Liang-yan Xue

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 Pcu20094, 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 26u2009kDa 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-xL, Bax, Bad, the voltage-dependent anion channel, or the adenine nucleotide translocator. Therefore, Bcl-2 is one target of PDT with Pcu20094, and PDT damage to Bcl-2 contributes to its efficient induction of apoptosis.

Bax is essential for mitochondrion-mediated apoptosis but not for cell death caused by photodynamic therapy

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 photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis 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. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF-7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.

Nuclear matrix proteins are crosslinked to transcriptionally active gene sequences by ionizing radiation.

Unirradiated, exponentially growing Chinese hamster cells contain a low level (less than 5%) of their DNA firmly bound to protein, as measured by a filter-binding assay. That fraction of DNA is highly enriched in sequences which hybridize to poly(A+)RNA or ribosomal RNA. After 60 Gy gamma irradiation, the additional crosslinked DNA is also enriched in transcriptionally active sequences compared to bulk DNA, while DNA crosslinked by uv radiation has a frequency of active sequences which is no higher than the bulk DNA. DNA crosslinked to protein by gamma radiation but not by uv is largely released during a 4-h postirradiation incubation. The DNA which remains bound to protein during that period becomes depleted in active sequences; this is followed by an apparent restoration of the active gene-enriched protein complex found in unirradiated cells. When nuclear matrix-associated DNA was isolated free of the majority (loop) DNA, an enrichment for active DNA sequences was found in the matrix-associated DNA, and the frequency of DNA-protein crosslinks was found to be 10- to 16-fold greater in the matrix fraction. Gel electrophoretic analysis of the crosslinking proteins identifies them as subset of proteins of the nuclear matrix. These data are consistent with known properties of the nuclear matrix and suggest that chromatin structure plays an important role in the formation and repair of gamma-radiation-induced DNA lesions.

Nuclear Structure and the Microdistribution of Radiation Damage in DNA

Evidence for the roles of proteins and metal ions in the microheterogeneity of DNA damage is reviewed. Decondensation of chromatin in hypotonic buffers markedly sensitizes the DNA to radiation, while treatment of nuclei with hypertonic buffers strips the DNA of histones and other nuclear proteins and enhances the radiosensitivity of the DNA with respect to double-strand break (dsb) formation. Addition of the radical scavenger DMSO reduces the yield of strand breaks, but dehistonized chromatin remains approximately 2.5 times more sensitive to radiation than does native chromatin at 0.1 M DMSO. DNA-protein crosslink (DPC) formation is relatively unaffected by the removal of the majority of histones from chromatin. Most DPC form at or near the nuclear matrix, and matrix is stabilized and radiosensitized by Cu++. To elucidate the role of Cu++, the induction of dsb and DPC by gamma-radiation has been compared with that by hydroxyl radical from Fe(++)-EDTA, or Cu++ catalysed Fenton reactions. Data comparing the size of DNA fragments produced, the effect of expanding or dehistonizing chromatin, and the effects of radical scavengers suggest that gamma-radiation and Fe(++)-EDTA produce dsb at open chromatin sites, whereas Cu(++)-generated dsb are similar to radiation-induced DPC in their location at the nuclear matrix. Both metal ions appeared to produce damage by site-specific generation of hydroxyl radicals. The nuclear matrix, the proteinaceous skeleton which anchors chromosomal loops and provides sites for DNA replication and transcription, binds metal ions and matrix-attachment DNA regions (MARs) consisting of 300 + bp of AT-rich DNA. The interaction of cloned MARs with isolated nuclear matrices has been found to be hypersensitive to crosslinking upon gamma-irradiation, in comparison with associations formed by similarly sized DNA fragments lacking MAR sequences. Thus, the non-random distribution of radiation damage is partially explained by the protection of DNA afforded by histones and chromatin structure and partially by the hypersensitivity of DNA-nuclear matrix associations.

INDUCTION OF DNA-PROTEIN CROSS-LINKS IN CHINESE HAMSTER CELLS BY THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo‐dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single‐strand breaks and DNA‐protein cross‐links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m2 of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy‐equivalents of single‐strand breaks. The repair of these breaks was slower than the repair of single‐strand breaks induced by m̀‐irradiation. The photodynamic action of CAPC also induces a large number of DNA‐protein cross‐links which, in contrast to m̀‐radiation‐induced DNA‐protein cross‐links, do not appear to be repaired during 4 h of post‐treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.

Induction of DNA Damage in γ-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.

ELEVATION OF GRP‐78 AND LOSS OF HSP‐70 FOLLOWING PHOTODYNAMIC TREATMENT OF V79 CELLS: SENSITIZATION BY NIGERICIN

Abstract— Chinese hamster V79 cells were treated with photodynamic therapy (PDT) sensitized by aluminum phthalocyanine (AlPc) or with the ionophore nigericin or with combinations of PDT and nigericin. We previously showed that PDT and nigericin interact synergistically in the killing of these cells; i.e. doses of PDT that kill no more than 10% of the cells in combination with nontoxic exposures to nigericin lead to a loss of clonogenicity of three to five orders of magnitude. Photodynamic therapy induces an enhanced rate of expression of the stress gene grp‐78 both at the transcriptional and trans‐lational levels and causes a decrease in the synthesis of the constitutive heat shock protein HSP‐70 as well as in expression of HSP‐70 mRNA. When the cells are exposed to PDT in the presence of nigericin, these effects are elicited at three‐ to four‐fold lower PDT doses. Thus, PDT in the presence of nigericin is much more effective in inducing the changes in gene expression than is PDT alone. In the absence of nigericin the PDT dose inducing a two‐fold increase in GRP‐78 accumulation causes little or no loss of clonogenicity. In the presence of nigericin, however, the PDT dose leading to a similar change in GRP‐78 level produces up to a 50% loss of clonogenicity. The fact that nigericin is dose‐modifying for both cell killing and stress responses suggests that nigericin either increases the yield of oxidative damage from a given dose of PDT or magnifies the cellular response to a constant level of oxidative stress.

Repair of Chromatin Damage in Glutathione-Depleted V-79 Cells: Comparison of Oxic and Hypoxic Conditions

We have assessed the effects of two radiomodifying conditions, glutathione (GSH) depletion and hypoxia, on the formation and repair of radiation-induced chromatin damage, specifically DNA-protein cross-links (DPC). As measured by a nitrocellulose filter-binding assay, untreated V79 cells contain a low level of DPC (1-1.5% of the cellular DNA). The background level of DPC is elevated in cells treated with L-buthionine sulfoximine (BSO), in hypoxic cells, and in cells treated with BSO and made hypoxic (2.98%, 2.82%, and 7.71%, respectively). The dose response for production of radiation-induced DPC is approximately 6.0% DNA bound per 100 Gy for cells irradiated in air, and the dose response is not significantly different for BSO-treated cells but increases by a factor of about 1.4 for hypoxic cells and 1.7 for BSO-pretreated hypoxic cells. DPC were also assayed by alkaline elution with or without proteinase K treatment. By this analysis, the yield of DPC appears to be elevated in irradiated hypoxic and irradiated GSH-depleted cells. It is not possible to assay for background DPC alone in unirradiated cells by alkaline elution. Cells not exposed to BSO repair 70-80% of the radiation-induced DPC in 4 h. BSO-treated cells are considerably less efficient in repair of DPC. As analyzed by alkaline elution, GSH depletion had little or no effect on the yield of radiation-induced single-strand breaks (SSB) but slowed their repair. The data suggest that depletion of GSH impairs an enzyme system(s) responsible for the turnover of both background and radiation-induced DPC and that hypoxia elevates both the background level of DPC and the ratio of radiation-induced DPC to SSB.

Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax+/−) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax+/− cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax+/− cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax+/− and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.

Chromatin compaction and the efficiency of formation of DNA-protein crosslinks in γ-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.