Abstract
ABSTRACT Proliferating cell nuclear antigen (PCNA), also known as a processivity factor of DNA polymerase delta and epsilon, is required for eukaryotic cell DNA synthesis and repair. Expression of PCNA gene is growth-regulated and stress-responsive. This thesis is composed of four chapters. In chapter 1, the general introduction of PCNA is provided. Previously, the rat PCNA promoter responsive to UV irradiation and serum stimulation has been localized to nucleotides -70 and it contains an ATF (nucleotides -51 to -44) and AP-1 sites (nucleotides -64 to -58). In chapter 2, the rat PCNA promoter, and the endogenous PCNA mRNA and protein are demonstrated to be UV inducible in serum or serum-free recovery, suggesting that this UV inducibility is ubiquitous in cycling and non-cycling cells but not an artifact. Similarly, cells synchronized at all stages show that the UV inducibility of rat PCNA promoter is increased in dose-dependent manner. Hence, the PCNA gene is firstly reported to be UV inducible in all cell stages. Subsequently, the regulating mechanisms for UV inducibility of PCNA gene are addressed. Concerned with the DNA damage induced by UV irradiation, the DNA-dependent protein kinase (DNA-PK) mutant cells (xrs-6) were found to be UV inducible as well as its parental CHO.K1 cells. Moreover, the DNA-PK down-stream pathways are known to involve the p53 and DNA-PK can phosphorylate the p53. However, UV inducibility of rat and human PCNA expression is found to be independent on the UV induced p53 expression (chapter 2 and 3, respectively). These observations suggest that DNA damage checkpoint pathways is not essential for UV inducibility of PCNA gene. Therefore, the cell signal pathways except the DNA damage are investigated in the followings. Given the thiol-rich nature of PCNA and its role in proliferation, PCNA might be affected by redox change. N-acetyl-L-cysteine (NAC), a GSH precursor, was used and inhibited this UV inducibility of PCNA promoter, suggesting that the UV inducibility of PCNA involves the oxidative stress. For comparison to rat PCNA expression as shown in chapter 3, the role of p53 in human PCNA gene expression is studied by infection of the human papillomavirus type 16 E6 oncogene into human fibroblasts (HF). The introduction of E6 to human fibroblasts abrogates the function of cellular p53 and p21WAF1 and the cell growth control in this system. For serum stimulation, the confluent E6-infected cells (E6-HF) but not the parental cells exhibited a marked induction of PCNA. For UV exposure, both parental and E6-infected cells show the PCNA induction at a modest level in the recovery of serum or serum-free containing medium. These observations suggest that p53 may be involved in serum stimulation but not in UV induction of human PCNA gene expression. Therefore, the UV inducibility of both rat and human PCNA expression shows the same characteristic of p53 independence. To define the transcription factors responsive to UV inducibility and serum responsiveness of PCNA promoter, the gel mobility shift assays (EMSA) were used in chapter 4. In serum stimulated or UV irradiated cells, nuclear extracts incubated with probe containing ATF and AP-1 sites also show the increasing formation of the DNA:protein complexes. Similarly, the AP-1 DNA binding ability is enhanced by serum stimulation or UV irradiation. Consisting with the finding, serum responsiveness and UV inducibility of rat PCNA promoter were shown to be AP-1 related by CAT assay previously. This is firstly reported that the DNA:protein interactions require the simultaneous involvement of ATF/CRE and AP-1 sites as either element can abrogate the complexes in the competition experiment, suggesting there are cross talk between these two sites when cells were serum-stimulated or UV-irradiated. Moreover, both the distance and the sequence are essential to complex formation in serum-stimulated cells. ATF-1, but not ATF-2 and CREB, in association with other factors is involved in regulating the serum stimulation of the rat PCNA promoter activity via the proximal ATF and AP-1 sites. In conclusion, this thesis establishes the UV inducibility of rat and human PCNA gene and provides the mechanisms for understanding the serum stimulation and UV induction of PCNA response. The most important contribution is to demonstrate that the oxidative stress, but not the DNA damage checkpoint pathways including DNA-PK and p53, is one of the factors for regulating the UV inducibility of PCNA gene.