Libby R. Friedman

The Induction of Partial Resistance to Photodynamic Therapy by the Protooncogene BCL-2

Abstract— Photodynamic therapy (PDT) is an efficient inducer of apoptosis, an active form of cell death that can be inhibited by the BCL‐2 oncoprotein. The ability of BCL‐2 to modulate PDT‐induced apoptosis and overall cell killing has been studied in a pair of Chinese hamster ovary cell lines that differ from one another by a transfected human BCL‐2 gene in one of them (Bissonnette et al., Nature 359,552–554, 1992). Cells were exposed to the phthalo‐cyanine photosensitizer Pc 4 and various fluences of red light. Pc 4 uptake was identical in the two cell lines. The parental cells displayed a high incidence of apoptosis after PDT, whereas at each fluence there was a much lower incidence of apoptosis in the BCL‐2‐expressing cells. Apoptosis was monitored by (a) observation of 50 kbp and oligonucleosome‐size DNA fragments by gel electrophoresis, (b) flow cytometry of cells labeled with fluores‐cently tagged dUTP by terminal deoxynucleotidyl transferase and (c) fluorescence microscopy of acridine orange‐stained cells. The time course of apoptosis varied with the PDT dose, suggesting that only after moderately high doses (> 99% loss of clonogenicity) was there a relatively synchronous and rapid entry of many cells into apoptosis. At PDT doses reducing cell survival by 90 or 99%, significant increases in apoptotic cells were found in the population after6–12 h. Clonogenic assays showed that BCL‐2 protein inhibited not only apoptosis but overall cell killing as well, effecting a two‐fold resistance at the 10% survival level. Thus, BCL‐2‐expressing cells may be relatively resistant to PDT.

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.

Gamma radiation as a probe of chromatin structure: damage to and repair of active chromatin in the metaphase chromosome.

Cobalt-60 gamma radiation has been employed as a means of preferentially damaging actively transcribing chromatin within interphase and metaphase Chinese hamster V79-379 lung fibroblasts. The single-strand size distribution and break frequency of bulk 3H-labeled DNA have been compared to those same parameters for active sequences, i.e., sequences complementary to 125I-labeled poly(A+)RNA. The results show that (a) sequences active during interphase are more sensitive than inactive sequences to single-strand break formation by gamma radiation even when the chromatin is condensed in metaphase, (b) repair of strand breaks in the bulk DNA is slower in metaphase than in interphase cells, but (c) during metaphase, repair is faster in active sequences than in the bulk DNA. Furthermore, this study demonstrates that chromatin structure can be probed within intact cells by a method which circumvents isolation of nuclei or chromatin and the use of exogenous nucleases.

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.

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.

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.

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.

POST-TREATMENT INTERACTIONS OF PHOTODYNAMIC and RADIATION-INDUCED CYTOTOXIC LESIONS

Abstract— The interaction of chloroaluminum phthalocyanine‐sensitized photodynamic treatment and γ‐irradiation was studied in confluent murine L929 fibroblasts. When the cells were given the combined treatments and immediately subcultured for determination of cell survival by colony formation, the data indicate independent actions of each modality. However, when subculture was delayed for 1 h, a substantial fraction of cells treated with a sub‐lethal dose of PDT followed by 5 Gy γ‐radiation detached from the monolayer. Most of these detached cells were no longer clonogenic. The mode of photosensitized cell killing was found to be different from that of ionizing radiation‐induced cell killing. Photosensitized cell killing was accompanied by morphological changes in the cells and extensive DNA degradation within one hour following the treatment. When chloroaluminum phthalocyanine pre‐treated cells were exposed to a sublethal fluence of light (6 kJ/m2) and a lethal dose of γ‐radiation (5 Gy), DNA degradation was enhanced, and about 20% of the cell population appeared to undergo the type of cell death typical of photodynamic treatment. Thus, although different initial lethal lesions are induced by photodynamic treatment and by ionizing radiation, interactions may occur during processing of the damage.

DNA-Protein Cross-Links: New Insights into their Formation and Repair in Irradiated Mammalian Cells

The production of strong binding between DNA and protein by radiations and chemicals has been known for many years. DNA-protein cross-links (DPCs) were first recognized as a distinct lesion in ultraviolet light (UV)-irradiated bacteria by Smith1 and by Alexander and Moroson.2 The importance of DPCs for cellular lethality was clearly demonstrated in E. coli.3 Smith has reviewed various aspects of this work on several occasions.4–6

Modification of DNA damage in transcriptionally active vs. bulk chromatin

Our previous experiments have demonstrated that regions of nuclear chromatin, containing transcriptionally active DNA sequences and associated with the nuclear matrix, are hypersensitive to the production of both single-strand breaks and DNA-protein cross-links upon gamma-irradiation of exponentially growing mammalian cells. In this study, we have irradiated Chinese hamster V79 cells in buffered saline with or without DMSO to scavenge hydroxyl radicals and in buffered salines of various tonicities to expand or condense chromatin. The yield of DNA-protein cross-links was assayed by a nitrocellulose filter binding technique and the DNA recovered from the cross-links hybridized to 125I-poly(A+)RNA to determine the relative frequency of transcriptionally active sequences in the cross-links compared to the bulk DNA. In all cases, the data show that active DNA is affected to a greater extent than bulk, primarily inactive DNA. The more extensive alteration of the level of ionizing radiation-induced damage in active DNA by the diffusible agents tested suggests that other agents, such as chemical sensitizers and protectors, which need to diffuse to the nuclear DNA, may also be acting primarily on active, matrix-associated DNA.