Jeannie Kibitel

UV-DNA Damage in Mouse and Human Cells Induces the Expression of Tumor Necrosis Factor α

Ultraviolet light induces the expression of tumor necrosis factor α (TNFα) in many mammalian cells. We have examined the signal for this induction in a human DNA repair‐deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene. When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source. Further, treatment of UV‐irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V in‐creased the level of repair of dimers and concomitantly reduced CAT gene expression. Membrane‐bound TNFα expression was increased by UV and reduced by repair of dimers. Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV‐irradiated cells did not increase CAT activity. These results show that DNA damage is a signal for the induction of TNFa gene expression in mouse and human cells.

Intracellular localization and intercellular heterogeneity of the human DNA repair protein O 6 -methylguanine-DNA methyltransferase

Abstractu2002O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O6-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3u2005h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and its depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.

Regulation of TNFα production and release in human and mouse keratinocytes and mouse skin after UV‐B irradiation

TNFα is a primary cytokine responsible for inflammatory and immunosuppressive responses in skin. After UV‐B irradiation of cultured human keratinocytes, we found that TNFα was released into the media, as monitored by ELISA, and was bound to cells, as observed by immunofluorescence microscopy. The release of TNFα into cell culture supernatant during the 24 h after UV‐B irradiation was augmented by the addition of IL‐1α to the cells. Further, we found this secretion was unaffected by rapamycin, and therefore independent of FRAP DNA‐protein kinase mediated signal transduction. However, UV‐B also induced expression of membrane‐bound TNFα, and this was dependent on FRAP signaling. In wild type mice, TNFα bound to skin increased immediately after irradiation, declined at 6 h, and then rose again at 12 h before falling by 24 h. This pattern of induction was confirmed by RT‐PCR of TNFα mRNA message in cultured epidermal cells. Induction of membrane‐bound TNFα was also found in c‐fos gene knockout mice deficient in the AP‐1 transcription factor, suggesting that, although AP‐1 containing c‐fos signaling is required for some UV responses, AP‐1 containing c‐fos is not required for this TNFα activation. However, in homozygous p53 knockout mice the basal level of TNFα bound to the epidermis was greatly elevated without UV irradiation. This level declined and remained constant following irradiation. This implies that p53 directly or indirectly represses TNFα gene expression and that modification of p53 mRNA stability or phosphorylation of p53 protein after UV may be responsible for TNFα induction in the membrane. Overexpression of the immunosuppressive cytokine TNFα in this locale may contribute to the carcinogen‐susceptibility of p53 knockout mice.

ENHANCEMENT OF ULTRAVIOLET-DNA REPAIR IN den V GENE TRANSFECTANTS and T4 ENDONUCLEASE V-LIPOSOME RECIPIENTS

The phage T4 deri V gene, coding for the pyrimidine‐dimer specific T4 endonuclease V, was transfected into human repair‐proficient fibroblasts, repair‐deficient xeroderma pigmentosum fibroblasts, and into wild type CHO hamster cells. Transfectants maintained den V DNA and expressed deri V raRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair‐proficient and ‐deficient human cells. The den V transfected clones and liposome‐treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both den V gene transfection and endonuclease V liposome treatment enhanced post‐UV survival in xeroderma pigmentosum cells but had no effect on survival in repair‐proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells.

Direct comparison of DNA damage, isomerization of urocanic acid and edema in the mouse produced by three commonly used artificial UV light sources

Abstract— Exposure to sunlight can result in a number of harmful effects, including sunburn, erythema, premature aging of the skin, immune suppression and skin cancer. Studies designed to understand the underlying mechanisms often depend upon the use of artificial sources of UV radiation. Unfortunately, conclusions from different laboratories using different lamps often conflict, and it is entirely possible that the different spectra of sunlights used in each may be a source of conflict. To minimize confounding variables, we employed two of the more commonly used UY light sources, fluorescent sunlamps, such as the FS‐40 and Kodacel‐filtered FS‐40 sunlamps, and a xenon arc solar simulator and compared, in one series of standardized experiments, the effects of each light source on DNA damage, urocanic acid isomerization and edema formation. The dose‐response curves, calculated by linear regression or curve fitting were compared. The data indicate that DNA damage and urocanic acid isomerization were more sensitive to shorter wavelengths of UV than longer wavelengths, and the biological endpoint of edema most closely correlated with the induction of DNA damage. The results emphasize the dominance of shorter wavelengths within the UV spectrum in damaging biological tissues, even when the solar simulator, which contains significant amounts of UVA, was used and demonstrate that each light source has a characteristic pattern of induction of biochemical and biological endpoints.