Facile nucleophilic addition to salophen coordinated to nitridoosmium(VI).
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Journal Article↗
Biomedical subjects
S M Chiu
Publications and source records attributed to S M Chiu.
Photochemical destruction of the Bcl-2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4.
Photodynamic therapy (PDT), utilizing a photosensitizer and visible light, causes localized oxidative damage. With the mitochondrial photosensitizer Pc 4, PDT induces apoptosis, yet its molecular targets are not known. Here, the anti-apoptotic protein Bcl-2 is shown to be highly sensitive to PDT, as judged on Western blots by the disappearance of anti-Bcl-2-reactive material from the position of the native 26 kDa protein. The loss of Bcl-2 was PDT dose dependent and was observed for both endogenous and overexpressed Bcl-2 in several cell lines, immediately after PDT, and with chilled cells. It was accompanied by a trace of a 23-kDa cleavage product as well as high-molecular weight products that may result from photochemical crosslinking. PDT-induced Bcl-2 loss occurred in MCF-7 cells that do not express caspase-3 or in the presence of protease inhibitors, but was prevented, along with the induction of apoptosis, by the singlet oxygen scavenger L-histidine. Loss of FLAG-Bcl-2 was observed with both anti-FLAG and anti-Bcl-2 antibodies, indicating loss of native protein rather than simple BCL-2-epitope destruction. Photochemical damage was not observed in Bcl-x(L), Bax, Bad, the voltage-dependent anion channel, or the adenine nucleotide translocator. Therefore, Bcl-2 is one target of PDT with Pc 4, and PDT damage to Bcl-2 contributes to its efficient induction of apoptosis.
Adenocarcinoma↗
Dissociation of mitochondrial depolarization from cytochrome c release during apoptosis induced by photodynamic therapy.
Photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 induces rapid apoptosis in mouse lymphoma (LY-R) cells, initiating with the release of cytochrome c from mitochondria. It has been proposed that the opening of the mitochondrial membrane permeability transition pores, which results in the dissipation of the mitochondrial membrane potential (Deltapsi(m)), is essential for the escape of cytochrome c from mitochondria into the cytosol as well as for apoptotic cell death. Therefore, we have assessed the correlation between the loss of Deltapsi(m)and the release of cytochrome c following PDT. Treatment of LY-R cells with 300 nM Pc 4 and 60, 90 or 120 mJ/cm(2)of red light resulted in apoptosis of 80-90% of the cells, accompanied by >20-fold elevation in caspase-3-like activity within one h. At all 3 doses of PDT employed here, the majority of the cytochrome c was released from mitochondria at 15 min after irradiation, as determined by an immunohistochemical method. In contrast, the loss of Deltapsi(m)following PDT, as monitored by the uptake of JC-1 or Rh-123, depended on the PDT dose and the post-treatment time. In spite of the release of cytochrome c at 15 min after each of the 3 doses, a corresponding loss of Deltapsi(m)was observed only for those cells that received the highest dose of PDT. Virtually all cells that received one of the lower doses of PDT (300 nM Pc 4 plus 60 or 90 mJ/cm(2)) maintained normal Deltapsi(m). Hence, our results support the conclusion that the release of cytochrome c from mitochondria resulting from Pc 4-PDT-induced photodamage is independent of the loss of Deltapsi(m). Therefore, it is important to consider a range of doses of this or other apoptotic stimuli in deciphering the relationship of metabolic responses that contribute to apoptosis.
Animals↗
Photodynamic therapy-induced death of MCF-7 human breast cancer cells: a role for caspase-3 in the late steps of apoptosis but not for the critical lethal event.
Photodynamic therapy (PDT) causes mitochondrial damage and induces apoptosis through release of cytochrome c and activation of caspase-3. To test whether caspase 3 is the sole executioner of apoptosis and its role in overall cell lethality, we compared the response of MCF-7c3 cells that express a stably transfected CASP-3 gene to that of parental MCF-7:SW8 cells transfected with vector alone (MCF-7v). Following photosensitization with the phthalocyanine Pc 4 and red light, cytochrome c was released from the mitochondria to equivalent extents in the two cell lines. However, the appearance of apoptotic indicators, such as active caspase-3 (DEVDase), cleavage of poly(ADP-ribose) polymerase, and oligonucleosomal DNA fragmentation, was observed only in MCF-7c3 cells during the first 6 h after photosensitization. Although production of 50-kb DNA fragments and chromatin condensation were found in PDT-treated MCF-7v cells by 20-24 h posttreatment, the rate and extent of apoptosis were much less than in MCF-7c3 cells. MCF-7c3 cells were more sensitive to photosensitization than were MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to photodynamic killing when evaluated by a clonogenic assay. These results show (a) the importance of assessing overall cell death by clonogenic assay; (b) that the critical lethal event is independent of caspase-3, perhaps at or near the release of cytochrome c from mitochondria; and (c) that the caspase-3-mediated events appear to be irrelevant in determining overall killing of cells.
Apoptosis↗
FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4.
Oxidative stress, such as photodynamic therapy with the silicon phthalocyanine Pc 4 (Pc 4-PDT), can induce apoptosis and tumor necrosis factor alpha (TNF) production. TNF receptors, as well as other death receptors, have been implicated in stress-induced apoptosis. To assess directly the role of FADD, a death receptor-associated protein, in induction of apoptosis post-Pc 4-PDT, embryonic fibroblasts from FADD knock out (k/o) and wild-type (wt) mice were used. Pc 4-PDT induced casp-3 activation and apoptosis in both cell types. In the presence of zVAD, a pancaspase inhibitor, Pc 4-PDT-induced apoptosis was abrogated in both cell lines. Fumonisin B1 (FB), an inhibitor of ceramide synthase, had no effect on apoptosis after Pc 4-PDT in either cell line. Similar to Pc 4-PDT, exogenous C6-ceramide bypassed FADD deficiency and induced zVAD-sensitive apoptosis. In contrast to Pc 4 photosensitization, TNF did not induce either apoptosis or ceramide accumulation in FADD k/o cells. In the absence of FADD deficiency, TNF-induced apoptosis was zVAD-sensitive and FB-insensitive. Induced ceramide levels remained elevated after cotreatment with TNF and zVAD in FADD wt cells. Taken together, these data provide genetic evidence for a lack of FADD requirement in Pc 4-PDT- or C6-ceramide-induced apoptosis. FB-sensitive ceramide production accompanies, but does not suffice, for apoptosis after Pc 4 photosensitization or TNF.
Adaptor Proteins, Signal Transducing↗
Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells.
Photodynamic therapy (PDT) is a novel cancer treatment utilizing a photosensitizer, visible light and oxygen. PDT with the silicon phthalocyanine Pc 4, a new photosensitizer, is highly effective in cancer cell destruction and tumor ablation. The mechanisms underlying cancer cell killing by PDT are not fully understood. Tumor necrosis factor alpha (TNF) is a multifunctional cytokine that has been implicated in photocytotoxicity. We asked whether recombinant human TNF (rhTNF) affects Pc 4-PDT cytotoxicity in A431 human epidermoid carcinoma cells. Co-treatment of A431 cells with various doses of Pc 4-PDT and a sub-lethal rhTNF dose led to a sub-additive reduction in cell survival. In addition, in the presence of Pc 4-PDT or rhTNF, caspase-3 activity and apoptosis were induced. The combined treatment, however, did not potentiate either caspase-3 activity or apoptosis. Similar to previous findings we observed that Pc 4-PDT initiated a time-dependent extracellular TNF accumulation. The data suggest that: a) PDT and rhTNF induce cancer cell killing through different mechanisms; and b) Pc 4-PDT-induced TNF production is a stress response that may not directly affect photocytotoxicity.
Apoptosis↗
Contribution of circulating renin to local synthesis of angiotensin peptides in the heart.
The activity of a local cardiac renin-angiotensin system (RAS) has long been suspected in the promotion of cardiac pathologies including hypertrophy, ischemia, and infarction. All of the components of the RAS cascade have been demonstrated to be synthesized within the heart with the possible exception of the first enzyme in the cascade, renin. In the current study, we provide direct evidence that circulating renin can contribute to cardiac-specific synthesis of angiotensin peptides. Furthermore, we demonstrate this effect is independent of blood pressure and that in animals of comparable blood pressure, elevated circulating renin significantly enhances cardiac fibrosis. These results may serve to explain some of the cardiac pathologies associated with the RAS.
Angiotensin I↗
Fumonisin B1 does not prevent apoptosis in A431 human epidermoid carcinoma cells after photosensitization with a silicon phthalocyanine.
Photodynamic therapy with the phthalocyanine photosensitizer Pc 4 (Pc 4-PDT), an apoptosis inducer, is associated with accumulation of ceramide in various cell lines. The role of ceramide in Pc 4-PDT-induced apoptosis was investigated in A431 cells. Caspase-3 (casp-3) was activated and TUNEL positive cells began to appear 30 and 60 min post-Pc 4-PDT, respectively. A rapid increase (10 min) in cellular ceramide levels was observed after Pc 4-PDT. Induced ceramide accumulation was maintained over 60 min, Acid sphingomyelinase, a ceramide-generating enzyme, was inhibited after photosensitization with Pc 4, suggesting that the enzyme was not required for stimulated ceramide accumulation. Co-treatment of A431 cells with fumonisin B1, a ceramide synthase inhibitor, and Pc 4-PDT led to a decrease in ceramide levels without any effect on induced casp-3 activity or apoptosis. In the presence of zVAD, a pan-caspase inhibitor, apoptosis was abolished, while ceramide levels remained elevated after Pc 4-PDT. Exposure of A431 cells to exogenous C6-ceramide for 22 h, led to induction of apoptosis, and the process was abrogated by zVAD. In conclusion, C6-ceramide-, like Pc 4-PDT-induced apoptosis, is zVAD-sensitive. Furthermore, Pc 4 photosensitization can lead to apoptosis without FB-sensitive elevation in ceramide levels upstream of caspases.
Acyltransferases↗
Phthalocyanine 4-photodynamic therapy induces ceramide generation and apoptosis in acid sphingomyelinase-deficient mouse embryonic fibroblasts.
Photodynamic therapy (PDT), a novel cancer treatment using a photosensitizer and visible light, produces an oxidative stress in cells that can lead to apoptosis. PDT with the phthalocyanine photosensitizer Pc 4 (Pc 4-PDT), causes increased generation of ceramide, a lipid mediator, and subsequent induction of apoptosis in various cell types. Formation of ceramide by acid sphingomyelinase (ASMase) in response to stress has been implicated in apoptotic cell death. We assessed the role of ASMase in photocytotoxicity using mouse embryonic fibroblasts (MEFs) isolated from ASMase knockout (k/o) and wild-type (wt) mice. Exposure of wt or k/o MEFs to Pc 4-PDT led to increased caspase-3 activity and subsequent apoptosis. Similarly, ceramide levels were elevated in both cell types post-PDT. We suggest that in MEFs, ASMase is dispensable for ceramide accumulation and induction of apoptosis after Pc 4-PDT.
Animals↗
Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation.
L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9 dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40-60%, and via Complex III by 50-90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumption via electron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.
Animals↗
Differential dependence on chromatin structure for copper and iron ion induction of DNA double-strand breaks.
The induction of DNA DSB (double-strand breaks) in isolated nuclear chromatin by Cu(II) or Fe(II)-EDTA in the presence of H2O2 and ascorbate has been compared to DSB induction by gamma-radiation. V79 nuclei embedded in agarose plugs were treated with each agent on ice, and the resultant DNA fragments were analyzed by pulsed-field gel electrophoresis. In the absence of low molecular weight radical scavengers, both irradiation and treatment with iron ion induced random DSB, as judged by the size distribution of DNA fragments, and the yield of DSB in each case was enhanced by either the expansion of chromatin (approximately 5-fold) or the removal of histones (21-25-fold) before treatment. In contrast, treatment with Cu(II) produced small DNA fragments of uniform size (approximately 100-200 kbp), independent of the yield of DSB. In addition, neither the DNA fragment size nor the yield of DSB produced by Cu(II) was affected by the prior removal of histones from chromatin. Deproteinized DNA was degraded randomly by Cu(II) but at a slower rate than observed for chromatin. In the presence of ascorbate, H2O2 was found to be essential for DSB induction by Fe(II)-EDTA but not by Cu(II), possibly because H2O2 can be produced from ascorbate and Cu(II) in the presence of oxygen. Despite the above differences between the production of DSB by the two metal ions, DSB induction in native chromatin by either metal ion was blocked by 0.1 M EDTA or 0.25 M thiourea but was resistant to the hydroxyl radical scavengers 0.25 M DMSO and 0.25 M mannitol.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Induction of DNA damage in gamma-irradiated nuclei stripped of nuclear protein classes: differential modulation of double-strand break and DNA-protein crosslink formation.
The influence of chromatin proteins on the induction of DNA double-strand breaks (dsb) and DNA-protein crosslinks (dpc) by gamma-radiation was investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaCl, whereas nuclear matrix proteins were not extractable even by 2.0 M NaCl. The yield of dsb increased with progressive removal of proteins from chromatin. Whilst removal of low molecular weight non-histone proteins and histone H1 resulted in small increases in the production of dsb, removal of histones H2A/H2B, all histones, or all proteins led to 18.4, 46.4 and 55.5-fold increases in the yield of dsb, respectively, relative to irradiated cells. Therefore, both histones and non-histone proteins contribute to the radioprotection of DNA, core histones being the major radio-protectors. In contrast, depletion of chromatin proteins caused little or no effect on the induction of dpc until the chromatin was extracted with > or = 1.4 M NaCl. However, our studies indicated no direct, quantitative correlation between the removal of histones and the induction of dpc. The data support our previous conclusion that nuclear matrix protein rather than the majority of the histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions.
Animals↗
Copper ion-mediated sensitization of nuclear matrix attachment sites to ionizing radiation.
Exposure of mammalian cells to ionizing radiation induces nuclear matrix proteins and their attached transcribing DNA sequences to form cross-links. To characterize the cellular and matrix components necessary for DNA-protein crosslink (DPC) formation, DPC yields have been examined in isolated nuclear matrices and in the intermediate steps during cell fractionation. It was found that, in both unirradiated and irradiated cells, all components of DPC are retained in isolated nuclei, and the formed DPC are retained as well during the cell fractionation procedure resulting in nuclear matrices. In contrast, nuclear matrices isolated from unirradiated cells are deficient in the ability to form DPC upon irradiation, indicating that elements necessary for DPC production have been disrupted or removed during the isolation procedure. When isolated nuclei were irradiated, the yield of radiation-induced DPC was about 2-fold higher than that for intact cells, presumably due to the removal of soluble cellular scavengers during the isolation procedure. Treatment of nuclei with Cu2+ to stabilize nuclear structural organization during the preparation of the nuclear matrix caused additional DNA, especially the matrix-associated newly replicated DNA, to become bound to protein. Such treatment also enhanced radiation-induced DPC production which was sensitive to OH radical scavengers. Moreover, radiation-induced DPC production in Cu(2+)-treated nuclei was more sensitive to EDTA and catalase than in untreated nuclei. It is therefore proposed that excess DPC induction in Cu(2+)-treated nuclei occurs preferentially at the sites of Cu2+ binding to chromatin where hydroxyl radicals are produced repeatedly through the Fenton reaction.
Animals↗
Chromatin compaction and the efficiency of formation of DNA-protein crosslinks in gamma-irradiated mammalian cells.
Chromatin has been prepared from Chinese hamster V79 cell nuclei by successive suspension and sedimentation in buffers of decreasing ionic strength. For buffer concentrations from 50 to 1 mM, the resultant chromatin maintained a normal histone content, nucleosomal organization, and attachment to the nuclear matrix; however, as the buffer concentration was reduced from 50 to 10 and 1 mM, the higher-order chromatin structures became increasingly relaxed. Fully expanded chromatin is 5- to 10-fold more susceptible to the induction of DNA-protein crosslinks (DPCs) by gamma radiation than is chromatin residing in living interphase cells. As much as 60-70% of expanded chromatin can be induced to form DPCs as compared to a maximum of about 20% of cellular DNA. For expanded chromatin, the maximum level of induced DPCs is two to three times higher than would be expected if only matrix-associated DNA were induced to form DPCs. Therefore, DNA in distal regions of chromatin loops must also be induced to form DPCs with histones or other nonhistone chromosomal proteins. The hypersensitivity of isolated chromatin to radiation-induced production of DPCs appears to be related to the expansion of chromatin conformation rather than to the removal of intracellular radical scavengers for the following reasons: (a) there is an inverse relationship between the buffer concentration in which the chromatin is suspended and DPC formation, and (b) the induction of a more compact 30-nm chromatin fiber from the expanded 10-nm chromatin fiber in the presence of a low concentration of MgCl2 results in a marked reduction in DPC formation. The formation of radiation-induced DPC seems to occur at maximum efficiency in fully expanded chromatin, since DPC formation cannot be further stimulated by the addition of Cu2+, which can catalyze the production of OH by Fenton chemistry. It is concluded that radiation-induced DNA damage production is greatly influenced by chromatin conformation, and that chromatin as it exists in the cell is a relatively poor substrate for DNA-protein crosslinking in comparison to completely expanded chromatin.
Animals↗
The fate of DNA-protein crosslinks formed in gamma-irradiated metaphase cells.
The induction of DNA-protein crosslinks (DPC) was compared in gamma-irradiated metaphase and asynchronous Chinese hamster V79 cells. Unirradiated metaphase cells were found to have a higher level of background DPC than unirradiated asynchronous cells, and the metaphase cells were less susceptible to radiation-induced DPC production than were asynchronous cells. SDS-PAGE analysis of crosslinked proteins prepared from the two cell populations, both irradiated and unirradiated, showed very similar protein patterns. Crosslinked DNA was isolated and probed with radioactively labelled interphase poly(A+)RNA. The results indicated that the hypersensitivity of interphase actively transcribing DNA sequences to radiation-induced DPC formation was maintained at metaphase when the chromosomes are highly condensed. In contrast to asynchronous cells, radiation-induced DPC formed in metaphase cells were not removed during a 4 h post-irradiation period. However, metaphase cells appear to be able to remove the active DNA involved in DPC as indicated by a depletion of the probed sequences in the unrepaired DPC. Cell size analysis as well as cytological examination of the irradiated metaphase cells showed an absence of cell division during post-irradiation incubation. Furthermore, about 50% of the irradiated metaphase cells grew into giant cells which contain multiple nuclei and micronuclei, an indication of aberrant chromosome segregation.
Animals↗
Radiation-induced binding of DNA from irradiated mammalian cells to hydroxyapatite columns.
In experiments designed to measure radiation-induced DNA damage using the DNA unwinding-hydroxyapatite chromatography technique, we observed that under some experimental conditions a significant proportion of the test DNA became tightly bound to the hydroxyapatite (HA) and could not be released even with a high concentration of phosphate buffer. Approximately 5-10% of DNA from unirradiated cells binds to the HA. With increasing radiation doses in air, the fraction of bound DNA increases, reaching about 30% at about 35 Gy. The binding exhibits many of the characteristics of a radiation-induced cell lesion: the proportion of DNA retained by the HA is less when cells are irradiated under hypoxic conditions or in the presence of the thiol radioprotector dithiothreitol; and the binding decreases when an incubation period is allowed between irradiation and harvest of the cells for assay. Studies to determine the nature of the lesion responsible for the binding demonstrated that lesion production requires a component found in cells since no binding was observed with irradiated isolated DNA or nuclear matrix; the binding is not a result of the production of DNA-protein crosslinks; and the bound DNA is single-stranded, based on its sensitivity to nuclease S1. Because of the dose dependence of the binding of DNA to HA, the slopes of the dose-response curves for DNA damage determined with this assay depend on the method used to calculate the fraction of double-stranded DNA. Our demonstration that the bound DNA is single-stranded guides the choice of the method for data analysis.
Animals↗
Comparison of DNA-protein cross-links induced by 4'-(9-acridinylamino)-methanesulfon-m-anisidide and by gamma-radiation.
The antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits topoisomerase II activity through the formation of a complex of DNA and covalently bound enzyme which, upon protein denaturation, yields DNA breaks (single strand breaks). In the present study, this complex served as a standard for analysis of radiation-induced DNA-protein cross-links (DPC). Following the treatment of exponentially growing mouse L929 cells with 0-100 ng/ml of m-AMSA for 1 h, a linear dose-dependent increase was found in the amount of DNA retained on nitrocellulose filters during subsequent analysis. This result indicates that the assay can detect DPC that have a single protein bound to each DNA fragment. The results of fractionation of nuclear DNA show that m-AMSA induces 20- to 45-fold more DPC in nuclear matrix-associated DNA than in the majority distal loop DNA, supporting the notion that topoisomerase II is located at the nuclear matrix. The frequency of single strand breaks induced by m-AMSA, which should be equal to the frequency of DPC, was determined by alkaline elution. Results of the alkaline elution assay could be correlated with the percentage of DNA retained on nitrocellulose filters; i.e., 1% DNA retention corresponded to 2560 DPC per log-phase L929 cell, which has been determined to have a DNA content of 22.25 pg. Using this standard curve, DPC induced by gamma-irradiation in air were estimated to be formed at a frequency of 133 DPC/cell/Gy, a frequency approximately 3% that of gamma-ray-induced single strand breaks. The radiation dose response for DPC production was unaffected by the high levels of DPC present in cells previously treated with m-AMSA. In addition, DPC induced by m-AMSA were rapidly reversed after the removal of the drug, in contrast to a slower removal of DPC induced by gamma-radiation. These observations suggest that although m-AMSA and gamma-radiation both preferentially induce DPC with matrix-attached DNA, they produce independent types of DPC.
Amsacrine↗
Formation and repair of DNA-protein crosslinks in newly replicated DNA.
The production and removal of gamma-radiation-induced DNA-protein crosslinks (DPC) in nuclear matrix-associated newly replicated DNA were examined, as well as the relationship of DPC to DNA replication. In unirradiated, exponentially growing Chinese hamster V79 cells, DNA pulse labeled with [3H]thymidine was observed to be bound preferentially to protein. The pulse-labeled DNA subsequently became dissociated from protein. After a 30- to 60-min chase period, the level of labeled DNA in DPC was reduced to the same level as for bulk DNA. The radiation dose response for the formation of DPC was similar in newly replicated DNA that had been chased for various times and in mature chromatin DNA. Labeled DNA, in the DPC formed after 60 Gy, was rapidly removed from protein during the postirradiation incubation period. However, no recovery of DNA synthesis was observed, even after the majority of DPC were released. Thus either DPC are not the sole cause of the inhibition of DNA synthesis or their removal is not sufficient for DNA synthesis to resume.
Animals↗