The Abnormal Expression Of P33lNG1b During Senescence Of HDFs And Its Efffects On Damaged DNA Repair And Apoptosis

Objective: The p33INGlb was first cloned in 1996. It involved in some biological functions, such as negative regulation of cell proliferation and in the control of cellular aging, anchorage dependence and apoptosis. Here we investigate if there are some differences between senescent and young human diploid fibroblasts(HDF) on expression of p33INGlb, damaged DNA repair and apoptosis.Methods: (1)Cloning the 5'-flanking 1.8kb-region of p33INGlb and constructing luciferase reporter plasmid named pGL3 -basic-1. A series of deletion was carried out on the pGL3-basic-l, so we got eight plasmids to transfect senescent or young HDFs. Luciferase assays were performed after transfection 24 hours. (2)We detected the expression of three isoforms of ING 1 in different cells, including young HDFs, senescent HDFs, premature senescent HDFs treated by H2O2, quiescent HDFs, Lovo and HEPG2 cell lines by RT-PCR analysis. (3)We cloned coding region of p33ING1b and constructed a recombinant adenovirus vector named Adeno-33. Cotransfeced the senescent and young cells by Adeno-33and UV radiated pRL-CMV, which has a Renilla luciferase reporter gene, and observed the effect of damaged DNA repair by luciferase assay. Single cell gel electrophoresis was performed to analysis the effect of genomic DNA repair in senescent or young HDFs transfected with Adeno-33. (4)Observation the apoptosis of p33INGlb in senescent and young HDFs transfected with Adeno-33 after H2O2 damaged.Results: (l)In the 5'-flanking regulation region, there is an element between 49bp and 336bp because of the upregulation of the p33INGlb promoter activity in young HDFs. Another negative control element is located in this sequence between 1505bp and 1667bp because of the significantly downregulation of the luciferase reporter gene expression in young cells. There is still a third negative element between 1667bp and 1870bp, for there is a slightly upregulation of luciferase activity from 336bp to 1870bp. (2)Expression of these three isoforms was upregulated in young HDFs. This result is corresponding to the analysis of the p33ING1b promoter activity. The expression of p33ING1b and p24ING1c was downregulated in tumor cell lines(Lovo and HEPG2). (3)The ectopic overexpression of p33INGlb enhanced repair of damaged DNA, either exogenous plasmid or genomic DNA. This effect was more evidently in young HDFs than in senescent cells. (4) The ectopic overexpression of p33INGlb made young HDFs more sensitive to H2O2 damage. The survival percent of young cells after treated by H2O2 was obviously low, but the caspase-3 activity was high. This mean that the low survival percent of cells was related to apoptosis. On the other hand, the senescent HDFs, undergoing or not undergoing H2O2 treatment, was resistant to apoptosis.Conclusions: The promoter activity of p33INGlb is upregulated in young HDFs. There is an element in the 5' flanking regulation sequence. The expressionof p33INGlb, p24INGlc and p47INGla was different in various cell lines. These three isoforms of INGl had upregulated expression in young HDFs. p33INGlb enhance damaged DNA repair both in senescent and young HDFs. Low PDs HDFs with overexpression of p33INGlb trend to apoptosis.