UVB-induced Apoptosis and p53 Expression in Cultured Normal Human Keratinocyte

Kim, Myeung Nam; Seo, Seong Jun; Hong, Chang Kwun; Ro, Byung In; Ro, Sung Wook; Cho, Sung In

Abstract

Cutaneous absorption of ultraviolet B(UVB) in the skin occurs primarily in keratinocyte, causing DNA and protein damage. p53 tumor suppressor gene appeared in the epidermis after UVB irradiation, and the wild type has been known to be responsible for apoptosis and plays an important role in excluding abnormal cells with significant DNA damage. While p53 has been implicated in both DNA repair and apoptosis, it is unclear whether the p53 protein is involved in both of these processes within the same cell. Therefore, UVB-induced apoptosis and changes in p53 expression were studied in cultured normal human keratinocyte to determine that the cellular response to UVB induced DNA damage(DNA repair or apoptosis) correlated with p53 expression. The cultured normal human keratinocytes were irradiated with the doses of UVB(25-150 mJ/cm2) and incubated for various times(3, 6, 12, 24 hour) after radiation. At UVB doses of 100 and 150 mJ/cm2, acridine orange/ethidium bromide(Ao/Eb) staining-positive cells and TUNEL (TdT mediated dUTP-biotin nick end labeling) staining-positive cells increased significantly after 3 hours and 6 hours postirradiation respectively. Twelve hour postirradiation, staining-positive cells increased at each level of UVB-radiation exposure. These results suggest that there were significant influences of UVB doses and time course after irradiation to the number of Ao/Eb and TUNEL staining-positive cells. To determine whether all Ao/Eb and TUNEL-positive cells were actually undergoing apoptosis, cellular DNA was extracted from keratinocytes at 12 hours after UVB irradiation and seperated by electrophoresis on an 2.5% agarose gel to detect the internucleosomal DNA fragmentation(DNA ladder). 'DNA ladder' occurred at every dose of UVB 12 hour after irradiation, but did not appear early after irradiation, suggesting that whether Ao/Eb and TUNEL-positive cells observed early after irradiation were not undergoing apoptosis. Activation of p53 and the response to DNA damage is not observed universally, but is dependent on tissue specificity, species specificity and type of genotoxic damage. To correlate p53 level with UVB-induced apoptosis at the dose of 100mJ/cm2 UVB, p53 levels were determined by western blot analysis. The accumulation of p53 protein was apparent after 6 hours postirradiation, and UVB irradiation caused a dramatic increase in p53 levels at 12 and 24 hours. These results demonstrate that p53 is required for UVB-induced apoptosis in cultured normal human keratinocyte and p53 has a time-dependent effect in the initiation of apoptosis. In this study, the results indicated that a low dose(25mJ/cm2) of UVB irradiation could induce apoptosis in human keratinocyte in vitro and UVB exerts a time-dependent effect on inducing apoptosis. And the results also give support to increasing evidence that p53 may play a role in UVB-induced DNA damage and the induction of apoptosis in cultured normal human keratinocyte and that p53 is involved in the decision process which determines the fate of keratinocyte after UVB -induced DNA damage.