Study Of Molecular Mechanism Of Keratinocyte Damage Induced By Ultraviolet B

Background Keratinocytes (KC) are main components of epidermis which are subject to a vast array of environmental insults. As ultraviolet B (UVB) component of the solar spectrum exposure is one of the leading causes of skin cancer, it is imperative to try to understand how KC responds to UVB irradiation. A hallmark feature of UV exposure in skin are so-called sunburn cells, which represent apoptotic epidermal KC. Previously, the functional role of these UVB-damaged KC was obscure, but recent evidence suggests that sunburn cells are not only markers for severe sun damage, and also this apoptotic process is important for preventing skin cancer. UVB damage KC via similar intracellular responses, jointly termed "the stress response", including genotoxic stress which is induced by direct DNA damage, and oxidative stress which is induced by reactive oxygen species (ROS). The inability of KC to UVB-induced stress responses can lead to the development of skin cancers.Cell cycle arrest is the first event in the process of UVB-induced stress response. Cell cycle checkpoints are thought to be protective because they allow additional time for injured cells to repair damaged DNA and other essential molecules. In absence of error-free DNA repair may giverise during DNA replication to mutations in key gene, these genes are involved in the maintenance of genetic integrity (caretaker genes) or govern signaling pathways implicated in the control of cellular proliferation, differentiation, and survival (gatekeeper genes). Apoptosis is the hallmark events when the UVB-damaged DNA cannot be repaired. Apoptosis represents a controlled scavenging mechanism which protects gene-mutated cells from malignant transformation. The UVB-induced stress response includes activation of particular signal transducing pathways and transcription factors, DNA repair enzymes, apoptotic molecules, as well as cytoskeletal changes. For apoptotic process, it involves activation of a series of biochemical events that underlie the morphological hallmarks of apoptosis. This includes internucleosomal DNA fragmentation and proteolytic degradation of specific substrates. Proteolysis is achieved through activation of cysteine proteases called caspases. For signal transducing pathways, tumor suppressor p53 is centrally involved in regulating the homoeostatic response to UVB-induced DNA damage. Several functions and features of p53 include its stability, cellular localization, and transcriptional activity to its downstream genes, such as p21, MDM2, Bax, and GADD45, et al. For transcription factors, nuclear factor-KB (NF-kB) seems to be involved in not only normal KC development and differentiation, but also carcinogenesis and UVB-induced stress response.The last but not the least, p16^INK4a, the tumor suppressor protein that control cell growth through the Rb-CDK4 and p53 pathways respectively is important regulator of cellular stress and proliferative behavior. Thus, to elucidate the multiple and complex signal transducing pathways will provide more information in UVB-induced skin carcinogenesis and the targets for molecular therapy of skin cancer.Methods Cultured normal human epidermal keratinocyte (NHEK) and immortalized Human Keratinocyte Cell Line—HaCaT cells (harboring mutant p53) were irradiated with UVB in dose of 20,40,60,80,100 and 120 mJ/cm2. The cell viability, cell cycle and apoptosis were simultaneously detected on the time of 2,12,24,48 and 72 hour after irradiation. The caspase-mediated apoptosis was detected by FITC-VAD-FMK which can irreversibly bind to activated caspases, cell membrane-mediated apoptosis was detected by FITC-Annexin V and PI double-stain, and cell nucleus-mediated apoptosis was detected by DNA ladder.The expression of p53, p21, MDM2, p16^INK4a, Bax, GADD45, and NF-kB following UVB irradiation of NHEK and HaCaT both at the mRNA(RT-PCR and real time quantitation RT-PCR) and the protein(Western blotting) levels. The activation of NF-icB/p65 was detected by Electromobility Shift Assay (EMSA). We also use the NF-kB specific inhibitor...