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Fibrosarcoma

Fibrosarcoma (fibroblastic sarcoma) is a malignant tumor derived from fibrous connective tissue and characterized by immature proliferating fibroblasts or undifferentiated anaplastic spindle cells. more...

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The tumor may present different degrees of differentiation: low grade (differentiated), intermediate malignancy and high malignancy (anaplastic). Depending on this differentiation, tumor cells may resemble mature fibroblasts (spindle-shaped), secreting collagen, with rare mitoses. These cells are arranged in short fascicles which split and merge, giving the appearance of "fish bone". Poorly differentiated tumors consist in more atypical cells, pleomorphic, giant cells, multinucleated, numerous atypical mitoses and reduced collagen production. Presence of immature blood vessels (sarcomatous vessels lacking endothelial cells) favors the bloodstream metastasizing.

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Investigation of cross-resistance to a range of photosensitizers, hyperthermia and UV light in two radiation-induced fibrosarcoma cell strains resistant
From Photochemistry and Photobiology, 1/1/01 by Mayhew, Stephen

Investigation of Cross-resistance to a Range of Photosensitizers, Hyperthermia and UV Light in Two Radiation-induced Fibrosarcoma Cell Strains Resistant to Photodynamic Therapy In Vitro

ABSTRACT

Two distinct photodynamic therapy-resistant variants of the murine radiation-induced fibrosarcoma (RIF) cell line have been isolated. One strain displayed relative resistance over the parental RIF-1 strain to treatment with the porphyrin-based compound, polyhaematoporphyrin (PHP), whereas the other strain displayed relative resistance over the RIF-1 strain to treatment using the cationic zinc (II) pyridinium-substituted phthalocyanine (PPC). The PHP-resistant strain did not display crossresistance to PPC-mediated treatment, and vice versa. In both PDT-resistant strains, the increased resistance could not be attributed to altered cellular growth rate, antioxidant capacity or intracellular sensitizer localization. The PHP-resistant strain displayed resistance to treatment with both short (1 h) and extended (16 h) sensitizer incubation periods, which may indicate that in this strain, the resistance has arisen through an alteration in a membrane component. Conversely, the PPC-resistant strain only displayed increased resistance over the parental cells to treatment involving the short drug incubation, which is likely to reflect the existence of a threshold effect caused by the alteration of an individual cellular target. Each resistant strain has been compared to the parental strain in terms of cellular sensitivity to treatment with a range of other photosensitizers, hyperthermia, LTV light and the anticancer agent cis-diamminedichloroplatinum. The PHP-resistant strain exhibited crossresistance to photosensitization treatment using exogenously added protoporphyrin IX, and also to treatment with the anionic phthalocyanine sensitizers, zinc (lT) tetrasulfonated phthalocyanine and zinc (II) tetraglycine-substituted phthalocyanine. The PPC-resistant strain did not display cross-resistance to any of the treatment strategies employed in this investigation. The results of this investigation indicate that there are at least two distinct mechanisms of PDT resistance in IF cells, and that the mechanism of PHP resistance may, to some

extent depend, upon the physical nature of the sensitizer molecule.

INTRODUCTION

Photodynamic therapy (PDT)t involves the administration of a photosensitizing compound which, when activated by visible light in the presence of molecular oxygen, results in tumor destruction via the production of reactive oxygen species (ROS), such as singlet oxygen (1). The only approved clinical photosensitizer, Photofrin is a porphyrin-based preparation (2); however, many compounds from a number of different classes such as chlorins (3) and phthalocyanines (4) are currently under investigation for potential clinical use.

Destruction of tumor tissue may occur by direct cell kill, or through vascular photosensitization resulting in tumor hypoxia (5). PDT-mediated cell death may proceed via either apoptosis or necrosis (6,7), the mode of death depending upon a number of factors, such as the sensitizer employed and the cell type involved and the PDT dose administered. Many cellular components have been shown to be susceptible to photosensitization (8-10); however, due to the relatively short diffusion distance of singlet oxygen (0. wm) (11), photodamage to a particular cellular structure is only likely to occur if the structure in question is closely associated with a photosensitizer molecule(s). This association is also sensitizer/cell-type specific. Owing to the number of different factors involved, a detailed understanding of the mechanisms) of PDT action, and of the precise cellular targets, is only just beginning to emerge.

Mitochondria have repeatedly been implicated as primary targets in PDT (8,12-14), and cell death due to photosensitization of this organelle may proceed by either apoptosis or necrosis, depending upon the factors mentioned above. Many photosensitizers demonstrate lysosomal affinity (15). This may be attributed to uptake by fluid-phase endocytosis and is therefore particularly true of water-soluble compounds. Lipophilic sensitizers, on the other hand, generally associate with membranous structures, such as the endoplasmic reticulum and the plasma membrane (16). The nucleus is not thought to be a major primary target for PDT, possibly due to its high capacity for repair, although a number of studies have reported photosensitization-mediated damage to this organelle (17-19).

A strategy involving the isolation and characterization of cells which display relative PDT resistance over the parental strain has been adopted by a number of researchers with a view of clarifying the mechanisms) of PDT cytotoxicity. Luna and Gomer (20) and Singh et al. (21) have both generated Photofrin-resistant marine cells which have been characterized in detail (22-26).

This study reports the isolation of two distinct PDT-resistant variants of the radiation-induced fibrosarcoma (RIF-1) cell line. Both PDT-resistant variants have been compared against the parental RIF-1 strain, in terms of susceptibility to treatment with other photosensitizers, in an attempt to investigate the individual modes of photosensitizer action. The Photofrin-resistant cells of Singh et al. (21) have previously demonstrated cross-resistance to UV radiation and to the anticancer agent, cis-diamminedichloroplatinum (cisplatin) (25,26), and therefore the PDT-resistant cells from the present study have been investigated for crossresistance to these agents, in an attempt to ascertain whether the mechanisms of PDT resistance are similar. It is important to appreciate that in this study PDT resistance has been used merely as a tool for investigating cellular function, and may not necessarily reflect any PDT resistance which may arise in the clinical situation.

MATERIALS AND METHODS

Photosensitizers and other compounds. Table 1 details the photosensitizers employed in this study. Polyhaematoporphyrin (PHP), a compound equivalent to Photofrin but prepared at the University of Leeds (27) was supplied at a concentration of 1 mg mL-1 in phosphate buffered saline (PBS), and stored at -20'C in working aliquots. Pyridinium-substituted- (PPC), tetrasulfonated- (TSPC) and tetraglycine-substituted- (TGly) zinc (II) phthalocyanine derivatives were synthesized as previously described (28) PPC is reported to have an average substitution corresponding to the dication while TSPC and TGly are tetrasubstituted. The 5,10,15,20-tetra(meta-hydroxyphenyl) chlorin (m-THPC) was kindly donated by Scotia QuantaNova (Surrey, UK) and solutions were freshly prepared before use according to the manufacturer's guidelines in polyethylene glycol/ethanol/water (2:3:5 vol/vol/vol). Protoporphyrin IX (PpIX), 5-aminolevulinic acid (ALA), meso-tetra(4-pyridyl) porphine (mTPyP) and cisplatin were obtained from Sigma (Dorset, UK) and solutions of each were freshly prepared before use. The mTPyP was dissolved in distilled water, the ALA was dissolved in PBS and the PpIX was mixed with tris base (1:1 wt/wt) and then dissolved in distilled water. The cisplatin was dissolved in PBS. All solutions were sterile filtered (0.22 wm acrodisc).

Cell culture. The RIF-1 cell line (kindly donated by Dr. J. Bremner, MRC Radiobiology Unit, Harwell, Oxford, UK) was used to isolate PDT-resistant variants, and also to act as the parental strain. Cultures were maintained at 37C in Roswell Park Memorial Institute 1640 culture medium, supplemented with 10% fetal calf serum, 1% glutamine and 0.1% gentamicin.

Photosensitization and cisplatin treatment conditions. Cells were plated in 96-well plates at a density of 1 X 104 cells per well for 18-24 h to allow attachment and growth. Sensitizer or cisplatin, prepared in fresh culture medium, was then added at a range of concentrations for 1-4 h (sensitizer) or 16 h (cisplatin). Following incubation, the cells were washed three times with PBS and then fresh medium was added. In the phototoxicity experiments, cells were then exposed to 10 mW cm-2 white light generated by a 500 W quartz-halogen bulb, which was IR filtered by a 250 turn path length water tank maintained at 37C. The total filtered output in each wavelength band is detailed in Table 2. Following illumination, the culture medium was removed and replaced with fresh medium. Cell survival was determined 24 It later using the 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (29). The MTT assay measures cytosolic and mitochondrial dehydrogenase activity, and gives comparable results to the clonogenic assay as a method of determining cell survival (30).

Isolation procedure for PDT-resistant cell strains. Two sets of PDT-resistant RIF-variants were isolated: one set which displayed relative resistance to PHP-mediated photosensitization (designated RIF-25R) and the other set which displayed relative resistance to PPC-mediated photosensitization (designated PlO). Both resistant strains were isolated following cycles of photosensitization treatment with increasing sensitizer concentrations and fixed light dose. Parental RIF-1 cells were seeded in 80 cm2 tissue culture flasks at a density of 1 X 105 cells per flask for 18-24 h to allow attachment and growth. Cells were then subject to a cycle of photosensitization involving 1 It incubation with PHP or PPC (2 ug mL-1 or 0.98 mM, respectively) and subsequent exposure to 10 mW cm-2 white light at a total dose of 3.0 J Cm-2. Following treatment, surviving cells were refed with fresh culture medium and allowed to grow. Surviving cells were then reseeded and subject to further cycles of photosensitization with increasing concentrations of PHP or PPC, respectively. Following 15 cycles of photosensitization, the RIF-25R strain displayed >50% survival following treatment with 25 ug mL-1 PHP and 1 h light exposure, and the PlO strain displayed >50% survival following treatment with 9.8 pM PPC and 1 h light exposure.

Photosensitizer uptake. Cells were plated in 35 turn Petri dishes at a density of 2 X 104 cells per dish for 18-24 h to allow attachment and growth. Sensitizer prepared in fresh culture medium was then added at a range of concentrations for I or 16 h. Following incubation in the dark at 37 deg C, the medium containing sensitizer was removed and the monolayers washed three times with cold PBS. Intracellular porphyrin (PHP and PpIX) levels were measured by solublizing cells in 0.1 M NaOH for 15 min and dissolving this cell homogenate in ethyl acetate:glacial acetic acid (4:1) (31). Porphyrins were then extracted into 1 M HCI and the concentrations determined by fluorescence as detailed below. For the analysis of PHP, the HCl layer was boiled prior to fluorescence measurement to hydrolyze ether and ester linkages (27). Intracellular mTHPC was measured using a similar method, however the extraction step into HCI was omitted and the fluorescence of mTHPC in the ethyl acetate was measured directly. Intracellular levels of PPC, mTPyP, TSPC and TGly were measured without the need for extraction, by five-fold dilution of the cell homogenate in 2% aqueous sodium dodecyl sulfate (SDS) prior to fluorescence measurement. Intracellular sensitizer levels were determined by fluorescence (PHP: k,x = 400 nm, hem = 596 nm; PPC: \ex = 605 nm, )se. = 682 nm; PpIX: kex, = 403 nm, hem = 602 nm; mTHPC: Xex = 418 nm, hem = 652 nm; TSPC: kex = 608 nm, )sem = 686 nm; TGly: rex = 614 nm, hem = 684 nm; mTPyP: kex = 408 nm, kem = 655 nm) using a Kontron SFM 25 spectrofluorometer, and the amount of sensitizer present was determined by interpolation from a standard curve of known concentration. The amount of cell-associated sensitizer was expressed as nanomoles per milligram of cellular protein. The protein content of each sample was determined using the Bio-Rad assay (32).

Hyperthermia and UV treatment conditions. Cells were plated in 35 mm Petri dishes at a density of 5 X 104 cells per dish for 18-24 h to allow attachment and growth. Hyperthermia treatments involved sealing the dishes with parafilm and immersing the dishes for 1 h in a temperature-controlled waterbath. For UV treatments, the culture medium was replaced with PBS. The UVA irradiation was performed using a predominantly 366 nm-emitting lamp exposing the cells to 0-200 J m-2 at a fluence rate of 215 mW cm-2. The UVC irradiation was performed using a predominantly 254 run-emitting germicidal lamp exposing the cells to 0-108 J m-2 at a fluence rate of 15 mW m-2. Both UV treatments were performed at a distance which did not cause heating. Cell survival was determined 24 h after treatment using the MTT assay.

Antioxidant capacity. Glutathione levels were measured using the method of Griffith (33) by following 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) reduction at 412 nm. The activities of glutathione reductase and glutathione peroxidase were measured using the methods described by Carlberg and Mannervik (34) and Paglia and Valentine (35), respectively. In each case the oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored at 340 nm. Glutathione S-transferase activity was measured by the method described by Habig et al. (36) using 1-chloro-2,4-dinitro-benzene (CDNB) as the second substrate. Superoxide dismutase (SOD) activity was measured by following the inhibition of reduced cytochrome c formation by SOD at 550 nm, as described by McCord and Fridovich (37).

Cell growth analysis. Cells in 35 mm Petri dishes were trypsinized, resuspended in isotonic buffer and counted on days 0-12 using a Coulter electronic counter. Cell doubling times were calculated from the exponential phase of cell proliferation.

Intracellular localization of sensitizer. Cells were seeded onto glass coverslips at a density of 1 X 101 cells per dish, and allowed to attach. The culture medium was replaced with fresh medium containing sensitizer (10 wg mL-I PHP, 10 lIM PPC) for I h. Following incubation, cells were washed three times with PBS and inverted onto a specialized chamber slide containing fresh medium at 37 deg C. Intracellular fluorescence was monitored directly using a Leitz Dialux 22 upright microscope. The excitation source was a 4 mW HeNe laser at 632 nm, and the emitted light was collected using 580750 nm interference filters. Captured images were processed using Visilog ImagerPlus software.

RESULTS

Isolation of PDT-resistant variants

Figure la,b shows the PDT-survival curves for both PDT-- resistant RIF-variants and for the parental RIF-1 cell line. The RIF-15R and P7.5 strains displayed an intermediate degree of resistance between the parental RIF-1 strain and the RIF-25R and PlO strains, respectively, which demonstrates a progressive and incremental increase in PDT resistance after each cycle of treatment and regrowth. At the 50% survival level the RIF-25R strain demonstrated a 5.7-fold increased resistance, and the PlO strain demonstrated a 7.1fold increased resistance over the parental strain to treatment with PHP and PPC, respectively. These values were the dose enhancement ratios at the 50% survival level (DER50) for the RIF-25R and P10 strains, when expressed in terms of increasing sensitizer concentration and fixed light dose. Figure 2a,b shows that the DER50 was significantly reduced in both RIF-25R and P10, to 1.7 and 1.8, respectively, when expressed in terms of fixed sensitizer concentration and increasing light dose. Figure 3 shows the cell survival profiles after treatment involving an extended incubation period of 16 h, expressed in terms of increasing sensitizer concentration and fixed light dose. The RIF-25R strain continued to display relative resistance to treatment with PHP (Fig. 3a), albeit with a lower DER50 of 3.2. Conversely, the PlO strain did not exhibit any increased resistance over the parental RIF-1 strain following treatment involving the extended incubation period with PPC (Fig. 3b).

There were no significant differences in drug uptake, cellular growth rate and cellular antioxidant capacity between the parental RIF-1 strain and both PDT-resistant strains (Tables 3 and 4). There was also no difference in the intracellular localization of PHP in RIF-1 and RIF-25R, or in the intracellular localization of PPC in RIF-1 and PIO (Fig. 4). This indicates that the increased PDT resistance in both RIF25R and P10 could not be attributed to alterations in these characteristics.

Cross-resistance studies

Figure 5a-d shows cell survival curves for RIF-I, RIF-25R and P10 following treatment with hyperthermia, UVA, UVC and the anticancer agent, cisplatin. Both PDT-resistant strains displayed comparable cell survival profiles to that of the parental RIF-1 strain, for each treatment protocol.

Figure 6a shows that the survival curve for the PPC-resistant strain (PlO) is comparable to that for the parental RIF-1 strain following PHP photosensitization; similarly, Fig. 6b shows that the survival curve for the PHP-resistant strain (RIF-25R) is comparable to that for RIF-1 following treatment with the cationic phthalocyanine, PPC. This indicates that the RIF-25R strain is not cross-resistant to PPC photosensitization, and vice versa. The PDT-survival curves for RIF-1, RIF-25R and P10 following photosensitization treatment with a range of compounds are shown in Fig. 7af. No cross-resistance was observed in either PDT-resistant cell strain following photosensitization with mTPyP, mTHPC or endogenously produced PpIX synthesized from extracellularly added ALA (Fig. 7a-c).

There was no difference in the PDT-survival curves for RIF-1 and P10 following photosensitization using exogenously added PpIX (Fig. 7d); however, the RIF-25R strain was crossresistant to exogenous PpIX, with a DER50 of 2.5 when expressed in terms of increasing sensitizer concentration and fixed light dose (Fig. 7d).

The RIF-25R strain also displayed a degree of crossresistance to photosensitization with the two anionic phthalocyanine compounds, TSPC and TGly (Fig. 7e,f), with DER50 of 2.2 and 2. 1, respectively, when expressed in terms of increasing sensitizer concentration and fixed light dose. It should be noted that for each sensitizer, the intracellular concentrations in the RIF-1 strain, the RIF-25R strain and the P10 strain were comparable (data not shown), indicating that any crossresistance observed was not due to altered drug influx/efflux.

DISCUSSION

Two PDT-resistant variants of the marine RIF tumor cell line have been isolated. When compared to the parental RIF1 cells, one strain (RIF-25R) displayed relative resistance to treatment with a Photofrin equivalent, and the other strain (PlO) displayed relative resistance to treatment with a cationic phthalocyanine,. The RIF-25R strain was not crossresistant to PPC photosensitization and vice versa, indicating that the PDT-resistant cell lines are distinct from one another. Luna and Gomer (20) and Singh et al. (21) have previously used cycles of photosensitization to isolate Photofrinresistant RIF variants. In the former study, a strategy of fixed Photofrin concentration and increasing light dose was used to isolate PDT-resistant cells designated CL-8, with a DER of 1.2-1.4 at the 1.0-0.1% survival level (20). In the latter study, cycles of photosensitization involving increasing Photoffin concentration and fixed light dose were employed to achieve a DER of 1.8 at the 10% survival level in cells designated RIF-8A (21). The DER50 of the RIF-25R strain isolated from the present study was somewhat higher than that observed for CL-8 and RIF-8A in terms of sensitizer concentration, although when expressed in terms of light dose it was more comparable. This effect may be explained by a number of factors such as the light being rate limiting or photochemical modification by the RIF-25R cells, and is the subject of continued investigation. It is as yet unclear whether the RIF-25R strain isolated from the present study is similar to either (or both) the CL-8 or the RIF-8A strain.

Interestingly, RIF-25R cells also displayed a degree of resistance, albeit not very pronounced, to treatment involving a 16 h PHP incubation. The short (1 h) PHP incubation has been primarily associated with plasma membrane damage, whereas extended (16 h) incubation is believed to result in damage to specific cellular organelles (20,38). Thus it appears that PHP-mediated cytotoxicity proceeds through both membrane and mitochondrial photosensitization. The PHP-resistance in RIF-25R cells was observed following both short and extended incubation periods, which fits with the hypothesis that resistance may be conferred through an alteration in a membrane component, thereby affording protection against PHP-mediated photodamage. This would explain why the DER50 of RIF-25R over RIF-1 was higher following the short incubation period where membrane damage predominates, than following the extended incubation period, where additional cytotoxic effects would be created by mitochondrial photosensitization.

The P10 cells did not display relative resistance over the parental RIF-1 strain to a 16 h PPC incubation strategy. The localization pattern of PPC is indistinguishable following 1 and 16 h incubation periods, therefore it seems unlikely that the absence of resistance at 16 h is due to the fact that different cellular targets are associated with short and extended periods of PPC incubation. More likely, the increased intracellular levels of PPC at 16 h may reflect a threshold effect whereby once a certain level of intracellular damage is sustained the P10 cells behave as RIF-1.

The cellular characteristics of the parental RIF-1 cells, the PHP-resistant RIF-25R cells and the PPC-resistant PIO cells were comparable. There were no differences in terms of doubling time, protein content, PHP and PPC uptake and intracellular glutathione and related enzyme levels among the three cell lines. Furthermore, the PHP resistance displayed by RIF-25R and the PPC-resistance displayed by P10 could not be explained in terms of altered subcellular localization, since the PHP localization pattern was comparable in RIF-I and RIF-25R, and the PPC localization pattern was comparable in RIF-1 and PIO. The diffuse fluorescence pattern of PHP in both RIF-1 and in RIF-25R was indicative of its association with membranous structures, whereas the punctate fluorescence of PPC in RIF-I and PlO suggested a lysosomal/mitochondrial association. These observations were consistent with previous reports (39).

There was no cross-resistance to hyperthermic treatment in either of the PDT-resistant strains which suggests a difference in the critical targets for PDT (with both PHP and PPC) and hyperthermia, and/or different pathways of repair of PDT- and heat-induced injury. This is consistent with the findings of Gomer et al. (40), who demonstrated that temperature-resistant RIF variants which contained elevated levels of the 70 kDa heat shock protein (HSP-70) did not display cross-resistance to Photofrin-mediated PDT. Further work reported that photosensitization treatment with monoL-aspartyl chlorin e6 and tin ethyl etiopurpurin could induce the expression of HSP-70 in RIF-1 cells; however, treatment with Photofrin failed to produce a cellular HSP response (41).

The absence of cross-resistance to UVA and to UVC in both PDT-resistant strains may reflect an increased capacity for recovery from specific PDT-mediated injury in these strains, and suggests that the pathways of repair of PDT-- induced injury and UV-induced injury are different. DiProspero et al. (25) have reported the cross-resistance of RIF-8A to UVC light using viral capacity as an indicator of cellular sensitivity to and recovery from treatment. Their results suggest an overlap in the types of cellular damage induced by UVC and PDT and/or an overlap in the pathways of repair of UVC- and PDT-induced damage in RIF-8A cells. Taken together with the results from the present study, this indicates that there may be more than one porphyrinresistant RIF-phenotype.

The absence of cross-resistance to the cationic porphyrin, mTPyP in both the RIF-25R strain and the PIO strain suggests that the PHP resistance of the RIF-25R strain is unlikely to be solely dependent on the porphyrin nature of the compound, and also that the PPC resistance of the P10 strain is unlikely to be solely due to the charge of the molecule. The RIF-1, RIF-25R and P10 strains all displayed similar sensitivities to m-THPC-mediated PDT, indicating that mTHPC acts via a different mechanisms) to that of PHP or PPC.

The P10 strain did not display cross-resistance to PpIXmediated photosensitization, either when endogenously produced from ALA or when added exogenously. The RIF-25R strain was not cross-resistant to PpIX produced from ALA; however, this strain did exhibit cross-resistance to exogenously added PpIX. Similar findings have been reported by Wilson et al. (24) in RIF-8A cells and have been explained in terms of the cellular localization of endogenous and exogenous PpIX. Endogenous PpIX is known to be produced in the inner mitochondrial membrane (42), and colocalization experiments with ALA-produced PpIX and Rhodamine123 (24) have demonstrated that there is no difference in the localization of endogenous PpIX between RIF-1 and RIF8A. This is consistent with the lack of cross-resistance to endogenously produced PpIX in the RIF-8A strain. Wilson et al. (24) also reported that exogenously added PpIX has a strong mitochondrial affinity, similar to that of Photofrin. A weaker mitochondrial association of exogenous PpIX (and Photofrin) was observed in the porphyrin-resistant RIF-8A strain compared to the parental RIF-1 strain. This correlates with the reduced sensitivity to exogenous PpIX observed in the RIF-8A strain and implicates the mitochondria as targets for porphyrin-mediated PDT. These observations suggest that an aspect of porphyrin transport (Photofrin and exogenous PpIX) to the mitochondria may be compromised in the RIF-8A strain, which could explain the increased resistance to Photofrin and the cross-resistance to exogenous PpIX.

The absence of cross-resistance to the anionic zinc (II) tetrasulfonated phthalocyanine in the PlO strain indicates that the mechanisms) of phthalocyanine-resistance is/are determined by factors other than the inherent structure of the phthalocyanine molecule.

In summary, the work presented here indicates that there are at least two distinct mechanisms of PDT-resistance displayed by RIF cells, and suggests that the mechanisms) of PHP-resistance is likely to depend, to some extent, upon the physical nature of the sensitizer molecule. Furthermore, studies involving short and extended photosensitizer incubation periods have indicated that PHP resistance may be a consequence of a membrane alteration in RIF-25R cells. These studies have also revealed that PPC-resistance could be due to the alteration of a single, as yet unidentified cellular target in P10 cells.

Acknowledgements-The authors would like to thank the Medical Research Council and Yorkshire Cancer Research for generous financial assistance.

Posted on the website on 22 November 2000.

^Abbreviations: ALA, 5-aminolevulinic acid; CDNB, 1-chloro-2,4dinitro-benzene; cisplatin, cis-diamminedichloroplatinum; DER50, dose enhancement ratio over the parental strain at the 50% survival level; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid); HSP-70, 70 kDa heat shock protein; MTT, 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate buffered saline; PDT, photodynamic therapy; PHP, polyhaematoporphyrin; PpIX, protoporphyrin IX; PPC, pyridinium-substituted zinc (II) phthalocyanine; RIF, radiation-induced fibrosarcoma; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase; m-THPC, 5,10,15,20-tetra(meta-hydroxyphenyl) chlorin; mTPyP, meso-tetra(4-pyridyl) porphine; TGly, tetraglycine-substituted zinc (II) phthalocyanine; TSPC, tetrasulfonated zinc (II) phthalocyanine.

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Stephen Mayhew, David I. Vernon, Jack Schofield, John Griffiths and Stanley B. Brown*

Center for Photobiology and Photodynamic Therapy, School of Biochemistry and Molecular Biology, University of Leeds, UK

Received 20 June 2000; accepted 17 October 2000

*To whom correspondence should be addressed at: Center tor Photobiology and Photodynamic Therapy, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. Fax: 44-113-233-3017; e-mail: s.b.brown@leeds.ac.uk

Copyright American Society of Photobiology Jan 2001
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