[Effects of ceruloplasmin/PTEN on histone modifications induced by silica].

OBJECTIVE: To explore the roles of ceruloplasmin (Cp) in histone modifications in human embryonic lung fibroblasts (HELFs) induced silica and the effects of phosphatase and tension homolog deleted on chromosome ten (PTEN) in this process. METHODS: HELFs were exposed to different concentrations of silica (50, 100, 200 microg/ ml) or Cp (10, 20, 30 microg/ml) for 24 h, and the level of protein lysine acetylation, of histone H2, H3 and H4 acetylation and histone H3 methylation were checked by werstern blot assay. HELFs and cells transfected with PTEN shRNA (PT) were treated with 200 microg/ml silica for 1 h, then added 30 microg/ml Cp for 24 h. Acetylation levels of protein lysine, histone H2, H3, H4 and methylation level of histone H3 were detected by werstern blot assay. RESULTS: Silica could induce the high level of protein lysine acetylation and acetyl-histone H2B (lys5/12), acetyl-histone H3 (lys9/14), acetyl-histone H4 (lys12) and the low level of methyl-histone (arg2), which could be reversed by Cp, in no exception for acetyl-histone H2B (lys5/12). Cp couldn't reverse histone modifications induced by silica when inhibited PTEN. CONCLUSION: Cp could reverse silica-induced the change of histone acetylation and histone methylation, and PTEN involved in this process.

[Roles of p53 in the interaction of p21 and cell cycle proteins induced by benzo [a] pyrene].

OBJECTIVE: To investigate the roles of p53 in the interaction of p21, cyclin D1 and CDK4 in human embryonic lung fibroblasts (HELFs) induced by benzo (a) pyrene. METHODS: p53-H group (cells transfected with p53 small interference RNA plasmid, p53 siRNA) and HELF/CMV group (cells transfected with CMV vector) were treated with 2 micromol/L B [a] P for 24 h, and HELF/CMV + PFT-alpha group (HELF/CMV cells were treated with p53 chemical inhibitor, Pifithrin-alpha) was treated with 2 micromol/L B [a] P and 20 micromol/L PFT-alpha for 24 h. The above three groups set up control groups, respectively. Western blot assay was used to check the levels of p53, phosphorylated p53 at 20 site (p53-ser20), p21, cyclin D1 and CDK4. Immunoprecipitation assay was used to investigate the roles of p53 in the interaction of p21, cyclin D1 and CDK4. RESULTS: After inhibition of p53 using PFT-alpha or siRNA, the high levels of p53, p53-ser 20 and p21 induced by B [a] P were markedly decreased. The change of cyclin D1 level was not obsevered and the level CDK4 was free of B [a] P. The combination of p21 and CDK 4 was increased after HELFs exposed to B [a] P, which can not be observed after inhibition p53. The combination of p21 and cyclin D1 was increased with or without the expression of p53 after HELFs exposed to B [a] P. The combination of cyclin D1 and CDK 4 was not affected by B [a] P. CONCLUSION: p53 can affect the combination of p21 and CDK4 in HELFs induced by B [a] P.

[Roles of ceruloplasmin in silica-induced JNK/ERK/AP-1 cell signaling pathway change].

OBJECTIVE: To investigate the roles of ceruloplasmin (Cp) in JNK/ERK/ AP-1 cell signaling pathway change in human embryonic lung fibroblasts (HELFs) induced by silica. METHODS: Cp stimulated HELFs in different time points (before 1 h, accompanied with or after 1 h of silica-adding). HELFs were divided into these groups: control group, silica(100 microg/ml for 24 h) group, Cp (30 microg/ml for 24 h) group and silica plus Cp (100 microg/ml silica plus 30 microg/ml Cp) group. DN-JNK cells and DN-ERK cells (cells were transfected with dominant negative mutant plasmid) contained these groups: control group, silica group, silica plus Cp group. MTT assay was used to detect the effects of Cp on silica-induced cell proliferation. Western blot assay was performed to detect the levels of JNK, ERK, c-Jun, c-Fos and their phosphorylated levels. RESULTS: Cp promoted cell proliferation induced by silica when silica stimulated HELFs 1 h then adding to Cp. Cp significantly increased silica-induced the high levels of JNK, ERK and phosphorylated JNK (p-JNK), p-ERK, p-c-Jun and p-c-fos protein. After inhibition of JNK or ERK, silica-and-Cp-induced cell proliferation was markedly decreased. When suppressing JNK protein, the increased levels of p-JNK, p-c-Jun and p-c-fos protein was not observed. The high levels of p-ERK, p-c-Jun and p-c-fos protein were decreased when inhibiting ERK protein. CONCLUSION: Cp could further strengthen silica-induced cell proliferation by JNK/c-Jun/c-Fos and ERK/c-Jun cell signaling pathway.

[The effects of ceruloplasmin in PI3K/PTEN cell signaling pathway change induced by silica].

OBJECTIVE: To investigate the roles of ceruloplasmin (Cp) in PI3K/PTEN cell signaling pathway change in human embryonic lung fibroblasts (HELFs) induced by silica. METHODS: HELFs transfected with pGenesil1.1 plasmid and pGenesil1.1 with PTEN shRNA (PT) plasmid were successfully established. 100 µg/ml silica and different concentrations of Cp (10, 20, 30 µg/ml) were used in this experiment and Cp were treated cells after exposed to silica for 1h. Three different cell lines (including HELFs, PT and cells were transfected with p85 dominant negative mutant plasmid (DN-p85)) were divided into control groups, silica groups and silica+different concentrations of Cp groups. MTT assay was used to detect the effects of Cp on silica-induced cell proliferation after inhibiting PTEN and p85. When suppressing the expression of PTEN and p85, western blot assay was performed to detect the levels of p85, p110, AKT308, AKT473 and ERK, JNK and their phosphorylated levels. RESULTS: After inhibition of PTEN, the high levels of p85 induced by 100 µg/ml silica with 30 µg/ml Cp were markedly decreased (P<0.05). When suppressing p85, the increased cell proliferation was not observed. And the high levels of AKT308, AKT473, ERK and phosphorylated JNK and ERK stimulated by 100 µg/ml silica with 30 µg/ml Cp were decrease (P < 0.05). CONCLUSION: Cp could further strengthened silica-induced cell proliferation by PI3K/AKT/MAPK cell signaling pathway, of which the level of p85 was regulated by PTEN.

[The roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts].

OBJECTIVE: To study the roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts (HELF). METHODS: Ku80 siRNA expression vectors were transfected into HELF by lipofectamine. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression level of Ku80, p53 and p21 proteins or the phosphorylation levels of p53-ser15 after cells were exposed to silica. RESULTS: The expression levels of Ku80 protein increased in concentration-dependent and time-dependent manners after cells were exposed to silica. The proportion of G1 phases in H-NC cells (controls) decreased from 89.28% +/- 2.19% to 68.93% +/- 3.79% after exposure to silica, and the proportion of G1 phases in HELF cells (H-Ku80) decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% after exposure to silica (P<0.05). The expression levels of Ku80, p53 proteins or p21 proteins or phosphorylation level of p53-ser15 were obviously suppressed in H-Ku80, as compared with H-NC. CONCLUSION: Ku80/p53 pathway plays a role in the cell cycle charges induced by silica in human embryo lung fibroblasts.

[Roles of DNA dependent protein kinase in silica-induced cyclin E and CDK2 expressions and cell cycle changes in human embryo lung fibroblasts].

OBJECTIVE: To study the roles of DNA dependent protein kinase (DNA-PK)in silica-induced cell cycle changes and expressions of CyclinE and CDK2 in human embryo lung fibroblasts (HELF). METHODS: The expressions of Ku80 and DNA-PKcs proteins were inhibited by siRNA plasmids, respectively. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression levels of CyclinE and CDK2 after cells were exposed to 200 microg/ml silica for 0, 3, 6, 12, 24 h. RESULTS: The proportion of G1 phases in negative control cells decreased from 83.53% +/- 2.24% to 69.11% +/- 3.12% after exposure to silica; the proportion of G1 phases in H-Ku80 and H-PKcs cells exposed to silica decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% and from 75.06% +/- 2.23% to 58.32% +/- 1.35%, respectively (P < 0.05). The exposure to silica resulted in the increasing protein expression levels of CyclinE and CDK2 in negative control cells, and the expression levels of CyclinE were obviously suppressed in H-Ku80 and H-PKcs as compared with control cells. However, the expression level of CDK2 protein did not change significantly. CONCLUSION: DNA-PK might play a role in silica-induced alternations of cell cycle and regulate silica-induced overexpression of CyclinE in human embryo lung fibroblasts.

Roles of the ERK, JNK/AP-1/cyclin D1-CDK4 pathway in silica-induced cell cycle changes in human embryo lung fibroblast cells.

Silica is a potent occupational fibrogenic agent capable of inducing lung fibrosis and many other lung diseases. Our current study focused on the signalling pathways regulating cell cycle changes in HELF (human embryo lung fibroblast) after silica (α-quartz) exposure. Our results showed silica exposure could lead to cell cycle changes. The cell cycle alternations were accompanied with overexpression of cyclin D1 and CDK4 (cyclin-dependent kinase 4) in a time-dependent manner. Silica exposure also decreased E2F-4 expression in HELF. These changes were blocked by overexpression of dominant-negative mutants of ERK (extracellular signal-regulated protein kinase) or the JNK (stress-activated c-Jun NH2-terminal kinase), respectively. Moreover, pretreatment of cells with curcumin, an activation of AP-1 (activator protein-1) inhibitor, inhibited silica-induced cell cycle alteration, the decreased expression of E2F-4 and overexpression of cyclin D1 and CDK4. Furthermore, both antisense cyclin D1 and antisense CDK4 can block silica-induced cell cycle changes. These results suggest that silica exposure can induce cell cycle changes, which may be mediated through ERK, JNK/AP-1/cyclin D1-CDK4-dependent pathway.

[Silica induce cell cycle changes by mitogen-activated protein kinases pathway].

OBJECTIVE: To investigate the roles of mitogen-activated protein kinases (MAPK) on silica-induced cell cycle changes. METHODS: After cells were treated with 200 microg/ml silica, Western blot and Immunofluorescence assays were utilized to detect the expression of cyclin D1, CDK4 and E2F-4, Flow cytometry was used to detect cell cycle progression, the dominant negative mutants techniques were used to investigate silica-induced signal pathway and the effects of which in silica-induced cell cycle changes. RESULTS: After cells were exposed to 200 microg/ml silica 24 h, the results of present study showed the proportion of cells in G1 phases was decreased. Silica-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutants of ERK or JNK, but not p38. It was found that ERK and JNK were involved in silica-induced cyclin D1 and CDK4 overexpression and the decreased expression of E2F-4. CONCLUSION: ERK and JNK, but not p38, mediated silica-induced cell cycle changes in human embryo lung fibroblasts.

[The roles of activated protein-1 and cell cycle protein in silica-induced cell cycle changes].

OBJECTIVE: To investigate the roles of cyclin D1 and CDK4 in the cell cycle changes of human embryonic lung fibroblasts (HELFs) exposed to silica. METHODS: HELFs were divided into 4 groups: control group, curcumin (20 µmol/L for 1 h) group, silica (200 µg/ml for 24 h) group and curcumin plus silica group, i.e. after exposure to 20 µmol/L curcumin for 1h, the HELFs were treated with 200 µg/ml silica for 24 h. Western blot and Immunofluorescence assays were utilized to detect the expression levels of cyclin D1, CDK4 and E2F1/4. Flow cytometry was used to detect the cell cycle progression, the RNA transfection technique was used to investigate the silica-induced signal pathway and the roles of which in silica-induced cell cycle changes. RESULTS: The expression levels of cyclin D1 and CDK4 significantly increased and the expression level of E2F-4 decreased obviously, but the expression level of E2F-1 did not significantly change in silica group. The proportion of G1 phase cells obviously decreased and the proportion of S phase cells significantly increased in silica group, as compared with control group (P < 0.05). When suppressing the expression of cyclin D1 or CDK4, the proportions of cells in G1 phase in anti-D1 plus silica group and anti-K4 plus silica group did not obviously change, as compared with control group. When suppressing AP-1 activity, the cyclin D1 and CDK4 expression levels decreased and the E2F-4 expression level increased in curcumin plus silica group, as compared with silica group. CONCLUSION: The results of present suggested that 200 µg/ml silica could induce the high expression of cyclin D1 and CDK4 and the low expression of E2F-4, resulting in the cell cycle changes by AP-1/cyclin D1 pathway in HELFs.

Silica induces cell cycle changes through PI-3K/AP-1 pathway in human embryo lung fibroblast cells.

Exposure to silica is associated with progressive pulmonary inflammation and fibrosis. Our previous study had demonstrated silica exposure could cause cell cycle alternation and activator protein-1 (AP-1) activation. This study showed that silica exposure induced phosphorylation of p70S6 kinase (p70S6K) and Akt in human embryo lung fibroblasts (HELFs). These changes were blocked by overexpression of dominant-negative mutants of phosphatidylinositol-3 kinase (Δp85) or Akt (DN-Akt), respectively. Moreover, pretreatment of cells with rapamycin, a specific p70S6K inhibitor, could inhibit silica-induced cell cycle alteration, AP-1 activation, and phosphorylation of p70S6K, but had no effect on Akt phosphorylation. This suggested that phosphatidylinositol-3 kinase (PI-3K)/AP-1 pathway was likely responsible for cell cycle changes. Furthermore, we observed the effect of the pathway on cell cycle regulatory proteins. Our results indicated that inactivation of PI-3K, Akt, or p70S6K could inhibit silica-induced overexpression of cyclin D1 and cyclin-dependent kinase 4 (CDK4) and decreased expression of E2F-4. Taken together, silica could induce cell cycle changes through PI-3K/ AP-1 pathway in HELFs.

[Roles of phosphatidylinositol 3 kinase in silica-induced DNA double strand breaks damage repair in human embryo lung fibroblasts].

OBJECTIVE: To study the role of Phosphatidylinositol 3 kinase (PI3K) in silica-induced DNA double strand break repair in human embryo lung fibroblasts (HELF). METHODS: Control HELF cells and DN-Deltap85 (HELF transfected with Dominant negative mutant of PI3K) were treated with 200 microg/ml silica for different times. The expression levels of phosphor-H2AX (H2AX), Ku70, Ku80 and DNA-PKcs were determined by Western blot. Furthermore, DNA double strand breaks were measured by neutral comet assay after cells were treated with 200 microg/ml silica for 0, 12 and 24 h. RESULTS: After treatment with 200 microg/ml silica for different times, the levels of H2AX were increased in a time-dependent manner and the expression levels of H2AX were obviously suppressed in DN-Deltap85 compared with control cells. The levels of Ku70 and Ku80 were also significantly suppressed in DN-Deltap85 (0.37 +/- 0.14, 0.55 +/- 0.17) compared with control cells (0.58 +/- 0.09, 0.95 +/- 0.21) after treatment with 200 microg/ml silica for 12 h (P < 0.05). Both the percentage of tail DNA in HELF and DN-Deltap85 increased significantly at 12 h (9.78 +/- 1.15, 11.79 +/- 4.90) compared with groups without treatment with silica (2.40 +/- 0.69, 3.31 +/- 1.35) and then decreased at 24 h (4.19 +/- 0.47, 7.58 +/- 4.32), but only the decrease of HELF at 24 h was significant compared with HELF at 12 h (P < 0.05). DNA repair competence of HELF was 75.74% and that of DN-Deltap85 declined to 49.64%. CONCLUSION: Silica dust can induce DNA double strand breaks in human embryo lung fibroblasts. PI3K might play a role in silica-induced DNA double strand break repair by regulating the expression levels of Ku70 and Ku80.

[Role of p53 in silica-induced cell cycle alternation and DNA double-strand break repair in human embryo lung fibroblasts].

OBJECTIVE: To study the role of p53 in silica-induced cell cycle alternation and DNA double strand breaks repair in human embryo lung fibroblasts (HELF). METHODS: Neutral comet assay was applied to detect silica-induced DNA double strand breaks. According to the neutral comet experimental result, the DNA repair competence was calculated. The expression levels and phosphorylation of protein in HELF were determined by Western blot. Cell cycle changes were identified by flow cytometry in HELF. RESULTS: After treatment with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h), the expression levels and phosphorylation of p53 increased in a time-dependent manner, reaching maximum at 12 h and then decreasing at 24 h. After treatment with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h, the expression levels and phosphorylation of p53 increased in concentration-dependent manner. After p53 expression was inhibited, silica-induced DNA damage repair competence was markedly increased (DRC = 87.68%), compared with the negative control cell induced by silica (DRC = 57.19%). Silica increased the percentage of S phase (31.8 +/- 1.1)% compared with the controls (24.3 +/- 3.8)% (P < 0.05). When p53 expression was inhibited, the number of S phase cells was significantly increased, (41.4 +/- 0.6)% compared with the controls (25.4 +/- 1.9)% (P < 0.05). CONCLUSION: The silica dramatically increases the expression levels and phosphorylation of p53. The increased expression of p53 mediates silica-induced cell cycle change and inhibits silica-induced DNA double strand breaks repair.

[Role of DNA-dependent protein kinase catalytic subunit in silica-induced DNA double-strand break repair in human embryo lung fibroblasts].

OBJECTIVE: To study the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in silica-induced DNA double-strand break repair in human embryo lung fibroblasts (HELF). METHODS: Two stable transfectants, HELF transfected with DNA-PKcs siRNA (HELF-PKcs) and with negative control siRNA (HELF-NC), were established. HELF cells were treated with 0, 25, 50, 100, 200, 300 and 400 microg/ml silica for 12 h and with 200 microg/ml silica for different times (0, 1, 2, 6, 12 and 24 h). HELF-PKcs and HELF-NC were treated with 200 microg/ml silica for 0, 12 and 24 h. The expression levels of DNA-PKcs and phosphor-H2AX (H2AX) were determined by Western blot. DNA double strand breaks were measured by neutral comet assay. RESULTS: After treatment with different doses of silica for 12 h, the levels of H2AX and the percentages of tail DNA increased in concentration-dependent manner. After treatment with 200 microg/ml silica for different times, the levels of H2AX increased in a time-dependent manner. The percentages of tail DNA increased significantly at 6 h, and reaching maximum at 12 h and then decreasing at 24 h. The expression level of DNA-PKcs was suppressed in HELF-PKcs. After treatment with silica at 12 h, the level of H2AX was lower in HELF-PKcs than in HELF-NC, and the percentages of tail DNA increased obviously in both HELF-PKcs and HELF-NC compared with non-treated cells, but no significant difference was found in the percentages of tail DNA between them. The percentages of tail DNA decreased markedly in silica-treated HELF-NC and was significantly lower than in HELF-PKcs at 24 h (P < 0.05). CONCLUSION: Silica can induce DNA double strand breaks in human embryo lung fibroblasts. DNA-PKcs might play a major role in silica-induced DNA double strand break repair. Silica-induced histone H2AX phosphorylation was dependent on DNA-PKcs.

[Decrease of cyclin D1 and CDK4 protein and their related factors induced by quartz in human embryonic lung fibroblasts].

OBJECTIVE: To study the expression level of cyclin D1-CDK4 protein in human embryonic lung fibroblasts (HELF) induced by quartz, and to study whether the expression level of cyclin D1-CDK4 protein mediated by mitogen activated protein kinase (MAPK)/(AP-1) signaling pathways. METHODS: Cells were harvested after stimulation 2 h for the detection of cytokines. Cyclin D1 and CDK4 (cyclin dependent kinase 4) proteins was measured by immunocytochemistry (IC) and Western blot (WB). RESULTS: The exposure of HELF to crystalline quartz for 2 hours could cause the decrease of cyclin D1 and cyclin dependent kinase 4 (CDK4) protein expression level, (7.91 +/- 0.29) x 10(3) and (5.17 +/- 0.28) x 10(4) respectively, which was lower than that of the HELF group (P < 0.05). AG126 (chemical inhibitor of the extracellular signal regulated protein kinase (ERK) signaling pathway) and the dominant negative mutant of ERK2 (molecular inhibitor of ERK2), prevented the decrease of cyclin D1 and CDK4 protein expression level. The chemical inhibitor of c-Jun NH2-terminal amino kinase (JNK), SP600125, could prevent both cyclin D1 and CDK4 protein expression level decrease. But SB203580, the chemical inhibitor of p38, prevented neither cyclin D1 nor CDK4 protein expression level decrease. Curcumin could prevent CDK4 protein expression level decrease but not cyclin D1 protein. CONCLUSION: ERKs and JNKs, but not p38, are responsible for the decrease of cyclin D1 and CDK4 protein expression level in HELF induced by quartz. AP-1 is responsible for the decrease of CDK4 expression level but not that of cyclin D1.

[Cell cycle alterations of human embryo lung fibroblasts induced by benzo(a)pyrene is positively regulated by cyclin D1 and CDK4].

OBJECTIVE: To study the effects of benzo(a)pyrene (B(a)P) on the cell cycle distributions and expression of cell cycle regulated protein cyclin D1 and CDK4 in human embryo lung fibroblasts (HELF), and to investigate the relationship between the expression of both cyclin D1 and CDK4 proteins and the cell cycle alterations. METHODS: Antisense cyclin D1 cDNA and antisense CDK4 cDNA were respectively transfected into HELF cells, and two stable transfectants were established. The protein expression levels of cyclin D1 and CDK4 were detected by western-blotting assay when HELF were treated with B(a)P at the doses of 0.1, 0.5, 2.5 and 12.5 micromol/L B(a)P for 24h. And the flow cytometry assay was used for detecting the cell cycle effects in HELF cells and two stable transfectants. RESULTS: Two cell lines respectively expressed antisense cyclin D1 RNA and antisense CDK4 RNA were successively established in the present study. The protein expressions of cyclin D1 were greatly increased by B(a)P treatment, whereas B(a)P had no significant effects on the protein levels of CDK4. The cell cycle distributions were significantly altered at the level of 2.5 micromol/L B(a)P for 24h treatment. The cell numbers in G1 phase significantly decreased. However, the cell numbers in S phase significantly increased. CONCLUSION: Cyclin D1 and CDK4 could be active genes and could positively regulate the cell cycle alterations induced by B(a)P.