RESISTANCE TO PHOTODYNAMIC THERAPY IN RADIATION INDUCED FIBROSARCOMA-1 and CHINESE HAMSTER OVARY-MULTI-DRUG RESISTANT CELLS in vitro

A degree of resistance to photodynamic therapy (PDT) has been induced in radiation-induced fibrosarcoma-1 (RIF-1) tumor cells by repeated photodynamic treatment with Photofrin (4 or 18 h incubation) in vitro to the 0.1-1% survival level, followed by regrowth from single surviving colonies. The resistance is shown as increased cell survival in the strain designated RIF-8A, compared to the wild-type RIF-1 cells, when exposed to increasing Photofrin concentration for 18 h incubation and fixed light exposure. No difference was found between RIF-1 and RIF-8A in the uptake of Photofrin per unit cell volume at 18 h incubation. Resistance to PDT was also observed in Chinese hamster ovary-multi-drug resistant (CHO-MDR) cells compared to the wild-type CHO cells, possibly associated with decreased cellular concentration of Photofrin in the former. By contrast, the PDT-resistant RIF-8A cells did not show any cross-resistance to Adriamycin, nor was there any significant drug concentration difference between RIF-1 and RIF-8A. These findings suggest that different mechanisms are responsible for PDT-induced resistance and multi-drug resistance.     

STRESS PROTEIN EXPRESSION IN MURINE TUMOR CELLS FOLLOWING PHOTODYNAMIC THERAPY WITH BENZOPORPHYRIN DERIVATIVE

Abstract— Photodynamic therapy (PDT) has been proven as a method of tumor eradication and is currently being used clinically to treat a wide variety of malignancies. Although it is understood that the interaction of light and sensitizer results in the production of potentially damaging oxygen species, the mechanism by which tumors are destroyed has yet to be defined fully. Using a new porphyrin sensitizer, benzoporphyrin derivative(BPD), we examined protein expression in murine tumor cells following treatment as an indication of molecular changes to target tissue concurrent with PDT-mediated damage. In order to assess the relevance of the results obtained using an in vitro PDT model, metabolic labeling of proteins synthesized subsequent to PDT was performed both in tumor cells grown and treated in tissue culture dishes and in cells explanted from PDT-treated solid tumors. We observed that the oxidative stress associated with PDT-resulted in the induction of a number or proteins corresponding to a set of heat-shock or stress proteins, and that the pattern of expression was similar when tumor cells were treated in vitro and in vivo . These results support the use of in vitro models in the dissection of the molecular erects of PDT and provide the foundation for future experiments that will examine the role of the immune system in tumor eradication by PDT.     

PHOTODYNAMIC INACTIVATION OF CHINESE HAMSTER CELLS

Abstract— Visible light exposures have been shown to kill acriflavine bound Chinese hamster cells. Such killing was enhanced when (a) dye was present in the medium during irradiation and (b) the pH of the medium was 8.5, instead of the normal 7.5 during the exposure. The induced killing could be suppressed by the presence of sodium azide during exposure. The results were taken to indicate that both DNA and non-DNA sites were involved in the cellular inactivation by visible light and that singlet oxygen was involved in the process.     

ACTIVATION OF NF-KB IN HUMAN SKIN FIBROBLASTS BY THE OXIDATIVE STRESS GENERATED BY UVA RADIATION

We have examined the role of the nucleus and the membrane in the activation of nuclear factor (NF)-KB by oxidant stress generated via the UVA (320–380nm) component of solar radiation. Nuclear extracts from human skin fibroblasts that had been irradiated with UVA at doses that caused little DNA damage contained activated NF-KB that bound to its recognition sequence in DNA. The UVA radiation-dependent activation of NF-KB in enucleated cells confirmed that the nucleus was not involved. On the other hand, UVA radiation-dependent activation of NF-KB appeared to be correlated with membrane damage, and activation could be prevented by a-tocopherol and butylated hydroxytol-uene, agents that inhibited UVA radiation-dependent peroxidation of cell membrane lipids. The activation of NF-KB by the DNA damaging agents UVC (200–290nm) and UVB (290–320nm) radiation also only occurred at doses where significant membrane damage was induced, and, overall, activation was not correlated with the relative levels of DNA damage induced by UVC/UVB and UVA radiations. We conclude that the oxidative modification of membrane components may be an important factor to consider in the UV radiation-dependent activation of NF-KB over all wavelength ranges examined.     

INACTIVATION AND MUTATION INDUCTION TO 6-THIOGUANINE RESISTANCE IN V79 HAMSTER FIBROBLASTS BY SIMULATED SUNLIGHT

Abstract— Risk estimates for an enhancement of ultraviolet radiation are frequently based on action spectra, in which case it is assumed that the different components of natural or artificial sunlight act independently and the overall effect can be derived by a simple summation procedure. Such an approach, however, may not always be justified and-as shown earlier-lead to a considerable underestimation of inactivation and mutation induction to 6-thioguanine resistance by filtered 'sunlamp'-radiation. Here we report on experiments employing different kinds of simulated sunlight. A good agreement between theoretical and experimental results was found. The apparent contradiction can be resolved by an analysis for the spectral regions primarily responsible for the overall effect.     

LASER-LIGHT INDUCED CHROMOSOME ABERRATIONS IN CHINESE HAMSTER CELLS

Wild-type Chinese hamster cells CHO Kl and their radiosensitive mutant xrs5 were irradiated at 308 nm, using light pulses of a XeCl excimer laser with total energy fluences of 0.1 kj/m² to 4.08 kj/m². Chromosome-type and chromatid-type chromosome aberrations have been observed at pulse irradiances of 2.5 × 10⁷ W/m² and 1.7 × 10⁸ W/m², indicating that in mammalian cells DNA double-strand breaks occur already in this irradiance range. The results obtained with laser irradiation are compared with X-ray irradiation.     

PTHALOCYANINE-INDUCED PHOTODYNAMIC CHANGES OF CYTOPLASMIC FREE CALCIUM IN CHINESE HAMSTER CELLS

Exposure to light of Chinese hamster cells preloaded with chloroaluminum phthalocyanine causes an immediate increase of cytoplasmic free calcium, [Ca2+], from about 0.2 microM to 1 microM within 5 min after illumination. This increase was dose-dependent within the biological dose range, reaching a plateau at a dose that kills 99.5% of the cells. Fluoride addition prior to light exposure protected against cell killing and reduced the increase of [Ca2+]i. These findings raise the possibility that changes in [Ca2+]i after photodynamic treatment may be relevant to cell killing and/or other biological responses of the cells, e.g. release of eicosanoids.     

PROTECTION BY THE FLUORIDE ION AGAINST PHTHALOCYANINE-INDUCED PHOTODYNAMIC KILLING OF CHINESE HAMSTER CELLS

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 AlPcCl, 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 inhibited 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-, and 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-.     

LARGE MUTAGENIC LESIONS ARE INDUCED BY PHOTODYNAMIC THERAPY IN MURINE L5178Y LYMPHOBLASTS*

Abstract— Mutagenic lesions at the thymidine kinase locus (tk) in mouse lymphoma L5178Y (LY) cells treated with red light and either Photofrin (PF) or chloroaluminurn phthalocyanine (AIPc) as the photosensitizer were compared in the relatively photodynamic therapy (PDT)-sensitive strain LY-R16 and the relatively resistant strains LY-S1 and LY-SR1. Southern blot analysis revealed that 92% (36/39) of the PDT-induced thymidine kinase (TK ^?/-) mutants of strains LY-R16 and LY-SR1 lost the entire active tk allele. (Strain LY-S1 lacks a known tk polymorphism and has not been analyzed for loss of the active tk allele.) A decrease in galactokinase (GK) activity in the TK^?/- mutants has been taken as an indication that the mutagenic lesion extends from the tk gene to the closely linked galactokinase gene (gk). Using PF as the photosensitizer, GK activity was decreased in 45% of the LY-R16 mutants and in 22% of the LY-S1 and LY-SR1 mutants. With photoactivated AIPc, 59% of the TK ^?/- mutants of strains LY-S1 and LY-SR1 showed GK inactivation. (LY-R16 mutants were not analyzed because of the low LY-R16 mutant frequency induced by PDT with AlPc.) Thus, many of the TK^?/- mutants of LY cells induced by PDT with either PF or AlPc harbor multilocus lesions.     

SYSTEMIC IMMUNOSUPPRESSION INDUCED BY PHOTODYNAMIC THERAPY (PDT) IS ADOPTIVELY TRANSFERRED BY MACROPHAGES

Some derivatives of hematoporphyrins are strongly retained by tumor tissue as compared to normal tissue, and exposure of these photosensitizers to radiation in the visible spectrum can cause serious biological damage. These properties have been exploited in the development of a new treatment for cancer termed photodynamic therapy (PDT). However, recent studies have also demonstrated that PDT can also induce a state of systemic immunosuppression. The purpose of this study was to determine whether PDT-induced suppression of contact hypersensitivity (CHS) responses was an active phenomenon that could be adoptively transferred by viable splenocytes from PDT-treated mice. Although induction of adoptively transferable suppressor cells in PDT-treated mice required exposure to antigen, the suppressor cells were found to be antigen nonspecific in their function. Furthermore, splenocytes from PDT-treated mice were capable of generating levels of allospecific cytotoxic T lymphocyte (CTL) activity which were comparable to those generated by normal control mice, but the ability of irradiated spleen cells from PDT-treated mice to stimulate a mixed lymphocyte response (MLR) was dramatically impaired. Finally, chromatographic separation of T cells, B cells and macrophages showed that the cell type which mediates adoptively transferable suppression of CHS responsiveness is in the macrophage lineage.     

THE PHOTODYNAMIC EFFECTS ONV–79 CHINESE HAMSTER CELLS

Abstract— Holding of acriflavine sensitizedV–79 cells in growth medium before visible light exposure decreases inactivation by visible light. The decrease depended upon the period of holding, indicating that there was release of cellular dye during this period. Exposures to visible light were done in two conditions: (a) with no dye in the medium during visible light exposure (washed) and (b) with dye in the medium during exposure (unwashed). Caffeine was found to slightly increase the sensitivity of the cells to visible light in the washed condition, whereas, in the unwashed condition no such effect was observed. Interaction studies with far UV did not reveal any correlation between photodynamic damage and UV damage. Visible light exposure of acriflavine sensitized cells was found to be mutagenic, as studied from the induction of 8-azaguanine resistant mutants. Inhibition of singlet oxygen production by sodium azide suppressed the induction of mutants. All these, taken together, have been discussed with respect to the relative importance of DNA and non-DNA damage in the photodynamic action of acriflavine.     

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 -irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to -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.     

PHOTODYNAMIC EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE UPTAKE OF RHODAMINE 123 BY MITOCHONDRIA OF INTACT MURINE L929 FIBROBLASTS AND CHINESE HAMSTER OVARY Kl CELLS

Abstract— It was shown that the cationic fluorescence probe rhodamine 123 accumulates in mitochondria of murine L929 fibroblasts and Chinese hamster ovary Kl epithelial cells due to the driving force of both plasma membrane and mitochondrial membrane potentials. Photodynamic treatment of L929 cells with hematoporphyrin derivative resulted in an increased uptake of rhodamine 123 and a diminished uptake of 1,1,3,3,3',3'-hexamethylindocarbocyanine iodide. This indicates a considerably increased mitochondrial membrane potential, which most likely is the result of a direct or secondary inhibition of the ATP-synthetase, and a decreased plasma membrane potential. The oxygen consumption rate and the ATP level decreased due to photodynamic treatment. Post-incubation of L929 cells subsequent to photodynamic treatment revealed that the uptake of rhodamine 123. the ATP content and the oxygen consumption rate were restored. For all parameters similar results were obtained with CHO-K1 cells, with the exception that during post-incubation the intracellular ATP content remained at the level reached after illumination. These results indicate that photodynamically induced disturbance of mitochondrial functions and the ATP level are not crucial for the loss of clonogenicity of L929 cells. In CHO-K1 cells however, the continuously lowered ATP level may have detrimental consequences for cell survival. The photodynamic stimulation of the rhodamine 123 uptake may be a rather general phenomenon. Because rhodamine 123 exhibits a much higher toxicity towards carcinoma cells than towards other cells, a synergistic interaction between this drug and photodynamic therapy (PDT) may be anticipated, if PDT also stimulates mitochondrial rhodamine 123 accumulation in carcinoma in vivo.     

ANOMALOUS DOSE-RESPONSE CHARACTERISTICS INDUCED BY CAFFEINE IN ULTRAVIOLET-IRRADIATED V79–79 CHINESE HAMSTER CELLS

Abstract— Cultured Chinese hamster cell line V79–79 exhibits an increase in survival with increasing UV fluence after a sharp decrease when exposed to 2.5 mM caffeine for 44 h after far-UV irradiation resulting in an anomalous maximum in the survival curve. No survival maximum is evident when either 0 or 1 mM caffeine is administered under the same conditions. The UV survival curve for 2.5 mM caffeine crosses the corresponding 1 mM curve and apparently becomes asymptotic to the OmM curve as UV fluence is increased. Chinese hamster cell lines V79–753B (related to V79–79 by derivation from the same parental line) and M3–1F3 (unrelated) exhibit only potentiation of post-UV lethality by the same concentration of caffeine and have no caffeine-induced anomalies in their survival curves. Xanthine. used alone or in combination with caffeine, only potentiated a slight amount of lethality and appears not to be a major causative factor of the anomaly.     

PHOTODYNAMIC THERAPY INDUCED ULTRASTRUCTURAL ALTERATIONS IN MICROVASCULATURE OF THE RAT CREMASTER MUSCLE

Abstract— Photodynamic therapy disrupts blood flow to tumors and produces tumor necrosis. These effects may be due to a localized generation of singlet oxygen. The current studies used direct observations of the rat cremaster microvasculature to examine the vascular effects of PDT. The objective of the morphological examination was to delineate the structural basis for the altered blood flow in photodynamic therapy. Dihematoporphyrin ether given 30 min or 48 h prior to the experiment was activated with green light (wavelength530–560 nm, 120 J/cm²). After the in vivo activation the tissues were prepared for electron microscopy. Light alone induced little or no change in the luminal content or vessel wall. On exposure to activating light both acute (30 min) and long term (48 h) dihematoporphyrin ether pretreated samples displayed formation of luminal aggregates, granulocyte margination and migration, and endothelial cell and smooth muscle cell damage. The latter was more pronounced in the arterioles than the venules. Perivascular changes included interstitial edema and damage to striated myocytes. Some of the alterations such as interstitial edema may be transient; however, smooth and skeletal muscle cell injury are important in normal and tumor tissue necrosis after photodynamic therapy.     

TUMOR DESTRUCTION IN PHOTODYNAMIC THERAPY

Abstract The effects of photodynamic therapy (PDT) on the tumor microvasculature in the first few hours after treatment was studied at the light microscope (LM) and electron microscope (EM) levels in DBA/2Ha mice bearing SMT-F tumors. Animals received intraperitoneal injections of 10 mg kg⁻ of Photofrin II and 24 h later tumors were treated with 100 J cm⁻² of light (630 nm). Animals were sacrificed and their tumors removed at time 0, 30 min, 1, 2, 4, 8, 16 and 24 h after treatment. The results indicate that the effects of PDT are initially direct destruction of the microfibrils in the subendothelial zone of the tumor capillaries with subsequent tumor cell death secondary to hemorrhage and vascular collapse.     

EFFECTIVE PHOTODYNAMIC ACTION BY RHODAMINE 123 LEADING TO PHOTOSENSITIZED KILLING OF CHINESE HAMSTER OVARY CELLS IN TISSUE CULTURE AND A PROPOSED MECHANISM

The effectiveness of rhodamine 123 (R123) as a photosensitizer of cell killing is relatively low and correlates with its inefficient production of singlet oxygen. The known selective retention of R123 in the mitochondria of epithelially derived carcinoma cells, however, is a selective feature that could lead to a more useful therapeutic ratio if photosensitizing effectiveness could be increased. Chinese hamster ovary (CHO) cells in tissue culture were therefore exposed to R123 shortly before and during illumination under conditions controlled for oxygen concentration and temperature. Effective photosensitization of cell killing, as judged by colony formation, was produced by 95% but not by 19% O₂ during illumination of cells at 5d?C or 37d?C, and this was additionally enhanced at the sublethal temperature of 42d?C. Two CHO cell lines were examined; one line, CHO-AA8, was proficient in the repair of DNA damage and the parent to the second line, CHO-EM9, that was deficient in the repair of DNA strand breaks. Cells of both lines incorporated R123 to a similar degree and were similarly photosensitized by the presence of igh oxygen concentration. Furthermore, plasma membrane damage as judged by teh exclusion of trypan blue was not observed immediately after illumination in the presence of R123, but was seen in the presence of meso-tetra-(4-sulfonatophenyl)-porphine (TPPS₄). The extent of damage to the plasma membrane by TPPS₄ was greater in the presence of 95% compared to 19% O₂ during illumination. Photodynamic action at the level of teh plasma membrane appears to contribute to photosensitization by TPPS₄ but not by R123 soon after exposure of cells to these sensitizers. It is hypothesized that photodynamic action by R123 is the primary mechanism causing the observed photosensitization of cell killing, and that mitochondria are teh site of photosensitized damage responsible for this killing.     

MEMBRANE DAMAGE INDUCED IN CULTURED HUMAN SKIN FIBROBLASTS BY UVA IRRADIATION

Irradiation of cultured human skin fibroblasts with ultraviolet light from 320 to 400 nm (UVA) leads to a decrease in the membrane fluidity exemplified by an enhanced fluorescence anisotropy of the lipophilic fluorescent probe 1-[4-trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene. This UVA-induced decrease in fluidity is associated with lactate dehydrogenase leakage in the supernatant. Vitamin E, an inhibitor of lipid peroxidation, exerts a protective effect on both phenomena. Therefore, this UVA-induced damage in membrane properties may be related to lipid peroxidation processes. Moreover, exponentially growing cells are more sensitive to these UVA-induced alterations than confluent cells.     

WAVELENGTH DEPENDENCE OF INACTIVATION AND MUTATION INDUCTION TO 6-THIOGUANINE-RESISTANCE IN V79 CHINESE HAMSTER FIBROBLASTS

Abstract The wavelength dependence of inactivation and mutation induction to 6-thioguanine-resistance in the range between 254 and 313 nm was investigated in V79 Chinese hamster fibroblasts. The action spectra agreed quite well with those reported by others for different effects on mammalian cells. There was no distinct difference between the wavelength dependencies of inactivation and mutation induction; the ratio of mutagenic to lethal action remained constant.