New Type Of Radiation-related Gene Screening And Functional Studies

Estrogenic chemicals, such as 523 and E838, are the active drugs used to prevent acute radiation diseases, so we hypothesized that some proteins in estrogen signal pathway may be associated with radiation sensitivity. It is well accepted that estrogen plays its role via estrogen receptors (ERs) and regulates the transcription of estrogen-responsive genes. The process is modulated by the proteins called ER co-regulators. Thus, we assumed that ER co-regulators may be involved in radioprotection. We screened many ER co-regulators, such as FHL1, FHL3, RPBMS, MEMO and HPIP. Fortunately, we identified HPIP and FHL1 as the targets.HPIP was originally identified from the fetal liver cDNA library through yeast two-hybrid-system with PBX1 as bait. Consequently, the protein was named hematopoietic PBX-interacting protein (HPIP). With ERβAF2 as a bait protein we found out HPIP by the same system. Immunoprecipitaion assays confirmed the interaction between ER and HPIP. When studying the relationship of HPIP with radiation, we know that HPIP protein level is highest after 10Gy of gamma irradiation, and using the same dose of irradiation, HPIP levels gradually increased with time. RT-PCR experiment indicated that the change tendency of HPIP mRNA was similar to that of the protein, suggesting that there may be a promoter sequence sensitive to irradiation. For this reason we detect a series of promoter fragments of different length. The results showed that the -782～-1 bp and -1282～-782area of the HPIP promoter was sensitive to irradiation. To research the relation of HPIP and radiosensitivity, we established the HPIP overexpression and knockdown stable MCF-7 cell lines. Growth curve assays showed that HPIP promoted the cell growth compared with empty vector, but after irradiation the function disappears. Colony formation also displayed the same conclusion. All above results suggest that HPIP is radiosensitive. With the purpose of investigating the molecular mechanism of HPIP, we detecte the changes in p53-positive cell lines, p53-negative cell lines, AT-defective cell lines, and BRCA1-defective cell lines. The results showed that in AT-defective and BRCA1-defective cell lines there was no obvious change in HPIP levels after irradiation. However, both in p53-positive and–negative cell lines, HPIP was still irradiation-inducible, indicating that radiation inducibility of HPIP has nothing to do with p53. We detected the p53, p21, pERK in the HPIP overexpression and knockdown stable MCF7 cell lines before and after radiation. The results demonstrated that HPIP could increase p21 level even without irradiation and p21 could be radiation-inducible. However, the magnitude of the p21 inducibility with HPIP overexpression was lower than that without HPIP overexpression. Regardless of HPIP overexpression, irradiation increased pERK. HPIP could not influence p53 before and after radiation. In order to confirm the mechanism, by colony formation we found that, through transfection of exogenous p21 into HPIP overexpression stable cell lines, the ability of colony formation is approximately equivalent to that of control cells. At the same time, using the pERK inhibitor PD98059 to inactivate pERK, we showed that it did not affect the ability of colony formation at different HPIP levels.As a member of the FHL family, FHL1 is the other target we chose. The growth curve assays indicated that FHL1 inhibited the cell proliferation without irradiation; while after radiation FHL1 could protect the cells from irradiation. Colony formation assays also confirmed that FHL1 is an anti-radiation gene.In conclusion, we identified two irradiation-associated genes, HPIP and FHL1, in estrogen signal pathway. The experiment results indicate that HPIP may function as a radiosensitizer and FHL1 a radioresister. These are the novel radio-associated genes isolated from ER coregulators and have different effects on radiosensitivity. Further research on their mechanisms will establish a solid foundation for us to develop radiation drugs of various purpose.