MODULATION OF CELLULAR SENESCENCE 85 INFLUENCE OF HEXAPEPTIDE-11 ON PREMATURELY SENESCENT DERMAL PAPILLAE CELLS Dermal papillae cells grow at the base of the hair shaft and are responsible for the growth of new hair fi bers in anagen phase hair growth cycles (27). Dermal papillae cells from balding and non-balding individuals have been grown ex vivo (5). It has been demon- strated that dermal papillae cells taken from balding areas of the scalp show a higher level of senescence cellular subpopulations compared to dermal papillae from non-balding ar- eas. For the purposes of this study, an in vitro senescence testing model was developed using UV to push dermal papillae cells into premature senescence. Dermal papillae cells were exposed to 20 mJ/cm2 UVB for a period of time previously determined to not be cytotoxic to the cells, but known to elicit expression of measureable quantities of SA- β-Gal. After the irradiation, the cells were incubated for 48 h with various concentrations of the Hexapeptide-11, after which SA-β-Gal activity was determined. Exposure of dermal Figure 4. Expression of ATM protein in normal dermal fi broblasts aged for 22 population doubling cycles and treated for the last 18 cycles with Hexapeptide-11. Data for the same fi broblasts then tested one week after removal of the peptide from the media. Figure 5. Expression of SA-β-Gal protein in normal dermal fi broblasts aged for 22 population doubling cycles and treated for the last 18 cycles with Hexapeptide-11. Data for the same fi broblasts then tested one week after removal of the peptide from the media.

JOURNAL OF COSMETIC SCIENCE 86 papillae cells to UV radiation causes an increase in the expression of SA-β-Gal indicating the cells are expressing biochemical signatures of premature senescence (Figure 6). Treatment with Hexapeptide-11 shows a dose-dependent, statistically signifi cant decline in SA-β-Gal indicating reduced senescence at the 0.5% and 1.0% treatment levels. CONCLUSION Senescence, whether replicative or stress-induced, can be detected in both fi broblasts and dermal papillae cells through analysis of ATM protein expression and SA-β-Gal activity. A hexapeptide originally isolated from S. cerevisiae fermentation lysates and later synthe- sized at high purity when applied to both intrinsically and extrinsically aged fi broblasts in vitro was found to reduce gene expression of ATM and SA-β-Gal proteins in a dose- dependent fashion. Application of Hexapeptide-11 reduced ATM protein expression in fi broblasts that have undergone SIPS using H2O2 and those grown through intrinsic ag- ing cycles. SA-β-Gal activity was reduced in intrinsically aged fi broblasts exposed to Hexapeptide-11 as well. A method of using UV light to induce SIPS in dermal papillae was also developed. It was shown that Hexapeptide-11 also decreased SA-β-Gal activity in UV-irradiated dermal papillae cells. This suggests that the hexapeptide can delay senescence in a second dermal cell line. The infl uence of Hexapeptide-11 on senescence-associated aging appears to be broadly applicable and reversible. To date, this may represent the fi rst known peptide to possess these attributes. The impact of these fi ndings is being further substantiated on ex vivo models. The results of these fi ndings will be reported at a later date. REFERENCES (1) J. A. Knight, The Biochemistry of aging, Adv. Clin. Chem., 35, 1–62 (2000). (2) L. Hayfl ick and P.S. Moorhead, The serial cultivation of human diploid cell strains, Exp. Cell Res., 25, 585–621 (1961). (3) L. Hayfl ick, The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res., 37, 614–636 (1965). Figure 6. Analysis of SA-β-Gal expression in UV-induced prematurely senescent human dermal papillae cells compared against nonirradiated cells.