Genome DNA hypomethylation in the process of crystalline nickel-induced cell malignant transformation / 中华预防医学杂志

<p><b>OBJECTIVE</b>To observe the effect of crystalline NiS on genome DNA methylation profile in in vitro cultured cells.</p><p><b>METHODS</b>16HBE Cells were treated with crystalline NiS at 0.25, 0.50, 1.00 and 2.00 µg/cm(2) for 24 h and three times at total. DAC treatment was given at 3 µmol/L for 72 h.5-mC immunofluorescence and SssI methyltransferase assay methods were applied to investigate if the hypomethylation of genome DNA involved.</p><p><b>RESULTS</b>The results of 5-mC immunofluorescence showed that the fluorescence intensity of NiS-treated cells were decreased in some degree, and transformed cells were decreased dramatically. By the SssI methylase assay, an average of (81.9 ± 7.3)% methylated CpG were found in negative control cells. By contrast, (77.9 ± 6.2)%, (75.3 ± 6.8)%, (59.5 ± 4.9)%, (67.4 ± 5.1)% methylated CpG were observed in cells treated with NiS for three times at dosage of 0.25, 0.50, 1.00 and 2.00 µg/cm(2) which were abbreviated as NiS0.25, NiS0.50, NiS1.00, NiS2.00 respectively. The ANOVA analysis results showed that there was a significant difference in the 5 groups above (F = 124.95, P < 0.01). The results of Dunnett-t test showed that the methylated CpG of both group NiS1.00 and NiS2.00 were significantly decreased compared with the negative control group (t values were 7.64, 4.89 respectively, P < 0.01). For methylated CpG, (46.2 ± 4.1)% and (43.6% ± 4.3)% were observed in NiS-transformed cells (NSTC1 and NSTC2) which were dramatically decreased compared with the negative control group (t values were 12.79, 13.56 respectively, P < 0.01).</p><p><b>CONCLUSION</b>Genomic DNA methylation levels were decreased during NiS induced malignant transformation.</p>

Relationship between polymerase eta expression and DNA damage-tolerance in human hepatic cells by hydroquinone / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of hydroquinone (HQ) on expression of Polymerase eta (Pol eta) and DNA damage in human hepatic cells (L-02), and to explore the role and possible mechanism of Pol eta involved in the process of DNA damage-tolerance.</p><p><b>METHODS</b>After L-02 hepatic cells were exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L) for 24 h, cell survival rate was detected by MTT assay; DNA impairment was detected by single cell gel electrophoresis (SCGE); Real-time fluorescent quantitative PCR and Western blotting methods were used to measure the expression of Pol eta at the mRNA and protein level in L-02 hepatic cells exposed to HQ with various concentrations (0, 5, 10, 20, 40, 80 and 160 micromol/L).</p><p><b>RESULTS</b>MTT assay showed that HQ with concentrations from 0 to 80 micromol/L had little effect on the survival rate of L-02 (P>0.05); whereas the survival rate of the group of 160 micromol/Lwas significantly higher than that of the control (P<0.01) after being treated with HQ for 24 h; the higher dose of HQ presented, the more degrees of DNA damage were produced. It was found that HQ in a low concentration (1-80 micromol/L) could induce the expression of Pol eta which was in proportion to the increasements of HQ concentration; the expression levels of mRNA and protein were reached to the maximum when treated with 80 micromol/L; the expression of Pol eta decreased (the relative quantity values were 2.32 +/- 0.16 and 1.20 respectively) once the concentration of HQ exceeded 160 micromol/L as compared with the group of 80 micromol/L, but it was higher than that of the control.</p><p><b>CONCLUSION</b>This study suggested that Pol eta might involve in the process of DNA damage-tolerance induced by HQ in the hepatic cells.</p>

Expression and sequence analysis of human MutS homologue 2 during different stages of human bronchial epithelial cells induced by cadmium chloride / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To explore the expression and sequence of human MutS homologue 2 (hMSH2) during different stages of human bronchial epithelial (16HBE) cells induced by cadmium chloride (CdCl2).</p><p><b>METHODS</b>Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining (SP method) were used to measure the hMSH2 mRNA and protein expression in 16HBE cells and its different passage cells treated by CdCl2 (the 5th, 15th, 35th passage, and neoplasm cells from nude mice's tumor tissue). hMSH2 exon 6, hMSH2 exon 7, hMSH2 exon 8, hMSH2 exon 9, hMSH2 exon 12 of the 16HBE cells and neoplasm cells from nude mice's tumor tissue were amplified by polymerase chain reactions (PCR). The amplified DNA strips were purified. Then the exons were detected by DNA analysis.</p><p><b>RESULTS</b>During the passages of 16HBE cells treated with CdCl2, the expression of hMSH2 gene were decreased gradually. The hMSH2 gene mRNA and protein expression levels of the CdCl2 transformed 35th 16HBE cells and tumorigenic cells of nude mice significant decreased compared with non-transformed 16HBE cells (P < 0.01). In the tumorigenic cells of nude mice induced by CdCl2, there were thymine (T) deletion in 1st, 2nd and 7th site of hMSH2 exon 8, there were adenine (A) deletion in 20th and 182th site of hMSH2 exon 9, there were adenine (A) insertion in 241st site of hMSH2 exon 12. All the mutations were frame shift mutation.</p><p><b>CONCLUSION</b>The expression decreased and the mutation of hMSH2 gene may be the possible carcinogenic mechanism for CdCl2.</p>

Breast cancer resistance protein expression and 5-fluorouracil resistance / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To filtrate breast cancer resistance protein (BCRP)-mediated resistant agents and to investigate clinical relationship between BCRP expression and drug resistance.</p><p><b>METHODS</b>MTT assay was performed to filtrate BCRP-mediated resistant agents with BCRP expression cell model and to detect chemosensitivity of breast cancer tissue specimens to these agents. A high performance liquid chromatography (HPLC) assay was established, and was used to measure the relative dose of intracellular retention resistant agents. RT-PCR and immunohistochemistry (IHC) were employed to investigate the BCRP expression in breast cancer tissue specimens.</p><p><b>RESULTS</b>MTT assay showed that the expression of BCRP increased with the increasing resistance of 5-fluorouracil (5-Fu) (P<0.05, n=3) in the cell model, while HPLC assay indicated that the intracellular retention dose of 5-Fu was significantly correlated with the expression of BCRP (r=-0.897, P<0.05, n=3). A total of 140 breast cancer tissue specimens were collected. BCRP-positive expression was detected in forty-seven specimens by both RT-PCR and IHC. As shown by MTT assay subsequently, the resistance index (RI) of 47 BCRP-positive breast cancer tissue specimens to 5-Fu was 7-12 times as high as that of adjacent normal tissue samples. BCRP expression was related to 5-Fu resistance (R2=0.8124, P<0.01).</p><p><b>CONCLUSION</b>Resistance to 5-Fu can be mediated by BCRP. Clinical chemotherapy for breast cancer patients can be optimized based on BCRP-positive expression.</p>

Possible role of DNA polymerase beta in protecting human bronchial epithelial cells against cytotoxicity of hydroquinone / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.</p><p><b>METHODS</b>DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-C1 were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 micromol/L to 120 micromol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone.</p><p><b>RESULTS</b>MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups.</p><p><b>CONCLUSIONS</b>Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.</p>

Alterations of FHIT gene and P16 gene in nickel transformed human bronchial epithelial cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis.</p><p><b>METHODS</b>16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting.</p><p><b>RESULTS</b>NiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P < 0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8.</p><p><b>CONCLUSIONS</b>The FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.</p>

Relationship between Characteristic of Symptoms and Executive Functions in Children with Tourette′s Syndrome / 实用儿科临床杂志

Objective To investigate the correlation between executive function(EF) and characteristic of symptom in children with Tourette′s syndrome(TS).Methods EF were measured with Wisconsin Card Sorting Test(WCST) in 53 patients with TS,and symptom characteristics were evaluated with Achenbach Child Behavior Checklist(CBCL) and Yale Global Tic Severity Scale(YGTSS).Results Compared with healthy control,TS had poor executive performances significantly in WCST(all P

Identification of protein peroxiredoxin 2 related to crystalline NiS-induced neoplastic transformation / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To provide evidence for illustrating the molecular mechanism of nickel carcinogenesis, and to identify the differential expression of protein in crystalline NiS-induced neoplastic transformation of human bronchial epithelial cell by proteomics technology.</p><p><b>METHODS</b>Two dimensional electrophoresis (2-DE) and the ImageMaster 3.10 software were used to analyze the differential expression of protein, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) combined with database search was applied to identify protein peroxiredoxin 2 (PDX2) related to malignant transformation.</p><p><b>RESULTS</b>The good 2-DE pattern including resolution and reproducibility was obtained. Nearly 700 expressed proteins per 2-D gel were isolated with molecular weights (MW) ranging from 14,400 to 94,000 KD and pI 3 - 10. A protein PDX2 with MW 21,890 KD, pI 5.66, which was highly expressed in malignantly transformed cell, was identified using MALDI-TOF-MS.</p><p><b>CONCLUSION</b>PDX2 was involved in malignant transformation of human bronchial epithelial cell induced by crystalline nickel sulfide.</p>

Crystalline nickel sulfide-induced genomic instability in transformed human broncho-epithelial cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To detect the genomic instability in the 16 human broncho-epithelial (16HBE) cells induced by crystalline nickel sulfide so as to provide the scientific basis for further study of nickel-induced cancer molecular mechanism.</p><p><b>METHODS</b>To analyse the genomic instability in transformed 16HBE cells induced by crystalline nickel sulfide by random amplified polymorphic DNA (RAPD).</p><p><b>RESULTS</b>All the 7 random primers selected could amplify 1 - 6 clear PCR bands. There were no significant differences between transformed 16HBE cells and negative control cells in the 4th, 5th, and 7th primers, but in the rest 4 primers there were significant differences, with special PCR bands for the same primer, indicating that genomic instability in transformed 16 HBE cells was induced by crystalline nickel sulfide.</p><p><b>CONCLUSION</b>Crystalline nickel sulfide could induce genomic instability in 16HBE cells.</p>

Antitransforming activity of chlorophyllin against trans-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of chlorophyllin (CHL) on trans-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) induced malignant transformation in human bronchial epithelial cell line (16HBE).</p><p><b>METHODS</b>10, 50 or 100 micro mol/L CHL were added into the media during the cells transformation induced by BPDE, and the malignant degree of transformed cells were identified by the ConA agglutination test and the assay for anchorage-independent growth and tumorigenicity.</p><p><b>RESULTS</b>After the cells were cultured for 25 times, the time of cells agglutination in groups treated with both CHL and BPDE was increased significantly; the colony formation efficiency in soft agar in groups treated with both CHL and BPDE (7.4 per thousand, 11.4 per thousand and 14.4 per thousand ) showed significant decrease (P < 0.05) in dose-dependent manner, as compared with that in group treated with BPDE alone (19.6 per thousand ). Cells treated with both CHL and BPDE or BPDE alone developed tumor in nude mice, a squamous carcinoma confirmed by histopathological examination. The volume of tumor in groups treated with both CHL and BPDE (0.43 +/- 0.13) cm(2), (0.22 +/- 0.04) cm(2) and (0.10 +/- 0.06) cm(3) was significantly smaller (P < 0.05) and dose-dependent, as compared with that in the group treated with BPDE alone (1.71 +/- 0.37) cm(3).</p><p><b>CONCLUSION</b>CHL showed significant antitransforming ability in human bronchial epithelial cell line induced by BPDE.</p>

Alteration of FHIT gene and p16 gene in malignant transformed cells induced by crystalline nickel sulfide / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To detect the alteration of fragile histidine triad (FHIT) gene and p16 gene during malignant transformation of immortal human bronchial epithelial cell line (16HBE) induced by crystalline nickel sulfide, and study the molecular mechanism of nickel carcinogenesis.</p><p><b>METHODS</b>Malignant transformed cells and tumorigenic cells were examined for the alteration of FHIT gene and p16 gene by RT-PCR, DNA sequencing and silver staining PCR-SSCP.</p><p><b>RESULTS</b>Compared with those of control 16HBE, neither mutation of exon2 or exon2-3, abnormal expression in p16 gene nor mutation of FHIT exon5, 6, 7 and 8, exon1-4 or exon5-9 were observed in transformed cells and tumorigenic cells. But aberrant transcript or FHIT gene expression loss were observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in FHIT gene, the deletion of exon6, exon7 and exon8 and an insertion of 36 bp sequence replacing exon6-8, was confirmed by sequencing.</p><p><b>CONCLUSION</b>FHIT gene, not p16 gene, could play a definite role in nickel carcinogenesis. Alterations of FHIT gene induced by crystalline NiS could be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation, and FHIT gene could be one of the important target genes activated by exotic carcinogens.</p>