Prognostic Impact of Cell Division Cycle Associated 2 Expression on Pancreatic Ductal Adenocarcinoma

Objective To examine the expression of cell division cycle associated 2(CDCA 2) in pancreatic ductal adenocarcinoma(PDAC) and investigate its role in prognosis of PDAC patients.Methods This retrospective study included 155 PDAC patients who underwent surgical treatment and complete post-operative follow-up.Clinicopathologic data were collected through clinical database.Tissue microarray was constructed and immunohistochemistry was performed to detect CDCA2 expression in the PDAC tumor tissues and adjacent non-tumor tissues.Clinicopathological characteristics between high and low CDCA2 expression were compared.Correlation of CDCA2 expressions with patients' survival was analyzed using Kaplan-Meier method and Cox regression analysis.Results Expression of CDCA2 in PDAC cells was significantly higher than that in adjacent non-tumor tissues(U=4056.5,P<0.001).Univariate analysis showed that CDCA2 expression [hazard ratio(HR)=1.574,95% confidence interval(CI)=1.014-2.443,P=0.043] and node metastasis(HR=1.704,95%CI=1.183-2.454,P=0.004) were significantly associated with prognosis.Cox regression analysis showed CDCA2 expression was not an independent prognostic risk factor(HR=1.418,95%CI=0.897-2.242,P=0.135) for PDCA patients.Stratification survival analysis demonstrated CDCA2 expression as an independent prognostic risk factor in male patients(HR=2.554,95%CI=1.446-4.511,P=0.003) or in non-perineural invasion patients(HR=2.290,95%CI=1.146-4.577,P=0.012).Conclusions CDCA2 is highly expressed in PDAC tumor tissue.Although CDCA2 is not an independent prognostic risk factor for PDAC patients,it might be used to help predict prognosis of male or non-perineural invasion patients of PDAC.

NCAPD2 serves as a potential prognostic biomarker for lung adenocarcinoma and promotes cell proliferation,migration,invasion and cell cycle in vitro

Objectives:The pro-oncogenic effects of NCAPD2 have been extensively studied across various tumor types;however,its precise role within the context of lung adenocarcinoma(LUAD)remains elusive.This study aims to elucidate the biological functions of NCAPD2 in LUAD and unravel the underlying mechanistic pathways.Methods:Utilizing bioinformatics methodologies,we explored the differential expression of NCAPD2 between normal and tumor samples,along with its correlations with clinical-pathological characteristics,survival prognosis,and immune infiltration.Results:In the TCGA-LUAD dataset,tumor samples demonstrated significantly elevated levels of NCAPD2 expression compared to normal samples(p<0.001).Clinically,higher NCAPD2 expression was notably associated with advanced T,N,and M stages,pathologic stage,gender,smoking status,and diminished overall survival(OS).Moreover,differentially expressed genes(DEGs)associated with NCAPD2 were predominantly enriched in pathways related to cell division.Immune infiltration analysis revealed that NCAPD2 expression levels were linked to the infiltration of memory B cells,naïve CD4+T cells,activated memory CD4+T cells,and M1 macrophages.In vitro experiments demonstrated that silencing NCAPD2 suppressed LUAD cell proliferation,migration,invasion,epithelial-mesenchymal transition(EMT),and cell cycle progression.Conclusions:In summary,NCAPD2 may represent a promising prognostic biomarker and novel therapeutic target for LUAD.

Meiotic nuclear divisions 1 suppresses the proliferation and invasion of pancreatic cancer cells via regulating H2A.X variant histone

Introduction:Among all malignant tumors of the digestive system,pancreatic carcinoma exhibits the highest mortality rate.Currently,prevention and effective treatment are urgent issues that need to be addressed.Methods:The study focused on meiotic nuclear divisions 1(MND1),integrating data from the Gene Expression Profiling Interactive Analysis(GEPIA)database with prognostic survival analysis.Simultaneously,experiments at cellular level were employed to demonstrate the effect of MND1 on the proliferation and migration of PC.The small-molecule inhibitor of MND1 was used to suppress the migration of PC cells by knocking down MND1 using small interfering RNA(siRNA)in Patu-8988 and Panc1 cell lines.Results:The results of Cell Counting Kit-8 indicated that the suppression of MND1 resulted in a decrease in cell proliferation.Wound healing and Transwell assays revealed that MND1 knockdown reduced cell migration and invasion.Flow cytometry revealed that inhibiting MND1 hindered the cell cycle.Furthermore,MND1 could stimulate the proliferation,migration,and invasion of Patu-8988 and Panc1 cells by increasing the expression of MND1.Notably,MND1 had a positive effect on H2AFX expression in PC cells.Elevated MND1 expression suggests the low overall survival rate of individuals diagnosed with PC.Conclusion:These findings suggest that MND1 has the potential to be a gene with the ability to accurately diagnose and treat PC.

Effects of Low Dose Radiation on Tumor Apoptosis, Cell Cycle and Apoptosis-Related Protein Bcl-2 in Tumor-Bearing Mice

To study the effects of low dose radiation (LDR) on tumor apoptosis, cellcycle progression and changes of apoptosis-related protein Bcl-2 in tumor-bearing mice. Methods:Male mice of Kunming strain were implanted subcutaneously with S180 sarcoma cells in the left inguenas an in situ experimental animal model. Seven days later, the mice were subjected to 75 mGywhole-body γ-irradiation. At 24 and 48 h after the irradiation, all mice were sacrificed. The tumorsizes were measured, and tumor cell apoptosis and cell cycle progression were analyzed by flowcytometry. The expression of apoptosis-related protein Bcl-2 and the apoptotic rate of tumor cellswere observed by immunohistochemistry and electron microscopy. Results: Tumors grew significantlyslower after LDR (P 【 0.05). The tumor cells were arrested in G1 phrase and the expression of Bcl-2protein decreased at 24 h. Apoptotic rate of tumor cells was increased significantly at 48 h afterLDR (P 【 0.01). Conclusion: LDR could cause a G1-phase arrest and increase the apoptosis of tumorcells through the low level of apoptosis-related protein bcl-2 in the tumor-bearing mice. Theorganized immune function and anti-tumor ability are markedly increased after LDR. Our studyprovides practical evidence of clinical application to cancer treatment.

Regulation of the cell cycle gene, BTG2, by miR-21 in human laryngeal carcinoma

MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific miRNAs and their target genes participate in the molecular pathogenesis of laryngeal carcinoma, oligonucleotide microarrays were used to assess the differential expression profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with normal tissues. The oncogeuic miRNA, microRNA-21 （miR-21）, was found to he npregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells, whereas overexpression of miR-21 elevated growth activity of the cells, as detected by the colony formation assay. The cell number reduction caused by miR-21 inhibition was due to the loss of control of the G1-S phase transition, instead of a noticeable increase in apoptosis. Subsequently, a new target gene of miR- 21, BTG2, was found to be downregulated in laryngeal carcinoma tissues. BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These findings indicate that aberrant expression of miR-21 may contribute to the malignant phenotype of laryngeal carcinoma by maintaining a low level of BTG2. The identification of the oneogenic miR-21 and its target gene, BTG2, in laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy.

Rice leaf inclination2, a VIN3-1ike protein, regulates leaf angle through modulating cell division of the collar

As an important agronomic trait, inclination of leaves is crucial Ior crop architecture and grain yields. 10 understand the molecular mechanism controlling rice leaf angles, one rice leaf inclination2 （1c2, three alleles） mutant was identified and functionally characterized. Compared to wild-type plants, lc2 mutants have enlarged leaf angles due to increased cell division in the adaxial epidermis of lamina joint. The LC2 gene was isolated through positional cloning, and encodes a vernalization insensitive 3-like protein. Complementary expression of LC2 reversed the enlarged leaf angles of lc2 plants, confirming its role in controlling leaf inclination. LC2 is mainly expressed in the lamina joint during leaf development, and particularly, is induced by the phytohormones abscisic acid, gibberellic acid, auxin, and brassinosteroids. LC2 is localized in the nucleus and defects of LC2 result in altered expression of cell division and hormone-responsive genes, indicating an important role of LC2 in regulating leaf inclination and mediating hormone effects.

Selective COX-2 inhibitor,NS-398,suppresses cellular proliferation in human hepatocellular carcinoma cell lines via cell cycle arrest

AIM: To investigate the growth inhibitory mechanism of NS-398, a selective cyclooxygenase-2 (COX-2) inhibitor, in two hepatocellular carcinoma (HCC) cell lines (HepG2 and Huh7). METHODS: HepG2 and Huh7 cells were treated with NS-398. Its effects on cell viability, cell proliferation, cell cycles, and gene expression were respectively evaluated by water-soluble tetrazolium salt (WST-1) assay, 4’-6-diamidino-2-phenylindole (DAPI) staining, flow cytometer analysis, and Western blotting, with dimethyl sulfoxide (DMSO) as positive control. RESULTS: NS-398 showed dose- and time-dependent growth-inhibitory effects on the two cell lines. Proliferating cell nuclear antigen (PCNA) expressions in HepG2 and Huh7 cells, particularly in Huh7 cells were inhibited in a time- and dose-independent manner. NS-398 caused cell cycle arrest in the G1 phase with cell accumulation in the sub-G1 phase in HepG2 and Huh7 cell lines. No evidence of apoptosis was observed in two cell lines. CONCLUSION: NS-398 reduces cell proliferation by inducing cell cycle arrest in HepG2 and Huh7 cell lines, and COX-2 inhibitors may have potent chemoprevention effects on human hepatocellular carcinoma.

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis

BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells

AIM:To investigate the anti-tumor effects of Paris chinensis dioscin(PCD)and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells.METHODS:Cell viability was analyzed by the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope(LSCM)using Annexin-V/propidium iodide(PI)staining,and the cell cycle was evaluated using PI staining with flow cytom-etry.Intracellular calcium ions were detected under fluorescence microscope.The expression of cell cycle and apoptosis-related proteins cyclin B1,CDK1,cytochrome C and caspase-3 was measured by immunohistochemical staining.RESULTS:PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose-and time-de-pendent manner.After treatment of SGC-7901 cells with PCD,apoptosis appeared in SGC-7901 cells.Morpho-logical changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining,and the cell number of the G0/G1 phase was decreased,while the number of cells in the G2/M phase was increased.Cell cycle-related proteins,such as cyclin B1 and CDK1,were all down-regulated,but caspase-3 and cytochrome C were up-regulated.Moreover,intracellular calcium accumulation occurred in PCD-treated cells.CONCLUSION:G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells.

Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As 2O 3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As 2O 3 induced cell apoptosis, K562 cells were cultured with As 2O 3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As 2O 3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G 2/M phase increased in proportion to As 2O 3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As 2O 3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G 2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As 2O 3-induced apoptosis.

Dexamethasone suppresses DU145 cell proliferation and cell cycle through inhibition of the extracellular signal-regulated kinase 1 /2 pathway and cyclin D1 expression

Aim： To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods： The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase （ERK）1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor （GR） antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results： Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion： Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Intrinsic apoptotic pathway and G2/M cell cycle arrest involved in tubeimoside I-induced EC109 cell death

Objective： Squamous esophageal carcinoma is highly prevalent in developing countries, especially in China. Tu Bei Mu （TBM）, a traditional folk medicine, has been used to treat esophageal squamous cell carcinoma （ESCC） for a long term. tubeimoside I （TBMS1） is the main component of TBM, exhibiting great anticancer potential. In this study, we investigated the mechanism of TBMS1 cytotoxic effect on EC109 cells. Methods： Comparative nuclear proteomic approach was applied in the current study and we identified several altered protein spots. Further biochemical studies were carried out to detect the mitochondrial membrane potential, cell cycle and corresponding proteins＇ expression and location. Results： Subcellular proteomic study in the nucleus from EC109 cells revealed that altered proteins were associated with mitochondrial function and cell proliferation. Further biochemical studies showed that TBMSl-induced molecular events were related to mitochondria-induced intrinsic apoptosis and P21-cyclin B 1/cdc2 complex-related G2/M cell cycle arrest. Conclusions： Considering the conventional application of TBM in esophageal cancer, TBMS1 therefore may have a great potential as a chemotherapeutic drug candidate for ESCC.

PADI3 induces cell cycle arrest via the Sirt2/AKT/p21 pathway and acts as a tumor suppressor gene in colon cancer

Objective:As a member of the peptidyl arginine deiminase(PAD)family,PADI3 is weakly expressed in colon cancer tissues and highly expressed in adjacent colon cancer tissues.However,the role of PADI3 in colon cancer is unclear.In this study,we investigated the function and molecular mechanism of PADI3 in colon cancer tumorigenesis.Methods:Western blot and real-time PCR were used to detect the expression levels of several genes.CCK-8,flow cytometry(FCM)and colony formation assays were used to examine cell proliferation,the cell cycle and colony formation ability.RNAsequencing analysis was used to study the molecular mechanism of PADI3 in tumorigenesis.A truncation mutation experiment was performed to determine the key functional domain of PADI3.Results:PADI3 overexpression inhibited cell proliferation and colony formation and led to G1 phase arrest in both HCT116(originating from primary colon cancer)and LoVo(originating from metastatic tumor nodules of colon cancer)cells.PADI3-expressing HCT116 cells had a lower tumor formation rate and produced smaller tumors than control cells.PADI3 significantly decreased Sirtuin2(Sirt2)and Snail expression and AKT phosphorylation and increased p21 expression,and Sirt2 overexpression partly reversed the effects induced by PADI3 overexpression.Immunocytochemistry showed that PADI3 is mainly localized in the cytoplasm.Truncation mutation experiments showed that the C-domain is the key domain involved in the antitumor activity of PADI3.Conclusions:PADI3 suppresses Snail expression and AKT phosphorylation and promotes p21 expression by downregulating Sirt2 expression in the cytoplasm,and the C-domain is the key domain for its antitumor activity.

Ubiquitin-conjugating enzyme E2T knockdown suppresses hepatocellular tumorigenesis via inducing cell cycle arrest and apoptosis

BACKGROUND Hepatocellular carcinoma(HCC)is now the most common primary liver malignancy worldwide,and multiple risk factors attribute to the occurrence and development of HCC.Recently,increasing studies suggest that ubiquitinconjugating enzyme E2T(UBE2T)serves as a promising prognostic factor in human cancers,although the molecular mechanism of UBE2T in HCC remains unclear.AIM To investigate the clinical relevance and role of UBE2T in HCC development.METHODS UBE2T expression in HCC tissues from the TCGA database and its association with patient survival were analyzed.A lentivirus-mediated strategy was used to knock down UBE2T in HCC cells.qRT-PCR and Western blot assays were performed to check the effect of UBE2T silencing in HCC cells.Cell growth in vitro and in vivo was analyzed by multiparametric high-content screening and the xenograft tumorigenicity assay,respectively.Cell cycle distribution and apoptosis were determined by flow cytometry.The genes regulated by UBE2T were profiled by microarray assay.RESULTS UBE2T was overexpressed in HCC tissues compared with paired and non-paired normal tissues.High expression of UBE2T predicted a poor overall survival in HCC patients.In vitro,lentivirus-mediated UBE2T knockdown significantly reduced the viability of both SMMC-7721 and BEL-7404 cells.In vivo,the xenograft tumorigenesis of SMMC-7721 cells was largely attenuated by UBE2T silencing.The cell cycle was arrested at G1/S phase in SMMC-7721 and BEL-7404 cells with UBE2T knockdown.Furthermore,apoptosis was increased by UBE2T knockdown.At the molecular level,numerous genes were dysregulated after UBE2T silencing,including IL-1B,FOSL1,PTGS2,and BMP6.CONCLUSION UBE2T plays an important role in cell cycle progression,apoptosis,and HCC development.

Stevenleaf from Gynostemma Pentaphyllum inhibits human hepatoma cell(HepG2)through cell cycle arrest and apoptotic induction

The anticancer activity of stevenleaf(SV)on the basis of cell viability,cell cycle,and apoptosis induction in HepG2 cancer cells were evaluated.SV controlled the growth of HepG2 cells with IC50 of 139.82μmol/L for 24 h,IC50 of 119.12μmol/L for 48 h and cell cycle arrested at G0/G1 phase,induced cell apoptosis and enhanced intracellular ROS generation.For cell cycle arrest,the mRNA expression levels of p21,p27 and p53 were up-regulated,while the expression levels of Cyclin A,Cyclin D1,Cyclin E and CDK1/2 were downregulated.SV efficiently up-regulated TNF R1,TRADD1 and FADD and down-regulated Caspase8 for cell death receptors;similarly,up-regulated Bax,Bak,Cytc,Apaf1,Caspase3 and Caspase9,and down-regulated Bcl2,Bcl xl and Bad for mitochondrial signal pathway.SV induced the mTOR-mediated cell apoptosis in HepG2 cells via activation of Akt and AMPK.The mechanistic explanation for the anticancer activity of SV as functional food can be derived from above results.

Smoc2 potentiates proliferation of hepatocellular carcinoma cells via promotion of cell cycle progression

AIM To determine the influence of Smoc2 on hepatocellular carcinoma(HCC) cell proliferation and to find a possible new therapeutic target for preventing HCC progression.METHODS We detected expression of Smoc2 in HCC tissues and corresponding non-tumor liver(CNL) tissues using PCR, western blot, and immunohistochemistry methods. Subsequently, we down-regulated and upregulated Smoc2 expression using siR NA and lentivirus transfection assay, respectively. Then, we identified the effect of Smoc2 on cell proliferation and cell cycle using CCK-8 and flow cytometry, respectively. The common cell growth signaling influenced by Smoc2 was detected by western blot assay. RESULTS The expression of Smoc2 was significantly higher in HCC tissues compared with CNL tissues. Overexpression of Smoc2 promoted HCC cell proliferation and cell cycle progression. Down-regulation of Smoc2 led to inhibition of cell proliferation and cell cycle progression. Smoc2 had positive effect on ERK and AKT signaling.CONCLUSION Smoc2 promotes the proliferation of HCC cells through accelerating cell cycle progression and might act as an anti-cancer therapeutic target in the future.

Msx2 plays a critical role in lens epithelium cell cycle control

AIM: To investigate the effects of Msx2 on lens epithelium cell cycle, and evaluate the changes of the proliferation, apoptosis of lens epithelium cells. METHODS: Mice lens epithelium cells were cultured and transfected with pEGFP-Msx2 and control. Msx2-deficient mice (Msx2(-/-)) lens tissue were isolated. Lens tissue and transfected cells were prepared for mRNA extraction using Trizol reagent. CyclinD1 and Prox1 expression were evaluated by real-time RT-PCR. BrdU incorporation and apoptosis rate were investigated by immunofluorescence and flow cytometry analysis. RESULTS: After transfected with pEGFP-Msx2, lens epithelium cells failed to incorporate BrdU and anti - phospho-histone-3 immunofluorescence failed to detect cell nuclei which GFP were positive. Msx2 over expression resulted in increasing apoptosis rate in lens epithelium cells. CyclinD1 and Prox1 expression increased significantly in Msx2 knockout mice by real time RT-PCR quantization and CyclinD1 expression decreased significantly in Msx2overexpressed cell. CONCLUSION: Msx2 has the effect of inhibiting proliferation and differentiation, triggering apoptosis on mice lens epithelium cells.

6-OHDA Induces Cycle Reentry and Apoptosis of PC12 Cells through Activation of ERK1/2 Signaling Pathway

This study investigated the effect and mechanism of cell cycle reentry induced by 6-hydrodopamine （6-OHDA） in PC12 cells. By using neural differentiated PC12 cells treated with 6-OHDA, the apoptosis model of dopaminergic neurons was established. Cell viability was measured by MTT. Cell apoptosis and the distribution of cell cycle were assessed by flow cytometry. Western blot was used to detect the activation of extracellular regulator kinasel/2 （ERK1/2） pathway and the phosphorylation of retinoblastoma protein （RB）. Our results showed that after PC12 cells were treated wtih 6-OHDA, the viability of PC12 cells was declined in a concentration-dependent manner. Flow cytornetry revealed that 6-OHDA could increase the apoptosis ratio of PC12 cells in a time-dependent manner. The percentage of ceils in G0/G1 phase of cell cycle was decreased and that in S phase and G2/M phase increased. Simultaneously, ERK1/2 pathway was activated and phosphorylated RB increased. It was concluded that 6-OHDA could induce cell cycle reentry of dopaminergic neurons through the activation of ERK1/2 pathway and RB phosphorylation. The aberrant cell cycle reentry contributes to the apoptosis of dopaminergic neurons.

Vitamin C Inhibits Benzo[a]pyrene-Induced Cell Cycle Changes Partly via Cyclin D1/E2F Pathway in Human Embryo Lung Fibroblasts

Objective To study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene （B[a]P）-induced changes of cell cycle in human embryo lung fibroblast （HELF） cells. Methods The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin DI, CDK4, E2FI, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. Results B[a]P significantly elevated the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D 1, E2F1, and E2F4 in B [a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C （10, 100, 500, 1000, and 5000 lamol/L） and the expression levels of cyclin D 1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2FI, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin DI and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin DI alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin DI and E2FI/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from GI to S phase. Both vitamin C and antisense cyclin DI suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin DI on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin DI alone. Conclusions B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D I/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.