus Integration

<b>OBJECTIVEb>To confirm the unscheduled in vivo and in vitro expression models of cyclin B1 in cancer cells so as to study the different profiles of cyclin B1 in G(1)-phase immortal cells under different culture states and culture conditions.

<b>METHODSb>Multiparameter flow cytometry (FCM) was used to correlate the expression of cyclin B1 with the position in cell cycle of immortal cells in vivo and in vitro using the MOLT-4 cell line as control. Cells which belonged to G(1)-phase were sorted by FCM according DNA diploidy, and then the expression of cyclin B1 was examined by confocal microscope to confirm the results. For further analysis, different subgroups in G(1) phase were sorted according to the fluorescent intensity of cyclin E, and then the exact period in G(1) phase when cyclin B1 was expressed, were assayed by Western blot.

<b>RESULTSb>Unscheduled expression of cyclin B1 expressed in G(1)-phase was found not only in synchronized leukemia cells MOLT-4 and in vivo transformed T-7 cells, but also in vivo tumor cells detached from clinical samples. In the synchronized growing cells, cyclin B1 was mainly detected in the early G(1) phase, while in transformed T7 cells, cyclin B1 was mainly detected in the late G(1) phase.

<b>CONCLUSIONb>The limitation of detecting cyclin B1 is due to its unscheduled expression, rending cyclin B1 being detected at different time-spots in the G(1) phase. This phenomenon may be related to the adjustment between the loss of control in cell proliferation and cell apoptosis, thereby leading to tumorigenesis.

Apoptosis ; physiology ; Cell Line, Transformed ; Cyclin B ; biosynthesis ; Cyclin B1 ; Flow Cytometry ; G1 Phase ; physiology ; Humans ; Tumor Cells, Cultured