Ultrastructural Changes in PAM Cells After PhotodynamicTreatment with Delta-Aminolevulinic Acid-InducedPorphyrins or Photosan

Sonja Radakovic-Fijan, Klemens Rappersberger,* Adrian Tanew, Herbert Honigsmann, and Bernhard Ortel†Division of Special and Environmental Dermatology and *Division of General Dermatology, University of Vienna, Austria; †Wellman Laboratories ofPhotomedicine, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, U.S.A.

Photodynamic therapy (PDT) is the combination of aphotosensitizing drug (Ps) with light in the presence ofoxygen leading to the generation of reactive molecularspecies and destruction of cancer cells. In this studywe compared PDT with two Ps, the hematoporphyrinderivative Photosan (Ph) and delta-aminolevulinic acid(ALA)-induced endogenous protoporphyrin IX, withrespect to mitochondrial function and ultrastrucuralalterations. The effects of PDT were investigated inPAM 212 cells after different Ps incubation times, lightdoses, and post-treatment periods. Both Ps induced alight dose-dependent impairment of the mitochondrialfunction with the dose–response curve being steep forALA and flat for Ph. The prolongation of the incub-ation time from 4 to 20 h resulted in an increased

Photodynamic therapy (PDT) is a treatment modalitythat employs the combined administration of a photo-sensitizing drug (Ps) with subsequent light irradiation(Dougherty et al, 1990; Moore et al, 1997). Systemicor local administration results in accumulation and

retention within malignant tissues. Irradiation of the tissue-localizedPs with light of appropriate wavelength leads to cell death andtumor necrosis (Henderson and Dougherty, 1992; Fisher et al,1995). One of the most commonly used Ps is hematoporphyrinderivative (HpD), which is a mixture of monomeric and oligomericforms of various hematoporphyrins. HpD is commercially availableas Photofrin or Photosan, and is used in fluorescence diagnosis andPDT of various internal neoplasms. In dermatology, PDT withtopically administered delta-aminolevulinic acid (ALA) has becomean established and increasingly used treatment modality in recentyears (Kennedy and Pottier, 1992). ALA is the first precursor inheme synthesis. The biosynthesis of ALA is regulated by a negativefeedback control by heme of ALA-synthetase. By addition ofexogenous ALA this negative feedback mechanism can be bypassed.This leads to an increased synthesis and accumulation of downstream

Manuscript received May 25, 1998; revised November 9, 1998; acceptedfor publication November 19, 1998.

Reprint requests to: Dr. Sonja Radakovic-Fijan, Division of Special andEnvironmental Dermatology, Department of Dermatology, University ofVienna Medical School, Wahringer Gurtel 18-20, A-1090 Vienna, Austria.

Abbreviations: ALA, aminolevulinic acid; HpD, hematoporphyrin deriv-ative; PDT, photodynamic therapy; Ph, Photosan; PP IX, protoporphyrinIX; Ps, photosensitizing drug.

0022-202X/99/$10.50 · Copyright © 1999 by The Society for Investigative Dermatology, Inc.

264

reduction of mitochondrial activity after ALA PDTbut not after Ph PDT. Treatment with an irradiationdose that decreased mitochondrial activity by 50%(IC50) led to early and profound changes of mitochon-drial morphology in ALA photosensitized cells,whereas photosensitization with Ph resulted in morepronounced alterations of lysosomes. We concludethat at bioequivalent sublethal PDT exposures of PAM212 cells, ALA-induced damage is primarily restrictedto mitochondria, whereas Ph-induced cytotoxicity ismediated by damage of the lysosomal system. Keywords: delta-aminolevulinic acid/mitochondrial activity/photodynamic therapy/Photosan/ultrastructure. J InvestDermatol 112:264–270, 1999

metabolites, mainly protoporphyrin IX (PP IX) (Malik and Lugaci,1987). Clinical studies have demonstrated the efficacy of ALA PDTin the treatment of basal cell carcinoma, Bowen’s disease, and solarkeratoses (Kennedy and Pottier, 1992; Wolf et al, 1993; Fijanet al, 1995).

In this study we applied two clinically relevant Ps and investigatedthe effects of PDT with ALA or Photosan (Ph) on mitochondrialfunction and ultrastructural morphology of PAM 212 cells underdifferent experimental conditions. We have chosen the murinekeratinocyte line PAM 212 because our prior work on ALA-induced PP IX formation and modification by iron complexationwas performed in these cells (Ortel et al, 1993) and was appliedclinically with consistent results (Fijan et al, 1995). Most importantly,we wanted to investigate the differential effects of two diversephotodynamic regimens on the cellular ultrastructure, but did notaddress differences between cell lines, which can be considerable(Iinuma et al, 1994; Ortel et al, 1998).

MTT assays and electron microscopy were performed after twodifferent Ps incubation times (4 and 20 h), increasing irradiationdoses and variable post-irradiation periods (1 and 24 h). To analyzethe effects of sublethal irradiations and to differentiate between ALAand Ph-specific subcellular targets, we determined the ultrastructuralchanges after PDT with a bioequvalent PDT dose that reducedthe mitochondrial activity by 50% (IC50).

MATERIALS AND METHODS

Cells PAM 212 is a clonogenic and tumorigenic cell line derived frommurine epidermal keratinocytes. The cells were grown continuously inRPMI 1640 medium (Gibco, Paisley, Scotland), supplemented with 10%

VOL. 112, NO. 3 MARCH 1999 ULTRASTRUCTURAL CHANGES IN PAM CELLS AFTER PHOTODYNAMIC TREATMENT 265

fetal calf serum, 100 µg streptomycin per ml, and 100 IU penicillin (Gibco)in a humidified 5% CO2 atmosphere.

Chemicals Delta-aminolevulinic acid hydrochloride (Fluka, Buchs,Switzerland) was dissolved to a final concentration of 1 mM in completemedium. Photosan (Seelab, Vienna, Austria) was diluted in phosphate-buffered saline (PBS, Gibco) and used at a final concentration of 7.5 µgper ml or 15 µg per ml in complete medium.

Radiation source A Coherent Medical 920 Argon Dye Laser (Coherent,Cambridge, U.K.) was used at 514 nm in the continuous wave mode. Theirradiance was measured at the level of the cells with a Model 210 powermeter (Coherent).

Quantitation of porphyrins The cellular content of PP IX and Ph wasdetermined after an incubation period of 4 and 20 h. The cells werewashed twice with PBS (without Ca211 and Mg211) and detached fromthe culture dishes using 1 ml of 0.1% trypsin/EDTA (Gibco). After dilutionwith 2 ml of cold PBS, 500 µl of the suspension was used for cell counting(Coulter Electronics, Luton, U.K.). The remaining 2.5 ml were centrifugedand the pellet was dissolved in 3 ml of 2% SDS in 0.1 M NaOH (NaOH/SDS). Fluorescence was measured in a LS 50B spectrofluorometer (PerkinElmer, Beaconsfield, U.K.). The excitation wavelength was set at 400 nm,the emission spectrum was recorded from 600 to 720 nm rather than at asingle wavelength to allow correction for background scattering and toexclude contribution from non-PP IX porphyrins. The peak height at631 nm was used for quantitation of PP IX according to a standard curve.The amount of PP IX was expressed as fg per cell. All these stepswere done under minimal lighting conditions. Photosan was quantitated

Figure 1. Reduction of MTT conversion after photosensitizationwith ALA or Ph and irradiation with an Argon dye laser (514 nm).n, 1 mM ALA per 4 h; u, 1 mM ALA per 20 h; s, 15 µg Ph per mlper 4 h; e, 7.5 µg Ph per ml per 20 h. Values are given as mean 6SD.

Table I. Synopsis of ultrastructural findings after different conditions of ALA and Ph PDT

ALA PDT Ph PDT

Incubation Time after Incubation Time afterLight dose time (h) PDT (h) Ultrastructural findings time (h) PDT (h) Ultrastructural findings

3 J/cm2 4/20 1 Pronounced cytoplasmic edema 4/20 1 Moderate cytoplasmic edemaVarious degrees of mitochondrial Large number of membraneswelling up to complete bound cytoplasmic vesiclesdestruction filled with granular materialModerate swelling of the Discrete damage ofendoplasmatic reticulum mitochondria

4/20 24 Progression of cell damage 4/20 24 Progression of cell damagecell death Cell death

IC50a 4 1 Slight to severe cytoplasmic edema 4 1 Moderate cytoplasmic edema

Mitochondrial condensation and Small number of cytoplasmicvarious degrees of hydropic vesicles filled with granularswelling materialOccasionally swelling of the Occasionally mitochondrialendoplasmatic reticulum swelling

4 24 Progression of cell damage 4 24 Large number ofCell death phagolysosomes

a Irradiation dose leading to 50% reduction of mitochondrial activity.

identically with the exception that the peak of fluorescence intensity usedfor quantitation was at 626 nm.

Photodynamic treatment Two 3 105 cells were plated into 60 mmculture dishes. Forty-eight hours later the medium was replaced by 2 mlof complete medium containing the Ps. To attain roughly equal intracellularlevels of Ph, the concentration of 15 µg per ml was used for the incubationperiod of 4 h, whereas the lower concentration of 7.5 µg per ml was usedfor the incubation period of 20 h. ALA was used at a concentration of1 mM for both incubation periods. After incubation with the Ps themedium was removed and the cells were rinsed twice with PBS, coveredwith 1 ml PBS, and exposed to 0.5–3.0 J per cm2 of 514 nm coherentlight radiation at a fluence rate of 50 mW per cm2. After a furtherincubation period for 20 h in fresh complete medium the mitochondrialfunction was determined. The irradiation dose that reduced the mitochon-drial activity by 50% was termed IC50. Untreated cells, cells incubatedwith ALA or Ph without light exposure, and unphotosensitized cells treatedwith laser light only served as control.

The ultrastructural studies were performed in two sets of experiments.In the first set the cells were incubated with ALA or Ph for 4 or 20 h andirradiated with a constant dose of 3 J per cm2. After a post-treatmentperiod of 1 or 24 h the samples were processed for electron microscopy.

In the second series of experiments the cells were incubated with ALAor Ph for 4 h and irradiated with the IC50. The post-treatment periodswere again 1 or 24 h.

MTT assay For the evalution of the mitochondrial function we slightlymodified the MTT conversion assay (Mosmann, 1983), which measuresmitochondrial dehydrogenase activity. Twenty hours after PDT the mediumwas removed and the cells were washed once with PBS at room temperature.One milliliter PBS containing 1.5 mg MTT [3-(4,5-dimethyl-thiaziol-2-yl)-2,5-diphenyl tetrazolium bromide, Sigma] per ml was added and thecells were incubated at 37°C for 4 h. The supernatant was replaced by2 ml dimethylsulfoxide (Merck, Vienna, Austria) and the dishes wereshaken at a moderate speed for 30 min. The optical density of each solutionwas measured at 576 nm and the values of treated cells were expressed aspercentage of untreated controls. Experiments were done in duplicates andrepeated six times.

Transmission electron microscopy Photosensitized cells were fixedin 2.5% glutaraldehyde for 5 h, washed in 0.1 M cacodylate at 4°C, post-fixed in 3% osmium tetroxide for 1 h, washed in distilled water, and leftin 0.5% uranylacetate-veronal buffer for 1 h at room temperature. Thecells were then dehydrated in a graded series of ethanol and subsequentlyembedded in Epon 812. Thin sections were cut on a Reichert Ultracut2000, stained with 1% uranyl acetate and 0.25% lead citrate, and examinedwith a transmission electron microscope (JEOL-EX 1200, Tokyo, Japan).

266 RADAKOVIC-FIJAN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

RESULTS

Similar cellular porphyrin levels after Ps incubation for 4 or20 h Spectrofluorometry of extracts from cells treated with ALAshowed a peak emission at 631 nm characteristic of PP IX. Thecellular PP IX content after ALA incubation for 4 and 20 h was193.5 6 24.2 fg per cell and 150.7 6 19.3 fg per cell, respectively(mean 6SD of six determinations). Control cells without ALA didnot yield measurable amounts of intracellular PP IX.

The fluorescence spectra from cells treated with Ph had a peakemission at 626 nm. The cellular content of Ph was 16.1 6 1.5 fgper cell after 4 h incubation with 15 µg Ph per ml and 19.8 62.1 fg per cell after 20 h incubation with 7.5 µg Ph per ml,respectively (mean 6SD of six determinations).

Mitochondrial activity is severely impaired after ALAPDT Incubation of PAM cells with ALA or Ph and subsequentlight irradiation resulted in a dose-dependent reduction of themitochondrial function as measured by MTT conversion (Fig 1).The slopes of the dose response curves were, however, clearlydifferent for the two Ps. With ALA PDT the slope was steep incontrast to a flat slope with Ph PDT.

Treatment with ALA for 4 h and the lowest irradiation dose of0.5 J per cm2 decreased the mitochondrial function by µ40%.After 20 h of ALA incubation the same light dose reduced theMTT converting ability by as much as 90%. Higher irradiationdoses led to a progressive impairment of the mitochondrial function,which was completely abolished after both incubation periods anda dose of 3 J per cm2. The IC50 was 0.62 J per cm2 after an ALAincubation period of 4 h and 0.3 J per cm2 after ALA incubationfor 20 h.

The reduction of mitochondrial activity was less pronouncedafter Ph PDT and only slightly influenced by the Ph incubationtime (Fig 1). The IC50 was 2.2 J per cm2 for the incubation periodof 4 h and 2.0 J per cm2 after Ph incubation for 20 h, which wasin accordance with the similar cellular porphyrin levels.

Mitochondrial swelling is an early result of ALA PDT,whereas Ph PDT induces lysosomal damage after high-doseirradiation (Table I) Four or 20 h incubation with ALAfollowed by irradiation with 3 J per cm2 and a post-irradiationperiod of 1 h led to similar degrees of cellular damage. A pronouncededema of the cytoplasm was seen that was primarily confined tothe periphery of the cells (Fig 2a). In addition, we detected amoderate swelling of the rough and smooth endoplasmatic reticu-lum and various degrees of hydropic swelling of the mitochondria,occasionally leading to complete destruction of these organelles(Fig 2b). In contrast, the Golgi apparatus, lysosomes, as well ascytoskeletal components such as keratin filaments and microtubules,appeared normal (Fig 2c).

Twenty-four hours after ALA PDT a progression of theseultrastructural changes and signs of incipient karyolysis weredetected.

Cells exposed to Ph for 4 or 20 h and irradiated with 3 J percm2 also disclosed similar ultrastructural changes 1 h after treatment.A moderate cytoplasmic edema was present with large numbers ofindividual ribosomes located at the periphery of the cytoplasm(Fig 3a). Between this cytoplasmic region and the nucleus, damagedlysosomes were found as membrane bound vesicles of differentsizes filled with an electron dense granular material (Ghadially,1988) (Fig 3b). These vesicles were never seen in Ph-treatedunirradiated control cells or cells after ALA PDT. All other cellorganelles were centralized and arranged around the nucleus. They

Figure 2. Ultrastructural findings 1 h after ALA PDT with 3 J percm2. (a) Pronounced edema (E) of the cytoplasm in the periphery of thecell; (b) various degrees of mitochondrial degeneration (M) with loss ofmitochondrial cristae and swelling of the rough and smooth endoplasmaticreticulum (ER); (c) lysosomes (L) and keratin filaments (KF) appear normal.Scale bars: (a) 1 µm; (b, c) 0.2 µm.

VOL. 112, NO. 3 MARCH 1999 ULTRASTRUCTURAL CHANGES IN PAM CELLS AFTER PHOTODYNAMIC TREATMENT 267

were morphologically well preserved with the exception of slightlycondensed mitochondria and discrete swelling of the endoplasmaticreticulum (Fig 3c).

Twenty-four hours after Ph PDT progression of the ultrastruc-tural changes and true cell death occurred.

Biologically comparable sublethal doses of ALA PDT andPh PDT result in different ultrastructural response patterns(Table I) One hour after ALA PDT with the IC50 the cellsdisplayed slight to moderate cytoplasmic edema (Fig 4a). Thenucleus was normal and surrounded by a rim of regular appearingcytoplasm that contained most if not all cell organelles. At theperiphery of the cells the cytoplasm contained ribosomes withoutevidence of other cell organelles. Again, the ultrastructural changesof the mitochondria were prominent with signs of condensationor hydropic swelling (Fig 4b). These changes, however, were lesspronounced than after irradiation with 3 J per cm2. Occasionally,the cells had a foamy appearance due to masses of membranebound vacuoles within the cytoplasm. Some of the vacuolescontained parts of mitochondrial cristae. Hydropic swelling ofthe endoplasmatic reticulum was also seen, but other subcellularstructures such as the cell cytoskeleton and Golgi apparatus appearedunaffected. Individual cells with pronounced cytoplasmic changesalso revealed margination of the chromatin indicative of earlykaryolysis.

Twenty-four hours after ALA PDT these ultrastructural changesclearly progressed and were characterized by a marked cytoplasmicedema, pronounced swelling of the mitochondria and endoplasmaticreticulum, and signs of frank karyolysis (Figs 4c, d).

One hour after Ph PDT with the IC50 we observed moderatecytoplasmic edema, many lipoidic vacuoles, and occasional con-densation or swelling of the mitochondria. Around the nucleus asmall number of altered lysosomes were present (Fig 5a).

An overall similar morphology was found 24 h after Ph PDTwith the notable exception of increased numbers of phagolyso-somes (Fig 5b).

DISCUSSION

This study was designed to determine the differences in functionaland ultrastructural changes after PDT with the exogenous HpDPh or ALA-induced endogenous PP IX in the murine keratinocytecell line PAM 212. In particular, we addressed the impact of Psincubation time, irradiation dose, and time interval after PDT onthe quantity and quality of ultrastructural alterations. The use ofthe IC50 allowed us to investigate the effects of sublethal irradiationand to compare the ultrastructural changes after equiphototoxicdoses of ALA PDT and Ph PDT.

ALA treatment resulted in high concentrations of PP IX afteran incubation period of 4 or 20 h. The intracellular PP IXconcentration was µ20% lower after ALA incubation for 20 h ascompared with 4 h, which might be due to the presence of 10%fetal calf serum in the cell medium. The presence of specialcomponents in the serum with high affinity for porphyrins cancause the translocation of PP IX from the cells to the medium(Fukuda et al, 1993).

Contrary to the kinetics of intracellular PP IX accumulation,ALA PDT-induced reduction of mitochondrial activity was greaterafter an incubation period of 20 h than after 4 h. This is in agreementwith other studies that demonstrated increased phototoxicity with

Figure 3. Ultrastructural findings 1 h after Ph PDT with 3 J percm2. (a) The periphery of the cytoplasm is filled with ribosomes (R). Thenucleus (N) is localized in the center and surrounded by cell organellesand intermediate filaments. Numerous membrane-bound vesicles (V) ofdifferent sizes filled with electron dense granular material are found in thecytoplasm. (b) Higher magnification of the membrane-bound vesicles (V)that are partly or completely filled with granular electron dense material.(c) Discrete damage of mitochondria (M) and endoplasmatic reticulum(ER). Scale bars: (a) 1 µm; (b) 0.2 µm; (c) 0.5 µm.

268 RADAKOVIC-FIJAN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 4. Ultrastructural findings after ALA PDT with the IC50. One hour after irradiation: (a) cytoplasmic edema (E) and mitochondrialcondensation (arrow); (b) higher magnification of mitochondrial condensation (arrow). Twenty-four hours after irradiation: (c) progression of cell damage;marked edema of the cytoplasm (E), karyolysis (N), and (d) pronounced swelling of the mitochondria (M) and endoplasmatic reticulum (ER). Scale bars:(a, c) 1 µm; (b, d) 0.5 µm.

prolonged ALA incubation but failed to find a correlation with theintracellular PP IX levels (Iinuma et al, 1994; Gibson et al, 1997).The reason for the incubation time-dependent increase in reductionof mitochondrial activity might be that PP IX redistributes withinthe mitochondria, leading to PDT-induced inhibition of differentlylocated mitochondrial enzymes (Gibson et al, 1989).

Incubation with 7.5 µg Ph per ml for 20 h resulted in µ20%higher intracellular concentrations of Ph than incubation with15 µg per ml for 4 h. The effects on mitochondrial activitycorrelated with the intracellular Ph levels at all except the highestirradiation doses and were substantially lower than those observedafter ALA PDT. These findings indicate that contrary to ALA PDTthe mitochondria were not the primary target of Ph photosensitiz-ation under our experimental conditions. This is in contrast toother studies with the related HpD compound Photofrin, in which

mitochondrial damage was the predominant ultrastructural findingafter PDT (Coppola et al, 1980; Milanesi et al, 1989). In this regard,besides the delivery system (Malik and Faraggi, 1992) the cell linemay have a major role as different subcellular Ps localizations andPs-induced mechanisms of cellular damage were observed indifferent cell lines (Berg and Moan, 1997).

ALA PDT with the high irradiation dose of 3 J per cm2 led toearly swelling of the mitochondria followed by swelling of theendoplasmatic reticulum and a dramatic progressive disintegrationof the cells. ALA-induced PP IX is synthetized in the mitochondriaand has a high affinity to membranes due to its pronouncedhydrophobicity. It is thus assumed that time-dependent translocationand accumulation of PP IX in cell organelles other than themitochondria results in their photodestruction (Malik and Lugaci,1987; Malik et al, 1989; Gaullier et al, 1995). This notion is

VOL. 112, NO. 3 MARCH 1999 ULTRASTRUCTURAL CHANGES IN PAM CELLS AFTER PHOTODYNAMIC TREATMENT 269

Figure 5. Ultrastructural findings after Ph PDT with the IC50. Onehour after irradiation: (a) mild cytoplasmic edema (E), many lipoidicvacuoles (LV), and cytoplasmic vesicles filled with granular material (V).Twenty-four hours after irradiation: (b) numerous phagolysosomes (PL),vesicles with granular content (V), and condensed mitochondria (M). Scalebars: (a) 1 µm; (b) 0.5 µm.

supported by fluorescence studies demonstrating a typical mitochon-drial pattern after short incubation with ALA (Iinuma et al, 1994;Liang et al, 1998) in contrast to a more diffuse pattern with largeperinuclear spots after ALA incubation for 42 h (Gaullier et al, 1995).

In our study we found that the morphologic changes after ALAPDT were largely independent of the ALA incubation time butincreased with the longer post-treatment period. This indicatesthat a 4 h ALA incubation period is sufficient for maximumphotosensitization, whereas a 1 h post-treatment time is too shortfor the full development of ultrastructural changes. Besides direct

photochemical reactions, PDT-mediated cell damage is supposedto involve enzymatic processes that require a longer time in exertingtheir effects (He et al, 1994).

After Ph PDT with the high irradiation dose of 3 J per cm2 anda post-treatment interval of 1 h we observed large numbers ofdamaged lysosomes. The mitochondria showed only discrete dam-age and the other cell organelles were well preserved. Twenty-fourhours after irradiation with 3 J per cm2 we found progression ofthe ultrastructural changes and signs of true cell death.

Several indications are found in the literature that lysosomesmight be a likely target for PDT using a HpD. Due to its mostlynegative charge HpD is difficult to concentrate in the mitochondriabut likely to accumulate in other parts of the cell. This notion isconsonant with the findings of fluorometric studies that showedthe absence of a mitochondrial fluorescence pattern after HpDtreatment (Bottiroli et al, 1992; Krammer et al, 1993). Incubationof cells with HpD in serum-containing culture medium, as wasdone in our experiments, promoted the delivery and accumulationof porphyrins in lysosomes (Malik and Faraggi, 1992; Geze et al,1993). This conceivably renders the lysosomes a primary target forHpD-induced photodynamic reactions. Further support for thepivotal role of lysosomes comes from our observation that thelysosomal changes after Ph PDT are morphologically similar tothose reported after PDT using Nile blue as photosensitizer, whichwas shown to localize in lysosomes (Lin et al, 1993).

Irradiation of ALA-photosensitized cells with the IC50 resultedin cellular alterations that were qualitatively similar albeit morediscrete than after irradiation with 3 J per cm2; however, with thelower irradiation dose the degree of cellular damage was muchmore variable between single cells as compared with high dose-treated cells where the cell damage was rather uniform. In addition,a small fraction of IC50-irradiated cells appeared unaffected by ALA-photosensitization and showed no or only minimal ultrastructuralalterations. This may reflect variable levels of ALA-induced PPIXwithin a subset of cells.

As was the case with ALA the irradiation of Ph-photosensitizedcells with the IC50 led to subcellular changes that were morediscrete but otherwise identical to those seen after irradiation with3 J per cm2. The prevailing finding 1 h after treatment with theIC50 was a small number of cytoplasmic vesicles filled with granularmaterial that represented damaged lysosomes. After the post-treatment period of 24 h in addition to these vesicles a largenumber of phagolysosomes was observed, reflecting increasedlysosomal activity and induction of autophagocytosis. This indicatesthe activation of a reparative process that sequesters and degradesdamaged parts of the cell (Zdolsek et al, 1990).

Summarizing our data we conclude that at least in PAM 212cells, (i) incubation with ALA results in marked and incubationtime-dependent photosensitization of the mitochondria, (ii) theeffect of Ph PDT on mitochondrial activity is much weaker thanthat of ALA PDT and only slightly affected by the Ph incubationtime, (iii) the functional effects of ALA PDT are paralleledby pronounced and predominant alterations of mitochondrialmorphology, and (iv) Ph PDT targets the lysosomal system andinduces autophagocytosis.

This work was supported by a grant from the ‘‘Fonds zur Forderung derWissenschaftlichen Forschung’’ (P 8532 MeD), Vienna, Austria.

REFERENCESBerg K, Moan J: Lysosomes and microtubules as targets for photochemotherapy of

cancer. Photochem Photobiol 65:403–409, 1997Bottiroli G, Croce AC, Ramponi R, Vaghi P: Distribution of di-sulfonated aluminium

phthalocyanine and Photofrin II in living cells: a comparative fluorometricstudy. Photochem Photobiol 55:575–585, 1992

Coppola A, Viggiani E, Salzarulo L, Rasile G: Ultrastructural changes in lymphomacells treated with hematoporphyrin and light. Am J Pathol 99:175–181, 1980

Dougherty TJ, Potter W, Bellnier D. Photodynamic therapy for the treatment of

270 RADAKOVIC-FIJAN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

cancer: current status and advances. In: Kessel D (ed.). Photodynamic Therapyof Neoplastic Disease. Boca Ration, FL: CRC Press, 1990, pp. 1–20

Fijan S, Honigsmann H, Ortel B: Photodynamic therapy of epithelial skin tumoursusing delta-aminolevulinic acid and desferrioxamine. Br J Dermatol 133:282–288, 1995

Fisher AMR, Murphree AL, Gomer CJ: Clinical and preclinical photodynamictherapy. Lasers Surg Med 17:2–31, 1995

Fukuda H, Batlle AM, Riley PA: Kinetics of porphyrin accumulation in culturedepithelial cells exposed to ALA. Int J Biochem 25:1407–1410, 1993

Gaullier JM, Geze M, Santus R, et al: Subcellular localization of and photosensitizationby protoporphyrin IX in human keratinocytes and fibroblasts cultivated with5-aminolevulinic acid. Photochem Photobiol 62:114–122, 1995

Geze M, Morliere P, Maziere JC, Smith KM, Santus R: Lysosomes, a key target ofhydrophobic photosensitisers proposed for photochemotherapeutic applications.J Photochem Photobiol B Biol 20:23–35, 1993

Ghadially FN: Ultrastructural Pathology of the Cell and Matrix, 3rd edn. London:Butterworths, 1988, pp. 589–765

Gibson SL, Havens JJ, Foster TH, Hilf R: Time-dependent intracellular accumulationof delta-aminolevulinic acid, induction of porphyrin synthesis and subsequentphototoxicity. Photochem Photobiol 65:416–421, 1997

Gibson SL, Murant RS, Chazen MD, Kelly ME, Hilf R: In vitro photosensitizationof tumour cell enzymes by Photofrin II administered in vivo. Br J Cancer59:47–53, 1989

He X-Y, Sikes RA, Thomsen S, Chung LWK, Jacques SL: Photodynamic therapywith Photofrin II induces programmed cell death in carcinoma cell lines.Photochem Photobiol 59:468–473, 1994

Henderson BW, Dougherty TJ: How does photodynamic therapy work? PhotochemPhotobiol 55:145–157, 1992

Iinuma S, Farshi SS, Ortel B, Hasan T: A mechanistic study of cellular photodestructionwith 5-aminolaevulinic acid-induced porphyrin. Br J Cancer 70:21–28, 1994

Kennedy JC, Pottier RH: Endogenous protoporphyrin IX, a clinically useful

photosensitizer for photodynamic therapy. J Photochem Photobiol B Biol 14:275–292, 1992

Krammer B, Huber A, Hermann A: Photodynamic effects on the nuclear envelopeof human skin fibroblasts. J Photochem Photobiol B Biol 17:109–114, 1993

Liang H, Shin DS, Lee YE, et al: Subcellular phototoxicity of 5-aminolaevulinicacid (ALA). Lasers Surg Med 22:14–24, 1998

Lin C-W, Shulok JR, Kirley SD, et al: Photodynamic destruction of lysosomesmediated by Nile blue photosensitizers. Photochem Photobiol 58:81–91, 1993

Malik Z, Ehrenberg B, Faraggi A: Inactivation of erythrocytic, lymphocyticand myelocytic leukemic cells by photoexcitation of endogenous porphyrins.J Photochem Photobiol Biol 4:195–205, 1989

Malik Z, Faraggi A: Ultrastructural damage in photosensitized endothelial cells:dependence on hematoporphyrin delivery pathways. J Photochem Photobiol BBiol 14:359–368, 1992

Malik Z, Lugaci H: Destruction of erythroleukaemic cells by photoactivation ofendogenous porphyrins. Br J Cancer 56:589–595, 1987

Milanesi C, Sorgato F, Jori G: Photokinetic and ultrastructural studies on porphyrinphotosensitization of HeLa cells. Int J Radiat Biol 55:59–69, 1989

Moore JV, West CML, Whitehurst C: The biology of photodynamic therapy. PhysMed Biol 42:913–935, 1997

Mosmann T: Rapid colorimetric assay for cellular growth and survival: applicationof proliferation and cytotoxicity assays. J Immunol Methods 65:55–63, 1983

Ortel B, Chen N, Brissette J, Dotto GP, Maytin E, Hasan T: Differentiation-specificincrease in ALA-induced protoporphyrin IX accumulation in primary mousekeratinocytes. Br J Cancer 77:1744–1751, 1998

Ortel B, Tanew A, Honigsmann H: Lethal photosensitization by endogenousporphyrins of PAM cells – modification by desferrioxamine. J PhotochemPhotobiol B Biol 17:273–278, 1993

Wolf P, Rieger E, Kerl H: Topical photodynamic therapy with endogenous porphyrinsafter application of 5-aminolevulinic acid. J Am Acad Dermatol 28:273–278, 1993

Zdolsek JM, Olsson GM, Brunk UT: Photooxidative damage to lysosomes ofcultured macrophages by acridine orange. Photochem Photobiol 51:67–76, 1990