Marian E. Clay

DNA LESIONS AND DNA DEGRADATION IN MOUSE LYMPHOMA L5178Y CELLS AFTER PHOTODYNAMIC TREATMENT SENSITIZED BY CHLOROALUMINUM PHTHALOCYANINE

Two closely related strains of mouse lymphoma L5178Y cells, LY‐R and LY‐S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY‐R is more sensitive to photodynamic cell killing than strain LY‐S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single‐strand breaks (which includes frank single‐strand breaks and alkali‐labile lesions), but substantially more DNA‐protein cross‐links were formed in strain LY‐R by CAPC and light. Repair of single‐strand breaks proceeded with similar kinetics in both strains for the first 30 min post‐irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY‐R. DNA degradation, as measured by the conversion of prelabled [14C] DNA to acid‐soluble radioactivity, was more rapid and extensive in strain LY‐R.

CYTOTOXIC AND MUTAGENIC EFFECTS OF THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT IN L5178Y CELLS

Abstract The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY‐R, deficient in the excision repair of UV‐induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY‐S, deficienl in the repair of DNA double‐strand breaks, was more sensitive than strain LY‐R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY‐S and LY‐R sub‐strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY‐SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 106 surviving cells. The induced frequency in strain LY‐S1 was much higher than in either tk+l‐heterozygous or ik+10 hemizygous strains of LY‐R. The rate and extent of incorporation of CAPC by the LY‐R strains was somewhat greater than for strain LY‐S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.

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.

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.

PHOTOFRIN II PHOTOSENSITIZATION IS MUTAGENIC AT THE tk LOCUS IN MOUSE L5178Y CELLS

Abstract— Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY‐S1 and LY‐R16 but not in strain LY‐R83 which is hemizygous at the tk locus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY‐S1 was similar to that of other mutagenic agents including x‐radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AIPcCI). Although both strain LY‐S1 and strain LY‐R16 were mutagenized by photosensitization with PFII, only strain LY‐S1 was mutagenized by photosensitization with AIPcCI. The non‐mutability of strain LY‐R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0 hemizygous strain, whereas the non‐mutability of strain LY‐R16 subjected to photodynamic treatment with AIPcCI may be because LY‐R16 cells sustaining mutagenic damage do not survive for reasons other than the loss of an essential gene.

Protection by the fluoride ion against phthalocyanine-induced photodynamic killing of Chinese hamster cells.

Abstract— When a dilute F‐ solution was added to a culture of Chinese hamster cells that had been preincubated with an aluminium phthalocyanine sensitizer derived from AlPcCI, the photosensitivity of the cells was markedly reduced compared to control cells not treated with F‐. Under the same treatment conditions, the reduction in [3H]thymidine incorporation into cellular DNA caused by light and this sensitizer and the production of DNA‐protein crosslinks caused by light and this sensitizer were also inhibited by F‐. In contrast, the killing of Chinese hamster cells, the reduction of thymidine incorporation by the cells, and the production of DNA‐protein crosslinks in the cells caused by the combination of light and either Photofrin II or the silicon phthalocyanine HOSiPcOSi(CH3)2(CH2)3‐N(CH3)2 were not inibited by F‐. We conclude that the aluminium phthalocyanine sensitizer used is largely or completely AlPc(OH)(H2O), that it is converted to a fluoro complex by F‐ that this compound probably is a less efficient generator of photochemical damage at a critical cellular target(s) than is AlPc(OH)(H2O). The inhibition of thymidine incorporation and DNA‐protein crosslink formation indicates that the effects of F‐ can be expressed at intracellular sites. It is further concluded that the silicon phthalocyanine sensitizer and Photofrin II do not interact significantly with F‐.

Effects of photodynamic treatment on DNA

A common feature of the photosensitizers in current or proposed use for photodynamic therapy (PDT) is their lipophilicity which promotes their accumulation in cellular membranes. In spite of the absence of observable photosensitizers in the nucleus, photodynamic activation of photosensitizer-loaded cells produces substantial amounts of DNA damage. With either porphyrins or phthalocyanines as photosensitizers, the yield of DNA single-strand breaks plus alkali-labile sites (SSB) is less than that resulting from an equitoxic dose of ionizing radiation; however, these same photodynamic treatments produce high yields of DNA-protein crosslinks (DPC), which are not repaired during post-treatment incubation of the cells, in contrast to the DPC produced by ionizing radiation. Initial yields of DPC after photodynamic treatment of murine lymphoma L5178Y cells sensitized by chloroaluminum phthalocyanine (AlPcCl) are greater in the relatively PDT-sensitive strain LY-R as compared to the relatively PDT-resistant strain LY-S. Photodynamic treatment sensitized either by AlPcCl or by Photofrin II is mutagenic at the thymidine kinase (tk) locus in one or more sub-strains of LY-R and LY-S. With Photofrin II, the induction of mutations has been observed in the tk+/- heterozygous strains LY-R16 and LY-S1, but not in the hemizygous tk+/0 strain LY-R83. This pattern of strain-specific mutagenesis is found for other agents, such a ionizing radiation, which produce a high proportion of multi-locus lesions. With AlPcCl, mutation induction is found in strains LY-S1 and LY-SR1, but not in either of the sub-strains of LY-R. Treatment of strains LY-R and LY-S with identical doses of AlPcCl and red light results in degradation of the DNA, which occurs earlier and to a greater extent in the more PDT-sensitive strain. Examination of the size of the DNA during the period of degradation revealed a series of fragments with sizes which were multiples of approximately 190 bp. This suggests that PDT treatment of L5178Y cells induces the process known as apoptosis, or programmed cell death, in which endonucleolytic scission of the DNA occurs in the internucleosomal linker region. DNA degradation is also stimulated by treatment of murine L929 fibroblasts with phthalocyanine and light but not by gamma- irradiation alone. Combined treatment with a minimally lethal dose of PDT and a dose of gamma-radiation producing 90% cell death results in the induction of a PDT-type cell death in a substantial portion of the cells. When the level of programmed cell death is very high, the recovery of mutants may be compromised. Thus, PDT appears to produce extensive DNA damage, but events at other cellular locations may alter the expression of that damage.

Photodynamic effects of silicon phthalocyanines in model cells and tumors (Invited Paper)

A series of silicon and aluminum phthalocyanines is being investigated in this laboratory for their potential as photosensitizers for photodynamic tumor therapy (PDT). Of these, one of the silicon phthalocyanines [SiPc(OH)OSi(CH3)2(CH2)3N(CH3)2] (Pc IV) has proven to be highly efficient in a series of in vitro assays and in PDT in vivo. When compared to sulfonated or non-sulfonated aluminum phthalocyanine and/or Photofrin II, Pc IV produced greater effects at lower concentrations in a clonogenic assay with V79 cells, and in photoenhancement of lipid peroxidation in human erythrocyte membranes. Physiological responses of treated cells in vitro appeared similar to those produced by PDT with other sensitizers; however, the responses, such as the induction of apoptosis in murine lymphoma, occurred with greater efficiency when Pc IV served as photosensitizer. In order to evaluate the efficacy of Pc IV in vivo, the dye was suspended in corn oil or incorporated into liposomes and injected intraperitoneally into C3H mice bearing RIF-1 tumors. Pharmacokinetic studies showed efficient uptake of Pc IV into the tumor, as well as into liver and kidney. For PDT, tumors were irradiated with 675 nm light from an argon-pumped dye laser. Treatment of tumors up to 100 mm3 with 1.0 mg/kg Pc IV and 135 J/cm2 produced ablation of the tumor within 48 hours. Tumors > 200 mm3 could be ablated with 2.0 mg/kg Pc IV. The data suggest that Pc IV may be a highly efficient photosensitizer for tumor PDT.

Interaction of phthalocyanine photodynamic treatment with ionophores and lysosomotrophic agents

Phthalocyanines are receiving increasing attention as second-generation sensitizers for photodynamic therapy (PDT). This paper discusses some of the investigations into the mechanism of the phototoxic responses of phthalocyanine-sensitized PDT exploiting the interaction of PDT with other metabolic modulators. Among the agents which interact strongly with PDT is the K+/H+ ionophore nigericin. Under the conditions studied with chloroaluminum phthalocyanine (AlPcCl), the Na+/H+ ionophore monensin, the Ca++ ionophore A23187, and the lysosomotrophic agent chloroquine, but not the K+ ionophore valinomycin, also potentiate photodynamic cell killing. None of the latter compounds interact with PDT as strongly as does nigericin. Both nigericin and monensin partially inhibit cellular respiration; however, KCN, which inhibits respiration completely, is less effective in potentiating PDT damage than is nigericin. Nigericin treatment alone does not deplete glutathione; however, the GSH level decreases after treatment of cells with PDT and nigericin. The potentiation of the PDT response is much greater at an extracellular pH (pHe) of 6.70 than at pHe 7.30. When nigericin is present at pHe 6.70, the intracellular pH (pHi) is equilibrated with pHe. None of the other ionophores tested was able to cause the acidification of the intracellular milieu as did nigericin. The evidence to date suggests that the lowering of pHi is an important component of the mechanism by which nigericin potentiates PDT.