Isolation and identification of psoralen plus ultraviolet A (PUVA)-induced genes in human dermal fibroblasts by polymerase chain reaction-based subtractive hybridization.
Premature aging of the skin is a prominent side-effect of psoralen photoactivation, a therapy used for a variety of skin disorders. Recently, we demonstrated that treatment of human dermal fibroblasts with 8-methoxypsoralen and ultraviolet A irradiation resulted in a permanent growth arrest with a switch of mitotic to postmitotic fibroblasts. Furthermore, an upregulation of matrix-degrading metalloproteinases and a high level of de novo expression of the senescence-associated beta-galactosidase was detected in the PUVA-treated postmitotic fibroblasts. The molecular basis for this PUVA-induced change in the functional and morphologic phenotype of fibroblasts resembling or mimicking replicative senescence is, however, unknown. Herein after, we have used a polymerase chain reaction-based subtractive hybridization protocol to identify human genes that are induced by PUVA treatment. Application of polymerase chain reaction-Select resulted in the cloning of four PUVA genes. Sequence analysis and homology searches identified three cDNA clones of known genes related to cell cycle regulation (p21waf1/cip1), stress response (ferritin H) and connective tissue metabolism (tissue inhibitor of metalloproteinases-3), whereas one cDNA clone represented a novel gene (no. 478). Northern blot analyses were performed to confirm a PUVA-dependent increase in specific mRNA levels in human dermal fibroblasts in vitro. This report on the identification of growth arrest related genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these known and novel genes in extrinsic and intrinsic aging processes on a molecular and cellular level.
Wlaschek
M
M
Department of Dermatology, University of Cologne, Cologne, Germany.
Hommel
C
C
Wenk
J
J
Brenneisen
P
P
Ma
W
W
Herrmann
G
G
Scharffetter-Kochanek
K
K
D002455:Cell Division / Q000187:drug effects; D002648:Child; D002675:Child, Preschool; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005796:Genes / Q000187:drug effects*; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods; D011701:PUVA Therapy; D016133:Polymerase Chain Reaction / Q000379:methods; D013382:Subtraction Technique