Domain-dependent photodamage to Bcl-2. A membrane anchorage region is needed to form the target of phthalocyanine photosensitization.
Photodynamic therapy using the photosensitizer Pc 4 and red light photochemically destroys the antiapoptotic protein Bcl-2 and induces apoptosis. To characterize the requirements for photodamage, we transiently transfected epitope-tagged Bcl-2 deletion mutants into DU-145 cells. Using confocal microscopy and Western blots, wild-type Bcl-2 and mutants with deletions near the N terminus were found in mitochondria, endoplasmic reticulum, and nuclear membranes and were photodamaged. A mutant missing the C terminus, including the transmembrane domain, spread diffusely in cells and was not photodamaged. Bcl-2 missing alpha-helices 5/6 was also not photodamaged. Bcl-2 missing only one of those alpha-helices, with or without substitutions of the singlet oxygen-targeted amino acids, behaved like wild-type Bcl-2 with respect to localization and photodamage. Using green fluorescent protein (GFP)-tagged Bcl-2 or mutants in live cells, no change in either the localization or the intensity of GFP fluorescence was observed in response to Pc 4 photodynamic therapy. Western blot analysis of either GFP- or Xpress-tagged Bcl-2 revealed that the photodynamic therapy-induced disappearance of the Bcl-2 band was accompanied by the appearance of bands indicative of heavily cross-linked Bcl-2 protein. Therefore, the alpha(5)/alpha(6) region of Bcl-2 is required for photodamage and cross-linking, and domain-dependent photodamage to Bcl-2 offers a unique mechanism for activation of apoptosis.
Usuda
Jitsuo
J
Department of Radiation Oncology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106,USA.
Chiu
Song-mao
SM
Murphy
Erin S
ES
Lam
Minh
M
Nieminen
Anna-Liisa
AL
Oleinick
Nancy L
NL
D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism / Q000528:radiation effects; D008297:Male; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D026082:Singlet Oxygen / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D008164:Luminescent Proteins; D017319:Photosensitizing Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D049452:Green Fluorescent Proteins; D026082:Singlet Oxygen