Roles Of Heat Shock Protein 70 In DNA Damage And Repair In A549 Cell Lines Caused By Ultraviolet C

Heat shock proteins (Hsps) are highly conserved proteins which are triggered in all organisms exposed to environmental stressors such as elevated temperature, nicotinamide, carbon monoxide, heavy metals, ionization, ischemia, hypoxia, and so on. Based upon their apparent molecular weight, Hsps are divided into many groups such as high-molecular-mass HSPs (≥100 kD), HSP90 (81 to 99 kD), HSP70 (65 to 80 kD), HSP60 (55 to 64 kD), HSP40 (35 to 54 kD), and small HSPs (≤34 kD). The HSP70 family have been extensively studied which are found to function as molecular chaperones, binding to the nascent polypeptides to assist them proper refolding, assembling and translocation, and facilitating the misfolding peptides to repair or targeting damaged polypeptides for degradation. Their overexpressions greatly change the ability of living things to be tolerant or susceptible to stressors such as physical, chemical and biological harmful stimuli. Increasing documents show that Hsp70 is relative to DNA damage and repair.In present study, we investigated the possible roles of Hsp70 in protection against DNA damage and for DNA repair in A549 cells by either inhibiting or overexpressing Hsp70 with the use of ultraviolet C (UVC) irradiation. UVC-induced DNA damage was measured by the comet assay. UVC-caused DNA repair were assessed with nuclear exision repair gene such as Xeroderma pigmentosum group A, C, B, F, G, and Excision Repair Cross Complementation Group 1 (ERCC1) detected by real time quantitative reverse transcriptase polymerase chain reaction. We tested the hypothesis that the levels of UVC-caused DNA damage and repair were correlated with levels of Hsp70 expression in A549 cells with inhibited or overexpressed Hsp70.Part I Establishment of Over-expressed and Inhibited Hsp70 in A549 CellsA549 cells were transfected with recombinant plasmid pcDNA3.0/hsp70 with lipofectemine 2000. After transfection, the positive clones were selected by 1000 μg/ml G418 (neomycin) for 4 weeks. Three positive clones were expanded and analyzed for the expression of Hsp70. A control population of A549 cells was transfected with a pcDNA3.0 plasmid containing the neomycin resistance gene but not hsp70 cDNA. We developed stably transfected A549 cell lines having increased expression of human Hsp70 (A549/hsp70 cells) and containing the neomycin resistance gene but not hsp70 cDNA (A549/pcDNA).To inhibit Hsp70 expression A549 cells. 50, 100, 150, 200μmol/L quercetin were used to treat cells at 37℃ for 6h, then at 42℃ water bathing for 1h. Compared with the normal group, there was a significant decrease of Hsp70 levels in 100 μmol/L (P < 0.05) and obviously decrease in 150umol/L and 200 μmol/L (P < 0.01). 150μmol/L quercetin were used to inhibit Hsp70 expression of A549 cells in following experiments according to both cell viability and Hsp70 levels caused by different concentrations of quercetin.Hsp70 siRNA (NPY-1 NPY-2, NPY-3, and NPY-4 plasmids) were transiently tranfected to knockdown Hsp70. NPY-4 transfection can result in 34.07% decrease for Hsp70 mRNA and 14.23% decrease for Hsp70 proteins.Cell models of overexpressed and inhibited Hsp70 in A549 cells were verified by Western-blot assay and immunofluorescence staining. Part II Roles of Hsp70 in DNA damage caused by ultraviolet CHsp70 has been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli, however, it is little known whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage in A549 cells increased in all cells after exposure to an increase dose of 10, 20, 40, and 80 J/m~2 whether Hsp70 was inhibited or overexpressed. This response was dose-dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/ m~2 with a maximum at 40 J/m~2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m~2. Our results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from DNA damage at UVC irradiation by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.Part III Roles of Hsp70 in changes of main enzyme in NER pathwaycaused by ultraviolet COur previous data have indicated that Hsp70 protects A549 cells against DNA damage caused by UVC. However, it is unknown whether Hsp70 has affects on DNA repair of cells exposed to UVC. DNA damage induced by UVC is mainly repaired by nucleotide excision repair (NER) pathway. After the cells with different levels of Hsp70 exposed to UVC, mRNA expression of Xeroderma pigmentosum group A, C, B, F, G and Excision Repair Cross Complementation Group 1 (ERCC1), major factors in NER pathway, were detected by real-time quantitative PCR. Association of Hsp70 and mRNA levels of XPA, XPC, XPB, XPF, XPG, and ERCC1 are analyzed. Our results showed that changes of mRNA levels: 1) XPA mRNA levels decrease in A549/hsp70 cells exposed to 40 J/m~2 UVC irradiation (P < 0.05); 2) XPC mRNA levels obviously decrease in A549/hsp70 cells exposed to 40 J/m~2 (P < 0.01); XPC mRNA levels decrease in A549/quercetin cells and increase in A549/hsp70 cells exposed to 80 J/m~2 (P > 0.05 respectly); 3) XPB mRNA levels obviously increase in A549/quercetin cells exposed to 10, 20, 40 and 80 J/m~2 (P < 0.01); There is no significant differences in A549/hsp70 cells exposed to UVC irradiation; 4) XPF mRNA levels obviously decrease in A549/quercetin cells and significantly increase in A549/hsp70 cells exposed to 40 J/m~2 (P < 0.01); XPF mRNA levels obviously increase in A549/hsp70 cells exposed to 80 J/m~2 (P < 0.01) but no significant differences are found in A549/quecetin cells exposed to 80 J/m~2(P > 0.01); 5) XPG mRNA levels obviously increase in A549/hsp70 cells exposed to 10 J/m~2, 40 J/m~2 and 80 J/m~2 compared to A549 or A549/quercetin group (P < 0.05); 6) ERCC1 mRNA levels obviously increase in A549/hsp70 cells exposed to 10 J/m~2 and 80 J/m~2(P < 0.05).Conclusions:1) Over-expressed or inhibited Hsp70 in A549 cells were established by transfected recombinant plasmid pcDNA3.0/hsp70 or 150μmol/L quercetin.2) NPY-4 plasmids are maybe potent to knowdown Hsp70 expression.3) Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from DNA damage at UVC irradiation by Hsp70.4) Hsp70 might interact with nuclear excision repair gene such as XPA, XPC. XPB, XPF and XPG5) Host cell reactivation is a useful tool to detect DNA repair capacity.6) Association of Hsp70 and mRNA levels of XPA, XPB, XPF, XPG and ERCC1 is found in A549 cells exposed to UVC irradiation. Perspectives:1) Special siRNA and stable tranfection should be developed to knockdown Hsp70then to investigate the functions of Hsp70.2) Interaction of Hsp70 and NER enzymes need be further verified using confocol microscope or chromatin immunoprecipitation (ChIP).3) Host cell reactivation will be developed to be used into population epidemilology.