Silica induce cell cycle changes by mitogen-activated protein kinases pathway / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the roles of mitogen-activated protein kinases (MAPK) on silica-induced cell cycle changes.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>ERK and JNK, but not p38, mediated silica-induced cell cycle changes in human embryo lung fibroblasts.</p>

The roles of activated protein-1 and cell cycle protein in silica-induced cell cycle changes / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the roles of cyclin D1 and CDK4 in the cell cycle changes of human embryonic lung fibroblasts (HELFs) exposed to silica.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Roles of DNA dependent protein kinase in silica-induced cyclin E and CDK2 expressions and cell cycle changes in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

The roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the roles of Ku80/p53 pathway in silica-induced cell cycle changes in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Ku80/p53 pathway plays a role in the cell cycle charges induced by silica in human embryo lung fibroblasts.</p>

Roles of phosphatidylinositol 3 kinase in silica-induced DNA double strand breaks damage repair in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the role of Phosphatidylinositol 3 kinase (PI3K) in silica-induced DNA double strand break repair in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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%.</p><p><b>CONCLUSION</b>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.</p>

Role of p53 in silica-induced cell cycle alternation and DNA double-strand break repair in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the role of p53 in silica-induced cell cycle alternation and DNA double strand breaks repair in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Role of DNA-dependent protein kinase catalytic subunit in silica-induced DNA double-strand break repair in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>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).</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>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.</p>

Decrease of cyclin D1 and CDK4 protein and their related factors induced by quartz in human embryonic lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

ERK, JNK/AP-1 pathway was involved in silica-induced cell cycle changes / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the alteration of activator protein-1 (AP-1) luciferase activity in human embryo lung fibroblasts (HELF) after exposed to silica, and the role of mitogen activated protein kinase (MAPK)/AP-1 pathway on silica-induced cell cycle changes.</p><p><b>METHODS</b>After HELF cells were treated with 200 microg/ml silica, immunofluorescence assays were employed to detect the translocation and the phosphorylation level of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK), flow cytometry was used to detect the distributions of cell cycle, the dominant negative mutant of ERK, JNK and p38 were applied to detect the upstream or downstream relationship of signaling pathways.</p><p><b>RESULTS</b>After HELF-AP-1 cells were exposed to 200 microg/ml silica 6, 12, 24 h respectively, silica exposure lead to AP-1 activation in a time-dependent manner, inducing significant AP-1 activation at 6 h, reaching a maximum activation at 12 h, and having a little decrease at 24 h. After silica exposure 1 h, phosphorylation level of ERK and JNK increased mainly in cytoplasm, however, after exposure 2 h, they translocated to nucleus. The proportion of cells in G1 phases was decreased from (63.80 +/- 9.57)% to (32.23 +/- 7.22)%, and the proportion of cells in S phases was increased from (35.17 +/- 10.33)% to (66.00 +/- 8.07)% after exposed to silica 24 h. Curcumin, a chemical inhibitor of AP-1, impaired the decrease of cells in G1 phases. Furthermore we found expression of dominant-negative mutant of ERK and JNK impaired silica-induced AP-1 activation, whereas, dominant-negative mutant of p38 did not show the effect.</p><p><b>CONCLUSION</b>These result suggested that 200 microg/ml silica exposure can induce AP-1 activation, induce cell cycle changes through ERK, JNK/AP-1-dependent pathway.</p>

Different patterns of cyclin D1/CDK4-E2F-1/4 pathways in human embryo lung fibroblasts treated by benzoapyrene at different doses / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the roles of the cyclin D1/CDK4 and E2F-1/4 pathways and compare their work patterns in cell cycle changes induced by different doses of B[a]P.</p><p><b>METHODS</b>Human embryo lung fibroblasts (HELFs) were treated with 2 micromol/L or 100 micromol/L B[a]P which were provided with some characteristics of transformed cells (T-HELFs). Cyclin D1, CDK4 and E2F-1/4 expressions were determined by Western blotting. Flow cytometry was used to detect the distribution of cell cycle.</p><p><b>RESULTS</b>After B[a]P treatment, the proportion of the first gap (G1) phase cells decreased. CDK4 and E2F-4 expression did not change significantly. In 2 micromol/L treated cells, a marked overexpression of cyclin D1 and E2F-1 was observed. However, in T-HELFs overexpression was limited to cyclin D1 only, and no overexpression of E2F-1 was observed. The decreases of G1 phase in response to B[a]P treatment were blocked in antisense cyclin D1 and antisense CDK4 transfected HELFs (A-D1 and A-K4) and T-HELFs (T-A-D1 and T-A-K4). After 2 micromol/L B[a]P treatment, overexpression of E2F-1 was attenuated in A-D1, and E2F-4 expression was decreased significantly in A-K4. In T-A-D1 and T-A-K4, E2F-4 expression was increased significantly, compared with T-HELFs. The E2F-1 expression remained unchanged in T-A-D1 and T-A-K4.</p><p><b>CONCLUSIONS</b>Cyclin D1/CDK4-E2F-1/4 pathways work in different patterns in response to low dose and high dose B[a]P treatment. In HELFs treated with 2 micromol/L B[a]P, cyclin D1 positively regulates the E2F-1 expression while CDK4 negatively regulates the E2F-4 expression; however, in HELFs treated with 100 micromol/L B[a]P, both cyclin D1 and CDK4 negatively regulate the E2F-4 expression.</p>

Activated protein 1-cyclin D1/E2F 1 pathways involved in cell cycle changes induced by benzo (a) pyrene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the roles of activated protein 1 (AP-1) in cell cycle changes on human embryo lung fibroblasts (HELF) induced by benzo (a) pyrene [B (a) P], and relationships between AP-1 and cyclin D1/CDK4-E2F-1/4.</p><p><b>METHODS</b>Cells transfected with AP-1 luciferase reporter plasmid (AP-H) were cultured with serum-free RPMI1640 for 48 h, and treated with 2 micromol/L B (a) P for 24 h. AP-1 relative activity was detected by luciferase assay. Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometer and Western blot assay.</p><p><b>RESULTS</b>After B (a) P was treated for 24 h, the ratio of G1 phase cells (71 +/- 2)% was decreased to (48 +/- 3)% (P < 0.05), and an increase was observed in the ratio of S phase. AP-1 activity and cyclin D1/E2F-1 expression were increased significantly, but CDK4/E2F-4 expression did not change after B (a) P treatment. When AP-1 activity was inhibited by curcumin, decreases of G1 phase in response to B (a) P treatment were blocked, and overexpression of cyclin D1/E2F-1 was attenuated, but CDK4/E2F-4 expression was not changed significantly.</p><p><b>CONCLUSION</b>AP-1 is involved in B (a) P induced cell cycle changes, and is the upstream signals of cyclin D1/E2F-1, but not CDK4/E2F-4.</p>

Benzo (a) pyrene-induced human embryo lung cell cycle alterations through positive regulation of mitogen-activated protein kinase signal pathways / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.</p><p><b>METHODS</b>The phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.</p><p><b>RESULTS</b>The phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.</p><p><b>CONCLUSION</b>ERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.</p>

The activation of MAPK/cyclin D1-CDK4 signaling pathway caused by quartz / 中华劳动卫生职业病杂志

<p><b>OBJECTIVES</b>To study the phosphorylation level of mitogen activated protein kinase (MAPK) in human embryonic lung fibroblasts (HELF), and the expression level of cyclin D1-CDK4 protein in S-HELF and whether the expression level of cyclin D1-CDK4 protein mediated by MAPK/AP-1 signaling pathway in S-HELF.</p><p><b>METHODS</b>Two kinds of treatment: (1) Cells were harvested after stimulation 2 h for the detection of cytokines. (2) Cells were stimulated by quartz for a long time (2 months) for transformation characters (S-HELF). The MAP kinase was detected by western blot. Cyclin D1 and CDK4 (cyclin dependent kinase 4) proteins was measured by immunocytochemistry. Flow cytometry was used to evaluate the alternation of cell cycle.</p><p><b>RESULTS</b>Crystalline quartz could cause the phosphorylation level of ERKs, p38K, and JNKs in HELF increase. However, activated levels of ERKs and p46 of JNKs increased in S-HELF, and p38K activation decreased, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Cyclin D1 and CDK4 protein expression levels increased in S-HELF as compared with parental HELF. Inhibition of ERKs activation by AG126, AP-1 by curcumin, and JNKs by SP600125 could reduced the induction of cyclin D1 and CDK4, whereas inhibition of p38K by SB203580 did not show any inhibitory effects on S-HELF.</p><p><b>CONCLUSIONS</b>The phosphorylation levels of ERK1/2, JNK1/2, and p38 increased in HELF exposed to quartz. The phosphorylation levels of ERK1/2 and JNK1 increased, but the phosphorylation level of p38 decreased in S-HELF. The expression level of cyclin D1-CDK4 protein increased in S-HELF. Overexpression of cyclin D1-CDK4 is due to the activation of ERKs, JNKs/AP-1 signaling pathway in S-HELF.</p>

c-Jun NH2-terminal kinase and extracellular signal-regulated protein kinase signaling pathways in regulation of benzo(a)pyrene-induced c-Jun activation in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of mitogen activated protein kinases (MAPKs) signaling pathways in the regulation of benzo(a)pyrene (B(a)P)-induced c-Jun activation in human embryo lung fibroblasts (HELFs).</p><p><b>METHODS</b>HELFs were cultured with 2.0 micromol/L B(a)P for various time (0, 3, 6, 12, 24 h) or with various concentration of B(a)P (0.0, 0.5, 1.0, 2.0 micromol/L) for 12 h. Western blot was performed to examine the effect of B(a)P on c-Jun activation. The dominant negative mutants of p38, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) were applied to establish stable transfectant, and to detect the relationship of MAPK signal molecules and c-Jun activation in B (a) P-treated cells.</p><p><b>RESULTS</b>B(a)P treatment resulted in a marked activation of c-Jun in time-dependent manner with a peak at 12 h (the densitometric ratios of phosphorylated c-Jun Ser63, Ser73 to actin were 20.1, 15.2 times for control respectively) and in dose-dependent manner. However, there was no evident change on total c-Jun expression in B(a)P-treated HELFs. Moreover, B(a)P-induced activation of c-Jun was inhibited by stable expression of dominant negative mutants of JNK or ERK, but not by dominant negative mutant of p38.</p><p><b>CONCLUSION</b>JNK and ERK signaling pathways, but not p38 pathway regulate B(a)P-induced c-Jun activation in HELFs.</p>

cyclin D1/E2F pathways involved in cell cycle changes of human embryo lung fibroblasts induced by benzo(a)pyrene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the roles of cyclin D1/CDK4-E2F-1/4 pathway in cell cycle changes of human embryo lung fibroblasts (HELF) induced by two different benzo(a)pyrene [B(a)P] treatment models.</p><p><b>METHODS</b>Two B(a)P treatment models: HELF were treated by 2 micromol/L B(a)P for 24 hours; HELF were treated by 100 micromol/L B(a)P three times 24 hours each and provide with some characteristics of transformed cells (T-HELF). Changes of cell cycle and the expression of cyclin D1, CDK4 and E2F-1/4 were checked using the flow cytometry and Western bolt analysis.</p><p><b>RESULTS</b>After 24 hours 2 microml/L B(a)P treatment, the HELFs in the G(1) phase was decreased. In HELF transfected with antisense cyclin Dl (A-Dl) and antisense CDK4 (A-K4), the expression of cyclin Dl and CDK4 blocked the cell cycle changes from the G(1) phase to the S phase induced by B(a)P. The overexpression of cyclin Dl and E2F-1 in HELF was induced by B(a)P. The E2F-1 overexpression in A-D1 induced B(a)P was inhibited. The E2F-4 expression was decreased and the CDK4 expression was increased significantly in A-K4 after B(a)P treatment. Most of T-HELF transfected with antisense cyclin Dl (T-A-Dl) and antisense CDK4 (T-A-K4) were retained in G(1) phase. The cyclin Dl expression in T-HELF was increased significantly compared with that in HELF. The E2F-4 expression in T-A-Dl and T-A-K4 was increased significantly compared with that in T-HELF.</p><p><b>CONCLUSION</b>B(a)P induces the cell cycle changes through cyclin D1/CDK4-E2F-1/4 pathway in HELF treated by 2 micromol/L B(a)P while it induces cell cycle changes through cyclin D1/CDK4-E2F-4 pathway in T-HELF.</p>

ERK and JNK/AP-1 pathways involved in benzo(a)pyrene induced cell cycle changes in human embryo lung fibroblasts / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the role of mitogen activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway in benzo(a)pyrene (B(a)P)-induced changes of cell cycle in human embryo lung fibroblasts (HELF).</p><p><b>METHODS</b>AP-1 luciferase activity was determined by the Luciferase reporter gene assay using a luminometer. The expression levels and activity of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle. The dominant negative mutant of ERK2, JNK1 and p38 were applied to detect the upstream or downstream relationship of signaling pathways.</p><p><b>RESULTS</b>B(a)P treatment resulted in a marked activation of AP-1 and its upstream MAPK, including ERK, JNK and p38 in human embryo lung fibroblasts (HELF). B(a)P exposure also led to increase the population of cells at S phase compared to control (P < 0.01) with a concomitant decline of cells at G(1) phase. B(a)P-induced cell cycle alternation was markedly impaired by stable expression of a dominant negative mutant of ERK2 or JNK1, but not p38. B(a)P-induced AP-1 transactivation was inhibited by the overexpression of dominant-negative mutant of ERK2 or JNK1, but not p38. Inhibition of the activation of AP-1 by curcumin, a chemical inhibitor of AP-1, significantly inhibited the cell cycle changes in response to B(a)P treatment.</p><p><b>CONCLUSION</b>ERK and JNK, but not p38, mediated benzo(a)pyrene-induced cell cycle changes by AP-1 transactivation in HELF.</p>

Vitamin C reverses benzo (a) pyrene-induced cell cycle changes by E2F pathway / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the role of E2F1/4 pathway in vitamin C reversing benzo (a) pyrene [B (a) P]-induced changes of cell cycle in human embryo lung fibroblasts (HELF) and the relationship between E2F1 and cyclin D1/CDK4.</p><p><b>METHODS</b>The stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established to detect the relationship of signaling pathway. Cells were cultured and pretreated with vitamin C before stimulation with B (a) P for 24 hours. The expression levels of cyclin D1, CDK4, E2F1 and E2F4 were determined by Western blot and the band intensity was analysed as the relative value to control by using the Gel-Pro 3.0 software. Flow Cytometric Analysis was employed to detect the distributions of cell cycle.</p><p><b>RESULTS</b>B (a) P significantly elevated the expression levels of cyclin D1, CDK4, E2F1 and E2F4 in HELF cells. Vitamin C decreased the expression levels of above proteins in B (a) P-stimulated HELF cells. The expression levels of these proteins in B (a) P-treated above transfectants were lower than those in B (a) P-treated HELF cells. The expression levels of above proteins with vitamin C combined with antisense cyclin D1 were decreased as compared to those with antisense cyclin D1 alone. B (a) P increased the percentage of S phase as compared to the controls [(41.1 +/- 0.2)% vs (33.5 +/- 3.2)%, P < 0.05]. Both vitamin C [(33.2 +/- 0.6)% vs (41.1 +/- 0.2)%, P < 0.05] and antisense cyclin D1 [(31.2 +/- 1.3)% vs (41.1 +/- 0.2)%, P < 0.05] suppressed the changes of cell cycle induced by B (a) P. Vitamin C combined with antisense CDK4 markedly suppressed B (a) P-induced changes of cell cycle as compared to those with antisense CDK4 alone.</p><p><b>CONCLUSION</b>Vitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.</p>

Vitamin C inhibits benzoapyrene-induced cell cycle changes partly via cyclin D1/E2F pathway in human embryo lung fibroblasts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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 D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle.</p><p><b>RESULTS</b>B[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, 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 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 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 D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone.</p><p><b>CONCLUSIONS</b>B[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.</p>

Inhibition of the pathway of benzo (a) pyrene-induced cell cycle changes by all-trans retinoic acid in lung fibroblast / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the reverse effect of all-trans retinoic acid (ATRA) on Benzo (a) pyrene (B (a) P)-induced cyclin D1, CDK4, E2F-1 and E2F-4 expression and cell cycle progression in human embryo lung fibroblast (HELF).</p><p><b>METHODS</b>After HELF cells was treated with ATRA, they were exposed to 2 micromol/L of B (a) P. Western blotting was employed to detect protein expression level; the RNA transfection techniques was used to investigate ATRA-induced signal pathway; flow cytometry was used to detect cell cycle progression.</p><p><b>RESULT</b>After treatment with 2 micromol/L B (a) P for 24 h, the expression of cyclin D1 and E2F-1 were both increased significantly in HELF; the expression of E2F-4 and CDK4 were not changed markedly; pretreatment with 0.1 micromol/L ATRA for 24 h could efficiently decrease B (a) P-induced overexpression of cyclin D1 and E2F-1; stimulation to antisense cyclin D1 or antisense CDK4 by B (a) P could significantly impair E2F-1 up-regulation; pretreatment with ATRA, cells with antisense cyclin D1 or antisense CDK4 showed a less decrease in B (a) P-induced overexpression of E2F-1 compared to similarly treated control cells; flow cytometry analysis showed B (a) P promoted cell cycle progression from G(1) phase to S phase, while pretreatment with ATRA could inhibit B (a) P-induced cell cycle progression by an accumulation of cells in the G(1) phase.</p><p><b>CONCLUSION</b>ATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.</p>

Role of cyclinD1 and CDK4 in the carcinogenesis induced by silica / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the role of cyclinD1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line (2BS) induced by silica.</p><p><b>METHODS</b>Recombination vectors with sense and antisense pXJ41-cyclinD1 and pXJ41-CDK4 were constructed, and then transfected into the malignant transformed cells induced by silica, respectively. At the same time, pXJ41-neo was used as the control.</p><p><b>RESULTS</b>During the progress of the malignant transformation of 2BS cells induced by silica, cyclinD1 and CDK4 were overexpressed. Antisense RNA suppressed cyclinD1 and CDK4 gene expression in the antisense pXJ41-cyclinD1 and pXJ41-CDK4 transfected cells. Antisense RNA led to cell cycle arrest, resulting in lengthened G1 phase (the percentages of cells in the G1 phase changed from 45.1% to 52.7% and 58.0% for cyclinD1 and CDK4 transfected cells, respectively), and eventually attenuated the increase of the proliferation of malignant transformed cells induced by silica. Compared with malignant transformed cells induced by silica, cells transfected with antisense pXJ41-cyclinD1 and pXJ41-CDK4 showed obviously reduced growth rates. On the 8th day, the suppression rates were 58.69 and 77.43% (the growth rate of malignant transformed cells induced by silica was 100%), doubling time changed from 21.0 h to 31.4 h and 21.0 h to 42.7 h, respectively, the growth capacities on soft agar of cells transfected by antisense pXJ41-cyclinD1 and pXJ41-CDK4 decreased obviously.</p><p><b>CONCLUSION</b>CyclinD1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.</p>