Roles Of Heat Shock Protein 70 In DNA Damage And DNA Repair Of A549 Cells Exposed To Benzo(a)pyrene

Cells respond to a variety of environmental stresses such as heat, hypoxia, chemicals and other stressors by rapidly synthesizing a group of highly conserved proteins known as heat shock or stress proteins (Hsps). Hsps function as molecular chaperones, binding to the nascent polypeptides to assist proper refolding, assembling and translocating, and activating specific regulatory proteins including transcription factors, replication proteins and kinases. They also participate in protein signalling, including steroid hormone activation. They facilitate the misfolding peptides to repair or target damaged polypeptides for degradation. Hsps have a critical role in the recovery of cells from stress and in cytoprotection. According to the apparent molecular weight, Hsps are divided into many families. The 70kDa heat shock protein family (HSP70, mainly including inducible Hsp70 and constitutive Hsc70) is one of the most abundant of Hsps, 1-2% of total cellular protein. Many studies had indicated that Hsp70 expression was correlated to the living thing tolerant or susceptible ability to environmental harmful factors. In fact, specific increased expression of Hsp70 protected cells from ultraviolet radiation, and inhibition of Hsp70 produced profound impairment of cellular integrity. However few reports had showed whether the inducible Hsp70 plays a role in protection from DNA damage and DNA repair when cells exposed to harmful environmental factors, such as benzo(a)pyrene (BaP).Benzo(a)pyrene (BaP) is the prototype of a multitude of polycyclic aromatic hydrocarbons (PAHs), which are products of incomplete combustion of organic matter and are widespread in the environment. Considerable experimental evidence suggests that BaP has carcinogenic and mutagenic effects documented in human, animals and mammalian cell systems. Cytochrome P450 can oxidize BaP to the ultimate electrophilic metabolite benzo(a)pyrene diol epoxide (BPDE), which can bind covalent to DNA and thus result in DNA damage. Major types ofdamage induced by BaP are removed by nucleotide excision repair (NER). From our past researches, the results showed a significant negative correlation of Hsp70 levels with DNA damage in workers who exposed to coke-oven emissions. These emissions contain many toxic chemicals like PAHs. Hsp70, the major Hsp, may play a role in protecting cells from DNA damage and DNA repair when cells exposed to benzo(a)pyrene.In present study, we increased the level of Hsp70 expression in A549 cells (a human lung adenocarcinoma cell line) by transfecting A549 cells with a recombinant plasmid pcDNA3.0/hsp70 containing human hsp70 cDNA, and inhibited Hsp70 expression in A549 cells with quercetin treatment. A549 cells were transfected with a pcDNA3.0 plasmid containing the neomycin resistance gene but not hsp70 cDNA as vector control. After establishing the models of overexpression and inhibition of Hsp70 in A549 cells, we exposed these kinds of cells to different concentrations of BaP. We detected DNA damage with alkaline single-cell gel electrophoresis, and measured cell viability by MTT assay, and detected Hsp70 expression using Western blot, to investigate whether Hsp70 plays the role in the protection from DNA damage by BaP. Moreover, we detected mRNA levels of the major nucleotide excision repair (NER) factors with real time RT-PCR when above cells were exposed to BaP. Our results showed that Hsp70 plays the role in the protection from DNA damage by BaP, and Hsp70 has some effects on mRNA levels of major NER genes. These results have three parts.Part I Establishment of cell models of overexpression and inhibition of Hsp70The expression vector containing human hsp70 cDNA was kindly provided by Dr. Liu L. A549 cells were transfected with recombinant plasmid pcDNA3.0/Hsp70 with lipofectemine 2000 reagent according to the manufacturer's instructions. After transfection, we selected the positive clones with antibiotic G418 (neomycin, 1000 μg/ml) for 4 weeks. Three positive clones were expanded and analyzed for the expression of Hsp70 by immunofluorescence staining. We chose the clone of the highest expression of Hsp70 for Western blot analysis. A control population of A549 cells was transfected with a pcDNA3.0 plasmid containing the neomycin resistance gene but not hsp70 cDNA. The results showed that A549 cells transfected with recombinant plasmid expressed obviously increased levels of Hsp70 compared withwild-type A549 and A549/pcDNA cells (transfected with empty plasmid pcDNA3.0). We developed a stably transfected A549 cell line having increased expression of human Hsp70 (A549/hsp70 cells).To establish the cell model of Hsp70 inhibition in A549 cells, we treated A549 cells with different concentrations of quercetin. The flavenoid quercetin has been shown to inhibit Hsp70 synthesis in a human colon carcinoma cell line after heat shock and in human monocyte-macrophages during erythrophagocytosis. We treated A549 cells with 50. 100, 150, 200umol/L quercetin at 37℃ for 6 h, then detected cell viability using MTT method. The results showed that cell viability decreased along with the increasing quercetin concentrations, from 50 100, 150 to 200umol/L. Compared with the group of normal culture, there was a significant decrease of cell viability in the group of 150 and 200μmol/L (P < 0.05) .And there was an obviously difference between the group of 150 and 200μmol/L (P < 0.05) .To observe the inhibition of Hsp70 by quercetin, we treated A549 cells with 50, 100, 150, 200μmol/L quercetin at 37℃ for 6 h, then at 42℃ water bath for 1h. Cells were cultured at 37℃ for 2h recovery. Normal cultured cells were as a normal control. Cells treated by 42℃ were as a positive control. Hsp70 levels were detected by western blot assay. Along with the increasing quercetin concentrations, from 50, 100, 150 to 200μmol/L, Hsp70 levels decreased. Compared with positive control, Hsp70 levels of A549 cells decreased significantly in the group of 100 (P < 0.05), 150 and 200umol/L (P < 0.01). There was no significant difference between 150μmol/L and 200μmol/L groups (P > 0.05). Taken together cell viability and Hsp70 inhibition of A549 cells exposed to different concentrations of quercetin, we chose the 150μmol/L quercetin treatment for cell model of Hsp70 inhibition.Part II Heat shock protein 70 protects cells against DNA damage caused by benzo(a)pyreneTo determine whether Hsp70 plays the role in the protection cells against DNA damage caused by BaP, we detected DNA damage and Hsp70 expression level of A549 cells exposed to the different concentration BaP groups. We investigated the relation between the degree of DNA damage and Hsp70 expression levels of A549 cells. Our results showed that cell viability of A549 cells exposed to BaP decreased along with the increase of doses. Comparedwith the control group (not exposed to BaP), cell viability of A549 cells decreased significantly at 10, 50, 100μM BaP groups (P < 0.01). We found the dose-dependent increases in DNA damage in A549 cells exposed to BaP. The OTM values in A549 cells exposed to 1, 5, 10, 50, 100μmol/L BaP all increased significantly compared with the group not exposed to BaP, respectively (P<0.01). We also found the dose-dependent decrease in Hsp70 expression in A549 cells exposed to BaP. Compared with the control group, Hsp70 expression level of A549 cells decreased significantly at 10, 50, 100μM BaP groups (P < 0.05). Moreover, we found a significant negative correlation of Hsp70 levels with DNA damage in A549 cells exposed to different concentrations of BaP. These results suggested that BaP inhibited inducible Hsp70, which might make A549 cells more susceptible to toxicity of BaP. To further confirm the effect of Hsp70 in the protection cells against DNA damage caused by BaP, we made use of the cell models of the first part, and exposed the cells with different expression levels of Hsp70 to the different concentrations of BaP for 24h. We detected DNA damage with alkaline single-cell gel electrophoresis, and measured the cell viability by MTT assay. Our results showed that A549/hsp70 cells had a significant increased in cell viability compared with A549 cells (P<0.05) in high concentrations of BaP (50μmol/L, 100μmol/L), and we found the dose-dependent increases in DNA damage in A549, A549/pcDNA, A549/hsp70 and A549/Quercetin cells exposed to BaP. The OTM values in A549, A549/pcDNA, A549/Quercetin cells exposed to 1, 5, 10, 50, 100μmol/L BaP all increased significantly compared with themselves control group cells (not exposed to BaP), respectively (P<0.05). The OTM values in A549/hsp70 cells exposed to 5, 10, 50, 100μmol/L BaP also increased significantly compared with the group not exposed to BaP (P<0.05). This indicated the threshold intensity of BaP in the transfected A549 cells overexpressing Hsp70 appeared to be shifted to high levels. Compared to A549 cells, OTM values in A549/hsp70 cells significantly decreased in all BaP treatment groups (P<0.05), but OTM values increased significantly in A549/Quercetin cells exposed to 5, 10, 50, 100μmol/L BaP, and no marked difference in A549/pcDNA cells. It was suggested Hsp70 protects cells against DNA damage caused by benzo(a)pyrene.Part III The effects of Hsp70 on mRNA level changes of major factors in nucleotide excision repair of the cells exposed to BaPThe before mentioned results have showed that Hsp70 is in protection cells from DNA damage caused by benzo(a)pyrene, but we have not known how Hsp70 plays a role in the DNA repair of cells exposed to BaP.BaP is a potent mutagen and carcinogen. Cytochrome P450 can oxidize BaP to the ultimate electrophilic metabolite benzo(a)pyrene diol epoxide (BPDE), which can bind covalent to DNA and thus result in DNA damage. Major types of damage induced by BaP are removed by nucleotide excision repair (NER). When the cells with different expression levels of Hsp70 were exposed to BaP, what happened to the major factors in nucleotide excision repair? In this study, we detected mRNA levels of major factors in NER of the cells with different expression levels of Hsp70 exposed to BaP by using real-time quantitative PCR, and studied the effects of Hsp70 in mRNA level changes of major factors in NER of the cells exposed to BaP. Our results showed that changes of XPA, XPC mRNA expression in the cells with different expression levels of Hsp70 are different when the cells were exposed to different concentrations of BaP. Compared with A549 cells exposed to BaP, the XPA mRNA levels of A549/hsp70 and A549/pcDNA cells had no significant change, but XPA mRNA levels of A549/Quercetin cells decreased significantly in 10μmol/L BaP groups (P<0.05). In 50μmol/L BaP groups, the XPA mRNA levels of A549/pcDNA cells had no significant change, but XPA mRNA levels of A549/hsp70 and A549/Quercetin cells decreased significantly compared with A549 cells(P<0.05). In 100μmol/L BaP groups, the XPA mRNA levels of A549/pcDNA, A549/hsp70 and A549/Quercetin cells increased significantly compared with A549 cells (P<0.05). Our results also showed that in 5μmol/L BaP groups, the XPC mRNA levels of A549/pcDNA cells decreased significantly (P<0.05), but XPC mRNA levels of A549/hsp70 and A549/Quercetin cells increased significantly compared with A549 cells (P<0.05). Compared with A549 cells, the XPC mRNA levels of A549/pcDNA cells had no significant change, but XPC mRNA levels of A549/hsp70 and A549/Quercetin cells increased significantly (P<0.05) in 10μmol/L BaP groups. In 100μmol/L BaP groups, the XPC mRNA levels of A549/pcDNA and A549/Quercetin cells had no significant change, but XPC mRNA levels of A549/hsp70 cells increased significantly compared with A549 cells (P<0.05).XPB is a subunit of TFIIH and plays a central role in NER. XPB and XPD exhibit DNA-dependent ATPase and helicase functions. XPB can unwind DNA in a 3'→5' direction, and XPD in the opposite direction. In 10μmol/L BaP groups, the XPB mRNA levels of A549/pcDNA and A549/Quercetin cells decreased significantly (P<0.05), but XPB mRNA levels of A549/hsp70 cells increased significantly compared with A549 cells (P<0.05). Compared with A549 cells, the XPB mRNA levels of A549/pcDNA cells decreased significantly (P<0.05), but XPB mRNA levels of A549/hsp70 and A549/Quercetin cells increased significantly in 100μmol/L BaP groups (P<0.05).The XPG gene product and ERCC1-XPF complex are structure-specific endonucleases, which cleave a variety of artificial DNA substrates, including bubbles, splayed arms and stem-loops. In NER, XPG makes the 3' incision and ERCC1-XPF makes the 5' incision, consistent with their cleavage polarities. The XPG-mediated 3' incision precedes the 5' incision made by ERCC1-XPF. XPG has a structural function in the assembly of the NER DNA-protein complex. Unlike XPG, ERCC1-XPF does not appear to have an architectural function in the NER protein-DNA complex. In 10μmol/L BaP groups, the XPG mRNA levels of A549/pcDNA, A549/hsp70 and A549/Quercetin cells all decreased significantly (P<0.05) compared with A549 cells. There was no significant change in XPG mRNA levels of A549/pcDNA and A549/hsp70 cells, but there was a significant decreased in XPG mRNA levels of A549/Quercetin cells (P<0.05) in 100μmol/L BaP groups compared with A549 cells. ERCC1-XPF makes the 5' incision consistent with their cleavage polarities in NER. It has an important role at cleavage stages of NER. In 1, 5, 10, 50μmol/L BaP groups, the XPF mRNA levels of A549/pcDNA, A549/hsp70 and A549/Quercetin cells all decreased significantly (P<0.05) compared with A549 cells. In 100μmol/L BaP groups, there was no significant change in XPF mRNA levels of A549/pcDNA and A549/hsp70 cells, but there was a significant increase in XPF mRNA levels of A549/Quercetin cells compared with A549 cells (P<0.05). In 1, 5, 10, 50μmol/L BaP groups, the ERCC1 mRNA levels of of A549/pcDNA, A549/hsp70 and A549/Quercetin cells all decreased significantly (P<0.05) compared with A549 cells. Compared with A549 cells, the ERCC1 mRNA levels of A549/pcDNA and A549/hsp70 cells decreased (P>0.05), but ERCC1 mRNA levels of A549/Quercetin cells increased significantly (P<0.05) in 100μmol/L BaP groups.In conclusion, our results suggested:(1) The experimental cell models of Hsp70 overexpression and inhibition could be established by transfected A549 cells with recombinant plasmid pcDNA3.0/Hsp70 and 150umol/L quercetin treatment for A549 cells.(2) BaP and its metabolic products inhibited inducible Hsp70 expression of A549 cells, which made A549 cells more susceptible to BaP. The results showed a negative correlation of Hsp70 levels with DNA damage in A549 cells exposed to BaP for 24h.(3) Hsp70 overexpression cells A549/hsp70 had a significant increased in cell viability compared with A549 cells (P<0.05) in high concentrations of BaP (50μmol/L, 100μmol/L). Compared to A549 cells, OTM values in A549/hsp70 cells significantly decreased in BaP treatment groups (P<0.05), and increased significantly in A549/Quercetin cells exposed to 5, 10, 50 100μmol/L BaP groups (P<0.05), but no marked difference in A549/pcDNA cells. These results suggested Hsp70 protects A549 cells against DNA damage caused by benzo(a)pyrene.(4) Under BaP treatment, Hsp70 may have effects on mRNA level changes of XPC and XPB in A549 cells. These possibly contribute to NER of DNA repair.Although some results have been received in this study, there still were some questions remained to be elucidated such as the special inhibition of Hsp70 using siRNA method, and whether Hsp70 having effects on NER, and how to protect human respiratory system against toxicity of BaP according to the character of Hsp70 expression.