Adolescent ; Adult ; Child ; Humans ; MELAS Syndrome ; diagnosis ; pathology ; Magnetic Resonance Imaging ; Male ; Tomography, X-Ray Computed

OBJECTIVETo 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.

METHODSThe 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.

RESULTSB[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.

CONCLUSIONSB[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.

Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; Blotting, Western ; methods ; Cell Cycle ; drug effects ; physiology ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; G1 Phase ; drug effects ; physiology ; Humans ; Lung ; cytology ; embryology ; RNA, Antisense ; genetics ; S Phase ; drug effects ; physiology ; Transfection ; methods

OBJECTIVETo study the role of cyclinD1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line (2BS) induced by silica.

METHODSRecombination 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.

RESULTSDuring 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.

CONCLUSIONCyclinD1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.

Carcinogens, Environmental ; toxicity ; Cell Line ; Cell Proliferation ; Cell Transformation, Neoplastic ; chemically induced ; Cyclin D1 ; genetics ; metabolism ; physiology ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; physiology ; Humans ; Plasmids ; RNA, Antisense ; metabolism ; RNA, Messenger ; analysis ; metabolism ; Silicon Dioxide ; toxicity

OBJECTIVETo genotype the RHCE gene of Hans, Xinjiang's Uigurs and Kazakstans in China, and to compare the results of RHCE genotyping with that of RhCc/Ee phenotyping.

METHODSRHCE genes of 98 Hans with RhD positive and 230 Hans, 72 Uigurs and 18 Kazakstans with RhD/RHD negative were genotyped with PCR-sequence specific primer (SSP) technique.

RESULTSThe results of RHE/RHe genotyping from samples with RhD positive and negative were in accord with that of phenotyping. It would result in 4.44% error using C-->G polymorphism at nt48 of RHCE gene to genotype RHCE, and 4.05% failure of detection using the 109 bp insertion to detectRHCE gene in Chinese Hans. The results of RHE/RHe genotyping in unrelated 72 Uigurs and 18 Kazakstans with RhD phenotype were consistent with that of phenotyping, and false positive and false negative were not found in genotyping in Uigurs and Kazakstans tested.

CONCLUSIONThe results of RHE/RHe and RHc genotyping were correct with PCR-SSP and accordant with that of phenotyping. Using the C48G polymorphism in exon 1 of RHCE to genotype RHC gene would result in false positive resulting from RHc mutation at this locus, and using the 109 bp insertion to genotype RHC gene would result in false negative because of the absence of the 109 bp. Therefore it is necessary to genotype RHC gene using more than two polymorphic loci.

Ethnic Groups ; genetics ; Genotype ; Humans ; Phenotype ; Polymorphism, Genetic ; Rh-Hr Blood-Group System ; blood ; genetics ; Serologic Tests ; methods

BACKGROUNDTo investigate the prognostic significance and role of coagulation factor V (CFV) levels in clinical diagnostic criteria for severe hepatitis.

METHODSThe CFV level and prothrombin activity (PTA) were tested by turbidimetry for 129 times in 58 patients with severe hepatitis. Comparative studies and clinical significance of CFV and PTA were analyzed by SPSS and SDAS softwares.

RESULTS1. The levels of CFV and PTA were 15.3%+/-9.7% and 23.5%+/-10.0%, respectively, at the onset of severe hepatitis. 2. The mortality of severe hepatitis gradually increased with the gradual decrease of CFV or PTA during the most severe stage of the illness (P=0.000). 3. The levels of CFV and PTA decreased continually and rapidly in patients who died but gradually increased in survivors. The decrease or increase of PTA preceded that of CFV on the exacerbation or convalescent stage. 4. Hepatic encephalopathy occurred in 14 cases (24.14%). In 10 cases, it occurred in the terminal stage of the illness, far later than the time of the decrease of CFV. 5. The level of CFV was closely related to PTA (the correlation coefficient was 0.812), the level of CFV was almost consistent with that of PTA.

CONCLUSION1. The level of CFV is an important prognostic indicator in severe hepatitis and is more specific than PTA. 2. Simultaneous determination of CFV and PTA may be helpful in earlier and more accurate diagnosis of severe hepatitis. 3. Possible use of CFV as one of the criteria for liver transplantation in patients with severe hepatitis should be studied.

Adult ; Aged ; Diagnostic Techniques and Procedures ; Factor V ; analysis ; metabolism ; Female ; Hepatitis ; diagnosis ; metabolism ; Humans ; Male ; Middle Aged ; Nephelometry and Turbidimetry ; methods ; Prognosis ; Prothrombin ; analysis ; metabolism ; Young Adult

OBJECTIVETo 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.

METHODSThe 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.

RESULTSB (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.

CONCLUSIONVitamin C might reserve the B (a) P-induced changes of cell cycle via intracellular signaling pathway of cyclin D1-CDK4/E2F-1/4.

Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; antagonists & inhibitors ; toxicity ; Cell Cycle ; drug effects ; Cyclin D1 ; metabolism ; E2F1 Transcription Factor ; metabolism ; E2F4 Transcription Factor ; metabolism ; Humans ; Lung ; cytology ; embryology ; Signal Transduction

OBJECTIVETo study the role of cyclin D1 in malignant transformation of human embryonic lung diploid fibroblasts (HELF) induced by quartz.

METHODSpXJ41-cyclin D1 expressing sense and antisense cyclin D1 RNA were transinfected into malignant transformed HELF induced by quartz with DNA recombination and gene transduction. The expression of cyclin D1 was detected with hybridization in situ and immunohistochemistry methods to analyze changes in cell growth, double multiplication time, distribution of cell cycles, colony forming ability on soft agar, etc., before and after cyclin D1 transduction.

RESULTSDuring the process of malignant transformation of HELF induced by quartz, cyclin D1 gene was overexpressed. Antisense pXJ41-cyclin D1 RNA could suppress the growth and proliferation of malignant transformed cells induced by quartz. Growth speed of antisense pXJ41-cyclin D1 transinfected cells decreased by 58.69% on the 8th day in culture, as compared to malignant transformed cells induced by quartz, and its double multiplication time prolonged from 21.0 h to 31.4 h. Antisense cyclin D1 RNA led to cell cycle arrest, resulting in lengthened G1 phase (proportion of cells in phase G1 increased to 52.7% from 45.1% and that of cells in phase S decreased to 33.1% from 40.3%). Colony forming rate reduced significantly and size of colony became smaller.

CONCLUSIONSAbnormal expression of cyclin D1 in cells related to their malignant transformation induced by quartz. Highly expressed cyclin D1 could play an important role in maintaining the transformed phenotype of malignant cells.

Cell Transformation, Neoplastic ; Cells, Cultured ; Cyclin D1 ; biosynthesis ; genetics ; Embryo, Mammalian ; Fibroblasts ; cytology ; Humans ; Lung ; cytology ; Quartz ; toxicity

OBJECTIVETo 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).

METHODSAfter 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.

RESULTAfter 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.

CONCLUSIONATRA could block B (a) P-induced cell cycle promotion through cyclin D1/E2F-1 pathway in HELF.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flow Cytometry ; Humans ; Lung ; cytology ; metabolism ; Signal Transduction ; drug effects ; Tretinoin ; pharmacology

OBJECTIVETo 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.

METHODSHuman 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.

RESULTSAfter 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.

CONCLUSIONSCyclin 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.

Benzo(a)pyrene ; pharmacology ; Cell Cycle ; drug effects ; Cell Line ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F4 Transcription Factor ; metabolism ; Fibroblasts ; drug effects ; enzymology ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; enzymology ; metabolism

OBJECTIVETo explore the role of telomerase in asbestos dust induced malignant transformation of human embryonic lung fibroblasts in vitro.

METHODSHuman telomerase catalytic subunit (hTERT) was transferred into human embryonic lung fibroblasts (HELF). Chrysotile dust at concentration of 2.5 microg/cm(2) was added to HELF transduced with and without hTERT (HELF-T+), respectively, and their transduced foci were separated. Biological characteristics of the cells, telomerase activity, length of telomere and cell growth curve were observed. Colony forming test was performed on soft agar to evaluate the nature of transformation.

RESULTSThe hTERT gene was transferred into HELF steadily, and HELF-T+ was established. Malignant transformation occurred in both HELF and HELF-T+ by asbestos stimulation. Asbestos dusts could induce higher rate of transformations in HELF-T+ [(2.08 +/- 1.08)/utensil] than in HELF [(1.08 +/- 0.10)/utensil], P < 0.05. Telomerase activity in both transformed malignant cells and HELF-T+ was higher, as well as the longer length of telomere in them.

CONCLUSIONRate of malignant transformation in cells with more activity of telomerase and longer length of the telomere was higher after stimulation with asbestos, indicating telomerase could play an important role in asbestos induced human cells malignant transformation.

Asbestos, Serpentine ; toxicity ; Cell Transformation, Neoplastic ; drug effects ; Cells, Cultured ; DNA-Binding Proteins ; Embryo, Mammalian ; Fibroblasts ; pathology ; Gene Transfer Techniques ; Humans ; Lung ; pathology ; Telomerase ; genetics ; metabolism