Abstract
ABSTRACT Although CHO.K1 cells are popularly chosen for biological research, the status of p53 gene in this cell is unclear. Recently, we have illustrated that p53 of CHO.K1 is UV inducible and functional as a transcription activator; although the sequencing characterization indicate that there is a missense mutation at amino acid residue 211. It has been noted that CHO.K1 cells fail to arrest at G1 after X-ray or gamma ray irradiation. The failure was suspected to be due to the dysfunction of p53. However, in our study, the cells after radiation still fail to arrest at G1 even with over-expression of wild type p53, suggesting that some component other than p53 is responsible for the phenomenon. A specific mechanism that results in the failure to accumulation of endogenous p21 (Waf1/Cip1) by over-expressing wild type p53 is involved. As an important environmental insult, UV was thought to activate p53 and related genes causing G1 arrest in many cells. Unlike other cell lines, CHO.K1 cells are characterized by arrest at G2 but not G1 after X-ray irradiation in the presence of colcemid. When CHO.K1 cells were treated in the same way except that the X-ray irradiation was replaced by a short-wavelength (254 nm) UV exposure, a massive cell death occurred. The massive death was also observed in the presence of other mitotic inhibitors and several cell cycle inhibitors. Besides, the massive cell death does not require cell cycle progression. Although the p21 (Waf1/Cip1) gene was present in the genome by southern blotting analysis, the expression of p21 (Waf1/Cip1) was undetectable in CHO.K1 cells in both protein and RNA levels. To gain insight into the effect of p21 (Waf1/Cip1) in UV-induced apoptosis in CHO.K1 cells, wild type human p21 (Waf1/Cip1) was ectopicly expressed. Over-expression of the wild type human p21 (Waf1/Cip1) in CHO.K1 cells prevented the apoptotic death. It suggests that the UV-induced apoptosis in CHO.K1 cells, which was exacerbated by various cell cycle inhibitors, may be result from the failure of p21 (Waf1/Cip1) expression. In second part of this dissertation, the focus is on the autoantibody of PCNA. A study was conducted using an ELISA screening of the anti-PCNA autoanntibodies in sera of patients with SLE. Eighteen out of 191 SLE patient sera were found to be positive, giving a frequency of nearly 10%. Among the positive sera, a sample with highest titer of anti-PCNA autoantibody preferentially recognizes the wild type PCNA as compared to the mutant Y114A, which contains a single amino acid substitution at 114 and fails to form the toroidal structure. Moreover, the autoantibody purified from this serum identifies only the free PCNA in crude mammalian cell extracts. This finding raises a possibility that the immunostaining with the human anti-PCNA autoantibodies might detect only part of total PCNAs in tissues. Besides, in order to examine the association of antibodies to PCNA in patients with HBV and HCV virus infection, various patients’ sera were tested for the presence of anti-PCNA antibodies by ELISA and immunoblotting, and the isotypes of anti-PCNA antibody were also identified. In ELISA screening, anti-PCNA antibodies were detected in 30 (12.3%) patients with chronic HBV infection, 71 (18.7%) patients with chronic HCV infection, and 5 (6.3%) patients with SLE. The inhibition of binding with these sera by purified recombinant PCNA was shown to exceed 71%. By immunoblotting, the positive frequency of anti-PCNA antibody in patients with chronic HBV and HCV infection was 17/243 (7%) and 41/379 (11%), respectively. Absorption studies on IIF showed that the typical nuclear speckled staining pattern by anti-PCNA sera was abolished by pre-incubation of sera with free PCNA. Anti-PCNA antibody was not detected in sera from patients with autoimmune diseases except SLE. The isotype of anti-PCNA antibodies in patients suffering from chronic HBV and HCV infection were predominantly IgG, indicating that anti-PCNA autoantibody may occur in the autoimmune response. These findings emphasize the correlation between autoimmune and viral infections.