Song Mao Chiu

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 α-helices 5/6 was also not photodamaged. Bcl-2 missing only one of those α-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 α5/α6 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.

Hypersensitivity of DNA in transcriptionally active chromatin to ionizing radiation

We have examined the size distribution of single-strand fragments of total 3H-labeled DNA and of DNA sequences complementary to specific probes in gamma-irradiated and unirradiated mouse L929 cells. Those DNA sequences which hybridize to rDNA or to poly(A+)RNA have lower number average molecular weights and sustain 5--6-times the number of single-strand breaks as do satellite DNA sequences or the bulk DNA. We therefore conclude that transcriptionally active DNA sequences are more susceptible to ionizing radiation-induced damage than are inactive sequences, and suggest that these differential susceptibilities are a likely consequence of differences in their chromatin organization.

Staurosporine-induced death of MCF-7 human breast cancer cells: a distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions

To test the role of caspase 3 in apoptosis and in overall cell lethality caused by the protein kinase inhibitor staurosporine, we compared the responses of MCF-7c3 cells that express a stably transfected CASP-3 gene to parental MCF-7:WS8 cells transfected with vector alone and lacking procaspase-3 (MCF-7v). Cells were exposed to increasing doses (0.15-1 microM) of staurosporine for periods up to 19 h. Apoptosis was efficiently induced in MCF-7c3 cells, as demonstrated by cytochrome c release, processing of procaspase-3, procaspase-8, and Bid, increase in caspase-3-like DEVDase activity, cleavage of the enzyme poly(ADP-ribose) polymerase, DNA fragmentation, changes in nuclear morphology, and TUNEL assay and flow cytometry. For all of these measures except cytochrome c release, little or no activity was detected in MCF-7v cells, confirming that caspase-3 is essential for efficient induction of apoptosis by staurosporine, but not for mitochondrial steps that occur earlier in the pathway. MCF-7c3 cells were more sensitive to staurosporine than MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to killing by staurosporine when evaluated by a clonogenic assay. A similar distinction between apoptosis and loss of clonogenicity was observed for the cancer chemotherapeutic agent VP-16. These results support our previous conclusions with photodynamic therapy: (a) assessing overall reproductive death of cancer cells requires a proliferation-based assay, such as clonogenicity; and (b) the critical staurosporine-induced lethal event is independent of those mediated by caspase-3.

Differential processing of ultraviolet or ionizing radiation-induced DNA-protein cross-links in Chinese hamster cells

The yield and repairability of DNA-protein cross-links have been compared after gamma- or U.V.-irradiation of Chinese hamster V79-379 lung fibroblasts. Using a filter-binding assay, cross-linked DNA can be specifically isolated after doses between 10 and 100 Gy of gamma-radiation and fluences between 20 and 300 J/m2 of U.V.-radiation. After ionizing radiation, the majority of DNA cross-linked to protein is released with biphasic kinetics, requiring 1 h for removal of 50 per cent of the cross-linked DNA and 24 h for 90 per cent release. In these cells, U.V.-induced cross-linked DNA is not removed; on the contrary, the yield of apparent DNA-protein complexes increases during postirradiation incubation. Prior gamma-irradiation, to initiate the associated repair system, does not stimulate release of U.V.-induced cross-linked DNA. Inhibition of protein synthesis by cycloheximide affects neither the removal of gamma-ray-induced cross-linked DNA nor the increase in U.V.-induced cross-linked DNA. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, slows the second phase of release after gamma-irradiation as well as the increase in apparent cross-links after U.V.-irradiation. Thus, even though both types of DNA-protein cross-links can be detected by the same assay, their structures or other factors must be substantially different, since the repair system for one type does not recognize the other.

Association Between the Photodynamic Loss of Bcl-2 and the Sensitivity to Apoptosis Caused by Phthalocyanine Photodynamic Therapy¶

Abstract We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT–induced apoptosis, whether overall cell killing is also sensitized or how upregulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2Δ33-54 and Bcl-2Δ37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2Δ210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2Δ33-54 or Bcl-2Δ37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells.

Photodamage to multiple Bcl-xL isoforms by photodynamic therapy with the phthalocyanine photosensitizer Pc 4

The antiapoptotic oncoprotein Bcl-2 is now a recognized phototarget of photodynamic therapy (PDT) with the phthalocyanine Pc 4 and with 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, no photochemical damage was observed to Bcl-xL, in spite of its similarity in size, sequence, location and function to Bcl-2. The original study used a commercial anti-Bcl-xS/L antibody. We have revisited this issue by examining Western blots developed using one of three epitope-specific anti-Bcl-xL antibodies from commercial sources, a polyclonal antibody generated to the entire protein, as well as the antibody used previously. All five Bcl-xL antibodies recognized bacterially expressed Bcl-xL, but not Bcl-2, whereas an anti-Bcl-2 antibody recognized Bcl-2 and not Bcl-xL. All five Bcl-xL antibodies recognized at least one protein migrating at ∼30 kDa; two of the antibodies recognized an additional band, migrating at ∼33 or ∼24 kDa. We now observe Pc 4-PDT-induced photodamage to all Bcl-xL-related proteins, except the 33-kDa species, in several human cancer cell lines. The results indicate that, in addition to the expected quantitative differences that may reflect exposure of individual epitopes, the antibodies also detect proteins of different apparent molecular weights that may be distinct isoforms or post-translationally modified forms of Bcl-xL. No evidence for PDT-induced phosphorylation or degradation was observed. Bcl-xL localized to mitochondria was considerably more sensitive to photodamage than was Bcl-xL in the cytosol, indicating that as previously found for Bcl-2, Bcl-xL must be membrane localized to be photosensitive.

Atg7 deficiency increases resistance of MCF-7 human breast cancer cells to photodynamic therapy

Photodynamic therapy (PDT) uses a photosensitizer, light, and oxygen to produce extensive oxidative damage to organelles housing the photosensitizer. Although PDT is an efficient trigger of apoptosis, it also induces autophagy in many kinds of cells. Autophagy can serve as both a cell survival and a cell death mechanism. Our previous study indicates that autophagy contributes to cell death after PDT, especially in apoptosis-deficient cells. Here, we provide further evidence to support the role of autophagy in cell killing after PDT. Autophagy was blocked by knockdown of one essential factor, LC3 or Atg7, in MCF-7 cells. The cells were exposed to a range of doses of PDT sensitized by the phthalocyanine Pc 4; steps in autophagy were monitored by western blotting for LC3-II and by fluorescence microscopy for the uptake of monodansylcadaverine or for the distribution of transfected GFP-LC3; and overall cell death was monitored by MTT assay and by clonogenic assay. We find that blocking autophagy increased the survival of MCF-7 cells after PDT and increased the shoulder on the dose-response curve. In response to Pc 4-PDT, Atg7-deficient MCF-7 cells remained capable of robust accumulation of LC3-II, but were defective in comparison to Atg7+ cells in the formation of autophagosomes. We conclude that apoptosis-deficient cells rely on autophagy for cell death after Pc 4-PDT and that the strong activation of LC3 maturation in response to PDT could occur even in cells with limited or no Atg7 expression.

Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4‐loaded cells, membrane components, especially Bcl‐2, are photodamaged and apoptosis, as indicated by activation of caspase‐3 and cleavage of poly(ADP‐ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three‐fold higher affinity constants for liposomes. The hydroxyl‐bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4‐PDT photodamages Bcl‐2 and Bcl‐xL, Pc 181‐PDT causes much less photodamage to Bcl‐2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase‐3‐dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl‐bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Promotion of Photodynamic Therapy-Induced Apoptosis by the Mitochondrial Protein Smac/DIABLO: Dependence on Bax¶

Abstract Photodynamic therapy (PDT) using the second-generation photosensitizer phthalocyanine (Pc) 4 causes mitochondrial damage and induces apoptosis through the release of cytochrome c to the cytosol. Another protein of the mitochondrial intermembrane space, Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), is also released to the cytosol in response to apoptotic stimuli and promotes caspase activation by binding IAP. To investigate the possible role of Smac/DIABLO in apoptosis induced by Pc 4-PDT, we transfected Smac/DIABLO (tagged at its C-terminus with green fluorescent protein [GFP]) into MCF-7c3 cells (human breast cancer MCF-7 cells stably transfected with procaspase-3) and DU-145 cells (human prostate cancer cells that express no Bax because of a frameshift insertion mutation). Confocal microscopy showed that recombinant Smac/DIABLO, like cytochrome c, localized to mitochondria and colocalized with MitoTracker Red. Three hours after exposure of MCF-7c3 cells to PDT (200 nM Pc 4 and 150 mJ/cm2 red light), Smac/DIABLO–GFP, as well as cytochrome c, was found largely in the cytosol. In contrast, for DU-145 cells, both Smac/DIABLO–GFP and cytochrome c remained in the mitochondria after PDT. By staining with Hoechst 33342, typical apoptotic nuclei were observed in MCF-7c3 cells, but not in DU-145 cells, after Pc 4-PDT. These results suggest that the release of Smac/DIABLO from mitochondria may be regulated by a Bax-mediated mechanism and that Smac/DIABLO may cooperate with the cytochrome c–dependent apoptosis pathway. In addition, in MCF-7c3 cells transfected by Smac/DIABLO–GFP, apoptosis induced by Pc 4-PDT was greater than in cells transfected with the GFP vector alone or in untransfected cells, as determined by flow cytometry. Thus, Smac/DIABLO promotes apoptosis after Pc 4-PDT in a Bax-dependent manner and may facilitate the passage of PDT-treated cells through the late steps of apoptosis.

The Death of Human Cancer Cells Following Photodynamic Therapy: Apoptosis Competence is Necessary for Bcl‐2 Protection but not for Induction of Autophagy†

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro‐apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase‐3 (human breast cancer MCF‐7v cells) and in apoptosis‐competent cells (MCF‐7c3 cells that stably overexpress human pro‐caspase‐3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl‐2. Autophagy was identified by electron microscopic observation of the presence of double‐membrane‐delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4‐PDT dose‐ and time‐dependent increase in the level of LC3‐II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl‐2. The presence of stably overexpressed Bcl‐2 in the cells protected against PDT‐induced apoptosis and loss of clonogenicity in apoptosis‐competent cells (MCF‐7c3 and CHO 5A100 cells). In contrast, Bcl‐2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis‐deficient cells (MCF‐7v and DU145 cells). Furthermore, 3‐methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis‐deficient than to apoptosis‐competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis‐competent cells are protected by Bcl‐2 against cell death.