In vitro and in vivo transfer of bcl-2 gene into keratinocytes suppresses UVB-induced apoptosis.
Bcl-2 is a member of the large Bcl-2 family and protects cells from apoptosis. Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes that is known as "sunburn cells." Previously we reported that UVB irradiation induces apoptosis accompanied by sequential activation of caspase 8, 3 and 1 in keratinocytes, and that the process is inhibited by various caspase inhibitors. Using bcl-2-expressing adenovirus vector we investigated the effect of Bcl-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bcl-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (1 x 10(6)) were transfected at 1 x 10(8) plaque-forming unit (PFU)/mL. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on the Bcl-2 expression level. Following UVB irradiation caspase 8, 3 and 9 activities were stimulated in NMK cells, whereas in bcl-2-transfected cells only caspase 8, but not caspase 3 or 9, activity was stimulated. In order to investigate the effect of Bcl-2 in vivo topical application of Ad-bcl-2 on tape-stripped mouse skin was performed. Following the application Bcl-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bcl-2 on the first day following the application of 1 x 10(9) PFU in 200 microL. The introduced Bcl-2 remained at least for 6 days. UVB irradiation (1250 J/m2) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Both bcl-2-transfected and topical caspase 3 inhibitor-treated mice skin were resistant to UVB-induced apoptosis. The suppressive effect of Bcl-2 was more potent than that of caspase 3 inhibitor application. Topical application of empty adenovirus vector alone had no effect on Bcl-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.
Takahashi
H
H
Department of Dermatology, Asahikawa Medical College, 2-1-1 Midorigaoka higashi, Asahikawa 078-8510, Japan. ht@asahikawa-med.ac.jp
Honma
M
M
Ishida-Yamamoto
A
A
Namikawa
K
K
Miwa
A
A
Okado
H
H
Kiyama
H
H
Iizuka
H
H
D000256:Adenoviridae; D000818:Animals; D017209:Apoptosis / Q000528:radiation effects*; D015153:Blotting, Western; D020170:Caspase 1 / Q000378:metabolism*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured / Q000378:metabolism / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000528:radiation effects; D004817:Epidermis / Q000378:metabolism / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000528:radiation effects*; D019254:Genes, bcl-2; D005822:Genetic Vectors; D006651:Histocytochemistry; D006801:Humans; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects; D051379:Mice; D008807:Mice, Inbred BALB C; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects; D014162:Transfection; D014466:Ultraviolet Rays / Q000009:adverse effects*
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D020170:Caspase 1