Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. The initiating events of PDT-induced apoptosis are poorly defined. It has been shown for other proapoptotic stimuli that the integral endoplasmic reticulum protein Bap31 is cleaved by caspases 1 and 8, but not by caspase-3. Further, a 20 kDa Bap31 cleavage fragment is generated which can induce apoptosis. In the current report, we sought to determine whether Bap31 cleavage and generation of p20 is an early event in PDT-induced apoptosis. The mitochondrial release of cytochrome c, involvement of caspases 1, 2, 3, 4, 6, 7, 8, and 10 and the status of several known caspase substrates, including Bap31, were evaluated in PDT-treated HeLa cells. Cytochrome c appeared in the cytosol immediately following light activation of the photosensitizer benzoporphyrin derivative monoacid ring A. Activation of caspases 3, 6, 7, and 8 was evident within 1^2 h post PDT. Processing of caspases 1, 2, 4, and 10 was not observed. Cleavage of Bap31 was observed at 2^3 h post PDT. The caspase-3 inhibitor DEVD-fmk blocked caspase-8 and Bap31 cleavage suggesting that caspase-8 and Bap31 processing occur downstream of caspase-3 activation in PDT-induced apoptosis. These results demonstrate that release of mitochondrial cytochrome c into the cytoplasm is a primary event following PDT, preceding caspase activation and cleavage of Bap31. To our knowledge, this is the first example of a chemotherapeutic agent inducing caspase-8 activation and demonstrates that caspase-8 activation can occur after cytochrome c release.z 1998 Federation of European Biochemical Societies.

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D 2 O) which enhances singlet oxygen ( 1 O 2 ) lifetime only increases necrosis without aecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identi®ed as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kB activation and apoptosis were clearly independent although NF-kB was shown to counteract apoptosis mediated by PPME photosensitization. Oncogene (2001) 20, 4070 ± 4084.

Inflammatory mediators of sepsis induce apoptosis in many cell lines. We tested the hypothesis that lipopolysaccharide (LPS) injection in vivo results in induction of early apoptotic and survival pathways as well as evidence of late-stage apoptosis in the heart. Hearts were collected from control rats and at 6, 12, and 24 h after LPS injection (4 mg/kg). Activation of an apoptotic pathway was identified by a 1,000-fold increase in caspase-3 activity at 24 h (P < 0.05). Confirmation of these results occurred when terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining identified myocardial cells undergoing DNA fragmentation with significant levels at 24 h post-LPS injection. LPS also caused early proapoptotic mRNA (Bax) to increase (16% at 24 h, P < 0.05), whereas the Bax protein initially decreased (35% at 6 h, P < 0.05) and then returned to baseline values by 24 h. Six hours after LPS injection, Bcl-2 (early prosurvival) mRNA levels increased, whereas its protein levels decreased (70%, P < 0.05) and then returned to baseline levels by 24 h. Mitochondrial cytochrome c levels decreased, suggestive of mitochondrial involvement. Thus involvement of proapoptotic and prosurvival pathways in the heart occurs during a septic inflammatory response.

Background-The mechanisms through which NOS2-mediated pathways regulate graft failure in acute cardiac rejection are ill defined. To determine whether apoptosis promoted by NOS2 may contribute, we used a heterotopic transplant model to study mouse cardiac allografts placed in recipients with targeted gene deletion of NOS2. Methods and Results-Using 5 different indexes of apoptosis, we showed that mouse cardiac allografts placed in NOS2 Ϫ/Ϫ recipients (nϭ7) had reduced apoptotic activity compared with those in NOS2 ϩ/ϩ controls (nϭ8). There were significantly fewer TUNEL-positive nuclei per high-powered field (PϽ0.01), less DNA fragmentation (antinucleosome ELISA; PϽ0.05), lower corrected transcript levels for caspase-1 and -3 ( 32 P reverse transcriptase-polymerase chain reaction; PϽ0.01), and reduced caspase-3 activity (cleavage of DEVD-pNA [PϽ0.001] and poly [ADP-ribose] polymerase) in grafts from NOS2 Ϫ/Ϫ recipients. This concordant reduction in apoptotic indexes paralleled the improved histological outcome of grafts transplanted into NOS2 Ϫ/Ϫ recipients (assessed as rejection scores; Pϭ0.012). To identify pathways controlled by NOS2, we compared intragraft transcript levels of potential triggers and regulators. Whereas Fas ligand/Fas and tumor necrosis factor (TNF)-␣/TNF receptor-1 levels were not altered by NOS2 deficiency, transcript levels for p53 were significantly lower in grafts from NOS2 Ϫ/Ϫ recipients, coinciding with a significant increase in the antiapoptotic Bcl-2/Bax balance and decrease in Bcl-X l levels. Conclusions-Using NOS2 knockout mice, we demonstrated that NOS2-mediated pathways can promote acute rejection, at least in part, by inducing apoptotic cell death. When NOS2 is present, p53 might control NOS2-mediated apoptosis by stimulating Bax and repressing Bcl-2 and Bcl-X l expression, which may activate the cell death program in the rejecting heart. (Circulation. 1999;99:836-842.)