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Thymidine dinucleotide (pTpT) stimulates melanogenesis in mammalian pigment cells and intact skin, mimicking the effects of UV irradiation and UV-mimetic DNA damage. Here it is shown that, in addition to tanning, pTpT induces a second photoprotective response, enhanced repair of UV-induced DNA damage. This enhanced repair results in a 2-fold increase in expression of a UV-damaged chloramphenicol acetyltransferase expression vector transfected into pTpTtreated skin fibroblasts and keratinocytes, compared with diluent-treated cells. Direct measurement of thymine dimers and (6-4) photoproducts by immunoassay demonstrates faster repair of both of these UV-induced photoproducts in pTpT-treated fibroblasts. This enhanced repair capacity also improves cell survival and colony-forming ability after irradiation. These effects of pTpT are accomplished, at least in part, by the up-regulation of a set of genes involved in DNA repair (ERCC3 and GADD45) and cell cycle inhibition (SDI1). At least two of these genes (GADD45 and SDI1) are known to be transcriptionally regulated by the p53 tumor suppressor protein. Here we show that pTpT activates p53, leading to nuclear accumulation of this protein, and also increases the specific binding of this transcription factor to its DNA consensus sequence.

Melanoma is the most fatal skin cancer, often highly resistant to chemotherapy. Here we show that treatment with an 11-base DNA oligonucleotide homologous to the telomere 3' overhang sequence (T-oligo) induces apoptosis of several established human melanoma cell lines, including the aggressive MM-AN line, whereas normal human melanocytes exposed to the same or higher T-oligo concentrations show only transient cell cycle arrest, implying that malignant cells are more sensitive to T-oligo effects. When MM-AN cells were briefly exposed to T-oligo in culture and injected into the flank or tail vein of SCID mice, eventual tumor volume and number of metastases were reduced 85-95% compared with control mice. Similarly, T-oligos administered intralesionally or systemically selectively inhibited the growth of previously established MM-AN tumor nodules in the flank and peritoneal cavity by 85 to 90% without detectable toxicity. We previously showed that T-oligos act through ATM, p95/Nbs1, E2F1, p16INK4A, p53, and the p53 homologue p73 to modulate downstream effectors and now additionally demonstrate striking down-regulation of the inhibitor of apoptosis protein livin/ML-IAP. We suggest that T-oligo mimics a physiologic DNA damage signal that is frequently masked in malignant cells and thereby activates innate cancer prevention responses. T-oligos may provide a novel therapeutic approach to melanoma.

Although the ability of UV irradiation to induce pigmentation in vivo and in vitro is well documented, the intracellular signals that trigger this response are poorly understood. We have recently shown that increasing DNA repair after irradiation enhances UV-induced melanization. Moreover, addition of small DNA fragments, particularly thymine dinucleotides (pTpT), selected to mimic sequences excised during the repair of UV-induced DNA photoproducts, to unirradiated pigment cells in vitro or to guinea pig skin The prokaryotic response to UV irradiation, the so-called SOS response, is well documented and is now known to include the induction of a set of >20 genes involved in DNA repair and cell survival (reviewed in ref. 1). In this case, the single-stranded DNA generated after UV irradiation interacts with and activates a protease, the Rec A protein (2). Activated Rec A protein then cleaves and inactivates the repressors of specific genes, leading to their induction (2).In eukaryotic cells, the existence of a UV-induced DNA damage-responsive SOS-like system mediated by one common transcription regulator has been the subject of considerable controversy. Although a variety of genes are known to be induced by DNA damage (3-6), many of these genes are also induced by agents such as phorbol esters (3, 7) and by metabolic or oxidative stress (8-10). Because UV irradiation is reported, like phorbol esters, to activate protein kinase C directly (11,12) and to produce oxidative damage through generation of free radicals from membrane lipids and otherThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. extranuclear cellular constituents, it cannot be determined whether the effects of UV are due directly to DNA damage or instead to other impacts on the cell. Indeed, two of the major UV-induced transcription factors, AP-1 and NF-KB, are now thought to initiate their responses at or near the plasma membrane (13,14).Perhaps the best-characterized example of DNA damagespecific gene induction involves a DNA repair enzyme, photolyase, encoded by the PHR1 gene in Saccharomyces cerevisiae (6). This gene is induced by a variety of DNA-damaging agents including UV light, methyl methanesulfonate (MMS) and 4-nitroquinoline 1-oxide (4-NQO). Up-regulation of PHR1 gene transcription is, at least in part, accomplished by the removal of a damage-responsive repressor which binds to a specific site in the 5' region of the gene (15).Another well-studied UV and DNA damage-inducible gene is the mammalian GADD45 gene. This gene is transcriptionally activated not only by UV irradiation but also by ionizing radiation and chemical agents that specifically cause base damage (10, 16). The induction of GADD45 by ionizing radiation is mediated by the p53 tumor suppressor protein and the ataxia telangiectasia gene product (17), but the UV-and base-damage responses are less well understood. ...