Possible Role of DNA Polymerase beta in Protecting Human Bronchial Epithelial Cells Against Cytotoxicity of Hydroquinone

Objective 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. Methods 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 μmol/L to 120 μmol/L） for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis （SCGE）] were performed respectively to detect the toxicity of hydroquinone. Results 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. Conclusions 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.

Alterative Expression and Sequence of Human Elongation Factor-1δ during Malignant Transformation of Human Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.

Transcriptional Factor Snail Mediates Epithelial-Mesenchymal Transition in Human Bronchial Epithelial Cells Induced by Silica

Epithelial-mesenchymal transition （EMT） plays an important role in fibrotic diseases. We have previously showed that silica induces EMT in human bronchial epithelial cells （BECs）; however, the underlying mechanism of silica-induced EMT is poorly understood. In the present study, we investigated the role of Snail in silica-induced EMT in human BECs in vitro. Human BECs were treated with silica at various concentrations and incubation times. Then MTr assay, western blot, electrophoretic mobility shift assay （EMSA）, and small interfering RNA （siRNA） transfection were performed. We found that silica increased the expression and DNA binding activity of Snail in human BECs. SNAI silica-induced expression siRNA upregulated the siRNA inhibited the of Snail. Moreover, SNAI expression of epithelial marker E-cadherin, but attenuated the expression of mesenchymal marker a-smooth muscle actin and vimentin in silica-stimulated cells. These results suggest that Snail mediates the silica-induced EMT in human BECs.

Malignant Transformation and Abnormal Expression of Eukaryotic Initiation Factor in Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 （eIF3 p36） in human bronchial epithelial （16HBE） cells induced by cadmium chloride （CdCl2）. Methods 16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdC12, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique （FQ-PCR）. Results The 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects （P〈0.01）. All Cd-induced transformed cell lines formed rumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control （P〈0.01）, and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.

Proteomics Study on the Differentially Expressed Proteins in c-fos-silenced Cells Exposed to PM2.5

Objective To investigate the effect of c-fos gene silencing on differentially expressed proteins(DEPs) in human bronchial epithelial(HBE) cells after exposure to fine particulate matter(PM2.5).Methods HBE cells and c-fos-silenced HBE cells were exposed to 50 μg/mL PM2.5, LC-MS/MS and tandem mass tag(TMT) labeling methods were combined with bioinformatics methods, and DEPs and interaction networks were identified.Results In the HBE group, 414 DEPs were screened, of which 227 were up-regulated and 187 downregulated. In the c-fos silenced HBE group, 480 DEPs were screened, including 240 up-regulated proteins and 240 down-regulated proteins. KEGG annotations showed that DEPs in the HBE group are mainly concentrated in the glycolysis/gluconeogenesis pathway and those in the c-fos silenced group are concentrated mainly in endoplasmic reticulum and the processing of proteins. Additionally, the abnormal expression of GPRC5 C, DKK4, and UBE2 C was identified in top 15 DEPs. After constructing the protein interaction network, 20 Hub proteins including HNRNPA2 B1, HNRNPL, RPS15 A, and RPS25 were screened from the HBE group and the c-fos silenced HBE group.Conclusion c-fos gene affected the expression of cancer-related proteins. Our results provided a scientific basis for further study of PM2.5-induced carcinogenesis mechanism.

Human bronchial epithelial cell injuries induced by fine particulate matter from sandstorm and non-sandstorm periods：Association with particle constituents

Epidemiological studies have demonstrated the exacerbation of respiratory diseases following sandstorm-derived particulate matter（PM） exposure.The presence of anthropogenic and biological agents on the sandstorm PM and the escalation of PM 〈 2.5 μm（PM2.5）pollution in China have led to serious concerns regarding the health effects of PM2.5during Asian sandstorms.We investigated how changes in PM2.5composition,as the weather transitioned towards a sandstorm,affected human airway epithelial cells.Six PM2.5samples covering two sandstorm events and their respective background and transition periods were collected in Baotou,an industrial city near the Gobi Desert in China.PM samples from all three periods had mild cytotoxicity in human bronchial epithelial cell line BEAS-2B,which was positively correlated with the contents of polycyclic aromatic hydrocarbons and several metals.All PM samples potently increased the release of interleukin-6（IL-6） and interleukin-8（IL-8）.Endotoxin in all samples contributed significantly to the IL-6 response,with only a minor effect on IL-8.Cr was positively correlated with both IL-6 and IL-8 release,while Si was only associated with the increase of IL-6.Our study suggests that local agricultural and industrial surroundings in addition to the sandstorm play important roles in the respiratory effects of sandstorm-derived PM.