The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL...The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20 %) MM patients and cyclin D, expression in 4/15 (27 % ) MM patients. BeLl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the BeL-1 gene was found in the 10 volunteers. It was concluded that Bel-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM.展开更多
Summary: The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were obs...Summary: The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.展开更多
Hepatocellular carcinoma(HCC)is one of the most common human cancers,and its incidence is still increasing in many countries.The prognosis of HCC patients remains poor,and identification of useful molecular prognostic...Hepatocellular carcinoma(HCC)is one of the most common human cancers,and its incidence is still increasing in many countries.The prognosis of HCC patients remains poor,and identification of useful molecular prognostic markers is required.Many recent studies have shown that functional alterations of cell-cycle regulators can be observed in HCC.Among the various types of cell-cycle regulators,p16 and p27 are frequently inactivated in HCC and are considered to be potent tumor suppressors.p16,a G1-specific cell-cycle inhibitor that prevents the association of cyclindependent kinase(CDK)4 and CDK6 with cyclin D1,is frequently inactivated in HCC via CpG methylation of its promoter region.p16 may be involved in the early steps of hepatocarcinogenesis,since p16 gene methylation has been detected in subsets of pre-neoplastic liver cirrhosis patients.p27,a negative regulator of the G1-S phase transition through inhibition of the kinase activities of Cdk2/cyclin A and Cdk2/cyclin E complexes,is now considered to be an adverse prognostic factor in HCC.In some cases of HCC with increased cell proliferation,p27 is overexpressed but inactivated by sequestration into cyclin D1-CDK4-containing complexes.Since loss of p16 is closely related to functional inactivation of p27 in HCC, investigating both p16 and p27 may be useful for precise prognostic predictions in individuals with HCC.展开更多
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, an...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 (IκBα M) 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.展开更多
Objective: To confirm feasibility of Cyclin D1 gene as a new target for cancer gene therapy and to verify the effectiveness of shRNA expression vector-mediated gene silencing. Methods: A RNA interference DNA templat...Objective: To confirm feasibility of Cyclin D1 gene as a new target for cancer gene therapy and to verify the effectiveness of shRNA expression vector-mediated gene silencing. Methods: A RNA interference DNA template targeting Cyclin D1 gene was designed and synthesized. By ligation, the fragment was inserted into Pgenesil-1-U6 to constract the recombinant plasmid Pgenesil-shRNA-Cyclin D1. The identified recombinant plasmid was transfected into ACHN cells with lipofactamine. Cyclin D1 mRNA and protein expression was analyzed by RT-PCR and western-blotting. MTT method was used for observing cell proliferation and drawing growth curve. The cell cycle and ratios of apoptotic cell were assessed by flow cytometric detection. The ability of invasion of cell migration was detected by Transwell chamber invasive models. Results: The plasmid was constructed successfully. After interference, The expression rate of Cyclin D1 mRNA decreased to 0.10±0.04 in Cyclin D1-shRNA(experimental) group and were significantly lower than Pgenesil-NC (negative) group (0.92±0.03) and ACHN (blank control) group(0.94±0.04)(P0.05). As well, the expression rate of Cyclin D1 protein was decreased evidently in experimental group. The results of flow cytometric detection showed that, including early and late apoptotic cells, the apoptotic ratio of experimental group increased to (37.26±0.60)% significantly, while, the negative group and blank control group were only (4.62±0.40)% and (5.95±1.20)%, respectively. The cell growth curves indicated that the proliferation of experimental group cells was inhibited significantly(P0.05) and Transwell results suggested that the abilities of invasion cells transfected with Pgenesil–CyclinD1-shRNA decreased conspicuously(P0.05). Conclusion: The shRNA can inhibit Cyclin D1 expression, specifically and persistently. The down-regulation of Cyclin D1 expression can inhibit the proliferation and induce the apoptosis of renal cell adenocarcinoma cell line ACHN.展开更多
To investigate the effects of Cyclin D1 antisense oligodeoxyneucleotides (ASODN) on the growth, cell cycle progression and expression of G 1 phase regulators in human gastric carcinoma cell lines SGC7901 and HS746T, ...To investigate the effects of Cyclin D1 antisense oligodeoxyneucleotides (ASODN) on the growth, cell cycle progression and expression of G 1 phase regulators in human gastric carcinoma cell lines SGC7901 and HS746T, phosphorothioate modified Cyclin D1 ASODN were encapsulated by LipofectAMINE2000 and transfected into gastric carcinoma cells. Dose dependent inhibitory effects were induced by Cyclin D1 ASODN in two gastric carcinoma cell lines. Treatment of gastric carcinoma cells with 0.2 μmol/L Cyclin D1 ASODN for 24 h could significantly inhibit their growth in vitro and in vivo , reduce expression of Cyclin D1mRNA to 26.3 % (SGC7901) and 17.3 % (HS746T) respectively. The percentage of cells in G 0/G 1 phase was increased as revealed by flow cytometry. Immunohistochemical staining showed that the expression of p21 was increased and the expression of Cyclin D1 and pRb was decreased in the two cell lines; the expression of p27 was increased in HS746T, but unchanged in SGC7901. Cyclin D1 ASODN could inhibit the growth and the expression of Cyclin D1 mRNA in gastric carcinoma cells, influence the cell cycle and expression of its regulators.展开更多
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 transfecta...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.展开更多
BACKGROUND: Previous studies have suggested that the hippocampus is one of the neurotoxic target sites for lead. However, the molecular mechanisms of action, including the effect of lead on cell-cycle arrest, remain ...BACKGROUND: Previous studies have suggested that the hippocampus is one of the neurotoxic target sites for lead. However, the molecular mechanisms of action, including the effect of lead on cell-cycle arrest, remain poorly understood. OBJECTIVE: To investigate the effects of different lead concentrations on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons. DESIGN, TIME AND SETTING: A randomized, controlled, in vitro experiment was performed at the China Medical University between July 2008 and May 2009. MATERIALS: Antibodies specific to cyclin D1 and actin were synthesized and purified by Santa Cruz Biotechnology, USA. FACStar flow cytometer was purchased from Becton Dickinson, San Jose, California, USA. METHODS: Wistar rat hippocampal neurons were primary cultured for 7 days. Neurons in the control group were treated with 0.01 mol/L phosphate buffered saline. Neurons in the 0.2, 1.0, and 10 umol/L lead acetate groups were subjected to 0.2, 1.0, and 10 umol/L lead acetate. Subsequently hippocampal neurons in each group were cultured for 24 hours. MAIN OUTCOME MEASURES: The effects of lead on cell cycle were measured by flow cytometry, DNA damage was measured using the comet assay, and cyclin D1 expression was measured using Western blot analysis. RESULTS: Treatment of hippocampal neurons with 0.2 umol/L lead acetate did not significantly alter cell cycle phase distribution, i.e., sub-G1, S, G0/G1, G2/M, whereas treatment with 1.0 and 10 umol/L lead acetate significantly increased the percentage of S and sub-G1 phase cells (P 〈 0.05). Olive tail moment in all lead-treated groups and the percentage of DNA in the tail in 1.0 umol/L and 10 umol/L lead acetate groups were significantly greater compared with the control group (P 〈 0.05). In addition, the percentage of tail DNA was greater in the 0.2 umol/L lead acetate group compared with the control group (P 〉 0.05). Following incubation with 0.2, 1.0, and 10 umol/L lead acetate for 24 hours, cyclin D1 expression gradually decreased with exposure to increasing lead acetate concentrations (1.0-10 umol/L). CONCLUSION: Lead exposure to primary cultured rat hippocampal neurons resulted in dose-dependently disturbed cellular homeostasis, including DNA damage, reduced cyclin D1 expression, and stagnation of cell-cycle progression.展开更多
Objective To investigate the significance of overexpresson of cyclin D1 and P53 protein in cervical squamous cell carcinomas.Methods Fifty cases of invasive cervical squamous cell carcinomas and 10 cases of normal c...Objective To investigate the significance of overexpresson of cyclin D1 and P53 protein in cervical squamous cell carcinomas.Methods Fifty cases of invasive cervical squamous cell carcinomas and 10 cases of normal cervical squamous epithelia were investigated with immunihistochemical technique. Results The overexpression of cyclin D1 and P53 in invasive cervical carcinomas was 70% and 50%, respectively. There was no overexpression of them in the control group. The overexpression of cyclin D1 in grade Ⅱ and Ⅲ was much higher than that in gradeⅠ(P<0.05). The overexpresson of cyclin D1 in stage Ⅲ of cervical carcinoma was significantly higher than that in stage Ⅱ (P<0.05). The overexpression of P53 in grade Ⅱ and grade Ⅲ of cervical carcinoma was remarkably higher than that in grade Ⅰ (P<0.05).Conclusion The action point of both cyclin D1 and P53 may be at G1/S transition. The overexpression of them was associated with development and progression of cervical carcinoma probably in different mechanisms and different pathways.展开更多
Objective: To prove the molecular mechanisms of Mahkota Dewa(Phaleria macrocarpa) in suppressing proliferation of human retinoblastoma cells through suppression of cell cycle's gene-regulators expression.Methods: ...Objective: To prove the molecular mechanisms of Mahkota Dewa(Phaleria macrocarpa) in suppressing proliferation of human retinoblastoma cells through suppression of cell cycle's gene-regulators expression.Methods: In this study, the molecular mechanism of anti-tumor effect of fractioned extract of Phaleria macrocarpa(DLBS1425) in human retinoblastoma cells Y-79 was investigated by measuring the tumor cells viability, the assessment of population profiles of tumor cells in the cell cycle, and the mRNA concentration of p16, p21, p53, cyclin D,cyclin E, and E2 F.Results: DLBS1425 showed an inhibition effects towards proliferation of Y-79 cell line.Inhibition of proliferation was shown by suppression of cell cycle progression.DLBS1425 downregulated cyclin E, a G1 phase regulator gene of cell cycle, in dosedependent manner without affecting p53–p21 pathway.In the other word, DLBS1425 inhibits cell proliferation through suppression of cyclin E independently towards conventional proliferation pathway.Conclusions: Our results suggest that DLBS1425 is a potential anticancer agent which targets genes involved in cell proliferation in human retinoblastoma cells which make it pharmacologically ideal for the prevention and/or treatment of retinoblastoma cancer.展开更多
The aim of this study is to investigate cyclin E, pl6ink4a and ki67 as possible diagnostic biomarkers for cervical preneoplasia using cervical exfoliated-cell specimens, and evaluate the significance for screening pat...The aim of this study is to investigate cyclin E, pl6ink4a and ki67 as possible diagnostic biomarkers for cervical preneoplasia using cervical exfoliated-cell specimens, and evaluate the significance for screening patients at high risk of de- veloping cervical carcinoma. The expression of cyclin E, p16ink4a and ki67 was examinated in 78 cervical exfoliated epithe- lial specimens diagnosed as atypical squamous cells of undetermined significance (ASCIIS) (12 cases), cervical intraepithe- lial neoplasia (CIN) of type 1 (17 cases), CIN2-3(38 cases) and invasive carcinoma (11 cases) using immunohistochemical analysis, and simultaneously, the DNA status of human papillomavirus (HPV) type 16/18 was detected by polymerase chain reaction (PCR) using type specific primers, cyclin E, pl6ink4a and ki67 were all overexpressed in CINs and invasive carci- noma, compared with little expression in ASCUS ( P < 0.005). Overexpression of cyclin E was observed in CIN1 (94.1 % , x2 = 21.16, P < 0.01), and pl6ink4a and ki67 were overexpressed in invasive carcinoma (100% and 90.9% respective- ly) . The degree of pl6ink4a and ki67 expression correlated well with that of epithelial lesions ( P < 0.005). HPV16/18 infec- tion was assessed in CINs and invasive carcinoma samples, and revealed a significant relationship with the degree of cervical epithelial lession. The expression level of pl6ink4a and ki67 seemed more closely associated with HPV16 infection than that of cyclin E ( r s = 1.0 vs rs = 0 . 4 ) . Only 1 case in CIN, and 4 cases in CIN2-3 of HPV18 positive samples were detected. Therefore no statistical significance was found by statistical analysis. Overexpression of cyclin E, p16ink4a and ki67 in CINs and invasive carcinoma cells demonstrates the potential use of cyclin E, p16ink4a and ki67 as diagnostic biomarkers for HPV-related cervical neoplastic lesions. In addition, this technique can be used for screening patients at high risk of devel- oping cervical carcinoma.展开更多
To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in...To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in VSMC cultured in different concentrations (0~100 nmol/L) of 17β-estradiol with or without serum.Results Under serum-stimulating conditions,17β-estradiol(1,10,100 nmol/L) promoted VSMC proliferation by accelerating their cell cycle progression from G1 to S phases,and the cell rates at S were (31.89±9.14)%(35.90±4.59)% and (30.77±1.20)% respectively,significantly higher than the corresponding values of control cells (21.63±1.80)%.This was accompanied by the significantly increased expression of Cyclin D1 and CDK4 proteins.In the cultures without serum,however,high concentrations (10,100 nmol/L) of E2 induced a cell cycle arrest at G1 phase,which was characterizsed by decreased cell rates at S phase [(9.93±1.43)% and (8.76±1.80)% respectively,P<0.05] as compared with the corresponding control values and a down-regulation of expressions of Cyclin D1 and CDK4 proteins.Conclusion E2 can either promote or inhibit VSMC proliferation depending upon the presence or absence of serum mitogens.The underlying mechanism may be associated with the hormone’s action on the expression of Cyclin D1 and CDK4 which act as the G1 phase regulators.4 refs.展开更多
文摘The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20 %) MM patients and cyclin D, expression in 4/15 (27 % ) MM patients. BeLl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the BeL-1 gene was found in the 10 volunteers. It was concluded that Bel-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM.
基金ThisprojectwassupportedbyagrantfromNationalNaturalSciencesFoundationofChina (No .30 0 70 32 6 ) .
文摘Summary: The pathogenesis of aplastic anemia (AA) was explored and the effects of AA serum on the expression of crucial cyclin D isoform (cyclin D3) in umbilical cord blood hematopoietic stem/progenitor cells were observed. The CD34+ cells were isolated from the cord blood with MIDI-MACS Semi-solid methylcellulose culture technique was used to measure the formation of CFU-GM; The expression level of cyclin D3 was assayed by semi-quantitative RT-PCR and Western-blot after the hematopoietic stem/progenitor cells were incubated in AA serum. The results showed that the AA serum could inhibit the formation of CFU-GM and down regulate the expression level of the cyclin D3 at the mRNA and protein level respectively. In conclusion, the AA serum could inhibit the proliferation of hematopoietic stem cells and down regulate level of cyclin D3, which might be one mechanism of hematopoiesis inhibition in AA.
文摘Hepatocellular carcinoma(HCC)is one of the most common human cancers,and its incidence is still increasing in many countries.The prognosis of HCC patients remains poor,and identification of useful molecular prognostic markers is required.Many recent studies have shown that functional alterations of cell-cycle regulators can be observed in HCC.Among the various types of cell-cycle regulators,p16 and p27 are frequently inactivated in HCC and are considered to be potent tumor suppressors.p16,a G1-specific cell-cycle inhibitor that prevents the association of cyclindependent kinase(CDK)4 and CDK6 with cyclin D1,is frequently inactivated in HCC via CpG methylation of its promoter region.p16 may be involved in the early steps of hepatocarcinogenesis,since p16 gene methylation has been detected in subsets of pre-neoplastic liver cirrhosis patients.p27,a negative regulator of the G1-S phase transition through inhibition of the kinase activities of Cdk2/cyclin A and Cdk2/cyclin E complexes,is now considered to be an adverse prognostic factor in HCC.In some cases of HCC with increased cell proliferation,p27 is overexpressed but inactivated by sequestration into cyclin D1-CDK4-containing complexes.Since loss of p16 is closely related to functional inactivation of p27 in HCC, investigating both p16 and p27 may be useful for precise prognostic predictions in individuals with HCC.
文摘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 (IκBα M) 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.
文摘Objective: To confirm feasibility of Cyclin D1 gene as a new target for cancer gene therapy and to verify the effectiveness of shRNA expression vector-mediated gene silencing. Methods: A RNA interference DNA template targeting Cyclin D1 gene was designed and synthesized. By ligation, the fragment was inserted into Pgenesil-1-U6 to constract the recombinant plasmid Pgenesil-shRNA-Cyclin D1. The identified recombinant plasmid was transfected into ACHN cells with lipofactamine. Cyclin D1 mRNA and protein expression was analyzed by RT-PCR and western-blotting. MTT method was used for observing cell proliferation and drawing growth curve. The cell cycle and ratios of apoptotic cell were assessed by flow cytometric detection. The ability of invasion of cell migration was detected by Transwell chamber invasive models. Results: The plasmid was constructed successfully. After interference, The expression rate of Cyclin D1 mRNA decreased to 0.10±0.04 in Cyclin D1-shRNA(experimental) group and were significantly lower than Pgenesil-NC (negative) group (0.92±0.03) and ACHN (blank control) group(0.94±0.04)(P0.05). As well, the expression rate of Cyclin D1 protein was decreased evidently in experimental group. The results of flow cytometric detection showed that, including early and late apoptotic cells, the apoptotic ratio of experimental group increased to (37.26±0.60)% significantly, while, the negative group and blank control group were only (4.62±0.40)% and (5.95±1.20)%, respectively. The cell growth curves indicated that the proliferation of experimental group cells was inhibited significantly(P0.05) and Transwell results suggested that the abilities of invasion cells transfected with Pgenesil–CyclinD1-shRNA decreased conspicuously(P0.05). Conclusion: The shRNA can inhibit Cyclin D1 expression, specifically and persistently. The down-regulation of Cyclin D1 expression can inhibit the proliferation and induce the apoptosis of renal cell adenocarcinoma cell line ACHN.
文摘To investigate the effects of Cyclin D1 antisense oligodeoxyneucleotides (ASODN) on the growth, cell cycle progression and expression of G 1 phase regulators in human gastric carcinoma cell lines SGC7901 and HS746T, phosphorothioate modified Cyclin D1 ASODN were encapsulated by LipofectAMINE2000 and transfected into gastric carcinoma cells. Dose dependent inhibitory effects were induced by Cyclin D1 ASODN in two gastric carcinoma cell lines. Treatment of gastric carcinoma cells with 0.2 μmol/L Cyclin D1 ASODN for 24 h could significantly inhibit their growth in vitro and in vivo , reduce expression of Cyclin D1mRNA to 26.3 % (SGC7901) and 17.3 % (HS746T) respectively. The percentage of cells in G 0/G 1 phase was increased as revealed by flow cytometry. Immunohistochemical staining showed that the expression of p21 was increased and the expression of Cyclin D1 and pRb was decreased in the two cell lines; the expression of p27 was increased in HS746T, but unchanged in SGC7901. Cyclin D1 ASODN could inhibit the growth and the expression of Cyclin D1 mRNA in gastric carcinoma cells, influence the cell cycle and expression of its regulators.
基金This work was supported by grants of National Natural Science Foundation of China (30371206, 30440420593), 973 National Key Basic Research and Development Program (2002 CB 512905) and Taishan Charitable Association LTD. HK.
文摘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.
基金the National Natural Science Foundation of China, No. 39970651
文摘BACKGROUND: Previous studies have suggested that the hippocampus is one of the neurotoxic target sites for lead. However, the molecular mechanisms of action, including the effect of lead on cell-cycle arrest, remain poorly understood. OBJECTIVE: To investigate the effects of different lead concentrations on cell-cycle arrest, DNA damage, and cyclin D1 expression in primary cultured rat hippocampal neurons. DESIGN, TIME AND SETTING: A randomized, controlled, in vitro experiment was performed at the China Medical University between July 2008 and May 2009. MATERIALS: Antibodies specific to cyclin D1 and actin were synthesized and purified by Santa Cruz Biotechnology, USA. FACStar flow cytometer was purchased from Becton Dickinson, San Jose, California, USA. METHODS: Wistar rat hippocampal neurons were primary cultured for 7 days. Neurons in the control group were treated with 0.01 mol/L phosphate buffered saline. Neurons in the 0.2, 1.0, and 10 umol/L lead acetate groups were subjected to 0.2, 1.0, and 10 umol/L lead acetate. Subsequently hippocampal neurons in each group were cultured for 24 hours. MAIN OUTCOME MEASURES: The effects of lead on cell cycle were measured by flow cytometry, DNA damage was measured using the comet assay, and cyclin D1 expression was measured using Western blot analysis. RESULTS: Treatment of hippocampal neurons with 0.2 umol/L lead acetate did not significantly alter cell cycle phase distribution, i.e., sub-G1, S, G0/G1, G2/M, whereas treatment with 1.0 and 10 umol/L lead acetate significantly increased the percentage of S and sub-G1 phase cells (P 〈 0.05). Olive tail moment in all lead-treated groups and the percentage of DNA in the tail in 1.0 umol/L and 10 umol/L lead acetate groups were significantly greater compared with the control group (P 〈 0.05). In addition, the percentage of tail DNA was greater in the 0.2 umol/L lead acetate group compared with the control group (P 〉 0.05). Following incubation with 0.2, 1.0, and 10 umol/L lead acetate for 24 hours, cyclin D1 expression gradually decreased with exposure to increasing lead acetate concentrations (1.0-10 umol/L). CONCLUSION: Lead exposure to primary cultured rat hippocampal neurons resulted in dose-dependently disturbed cellular homeostasis, including DNA damage, reduced cyclin D1 expression, and stagnation of cell-cycle progression.
文摘Objective To investigate the significance of overexpresson of cyclin D1 and P53 protein in cervical squamous cell carcinomas.Methods Fifty cases of invasive cervical squamous cell carcinomas and 10 cases of normal cervical squamous epithelia were investigated with immunihistochemical technique. Results The overexpression of cyclin D1 and P53 in invasive cervical carcinomas was 70% and 50%, respectively. There was no overexpression of them in the control group. The overexpression of cyclin D1 in grade Ⅱ and Ⅲ was much higher than that in gradeⅠ(P<0.05). The overexpresson of cyclin D1 in stage Ⅲ of cervical carcinoma was significantly higher than that in stage Ⅱ (P<0.05). The overexpression of P53 in grade Ⅱ and grade Ⅲ of cervical carcinoma was remarkably higher than that in grade Ⅰ (P<0.05).Conclusion The action point of both cyclin D1 and P53 may be at G1/S transition. The overexpression of them was associated with development and progression of cervical carcinoma probably in different mechanisms and different pathways.
基金Supported by The Dexa Laboratories of Biomolecular Sciences(DLBS),PT.Dexa Medica,Cikarang,West Java,Indonesia,Grant No.135/MP/DLBS/2016
文摘Objective: To prove the molecular mechanisms of Mahkota Dewa(Phaleria macrocarpa) in suppressing proliferation of human retinoblastoma cells through suppression of cell cycle's gene-regulators expression.Methods: In this study, the molecular mechanism of anti-tumor effect of fractioned extract of Phaleria macrocarpa(DLBS1425) in human retinoblastoma cells Y-79 was investigated by measuring the tumor cells viability, the assessment of population profiles of tumor cells in the cell cycle, and the mRNA concentration of p16, p21, p53, cyclin D,cyclin E, and E2 F.Results: DLBS1425 showed an inhibition effects towards proliferation of Y-79 cell line.Inhibition of proliferation was shown by suppression of cell cycle progression.DLBS1425 downregulated cyclin E, a G1 phase regulator gene of cell cycle, in dosedependent manner without affecting p53–p21 pathway.In the other word, DLBS1425 inhibits cell proliferation through suppression of cyclin E independently towards conventional proliferation pathway.Conclusions: Our results suggest that DLBS1425 is a potential anticancer agent which targets genes involved in cell proliferation in human retinoblastoma cells which make it pharmacologically ideal for the prevention and/or treatment of retinoblastoma cancer.
文摘The aim of this study is to investigate cyclin E, pl6ink4a and ki67 as possible diagnostic biomarkers for cervical preneoplasia using cervical exfoliated-cell specimens, and evaluate the significance for screening patients at high risk of de- veloping cervical carcinoma. The expression of cyclin E, p16ink4a and ki67 was examinated in 78 cervical exfoliated epithe- lial specimens diagnosed as atypical squamous cells of undetermined significance (ASCIIS) (12 cases), cervical intraepithe- lial neoplasia (CIN) of type 1 (17 cases), CIN2-3(38 cases) and invasive carcinoma (11 cases) using immunohistochemical analysis, and simultaneously, the DNA status of human papillomavirus (HPV) type 16/18 was detected by polymerase chain reaction (PCR) using type specific primers, cyclin E, pl6ink4a and ki67 were all overexpressed in CINs and invasive carci- noma, compared with little expression in ASCUS ( P < 0.005). Overexpression of cyclin E was observed in CIN1 (94.1 % , x2 = 21.16, P < 0.01), and pl6ink4a and ki67 were overexpressed in invasive carcinoma (100% and 90.9% respective- ly) . The degree of pl6ink4a and ki67 expression correlated well with that of epithelial lesions ( P < 0.005). HPV16/18 infec- tion was assessed in CINs and invasive carcinoma samples, and revealed a significant relationship with the degree of cervical epithelial lession. The expression level of pl6ink4a and ki67 seemed more closely associated with HPV16 infection than that of cyclin E ( r s = 1.0 vs rs = 0 . 4 ) . Only 1 case in CIN, and 4 cases in CIN2-3 of HPV18 positive samples were detected. Therefore no statistical significance was found by statistical analysis. Overexpression of cyclin E, p16ink4a and ki67 in CINs and invasive carcinoma cells demonstrates the potential use of cyclin E, p16ink4a and ki67 as diagnostic biomarkers for HPV-related cervical neoplastic lesions. In addition, this technique can be used for screening patients at high risk of devel- oping cervical carcinoma.
文摘To study the effects of 17β-estradiol(E2) on the growth of cultured rat vascular smooth muscle cells (VSMC).Methods The cell cycle and the expressions of Cyclin D1 and CDK4 proteins were examined by flow cytometry in VSMC cultured in different concentrations (0~100 nmol/L) of 17β-estradiol with or without serum.Results Under serum-stimulating conditions,17β-estradiol(1,10,100 nmol/L) promoted VSMC proliferation by accelerating their cell cycle progression from G1 to S phases,and the cell rates at S were (31.89±9.14)%(35.90±4.59)% and (30.77±1.20)% respectively,significantly higher than the corresponding values of control cells (21.63±1.80)%.This was accompanied by the significantly increased expression of Cyclin D1 and CDK4 proteins.In the cultures without serum,however,high concentrations (10,100 nmol/L) of E2 induced a cell cycle arrest at G1 phase,which was characterizsed by decreased cell rates at S phase [(9.93±1.43)% and (8.76±1.80)% respectively,P<0.05] as compared with the corresponding control values and a down-regulation of expressions of Cyclin D1 and CDK4 proteins.Conclusion E2 can either promote or inhibit VSMC proliferation depending upon the presence or absence of serum mitogens.The underlying mechanism may be associated with the hormone’s action on the expression of Cyclin D1 and CDK4 which act as the G1 phase regulators.4 refs.