Stimulation of melanogenesis by DNA oligonucleotides: effect of size, sequence and 5 ' phosphorylation

It has been shown that the small DNA fragment thymidine dinucleotide, (pTpT ) induces photoprotective responses in cultured cells and intact skin. Thes e responses include increased melanogenesis, enhanced DNA repair, and induc tion of TNF-alpha, and are accomplished, at least in part, through the indu ction and activation of the p53 tumor suppressor and transcription factor. Here it is reported that other, but not all, larger oligonucleotides induce the pigmentation response even more efficiently than pTpT. A 9 base oligon ucleotide (p9mer) stimulated pigmentation in Cloudman S91 murine melanoma c ells to 6-times the level of control cells while a 5 base oligonucleotide ( p5mer # 1) was inactive. In addition, the p9mer increased p21 mRNA levels a nd inhibited cell proliferation to a greater degree than did pTpT, consiste nt with the presumptive mechanism of action involving p53. Smaller, truncat ed versions of the p9mer also stimulated pigmentation, although to a lesser extent than did the p9mer. The ability of these oligonucleotides to stimul ate pigmentation was highly dependent on the presence of a 5' phosphate gro up on the molecule, which was shown by confocal microscopy and fluorescent activated cell sorter (FACS) analysis to greatly facilitate the uptake of t hese oligonucleotides into the cells. Although the melanogenic activity of the oligonucleotides was directly related to increased length and 5' phosph orylation, nucleotide sequence is also critical because a p20mer was effici ently internalized yet was a poor inducer of pigmentation. (C) 2001 Elsevie r Science Ireland Ltd. All rights reserved.