Reference #: AND-1019-510516
Submit Date: 04/22/2002 15:43:33-0500

Deciphering endoplasmic reticulum and mitochondria specific pathways of apoptosis by selective targeting of Bcl-2

ABSTRACT:
In cells the anti-apoptosis protein Bcl-2 is found at both mitochondria and endoplasmic reticulum. To identify the function(s) of Bcl-2 at each organelle, we created mutants with restricted sub-cellular localization. At the endoplasmic reticulum Bcl-2 prevents cell death due to a subset of insults including serum starvation, ceramide and taxol. At the mitochondria Bcl-2 prevents apoptosis induced by these agents and also etoposide and doxorubicin. Cell death due to etoposide and doxorubicin are not blocked by Bcl-2 at the endoplasmic reticulum. We have used cell lines expressing these mutants to dissect two pathways for apoptosis that differ in regulation of cell-cell adhesion, activation of the pro-apoptosis protein Bax, cytochrome c release, and mitochondrial transmembrane potential. We conclude that Bcl-2 regulates two different but convergent pathways for apoptosis that are spatially restricted to either the endoplasmic reticulum or mitochondria.

Keywords: Bcl-2, Apoptosis, endoplasmic reticulum, mitochondria

Reference #: KOC-1017-517941
Submit Date: 03/30/2002 12:56:10-0500

Singlet oxygen initiated pro-and anti-apoptotic processes at the plasma membrane

ABSTRACT:
Signaling processes leading to cell survival and growth as well as those initiating apoptosis occur at the plasma membrane in response to singlet oxygen. The balance between these pathways may determine the fate of the cell. The results of two studies will be contrasted. Singlet oxygen activated the cell survival protein, protein kinase B (PKB; also called Akt), in a time and light-dose dependent manner in fibroblasts. Phosphorylation of PKB, was observed at 5 min, detectable until at least 2 h and dependent on activation of PI3-kinase. Inhibition of PI3-kinase enhanced singlet oxygen-induced cell death demonstrating that this survival pathway is functionally significant. Singlet oxygen induced covalent dimerization, with minimal phosphorylation, of platelet-derived growth factor receptor (PDGFR), did not activate SH-PTP2, a substrate of growth factor receptors. Inhibition of PDGFR did not affect Akt phosphorylation. These results and others suggest that Akt activation by singlet oxygen is independent of growth factor receptors even though Akt provides a survival signal that protects cells following singlet oxygen-induced damage. In another study, we examined whether pro-survival signaling from the epidermal growth factor receptor (EGFR) is altered during singlet oxygen-induced apoptosis in human keratinocytes. Conditions causing apoptosis produced time-dependent loss of EGFR, first observable at 5 min. EGFR degradation was not blocked by either inhibition of receptor internalization or by agents interrupting the major proteolytic pathways. However, pre-treatment with a caspase-3 inhibitor blocked EGFR degradation, and singlet oxygen-induced cleavage of caspase-3 was detected within 5 min. In addition, recombinant active caspase-3 completely cleaved EGFR in a membrane fraction. The singlet oxygen-induced loss of EGFR was accompanied by dephosphorylation of EGFR as well as of Akt and ERK1/2, downstream effectors of EGFR that promote cell survival and proliferation. These results indicate that singlet oxygen rapidly activates caspase-3 activitywhich subsequently degrades EGFR but is not involved in dephosphorylation of EGFR, Akt and ERK1/2. These results and others suggest that inhibition of pro-survival pathways may contribute to ROS-induced apoptosis in human keratinocytes.

Keywords: singlet oxygen, apoptosis, survival, growth factor receptors

Reference #: 095719
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Reference #: REI-1019-153688
Submit Date: 04/18/2002 08:45:44-0500

Lysosomal Photodamage and Bid-initiated Apoptosis

ABSTRACT:
Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to the loss of mitochondrial membrane potential. The mechanism by which lysosomal disruption initiates apoptosis is not known. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes of the adherent murine hepatoma cell line 1c1c7. Irradiation of cultures preloaded with NPe6 induced a very rapid disruption of the lysosomes, release of lysosomal proteases, and subsequent cleavages and activations of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of mitochondrial membrane potential. Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. A variant of the 1c1c7 cell line was as susceptible as the parental cells to the induction of apoptosis by staurosporine or doxorubicin, but underwent necrosis, instead of apoptosis, following PDT with NPe6. Lysosomal disruption occurred in the variant line following PDT. However, lysosomal disruption was not accompanied by Bid cleavage, cytochrome c release, or pro-caspase activation. Extracts of purified lysosomes from the variant line did not catalyze the in vitro cleavage of Bid. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that cause the activation of Bid. This work was supported in part by NIEHS grant ES09392.

Keywords: apoptosis, pdt, bid, lysosomes

Reference #: OLE-1017-079188
Submit Date: 03/25/2002 11:47:39-0500

Determinants of Bcl-2 photosensitivity and possible role in PDT-induced cell death.

ABSTRACT:
Photodynamic therapy (PDT) causes photooxidative damage to membrane lipids and proteins that reside within a few nm of the photosensitizer binding sites. Identification of specific target molecules can help elucidate detailed mechanisms for how photodamage induces subsequent biochemical responses, including cell death. With the phthalocyanine photosensitizer Pc 4, as well as with other photosensitizers that act on mitochondria, the endoplasmic reticulum (ER), and the nuclear membrane, a few protein phototargets have been defined. The anti-apoptotic protein Bcl-2, as well as some isoforms of its homolog Bcl-xL, are found in the above organellar membranes and are sensitive phototargets of PDT. A group of ER membrane proteins, including the ER calcium ATPase, may also be phototargets. We know most about photodamage to Bcl-2. Oxidation of critical residues leads to crosslinking of Bcl-2 to itself or to other neighboring proteins. To help reveal the sensitive regions of Bcl-2, a series of mutants has been constructed in which various regions of the Bcl-2 protein are deleted. Two regions were found to be the most critical: the C-terminal transmembrane domain, which is necessary to properly anchor the protein to the organellar membranes; and an internal region harboring two alpha helices that are thought to form a secondary membrane-anchoring site essential for pore formation and for photodamage. Among the consequences of Bcl-2 photodamage are the recruitment of the pro-apoptotic Bax to the mitochondria and the release of cytochrome c, which triggers subsequent steps in apoptosis. Study of specific photosensitizer-protein interactions will be important for further defining the mechanism of the initial photodamage to cells by PDT.

Keywords: photodynamic therapy, apoptosis, phthalocyanine, Bcl-2

Reference #: KES-1016-209841
Submit Date: 03/15/2002 10:28:46-0500

Sub-cellular targets for PDT

ABSTRACT:
Photodynamic therapy (PDT) has been employed for treatment of neoplasia, macular degeneration and atherosclerotic disease. We have examined a variety of photosensitizing agents and found that the mode of cell death depends on site(s) of initial sensitizer localization. Based on studies with a variety of tumor cells in culture, we have classified PDT sensitizers into six major groups. Class I sensitizers target the anti-apoptotic protein bcl-2; Class II = lysosomes & bcl-2; Class III = lysosomes only; Class IV = lysosomes & plasma membrane, Class V = the plasma membrane; Class VI = bcl-2, lysosomes & plasma membrane. When bcl-2 is the primary target, there is a rapid apoptotic response. Lysosomal photodamage induces apoptotic cell death by releasing lysosomal enzymes that catalyze bid cleavage, resulting in promotion of bax oligomerization, an effect that results in the release of cytochrome c from mitochondria. This is a slow process, compared with the much more rapid caspase activation when Class I or II sensitizers are used. Agents that target the plasma membrane are associated with a greatly delayed apoptotic response. This derives from relocalization of sensitizers to the cytosol during irradiation, resulting in photodamage to procaspase- and caspase-3. The Class I and Class II agents we have examined to date (mTHPC, SnET2, the porphycene CPO) are highly selective for bcl-2, leaving unaffected both bax and the bcl-2 analog bcl-x_L. This selectivity likely relates to the sub-cellular site(s) of sensitizer localization since protoporphyrin derived from ALA initially resides in mitochondria, and causes photodamage to both bcl-2 and bcl-x_L as does the phthalocyanine Pc4 (as recently reported by Oleinick's group). Pc 4 shows some mitochondrial localization. Effects of Class I sensitizers can be mimicked by the synthetic bcl-2/bcl-x_L antagonist HA14-1. These include a rapid loss of the mitochondrial membrane potential, translocation of cytochrome c to the cytosol, appearance of an apoptotic morphology within 60 min, and a marked promotion of caspase activation by the bile acid UDCA or the PKC inhibitor staurosporin. We suggest that, in addition to its therapeutic value, PDT may be a useful tool for analyzing apoptotic phenomena.

Keywords: photodynamic, apoptosis, photosensitization