David C. Gan

Green Tea Phenol Extracts Reduce UVB‐induced DNA Damage in Human Cells via Interleukin‐12†

Green tea chemoprevention has been a focus of recent research, as a polyphenolic fraction from green tea (GTP) has been suggested to prevent UV radiation‐induced skin cancer. Recently, it was demonstrated that GTP reduced the risk for skin cancer in a murine photocarcinogenesis model. This was accompanied by a reduction in UV‐induced DNA damage. These effects appeared to be mediated via interleukin (IL)‐12, which was previously shown to induce DNA repair. Therefore, we studied whether GTP induction of IL‐12 and DNA repair could also be observed in human cells. KB cells and normal human keratinocytes were exposed to GTP 5 h before and after UVB. UVB‐induced apoptosis was reduced in UVB‐exposed cells treated with GTP. GTP induced the secretion of IL‐12 in keratinocytes. The reduction in UV‐induced cell death by GTP was almost completely reversed upon addition of an anti‐IL‐12‐antibody, indicating that the reduction of UV‐induced cell death by GTP is mediated via IL‐12. The ability of IL‐12 to reduce DNA damage and sunburn cells was confirmed in “human living skin equivalent” models. Hence the previously reported UV‐protective effects of GTP appear to be mediated in human cells via IL‐12, most likely through induction of DNA repair.

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures

Normal human dermal fibroblasts have a limited life‐span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase‐3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

The Cytoprotective Effects of Exogenous DNA Fragments

Ultraviolet irradiation of normal human keratinocytes induces a cytotoxic effect. The chromophore for this effect is believed to be genomic DNA. However, DNA damage is known to be repaired in UVB irradiated keratinocytes. The trigger for this DNA repair is potentially damaged DNA itself. To test the hypothesis that damaged DNA can induce the host cell’s own DNA repair mechanism, we treated the keratinocytes with the damaged DNA and evaluated its cytoprotective effects. We have observed that fragmented calf thymus DNA irradiated and damaged with a UVC light can induce a protective effect in cultured human keratinocytes. Keratinocytes treated with UVC damaged DNA fragments are less susceptible to UVB irradiation-induced cell death as measured by neutral red uptake. Unirradiated exogenous DNA did not induce this protective effect. Similar protective effects can be seen with irradiated salmon sperm DNA. UVC damaged DNA fragments induced 60% increase in protection in human HaCaT keratinocyte in culture to the cell death induced by UVB. Similar protection was observed with UVC irradiated oligothymidylic acid (dT3-dT5) which increased the survival of human HaCaT keratinocytes after UVB irradiated by 50%. Isolated mononucleotides, irradiated or not, do not increase UVB survivability. Cellular DNA synthesis was greatly inhibited by UVB, becoming undetectable at 40 mJ/cm2. Exogenous treatment with damaged fragments causes immediate and significant inhibition of total cellular DNA synthesis. This inhibition was dose dependent. Cells that undergo damage to their DNA are known to inhibit endogenous DNA synthesis via p53 suppressor gene activation. This is believed to allow them sufficient time to repair the host DNA. The cellular response to exogenous damaged DNA may be a similar mechanism.