Sites of photodamage induced by photodynamic therapy with a chlorin e6 triacetoxymethyl ester (CAME)

The acetoxymethyl ester of chlorin e6 (CAME) was initially designed to be a hydrophobic photosensitizing agent that would be recognized by an endocytic pathway and initially accumulated in lysosomes. This was expected to lead to hydrolysis of the ester groups, followed by redistribution of the free chlorin to other subcellular sites. In this study, we examined the patterns of localization of CAME and of subsequent photodamage in murine leukemia L1210 cells. The drug was initially localized at intracellular sites, yielding a pattern similar to that obtained with a fluorescent probe for acidic intracellular vesicles and endosomes. A brief (30 min) incubation with 10 microM CAME followed by irradiation led to mitochondrial photodamage and apoptotic cell death. At a higher drug level, or with a longer incubation time, we observed additional photodamage to the plasma membrane and to lysosomes. The higher photodynamic therapy dose led to inhibition of apoptosis, with cell death likely occurring via a necrotic process. Distribution of CAME among the components of human plasma was to albumin > high-density lipoprotein > low-density lipoprotein. These results have implications concerning the likely mechanism of CAME accumulation and subcellular distribution.     

Rapid Initiation of Apoptosis by Photodynamic Therapy

Abstract— Photodynamic therapy (PDT) of neoplastic cell lines is sometimes associated with the rapid initiation of apoptosis, a mode of cell death that results in a distinct pattern of cellular and DNA fragmentation. The apoptotic response appears to be a function of both the sensitizer and the cell line. In this study, we examined photodynamic effects of several photosensitizers on murine leukemia P388 cells. Two drugs, a porphycene dimer (PcD) and tin etiopurpurin (SnET2), which localized at lysosomal sites, were tested at PDT doses that resulted in 50% loss of viability (LD₅₀), measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. An oligonucleosomal pattern of DNA degradation was observed within 1 h after irradiation. Neither sensitizer antagonized PDT-mediated internucleosomal DNA cleavage by the other. Very high PDT doses with either agent abolished this rapid internucleosomal cleavage. Exposure of cells to high concentrations of either sensitizer in the dark also resulted in rapid DNA fragmentation to nucle-osomes and nucleosome multimers; this effect was not altered by the antioxidant 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox), although the latter could protect cells from cytotoxicity and apoptotic effects caused by LD₅₀ PDT doses. Photodamage from two cat-ionic sensitizers, which localized at membrane sites, caused rapid DNA cleavage to 50 kb particles; however, no further fragmentation was detected after 1 h under LD₁₀, LD₅₀ or LD₉₅ PDT conditions. Moreover, the presence of either cationic sensitizer inhibited the rapid internucleosomal cleavage induced by SnET2 or PcD photodamage. The site of photodynamic action may therefore be a major determinant of the initiation and rate of progression of apoptosis.     

Solvothermal synthesis and crystal structure of a 3-D hexagonal metal-porphyrin coordination network

A hexagonal-shaped hybrid metal-porphyrin framework containing 5,15-di(4-pyridyl)-10,20-diphenylporphyrin (trans-H₂DPyP) and Zn(NO₃)₂6H₂O in DMF has been self-assembled solvothermally. The solvothermal reaction of Zn(II) with conformationally versatile 5,15-di(4-pyridyl)-10,20-diphenylporphyrin, which can coordinate axially to two ligands, resulted in metalation of the porphyrin core and then self-coordination of the metalated porphyrin-produced single crystals of 3-D polymeric arrays. The trans-ZnDPyP framework is thermally stable at 450?°C and allows solvent exchange without losing the crystal structure.     

Subcellular localization of merocyanine 540 (MC540) and induction of apoptosis in murine myeloid leukemia cells

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m2 (LD90 for JCS and LD86 for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.     

SITES OF PHOTODAMAGE in vivo and in vitro BY A CATIONIC PORPHYRIN

Abstract— Localization and photodynamic efficacy of a monocationic porphyrin (MCP) were assessed using murine leukemia cells in culture. This sensitizer localized at surface membrane loci and catalyzed selective photodamage to membrane structures. Although both cationic and hydrophobic, this porphyrin was not recognized by the multidrug transporter, which excludes many cationic agents from cells that express multidrug resistance. Photodynamic studies with the murine radiation-induced fibrosarcoma tumor model indicated moderate photosensitization of neoplastic lesions in vivo at 3 h, but not at 24 h after sensitizer administration. Pharmacokinetic studies indicate that plasma levels, not tissue levels were the major determinant of photodynamic therapy (PDT) response. Consistent with this observation, vascular damage and disturbances of tissue perfusion followed PDT. These effects were more pronounced in tumor-bearing skin than in normal skin. The therapeutic response to MCP appeared to be related mainly to secondary, probably vascular, effects.     

5-aminolevulinic acid-based photodynamic therapy in leukemia cell HL60

A study to explore the optimal experimental parameters and the photosensitization of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) in promyelocytic leukemia cell HL60 has been conducted, in which HL60 cells and their control groups, peripheral blood mononuclear cell (PBMC), first are incubated with different concentrations of ALA in dark for different periods of time and then followed by irradiating with different wavebands for different fluences. Fluorescence microscope and spectrofluorometer have been used to detect the fluorescence of protoporphyrin IX (PpIX) endogenously produced by ALA. The response of the cells to ALA-PDT was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay (interval between irradiation and the MTT assay is 24 h) and by flow cytometry (the length of time between irradiation and the flow assay is 30 min). MTT results will reflect the relative number of metabolically active mitochondria in the population. Propidium iodide uptake in flow cytometry will test for membrane damage. The results of parameter experiments were obtained: 1 x 10(5)/mL HL60 cell was first incubated with 1 mmol/L ALA in dark for 4 h and the maximum fluorescence of PpIX level appeared; then irradiated with 410 nm (4 mW/cm2) for 14.4 J/cm2 and maximum photodamage to membrane and mitochondrial function of HL60 cell resulted. With the normal granulocytes, such response was not detected. Therefore a hypothetical idea can be brought forward that ALA-based PDT can be used for inactivation of leukemia cell HL60 and these optimal parameters may be useful for clinical application.     

Merocyanine 540 Solubilized as an Ion Pair with Cationic Surfactant in Nonpolar Solvents: Spectral and Photochemical Properties

Merocyanine 540 (MC) is an anionic dye that is used to photopurge the bone marrow of leukemia cells. Under these conditions it is localized mostly in cell membranes, which may affect its photochemical reactivity. We investigated the photochemistry of MC dissolved as a hydrophobic ion pair with a hexadecyltrioctadecylammonium cation in cyclohexane, trimethylpentane and toluene as well as in propylene carbonate, CH3CN, C2H5OH and D2O. In organic solvents, the absorption and fluorescence spectra of MC were strongly red-shifted compared with aqueous solutions. The fluorescence was also more intense despite aggregation that occurred in some solvents. Aggregation strongly affects the spectral and photochemical properties of MC, especially in aliphatic hydrocarbons in which distinctive H-type aggregates are formed. Hydrophobic MC is a moderate photosensitizer of singlet molecular oxygen (1O2). The following values for 1O2 quantum yields were calculated based on 1O2 phosphorescence relative to 1O2 generation by Rose Bengal: approximately 0.12 in trimethylpenthane, approximately 0.13 in cyclohexane, 0.045 in EtOH, 0.039 in toluene, 0.007 in CH3CN and approximately 3 x 10(-4) in D2O. The H-aggregates of MC in cyclohexane and trimethylpentane are better 1O2 producers than monomeric MC. The above 1O2 quantum yields are corrected for self-quenching because MC is an efficient 1O2 quencher (17 x 10(7) M-1 s-1 in CH3CN, 6.8 x 10(7) M-1 s-1 in D2O, 5.2 x 10(7) M-1 s-1 in EtOH, and 1.4 x 10(7) M-1 s-1 in toluene). Merocyanine undergoes photodegradation, a solvent-dependent process that proceeds faster when the dye is aggregated. The initial photodegradation rate is much slower in organic solvents than in water, but photodegradation products accumulated during longer irradiation may increase the rate in most solvents. Higher photostability and better photosensitization by MC in hydrophobic nonpolar solvents suggest that the killing of leukemia cells via a photodynamic mechanism may operate mostly in cell membranes. In contrast, any cytotoxic products from photodecomposition may be important in hydrophilic cell compartments. Our data show the spectral and photochemical properties of MC in a pure hydrophobic environment.     

3-Cyclobutenyl-1,2-dione-substituted porphyrins. A general and efficient entry to porphyrin-quinone and quinone-porphyrin-quinone architectures

A new and efficient synthesis of meso-linked porphyrin-quinone dyads and quinone-porphyrin-quinone triads has been developed via the intermediacy of porphyrins bearing 3-cyclobutenyl-1,2-dione and 3-(1-ethenyl)cyclobutenyl-1,2-dione substituents at one or two nonadjacent meso-positions. The free-base porphyrins 5-bromo-10,20-diphenylporphyrin and 5,15-dibromo-10,20-diphenylporphyrin undergo facile palladium-catalyzed Stille coupling with 3-isopropoxy-2-tri-n-butylstannyl-cyclobutene-1,2-dione to produce the corresponding mono- and bis(3-cyclobutenyl-1,2-dione)-substituted porphyrins in good yields. In contrast, the zinc bromoporphyrins reacted with the same tin reagent only slowly and with the formation of side products. The free-base bromoporphyrins also were coupled with tri-n-butylvinyltin to afford vinylporphyrins in very good yields. 5,15-Diphenyl-10-vinylporphyrin was converted into trans-bromovinylporphyrin, which underwent facile Stille coupling with 3-isopropoxy-2-tri-n-butylstannylcyclobutene-1,2-dione to afford the vinylogous 3-cyclobutenyl-1,2-dione-substituted porphyrin. The molecular structure of 5,15-bis(3-cyclobutenyl-1,2-dione)-10,20-diphenylporphyrin(Z n) was determined by X-ray crystallography. Although the data revealed a fairly large dihedral angle between the cyclobutenedione and the porphyrin rings (57 degrees), the UV-vis spectra of both the mono- and bis(3-cyclobutenyl-1,2-dione)-substituted porphyrins showed B- and Q-band red shifts indicative of strong electronic coupling between the porphyrin and cyclobutenedione chromophores in solution. Introduction of a double bond between the cyclobutenedione and porphyrin rings resulted in a significant red shift of both the B- and Q-bands compared to those of the nonvinylogous system. All porphyrinic cyclobutenediones were metalated with zinc and then, using established cyclobutenedione chemistry, converted into a variety of porphyrin-quinones in excellent yields with aryllithium and vinylic Grignard reagents. From the mono(3-cyclobutenyl-1,2-dione)-substituted porphyrin, 7, a variety of directly linked monoquinone-porphyrin dyads were easily synthesized. Substituents could also be introduced at the free meso-position of 7 by bromination followed by palladium-catalyzed cross-coupling reactions, and additional porphyrinic monoquinones were then prepared from these starting materials. The vinylogous squarylporphyrin was converted into a double bond linked porphyrin-quinone via reaction with phenyllithium followed by thermal rearrangement and oxidation. As a result of the hindered rotation around the C-C bond between the porphyrin and the quinone, pairs of stable, separable, and thermally interconvertable atropisomers of porphyrin-quinones were obtained from 5,15-bis(3-cyclobutenyl-1,2-dione)-10,20-diphenylporphyrin(Z n). The structure of one of the atropisomers was determined by X-ray crystallography.     

Studies on mesoionic compounds,II. The solvent effect on the electronic absorption spectra of mesoionic 2,3-diaryl-2H-tetrazolium-5-thiolate derivatives

The electronic absorption spectra of 2,3-di(2-fluorophenyl)-, 2,3-di(4-fluorophenyl)-, 2,3-di-(2-chlorophenyl)-, 2,3-di(2-methylphenyl)-, 2,3-di(4-methylphenyl)- and 2,3-di(2-methoxyphenyl)-2H-tetrazolium-5-thiolates have been measured in pure and mixed solvents. They were found to exhibit three characteristic absorption bands at 480-380 nm, near 250 and near 210; the longest wavenlength band of which was assigned to an n → π* transition.     

Effects of HPV Status on Responsiveness to Ionizing Radiation vs Photodynamic Therapy in Head and Neck Cancer Cell lines

Efficacy of ionizing radiation (I/R) was compared with phototoxic effects of photodynamic therapy (PDT) in vitro using two cell lines derived from patients with head and neck squamous cell carcinoma (HNSCC). A cell line derived from a donor with a human papilloma virus (HPV) infection was more responsive to I/R but significantly less responsive to PDT than a cell line derived from an HPV-free patient. Cell death after I/R in the HPV(+) cell line was associated with increased DEVDase activity, a hallmark of apoptosis. The HPV(−) line was considerably less responsive to I/R, with DEVDase activity greatly reduced, suggesting an impaired apoptotic program. In contrast, the HPV(−) cells were readily killed by PDT when the ER was among the targets for photodamage. While DEVDase activity was enhanced, the death pathway appears to involve paraptosis until the degree of photodamage reached the LD₉₉ range. These data suggest that PDT-induced paraptosis can be a death pathway for cells with an impaired apoptotic program.     

新颖的5,15-二(4-(5-乙酰基硫戊氧基)苯基)卟啉化合物的结构和性质的密度泛函理论计算

通过密度泛函理论计算比较性地研究了5,15-二(4-(5-乙酰基硫戊氧基)苯基)自由卟啉及其锌配合物的分子结构、电荷性质、分子轨道、电子吸收光谱和红外光谱.这类化合物具有在卟啉相对的两个中位的苯环上连有5-乙酰基硫戊氧基的新颖结构.模拟得到的这两个化合物的分子结构和电子吸收光谱以及红外光谱都与实验测得的符合得很好.通过与未取代的自由卟啉和卟啉锌的结构和性质进行比较,研究了中位取代基、极性溶剂和中心金属取代对此类卟啉化合物结构和性质的影响规律.对化合物的电子吸收光谱中的电子跃迁本质进行了归属,并通过基于正则坐标分析产生的动画对红外光谱的振动模式进行了指认.目前的工作将对理解此类新颖卟啉化合物的结构和性质以及取代基和溶剂效应提供很大的帮助.     

Mitochondrial localization and photodamage during photodynamic therapy with tetraphenylporphines

The subcellular localization sites of TPPS4 and TPPS1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS4 favors an aqueous environment, while TPPS1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS1 in the peri-nuclear region, while TPPS4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na(+)/K(+)-ATPase activity. As a result, cytosolic concentrations of Na(+) and Ca(2+) increased, and the level of K(+) decreased. In contrast, the lipophilic TPPS1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS1 has no effect on the mitochondria.     

Sites of photosensitization by protoporphyrin and tin protoporphyrin in leukemia L1210 cells

Studies with protoporphyrin (PP) and tin protoporphyrin (SnPP) were carried out to assess the effects of tin insertion on the sites of dye localization. Fluorescence emission spectra and studies on the sites of photodamage were consistent with a concentration of PP at membrane loci. In contrast, SnPP photodamage involved an intracellular site.     

Apoptosis mechanisms related to the increased sensitivity of Jurkat T-cells vs A431 epidermoid cells to photodynamic therapy with the phthalocyanine Pc 4

To examine the clinical applicability of Pc 4, a promising second-generation photosensitizer, for the photodynamic treatment of lymphocyte-mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte-derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose-response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4-PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl-2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl-2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl-1 was extensively cleaved in a dose- and caspase-dependent manner in Jurkat, but not in A431, cells exposed to Pc 4-PDT. Thus, the greater killing by Pc 4-PDT in Jurkat compared with A431 cells correlated with greater Bcl-2 photodamage and more strongly to the more extensive Mcl-1 degradation. Pc 4-PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T-cell-mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl-2 family proteins, especially Mcl-1, in the therapeutic response.     

Reversible Effects of Photodamage Directed Toward Mitochondria

When the initial effect of photodynamic therapy (PDT) involves mitochondrial photodamage, an early effect is loss of the mitochondrial membrane potential (ΔΨ_m). Using murine hepatoma 1c1c7 cells and a photosensitizing agent known to target mitochondria, we examined loss of ΔΨ_m, initiation of apoptosis and loss of viability as a function of time and light dose. There was a correlation between loss of viability and the rapid disappearance of ΔΨ_m, as detected by the potential‐sensitive probe Mitotracker Orange (MTO). Loss of ΔΨ_m was, however, reversible even with a substantial loss of viability. Unless there was a supralethal level of photodamage, 1c1c7 cells recovered their mitochondrial membrane potential, even if the cell population was on the pathway to apoptosis and cell death. These results indicate that when mitochondria are the initial PDT target, a qualitative estimate of photokilling can be provided by assessing the initial loss of ΔΨ_m.     

Porphyrin-fullerene C60 dyads with high ability to form photoinduced charge-separated state as novel sensitizers for photodynamic therapy

The photodynamic activities of a porphyrin-C60 dyad (P-C60) and its metal complex with Zn(II) (ZnP-C60) were compared with 5-(4-acetamidophenyl)-10,15,20-tris(4-methoxyphenyl)porphyrin (P), both in homogeneous medium-bearing photooxidizable substrates and in vitro on the Hep-2-human-larynx-carcinoma cell line. This study represents the first evaluation of dyads, with a high capacity to form a photoinduced charge-separated state, to act as agents to inactivate cells by photodynamic therapy (PDT). Absorption and fluorescence spectroscopic studies were performed in toluene and N,N-dimethylformamide (DMF). The emission of the porphyrin moiety in the dyads is strongly quenched by the attached fullerene C60 moiety. The singlet molecular oxygen, O2(1delta(g)), productions (phi(delta)) were determined using 9,10-dimethylanthracene (DMA). The values of phi(delta) were strongly dependent on the solvent's polarity. Comparable phi(delta) values were found for dyads and P in toluene, while O2(1delta(g)) production was significantly diminished for the dyads in DMF. In more polar solvent, the stabilization of charge-transfer state takes place, decreasing the efficiency of porphyrin triplet-state formation. Also, both dyads photosensitize the decomposition of L-tryptophan in DMF. In biological medium, no dark cytotoxicity was observed using sensitizer concentrations < or = 1 microM and 24 h of incubation. The uptake of sensitizers into Hep-2 was studied using 1 microM of sensitizer and different times of incubation. Under these conditions, a value of approximately 1.5 nmol/10(6) cells was found between 4 and 24 h of incubation. The cell survival after irradiation of the cells with visible light was dependent upon light-exposure level. A higher photocytotoxic effect was observed for P-C60, which inactivates 80% of cells after 15 min of irradiation. Moreover, both dyads keep a high photoactivity even under argon atmosphere. Thus, depending on the microenvironment where the sensitizer is localized, these compounds could produce biological photodamage through either an O2(1delta(g))-mediated photoreaction process or a free-radicals mechanism under low oxygen concentration. These results show that molecular dyads, which can form a photoinduced charge-separated state, are a promising model for phototherapeutic agents, with potential applications in cell inactivation by PDT.     

Hypericin-mediated photodynamic antimicrobial effect on clinically isolated pathogens

The aim of this study was to determine the photodynamic antimicrobial effect of hypericin on clinically isolated Staphylococcus aureus and Escherichia coli cells. Bacterial cells (10(8) cells per mL) were incubated with hypericin (0-40 μM) for 30 min and followed by light irradiation of 600-800 nm at 5-30 J cm(-2). Cell survival was determined by colony counting, cellular hypericin uptake examined by flow cytometer, and cell membrane damage examined by scanning electron microscopy and leakage assay. The effectiveness of hypericin-mediated photodynamic killing was strongly affected by cellular structure and photosensitizer uptake. The combination of hypericin and light irradiation could induce significant killing of Gram positive methicillin-sensitive and -resistant S. aureus cells (>6 log reduction), but was not effective on Gram negative E. coli cells (<0.2 log reduction). The difference was caused by different cell wall/membrane structures that directly affected cellular uptake of hypericin.     

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.     

SITES OF PHOTOSENSITIZATION BY DERIVATIVES OF HEMATOPORPHYRIN

Leukemia L1210 cells were incubated in vitro with the tumor-localizing product HPD (hem-atoporphyrin derivative) for 0.5. 4 and 18 h. Effects of subsequent irradiation on viability, membrane transport and integrity, DNA synthesis and intracellular ATP concentration were assessed. Intracellular porphyrin pools were analyzed by HPLC. A 30 min incubation led to concentration of a readily-exchangeable pool of monomeric HPD components at plasma membrane loci; irradiation resulted in photodamage to membrane transport and a loss in capacity for dye exclusion. In contrast, increasing the incubation time led to a corresponding increase in the size of a non-exchangeable intracellular pool of other HPD components. Subsequent irradiation led to depletion of intracellular ATP and loss of capacity for biosynthesis of DNA, but little plasma membrane damage.     

PORPHYRIN PHOTOSENSITIZATION OF MULTI-DRUG RESISTANT CELL TYPES

The P388 murine leukemia and P388/ADR, a subline expressing the multi-drug resistance (MDR) phenotype, were examined with regard to the role of MDR as a determinant of responsiveness to photodynamic therapy in vitro. Mesoporphyrin was used as a model substrate. We found no differences in porphyrin accumulation nor transport alterations associated with exposure of P388/ADR cells to the verapamil analog DMDP. There was a significant correlation between photodamage to mitochondria vs loss of cell viability in both cell lines, and LD50 sensitizer levels were not significantly different in P388 vs P388/ADR. P388/ADR cells were partly resistant to porphyrin-catalyzed photodamage to amino acid transport, but this result was not associated with differences in sensitizer localization, as indicated by fluorescence studies. Moreover, photodamage to membrane transport was not associated with loss of viability. These studies suggest that cells which express the MDR phenotype are unlikely to be cross-resistant to photodynamic therapy.