Reference #: CHI-1017-267485
Submit Date: 03/27/2002 15:45:04-0500

The Pro-apoptotic Protein Bax is Essential for Mitochondrion-Mediated Apoptosis but not for Cell Death After Photodynamic Therapy

ABSTRACT:
The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that PDT with the photosensitizer Pc 4 induced Bax translocation from the cytosol to mitochondria prior to cytochrome c release and caspase activation in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. As a second approach, Bax-deficient human prostate cancer DU-145 cells were studied. Following PDT, the appearance of hallmarks of apoptosis, including cytochrome c release, caspase-3 (DEVDase) activity, and condensed chromatin, were blocked. However, after transient transfection of these cells with an expression plasmid encoding Bax-GFP fusion protein, flow cytometric analysis indicated induction of apoptosis exclusively in the Bax-GFP-positive cells. Thus, the resistance of DU-145 cells to PDT-induced apoptosis seems due to the lack of Bax rather than another defect in the apoptotic machinery. Bak, a related pro-apoptotic protein which can compensate for Bax in other systems, is present in both cell lines but was apparently not activated by PDT to form higher-order oligomers. When overall cell killing was monitored by clonogenic assay, Bax-deficient DU145 cells were about as photosensitive as Bax-replete MCF-7c3 cells, and overexpresssion of Bcl-2 did not alter the photocytotoxicity of DU145 cells. Thus, it appears that the Bax/Bcl-2 ratio is not always a determinant of cytotoxicity. (Research supported by NIH grants P01CA48735, R01CA83917, and P30CA43703).

Keywords: apoptosis, PDT, Bax

Reference #: USU-1017-270969
Submit Date: 03/27/2002 17:02:18-0500

Identification of the region of Bcl-2 photodamaged by Pc 4-PDT

ABSTRACT:
We have reported that photodynamic therapy (PDT) sensitized by the phthalocyanine Pc 4 destroys the anti-apoptotic protein Bcl-2. To elucidate the mechanism of the photodamage, we constructed Bcl-2 mutants in which regions of the protein are deleted. We transfected plasmids expressing His-tagged wild-type (239 amino acids) or mutated Bcl-2 into human prostate cancer DU-145 cells, which have little endogenous Bcl-2 protein. The intracellular localization of the expressed proteins was observed by confocal microscopy, and the extent of photodamage was assessed on western blots after a PDT dose (200 nM Pc 4 + 200 mJ/cm(2) red light) causing 90% loss of clonogenicity of non-transfected cells. Wild-type Bcl-2 protein localized in mitochondria, endoplasmic reticulum and the nuclear envelope, as previously reported. Mutants with deletions (del) in the N-terminal half of Bcl-2 (del 33-54, 37-63, or 10-125) localized similarly to wild-type Bcl-2 and were readily photodamaged by PDT. Thus, Asp-34, a caspase cleavage site, is not required for photodamage, and there are no essential target sites in the N-terminal half of Bcl-2. A mutant missing the C-terminal region (del 210-239), including the transmembrane (TM) domain, localized in a diffuse non-mitochondrial pattern and was not destroyed by Pc 4-PDT, indicating the importance of membrane anchorage for photodamage. Bcl-2 (del 153-179), in which the region between the BH1 and BH2 domains is deleted, was found only in mitochondria but was not photodamaged, in spite of having a TM domain. This region contains two alpha helices, which form a secondary membrane-anchoring site essential for pore formation. However, Bcl-2 proteins missing only one of the alpha helices (del 153-168 or 168-179) behaved like wild-type Bcl-2. These results suggest that Pc 4 binds to the membranes very near to the secondary insertion, pore-forming site of Bcl-2. (Research supported by NIH grants P01CA48735, R01CA83917, and P30CA43703).

Keywords: PDT, Pc 4, Bcl-2, photodamage

Reference #: XUE-1017-334451
Submit Date: 03/28/2002 10:36:07-0500

A Second Protein Target of Photodynamic Therapy on the Mitochondrial Membrane: the Anti-apoptotic Bcl-2 Homolog, Bcl-XL

ABSTRACT:
The anti-apoptotic oncoprotein Bcl-2 is now a recognized phototarget of PDT with the phthalocyanine Pc 4 and with certain other mitochondrion-targeting photosensitizers. Photodamage, observed on western blots as the loss of the native 26-kDa Bcl-2 protein, is PDT dose dependent and occurs in multiple cell lines, in the cold, and immediately upon photoirradiation. In our initial study (Xue et al., Oncogene 20: 3420-3427, 2001), no photochemical damage was observed in Bcl-XL, in spite of its similarity in size, sequence, and location to Bcl-2. The original study used a Bcl-XL/S antibody provided by Santa Cruz (cat. no. sc-634). To explore further, we have revisited this issue by examining western blots with alternative antibodies. We now find that photodamage to Bcl-XL is also observed upon PDT to several cell lines, including DU145, DU145 overexpressing Bcl-2, MCF-7, MDA-MB468 and A431, if the proteins are probed with an anti-Bcl-X antibody from Transduction Laboratories (cat. no. 610746). In contrast, the Bcl-XL band detected on western blots by an antibody from Pharmingen (cat. no. 66461A) was shifted in position when the proteins derived from PDT-treated cells. To assess the specificity of these antibodies, they were tested against bacterially expressed Bcl-2 and Bcl-XL proteins. All three Bcl-XL antibodies recognized Bcl-XL but not Bcl-2; whereas the anti-Bcl-2 antibody recognized only Bcl-2 and not Bcl-XL. Finally, the three Bcl-XL antibodies detected proteins of different sizes (24-31 kDa). The results suggest that the antibodies detect different isoforms of Bcl-XL, at least one of which is photodamageable. Based on information from studies of Bcl-2, it is possible that the differential photosensitivity of the isoform(s) is due to their differential localization within the mitochondrial outer membrane and other sites. (Research supported by NIH grants P01 CA48735, R01 CA83917, and P30 CA43703).

Keywords: PDT, Bcl-XL, apoptosis

Reference #: KOC-1017-519927
Submit Date: 03/30/2002 13:58:02-0500

UVA initiates rapid apoptosis in HL-60 cells by the Fas/FADD/caspase-8 pathway

ABSTRACT:
The mechanism for immediate apoptosis induced by UVA radiation is not well defined. Apoptosis in HL-60 cells, as detected by nuclear morphology and internucleosomal fragmentation of DNA, occurs as early as 2 h after treatment with 5 -15 J/cm2 UVA (364 nm, argon laser). A broad caspase inhibitor, VAD-FMK, but not neutralizing anti-Fas antibody, blocked UVA-induced apoptosis. UVA treatment induced the cleavage of caspase-8 and subsequent processing of Bid and caspase-3-like proteases. Inhibition of caspase-8 by IETD-FMK completely blocked caspase-3 cleavage and apoptosis. UVA treatment stimulated Fas clustering , as observed by immunofluorescence microscopy, and aggregation of FADD to Fas, as demonstrated by immunoprecipitation. In addition, an increase in caspase-8 was detected with Fas immunoprecipitation. These data suggest that rapid formation of the Fas/FADD/caspase-8 complexy induced by UVA treatment is independent of Fas ligand and leads to activation of the apoptotic pathway in HL-60 cells.

Keywords: apoptosis, UVA, plasma membrane, Fas