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In Vitro and In Vivo Transfer of bcl‐2 Gene into Keratinocytes Suppresses UVB‐induced Apoptosis ¶
Author(s) -
Takahashi Hidetoshi,
Honma Masaru,
IshidaYamamoto Akemi,
Namikawa Kazuhiko,
Miwa Akiko,
Okado Haruo,
Kiyama Hiroshi,
Iizuka Hajime
Publication year - 2001
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/0031-8655(2001)0740579ivaivt2.0.co2
Subject(s) - in vivo , apoptosis , in vitro , gene transfer , chemistry , microbiology and biotechnology , gene , cancer research , biology , biochemistry , genetics
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 × 10 6 ) were transfected at 1 × 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 × 10 9 PFU in 200 μL. The introduced Bcl‐2 remained at least for 6 days. UVB irradiation (1250 J/m 2 ) 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.