150 (Suppl.

), S146-S156 (1998)
RESEARCH
RADIATION
0033-7587/98 $5.00
?1998 by Radiation Research Society.
All rights of reproduction in any form reserved.

The Photobiologyof PhotodynamicTherapy:

CellularTargetsand Mechanisms
Nancy L. Oleinick and Helen H. Evans

Department of Radiation Oncology, and the CWRU/Ireland Comprehensive Cancer Center, Case Western Reserve University
School of Medicine, Cleveland, Ohio 44106-4942

including cancer, and the testing of new photosensitizers

Oleinick, N. L. and Evans, H. H. The Photobiology of Photo- that improve upon the first-generation photosensitizer,
dynamic Therapy: Cellular Targets and Mechanisms. Radiat. Photofrin? (PF). There is also increased interest in elucidat-
Res. 150 (Suppl.), S146-S156 (1998).
ing the mechanisms by which PDT causes its effects in cells
Photodynamictherapy(PDT) is dependenton the uptakeof a and tissues. PDT is a three-component treatment. It requires
photosensitizingdye, often a porphyrin-relatedmacrocycle,by a photosensitizer that accumulates somewhat preferentially
the tumoror other abnormaltissue that is to be treated,the sub- in abnormal tissue, including tumors. The second compo-
sequentirradiationof the tumorwith visiblelight of an appropri- nent is nonthermal visible light of a wavelength matching
ate wavelengthmatched to the absorptionspectrumof the dye, the absorption spectrum of the photosensitizer and generally
and molecularoxygento generatereactiveoxygenintermediates. in the red region of the spectrum (since longer wavelengths
The initial oxidative reactions lead to damage to organelles in of light penetrate human tissue best). The third component
which the dye is bound, culminating in cell death and destruc-
is molecular oxygen. Photodynamic action is defined as oxy-
tion of the tumor or abnormal tissue. Apoptosis is a common
mechanism of cell death after PDT both in vitro and in vivo. gen-requiringphotosensitized reactions.
PDT also triggers the activation of several signal transduction
pathwaysin the treated cells; some of these are stress responses HISTORY
aimed at cell protection,while others are likely to contributeto
the cell death process.The photosensitizersof greatestinterestin Photodynamic action was discovered a century ago at
PDT bind to various cytoplasmicmembranesbut are not found about the same time as the discovery of radium by Madame
in the nucleusand do not bind to DNA. Nevertheless,some DNA Curie. In the first published report of photodynamic action
damage is producedthat can lead to mutagenesis,the extent of in 1888, Marcacci claimed without presenting data that the
which is dependent on the photosensitizer, the cellular repair
toxicity of quinine and cinchonamine to enzymes, plants
propertiesand the target gene. Thus, in spite of generatingsome and frog eggs was greater in the light than in the dark (1).
responses common to ionizing radiation and other oxidative Since he apparently did not realize the significance of his
stresses,PDT is uniquein the subcellularlocalizationof damage,
the combinationof signaling pathways that are activated, and observations, the real history of photodynamic action did
not begin for another decade. While conducting his doc-
rapid kinetics of the induction of cell death processes. ? 1998by
Radiation Research Society toral research in the laboratory of Professor Hermann von
Tappeiner, Oscar Raab observed that the toxicity of acri-
dine to Paramecium increased with the amount of sunlight
INTRODUCTION in the laboratory (2). After his dissertation was published in
1900, Raab's name was never seen again in the literature of
Photodynamic therapy (PDT)1 has received increasing phototoxic effects. However, his professor actively followed
attention in recent years as a result of the first approvals up on the observations, coined the German term that trans-
granted by the U.S. Food and Drug Administration (FDA), lates to "photodynamic action", and conducted the first
recognition of the efficacy of PDT in a range of diseases, clinical test of PDT with eosin for skin cancer and other
skin conditions (3). After an initial flurry of activity, there
was little interest in these effects until mid-century, when it
1Abbreviations acid;APAF1, apoptosis- was reported that injected hematoporphyrin accumulated
used:ALA, 5-aminolevulinic
activatingfactor1; BPD, benzoporphyrinderivative;CoA, coenzymeA; in tumors in rats and, upon illumination, led to necrosis of
DHE, dihematoporphyrinether/ester;HPD, hematoporphyrinderiva- the
tumor, and when Schwartz,Winkelman and Lipson pub-
tive; LuTex,lutetiumtexaphyrin;MPT,membranepermeabilitytransi-
tion; PBR, peripheralbenzodiazepinereceptor;Pc 4, phthalocyanine4;
lished a series of papers demonstrating that the fluorescence
PDT, photodynamictherapy;PF, Photofrin?,porfimersodium;PPIX, of porphyrin photosensitizers, specifically the preparation
protoporphyrinIX; SnET2,tin-etiopurpurin. known as hematoporphyrin derivative (HPD), could be
S146

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 PHOTOBIOLOGYOF PDT S147

used to detect tumors (4). The fascinatingearly history of number of porphyrinand porphyrin-relatedcompounds
the fieldhas been reviewedpreviously(4,5). have been synthesizedand studiedas potentialnew photo-
Modernphotodynamictherapywas initiatedin the 1970s sensitizers for PDT. Some of these have entered clinical
and was given its greatest impetus by Dr. Thomas J. trials,and others have been tested and found to be effica-
Dougherty of Roswell Park Cancer Institute, who recog- cious in animal studies (16). Figure 1 presents the struc-
nized the potential of PDT for tumor treatment and who tures of four of the second-generationphotosensitizersof
demonstratedthat HPD had efficacy for the treatmentof interest:benzoporphyrin derivative(BPD), tin-etiopurpurin
humantumorsmetastaticto the skin (6, 7). HPD is a com- (SnET2), lutetium texaphyrin (LuTex) and phthalocya-
plex mixtureof porphyrinmonomersand oligomersderived nine Pc 4 (Pc 4). These and other newer photosensitizers
from hematoporphyrin,the componentsof whichhave not have stronger absorption maxima at longer wavelengths
been fullydefined.A partiallypurifiedpreparationcontain- (X > 650 nm) than does PF (X = 630 nm).
ing the more hydrophobic fractions of HPD was subse- In addition to these photosensitizers, in recent years
quentlytermeddihematoporphyrin ether/ester(DHE) and, there has been considerableinterest in the endogenously
after additional name changes, is now referred to as synthesized photosensitizer, protoporphyrinIX (PPIX)
Photofrin?(porfimersodium).This preparationis the only (Fig. 1). PPIX is the penultimate product of the heme
one currentlyapprovedfor clinicalPDT by the U.S. Food biosynthesispathway.The first (rate-limiting)step in this
and Drug Administration. pathwayis the synthesis of 5-amino levulinic acid (ALA)
This paper will focus on a selected review of PDT and from succinyl-CoAand glycine.Introductionof exogenous
the photobiologyon whichit is based,with emphasison cel- ALA bypassesthe rate-limitingstep and providessubstrate
lulartargetsand mechanismsand with limitedreferenceto for the synthesisof excess PPIX whichis subsequentlycon-
tissue-basedmechanismsandclinicalapplications. verted to heme by ferrochelatasein the presence of Fe+.
When availabilityof Fe+ is limited,as it is in manytumors
CLINICALAPPLICATIONS and other fast-growingtissues, PPIX accumulatesand the
tissue is photosensitiveuntilPPIX is lost by effluxfromthe
Photodynamictherapy has been tested for the treatment tissue or by metabolicconversionto heme (17).
of many types of tumors,and selection of potential tumor
sites is limitedonly by the abilityto directlightto the tumor. PHOTOCHEMISTRY
The most commonsite is the skin,wherePDT is beingstud-
ied for treatmentof primarybasal and squamouscell carci- Upon absorptionof a photon,the photosensitizeris acti-
nomas, breast cancer metastatic to the skin, cutaneous vated to an excitedsingletstateandthen,by intersystemcross-
Kaposi's sarcoma, and cutaneous T-cell lymphoma(7-9). ing, an excited tripletstate. The tripletcan undergotwo
to
PDT with PF is now approved in the U.S. for advanced types of reactions.Type I photochemistryinvolveselectron
obstructingesophagealcancerand earlylung cancer,and in transferbetween the photosensitizertriplet and a nearby
other countries for additionalsites, and PDT with newer molecule,e.g. a membranelipid.After this oxidation-reduc-
photosensitizersis in clinicaltrialsfor cancersof the bladder, tion reaction,the resultantsubstrateradicalmay reactwith
brain, head and neck, eye, ovary and lung. PDT has been oxygento generatea peroxylradicalwhichwillundergotypi-
tested for purgingtumorcells frombone marrowor periph- cal radicalchainreactions.Or the redoxreactionmay occur
eral blood stem cell populationspriorto autologoustrans- directlywith oxygen to generatean oxygen radical,such as
plantation.In addition,treatmentof preneoplasticlesions, superoxide(02). Type II photochemistryinvolves energy
such as actinickeratoses,benign prostatichyperplasiaand transferfromthe photosensitizer tripletto ground-statemolec-
Barrett'sesophagus,is underinvestigation,as is the treat- ularoxygento generatesingletoxygen(102),a nonradicalbut
ment of manynoncancerousconditions,such as atheroscle- highlyreactiveformof oxygen.Both singletoxygenandmany
rosis,vascularrestenosis,age-relatedmaculardegeneration, of the oxygenradicalscan producedamageto cellularstruc-
rheumatoidarthritisand blood sterilization(7, 9-15). The tures.All of the photosensitizers in or nearingclinicaltrialsfor
advantages of PDT for treatment and cure of human disease PDT are efficient of
generators singletoxygen.Althoughit has
are becomingincreasinglyappreciated. not beenpossibleto detectsingletoxygenin tissue,becauseof
its high reactivity,it is generallyassumed,based on known
PHOTOSENSITIZERS photochemistryin chemical systems and on the products
formed,thatsingletoxygenaccountsfor a substantialportion
In spite of the proven efficacy of PF in cancer treat- of the biologicaldamageproducedduringPDT (18-22).
ment, it has certain limitations, i.e. a complex chemical
composition, low extinction for tissue-penetrating red MECHANISMSOF PDT RESPONSESIN TUMORS
light, and tendency to accumulatein skin. The last prop-
erty results in a lingering cutaneous photosensitivity in Treatmentof tumorswith PDT can causetumorablation
patients for 1-2 months after a single administration.The within a few days. The results of many studies in rodents
limitationsof PF have created an industryin whicha large have provided evidence for three types of mechanism that
S148 OLEINICKAND EVANS

OH

OH OH

Derivative
Benzoporphyrin PhthalocyaninePc 4 ProtoporphyrinIX

'O 0O 0 OCH3

LutetiumTexaphyrin
Tin-etio-purpurin

FIG.1. Structuresof some second-generation

photosensitizersfor PDT.

may be involved in the rapid response of tumors to PDT or endocytosis.This distributionmay changeduringor after
(20-22). First,PDT may damagethe malignantcells of the illuminationif the lysosomesinternalizeorganellescontain-
tumordirectly.Second,PDT mayproduceprofoundchanges ing photosensitizers or if the photodynamic treatment
in the tumorvasculature,includingblood flow stasis,vascu- inducesmembranedamageleadingto the loss of photosen-
lar collapseand/orvascularleakage.Third,PDT causesthe sitizerfromorganelles.The intracellulardistributionas well
release of cytokines and other inflammatorymediators as the pharmacokineticsof some photosensitizershave been
from treated cells that can produce an inflammatory reviewed extensively by Boyle and Dolphin (23) and in a
response and recruit additional host (lymphocyte and symposium-in-printintroducedby Kessel(24).
phagocytic) cells to the tumor. The contributionof each
mechanismto the overall tumorresponse is dependenton APOPTOSISCOMPAREDTO NECROSISIN THE
the photosensitizerand the tumor.In general,PF-PDThas RESPONSETO PDT
been shown to work predominantlythroughvascularcol-
lapse and inflammation,whereasdirectkillingof malignant After PDT in vivo, there is evidence for both apoptosis
cells plays a large role in the case of the phthalocyanines and regions of necrosis in tumor biopsies (25-28). It is still
(20-22). However, with any of the photosensitizers, it is not clear how important the induction of apoptosis is to the
likely that the rapid and efficient tumor ablation depends overall tumor response or whether apoptosis is the result
on more thandirectkillingmechanismsalone. of direct damage, a secondary result of vascular and
inflammatoryeffects or a combinationof these effects.It is
INTRACELLULARDISTRIBUTIONOF also not certainthat all of the apoptosis observed derives
PHOTOSENSITIZERS from dying malignantcells or if some apoptosisin tumors
is the result of the death of host cells, such as endothelial
Porphyrin-related photosensitizers generally localize in cells, lymphocytesand macrophages.
the membranesof cellular organelles, includingthe lyso- Apoptosisis a prominentformof cell deathin responseto
somes, the mitochondria,the endoplasmicreticulumand PDT for many, but not all, cells in culture (29-37), and in this
Golgi apparatus,and the nucleus, as well as the plasma situation,the effectsmustbe causedby directPDT damage
membrane.The specificlocalizationand the kineticsof the to the culturedcells. As judgedby assaysmeasuringeither
intracellulardistributionof each sensitizerdependupon its the fragmentation of DNA or the condensation of chro-
hydrophobicity, the type andnumberof charges,the charge- matin, most cells that have been investigated have been
to-mass ratio, the type and number of ring and core sub- found to undergo apoptosis in response to PDT. Notable
stituents, and whether entry into the cell occurs by diffusion exceptions are cells of one of three carcinoma lines exposed
 PHOTOBIOLOGYOF PDT S149

to PF-PDT (30), the radiation-inducedRIF-1 fibrosarcoma function. Thus photodynamic action and the inhibitors
cells exposedto Pc 4-PDTin vitro(37), andCV-1cellsirradi- inducethe formationof micronucleiand giantcells and the
ated aftera shortincubationwith PF allowingaccumulation accumulationof cells in mitosis(reviewedin ref. 41). Sporn
of the photosensitizerprimarilyin the plasma membrane and Foster (42) have shownthat depolymerizationof tubu-
(31). With ALA-PDT, Noodt et al. (32) found apoptosisin lin results 15 min after illuminationand is reversibleafter
ChinesehamsterV79 cells andnecrosisin WiDrhumanade- low fluencesbut irreversibleafterhighfluences.The authors
nocarcinomacells. The particularmode of cell death in suggestthat the depolymerizationis causedby an increase
responseto PDT dependson experimentalconditions,such in intracellularcalcium. Actin filaments remained unaf-
as the dose of PDT (34,38) andthe subcellularlocalizationof fected. At higherPDT doses, however,Wiemanet al. (43)
the photosensitizers(39, 40). It shouldbe kept in mindthat reportedmicrofilamentdisruption.
the definitionof apoptosisis itselfnot firmlyestablished,and
someof the exceptionsmayturnout to be a formof apoptosis Lysosomes
in whichthe nucleareventsarenot observed. Although a variety of photosensitizers localize in the
The sensitivityof cells to PDT can varybetween closely lysosomes, it appears that lysosomal disruptionmay not
related cell lines as a functionof the expressionof defined always lead to cell killing. Lin and colleagues have
genes that have a role in controllingapoptosis.Thus HL60 reported that the degree of lysosomal damage caused by
cells expressingwild-type p53 were more sensitive to cell PDT was not correlatedwith the photocytotoxicity(44, 45).
killingby PDT, with eitherPF or SnET2as the photosensi- Some cells survivepartiallysosomal disruption,probably
tizer, than were HL60 cells in which the p53 genes were because lysosomal enzymes are inactivated by the treat-
deleted or inactive. All of these cell lines underwentapo- ment, as well as by cytosolicinhibitors(46).
ptosis rapidly in response to PDT (35). Chinese hamster
ovary cells expressinga humanBCL2 gene were partially Mitochondria
resistant to apoptosis and to overall cell death after PDT There is now considerableevidence that mitochondria
sensitizedby the phthalocyaninePc 4 (36). are important targets in the cytotoxicity of PDT. Early
studies with PF showed a promptpost-PDT inhibition of
TARGETS respiration,measuredboth in intact cells and in isolated
mitochondria,inhibitionof the actionof electrontransport
Assumingan adequateoxygensupplyand lightintensity, components,suchas succinatedehydrogenase andcytochrome
the site of photodamage depends on the location of the c-oxidase,and disruptionof the mitochondrialelectrochem-
photosensitizerat the time of irradiationwith visible light. ical gradient,all of which occurredearlierthan loss of the
Secondarily,it can depend upon movement of the photo- abilityof the cells to exclude trypanblue (47-49). Because
sensitizerto a secondarysite duringcontinuingirradiation. PPIX is producedin mitochondria,mitochondrialdamage
Nearly all of the photosensitizerscurrentlybeing studied is prominentwhen cells are irradiatedat earlytimes (-4 h)
are hydrophobicmoleculesthat tend to bind to membranes after introducingthe metabolicprecursorALA; however,
and are excludedfromthe nucleus. becausePPIX can diffuseout of the mitochondria,damage
Photodynamictherapyof cells resultsin extensivedam- is foundat othersites as well (17, 50-52). Cationiclipophilic
age to cell structures.Depending on the photosensitizer, compounds,such as the krytocyaninedye EDKC and rho-
overall dose and experimentalprotocol, damage hasbeen damine-123,accumulatein mitochondriaas a result of the
observedin microtubules,membranous organelles,especially highly negative electrochemical potential of the active
lysosomes and mitochondria,plasma membrane and the inner mitochondrialmembrane(53). However,some por-
nucleus.Muchof the earlywork in this area was done with phyrin-relatedphotosensitizersthat are negativelycharged,
PF or one of its precursors,but since these are complex suchas PF, or neutral,suchas Pc 4, also accumulatein mito-
mixtures,it is impossibleto distinguishthe location of the chondria2(49), possiblybecauseof bindingto specificmito-
active comparedto the inactive components.More recent chondrialconstituents.Ratcliffe and Matthews(52) found
studies have focused on chemicallypure photosensitizers, that the mitochondrialeffects of photoactivatedPPIXwere
identifyingsites of bindingby fluorescencemicroscopyin inhibitedby ligandsof the peripheralbenzodiazepine receptor
comparison to probes of specific organelles. Studies of (PBR), a mitochondrialpore-formingprotein. Porphyrins
some of these targetswill be summarizedbelow. are endogenousligandsfor this receptor(54), and Morgan
and Oseroff3have recentlydemonstratedthat the binding
Cytoskeletal Elements of a specificPBR ligand(PK11195)could be competitively
Berg and Moan (41) have suggested that nonpolymer- inhibitedby pyropheophorbidephotosensitizersthat local-
ized tubulin may be a target for cytotoxicity induced by ize to mitochondria.Wilson et al. (55) demonstratedthe
photodynamicaction.Some of the photosensitizersbind to
tubulin, and after illumination its polymerizationis pre-
vented. Tubulinis affected by low doses of photodynamic 2N. L. Oleinick, A. L. Nieminen and N. Trivedi, unpublishedobservation.
action similarly to treatment with inhibitors of microtubulin 3j. MorganandA. R. Oseroff,unpublishedobservation
S150 OLEINICKAND EVANS

abilityof a cardiolipinprobe, lON-nonylacridineorange,to generateddamage.Alternatively,it is possiblethat relocal-

competitivelyinhibit the uptake of PF into mitochondria, ization of the sensitizers to the nucleus occurs during
indicatingthat some photosensitizersmight bind to cardi- illuminationand is responsiblefor the inductionof DNA
olipinsof the innermitochondrialmembrane. damage. Relocalization has been reported for several
The PBR is a complex of several proteins that includes photosensitizers(71-75).
the 18-kDa receptor in the mitochondrial outer mem- Photodynamictherapyof varioustypes of cells resultsin
brane linked to the voltage-dependentanion channel and DNA lesionssuchas single-strand breaksand/oralkali-labile
the adenine nucleotide translocator that is responsible sites (76-78). In two closelyrelatedL5178Y(LY) cell lines,
for ADP/ATP exchange (56, 57). The latter two compo- the cytotoxicityof PDT did not correspondto the induction
nents of the PBR are also thought to be components of or rejoiningof single-strandbreaks,but did correlatewith
the membranepermeabilitytransition (MPT), a nonspe- the numberof DNA-protein crosslinksand with the extent
cific ion channel that brings the outer and inner mito- of DNA degradationcausedby the treatment(78). Chromo-
chondrial membranes into contact (58, 59). Normally, some aberrationsand sisterchromatidexchangeshave also
passage of ions and molecules across the inner mem- been observedin CHO andNHIKcellstreatedwithhemato-
brane is tightly regulated by the membrane potential. porphyrinandredlight(70,77, 79).
However, the MPT senses changes in redox state, pH, The mutagenicity of PDT varies with the photosensi-
etc. and can open to permit depolarization of the mem- tizerand the cell line, as well as the targetgene. Noodt et al.
brane, inhibition of oxidative phosphorylationand efflux (80, 81) comparedthe mutagenicityof PF and three closely
of Ca+. The results of a recent study of PDT with related porphyrins at the Hprt locus in V79 cells. PDT
hematoporphyrin on isolated rat liver mitochondria was significantly mutagenic when sensitized by the
showed that photodynamic action probably degrades lipophilic photosensitizers,PF or 3THPP, but not by the
critical histidines of the MPT, leading to site-selective hydrophilicphotosensitizers, TPPS4 and TmPyPh2.The
inactivationof discrete pore functions (60). differences in mutagenicitywere not correlated with dif-
The mitochondrionis a criticalsite for release of factors, ferences in the efficiency of induction of DNA strand
especiallycytochromec, that triggerthe finalstagesof apo- breaks, nor could they be explained by the intracellular
ptosis (61, 62). It has been proposedthat cytochromec, in distributionof the photosensitizers.No significantmuta-
combinationwith the cytoplasmicprotein, apoptosis-acti- genicitywas detected at the Hprtor Na+/K+ATPase loci in
vating factor 1 (APAF1), directlyactivatesthe cascade of Chinese hamsterV79 or CHO cells treated with hemato-
caspases (cysteine proteases acting on asparticacid) that porphyrinand red light (76, 79, 82), nor was morphologi-
carryout the final stages of apoptosis (63). Such caspases cal oncogenictransformation inducedundertheseconditions
are rapidly activated in PDT-treated L5178Y-R, HL60, in C3H 101/2cells (82).
P388 and L1210 cells (34, 64, 65). Expression of BCL2, a We havestudiedthe mutagenicityof PF andtwo phthalo-
proteinof the mitochondrialouter membrane,preventsthe cyanines(AlPcandPc 4) in murineandhumanlymphoblasts,
release of the mitochondrialfactors (66, 67), partiallypro- all heterozygousfor the autosomalthymidinekinase gene
tects cells from PDT-inducedapoptosis, and endows cells (Table I). TK6 and WTK1 human lymphoblastsare simi-
with a partialresistanceto PDT (36). Furthermore,Kessel larlysensitiveto the cytotoxiceffects of PDT with AlPc or
and Luo have shown that photosensitizers that bind to PF, but the mutabilityof these two cell lines differs:WTK1
mitochondria induce apoptosis upon photoirradiation, cells are significantlymutagenizedby PDT with either sen-
whereas those that bind to the plasmamembraneor lyso- sitizer. However, TK6 cells are poorly mutagenized by
somes,but not to mitochondria,kill cells less efficientlyand PDT. The mutabilityof TK6 cells is also significantlylower
by a mechanismnot involvingapoptosis(39, 40). thanthat of WTK1cells aftertreatmentwithUVC or ioniz-
ing radiation(TableI). TK6cellspossessan activep53 gene,
TheNucleus:DNA Alterationsand Mutagenicity while p53 is inactive in WTK1 cells (92, 93). TK6 cells
As indicatedabove, the lipophilicPDT photosensitizers show higherlevels of PDT-inducedapoptosis(83) and are
generally localize in membranes, including the nuclear more deficient in recombination(94) and the rejoiningof
membrane (68). Although these photosensitizers do not DNA double-strandbreaks than WTK1cells (83, 85, 95).
appearto enter the nucleus, illuminationof cells preincu- Any one or a combinationof these characteristicscould
bated with the photosensitizerscauses DNA damage that explainthe low mutabilityof TK6cells.
subsequentlyresultsin chromosomeaberrationsand muta- The frequencyof PDT-inducedmutantsis muchhigherin
tions. Singlet oxygen, a form of reactiveoxygen generated the murineLY-S1cell line thanin eitherof the humanlym-
by PDT, has a rangeof -0.1 pm and a lifetime of less than phoblasticlines or the LY-R16cell line. Both LY cell lines
1 s (69), but may reach DNA closely positioned near the also harborinactivep53 genes (96). In the murinecells and
nuclear membrane. In agreement with this possibility, in human WTK1 cells, PDT with PF resulted in a higher
Evenson and Moan (70) have reporteddata suggestingthat mutant frequency than did AlPc-PDT, while Pc 4-PDT
the chromosomalcentromericand telomericregionswhich inducedthe lowestmutantfrequencyof the threephotosen-
bind to the nuclearmembraneare quite sensitive to PDT- sitizers tested when measured at the F37doses (Table I).
 PHOTOBIOLOGYOF PDT S151

TABLEI
Mutagenicityof PDT, X Radiationand UVC Radiationin Humanand MurineCells at the ThymidineKinaseLocus
TK/- mutantfrequency
Cell line Agent Unit D37 or F37a X 106at D37or F37 Reference(s)
TK6 AlPc, red light kJ/m2 4 0 83
PF, red light kJ/m2 44 1 83
UVC radiation J/m2 8 33 83
X radiation Gy 0.66 30 84-86
WTK1 AlPc, red light kJ/m2 4 30 83
PF, red light kJ/m2 58 74 83
Pc 4, red light kJ/m2 2.2 20 87
UVC radiation J/m2 8 94 83
X radiation Gy 1.2 320 83-84
L5178Y-S1 AlPc, red light kJ/m2 12.3 1100 88
PF, red light kJ/m2 96 1400 89
Pc 4, red light kJ/m2 4.1 56 87
UVC radiation J/m2 8 830 90
X radiation Gy 0.7 1700 91
L5178Y-R16 AlPc, red light kJ/m2 9.5 83 88
PF, red light kJ/m2 52 500 89
Pc 4, red light kJ/m2 1.1 4 87
UVC radiation J/m2 2.6 500 90
X radiation Gy 1.5 2750 91
Notes.This table is a combinedand modifiedversionof previouslypublishedtables (83, 87). In these experimentsthe finalconcentrationsof the
photosensitizersin the mediumwere 1 tMAlPc, 0.5 pMPc 4 and25 pg/mlPF.
aF37is the fluencethatreducedsurvivalto 37%in the presenceof the indicatedphotosensitizerat the specifiedconcentration.

The high mutant frequency obtained at the thymidine and LY-R16cells, assumingthat the TK'- mutantsof these
kinaselocus comparedto the Hprtand Na+/K+ATPase loci cell lines are more sensitivethanthe TK'- cells.
is most probablycausedby the higherrecoveryof mutants Our results show that the mutagenicityof PDT varies
withintergenicmutationsin the case of the thymidinekinase greatly with the target gene and the cell line. The mutant
autosomallocus. When the target gene is in a hemizygous frequency at a particulartarget gene appears to depend
target region (i.e. the Hprt gene on the X chromosome), upon the essentialgenes in its vicinity,as well as the activ-
mutants with intergenic mutations are poorly recovered ity of these target genes on the homologouschromosome,
because of the inactivationof essentialgenes linked to the and thereforewould be expectedto varywith the arrange-
target.In contrast,when the targetgene is on an autosomal ment and activity of genes on the chromosomes of each
chromosome (such as the thymidine kinase gene), the particularcell line. Because of the extensive genetic vari-
homologouschromosomecan usuallysupplyan activecopy ation in the humangenome,it is probablethat humancells
of the linked essential gene. Since multilocus lesions are may varygreatlyin theirmutabilityfromindividualto indi-
induced by PDT (97), the location of the target gene can vidual,from cell type to cell type, and from locus to locus.
explainthe highermutantfrequencyinducedby PDT at the Use of PDT for the treatmentof benign conditionsshould
thymidinekinase comparedto the Hprt locus. Use of the thereforebe undertakenwith caution.
co-dominantgene for Na+/K+ATPase also does not detect
large or even small deletions,because the sequencecoding SIGNALTRANSDUCTIONPATHWAYS
for the ouabain-bindingsite (the mutationof whichis nec- ACTIVATEDBY PDT
essary for ouabain resistance) very closely neighbors the
sequencefor the ATP-bindingsite. Thusdeletionsaffecting Photodynamictherapyactivatesmanysignalingreactions
the ouabain-bindingsite almostalwaysinactivatethe ATP- that have been characterizedin responseto other oxidative
binding site, resulting in inactivation of the resistant stresses or physiological stimuli. Table II lists the major
enzyme. reactionsthat have been reportedto occurin one or more
The high mutabilityof the LY-S1 cells comparedto the PDT-treatedcell lines in vitro,alongwith the cell lines and
humancells maybe causedby a higherfrequencyof regions photosensitizersused experimentally.It is apparentthat
of hemizygosityin essentialgenes neighboringthe TK gene PDT causes the release of a variety of lipid and inorganic
in the WTK1and TK6 humancells (98). Alternatively,the second messengers,as well as the release of calciumions
higher sensitivity of the human cells (and of the LY-R16 fromintracellularstores.Eitherindependentlyor as a result
cells) to the cytotoxic effects of PDT comparedto LY-S1 of stimulationby one or more second messengers,several
cells could also explain the lower mutabilityof the human protein kinase signaling cascades are activated, some of
S152 OLEINICKAND EVANS

which (stress kinases) are presumed to lead to apoptosis For example, as noted above, photoactivation of a sensitizer
(121, 122), whereas others (NF-KB, HS1) are presumed to in the mitochondria leads to rapid apoptosis, presumably as
promote cell survival (106, 123). PDT is also a strong a result of the release of cytochrome c into the cytoplasm
inducer of the expression of a range of stress response genes where it can interact with APAF1 and activate the caspases
that are also thought to enhance survival after oxidative involved in apoptosis. However, inhibition of p38/HOG by
stress (Table II). Interestingly, it has been demonstrated the chemical inhibitor SB202190 (124) can inhibit PDT-
recently that up-regulation of GRP78, an endoplasmic retic- induced apoptosis (108). This observation suggests that the
ulum-localized stress protein, can either protect or sensitize pathway to apoptosis involves the intersection of the stress
cells exposed to PDT, depending on the subcellularlocaliza- kinase pathway with mitochondria-derived factors. The
tion of the photosensitizer (115, 118). Furthermore, the mechanism of these interactions and others will be of great
expression of cytokines may promote either cell death or cell interest in elucidating cell responses to PDT.
survival or, in vivo, an inflammatoryresponse.
Interpretation of the data summarized in Table II must CONCLUSION
be made with caution. It is unlikely that there is a single
universally applicable mechanism for the response of cells Tumors respond rapidly to PDT, as a result of damage to
to PDT. The responses vary with the cell type and its both the malignant cells and cells of the tumor vasculature,
genetic and metabolic potential, as well as the photosensitizer and both apoptosis and necrosis are prominent as tumor
and its subcellular localization. The responses also vary ablation proceeds. At the cellular level, photodynamic dam-
with the overall dose of PDT (e.g. sublethal or lethal), since age occurs initially at sites of photosensitizer localization in
gene induction that requires many hours after PDT is cellular membranes. Considerable attention has been
unlikely to occur if the cells undergo apoptosis within 1-2 h. directed to the mitochondrion as a target of PDT. Because of
Furthermore, PDT appears to stimulate multiple reactions the importance of mitochondria in apoptosis, the localization
for both cell death and survival simultaneously, and the of efficient photosensitizers in these organelles may partially
interaction of these pathways is likely to determine cell fate. explain the rapid progress of apoptosis in treated cells.

TABLE II
Some Signaling Reactions Activated by PDT
Reactionactivated Cell line(s) Photosensitizer Reference(s)
A. Synthesisandreleaseof secondmessengers
PhospholipasesC, A2 LY-R AlPc 99
(inositoltrisphosphate,arachidonicacidrelease)
Sphingomyelinase(ceramiderelease) LY-R Pc 4 100
U937, CHO,RIF, A431, Pc 4 37
humanlymphoblasts
ProstaglandinE2 release RIF,murinemacrophages PF 101
Calciumion release CHO AlPc 102,103
LY-R AlPc 99
Nitricoxide release A431 Pc 4 104
B. Activationof proteinkinasecascades
NF-KBactivation L1210 PF 105
HS1 phosphorylation LY-R Pc 4 106
Stresskinaseactivation humankeratinocytes BPD 107
(SAPK/JNK,p38/HOG1) LY-R,CHO Pc 4 108
C. Inductionof stressgene andcytokineexpression
Earlyresponsegenes
Fos,Jun,Myc,Egrl RIF NPe6, PF 109
FOS,JUN HeLa PF 110
Cytokines: TNF-a murinemacrophages PF 111
TNF-a murinekeratinocytes Pc 4 112
IL-6 HeLa PF 113
IL-6,IL-10 EMT6 PF 114
Glucose-regulatedproteins(e.g. GRP78) RIF PF 115
M1 BPD 116
V79 Pc 4 117
FaDu, CHO VBBO 118
Heat-shockproteins(e.g. HSP70) M1 BPD 116
RIF NPe6, SnET2 119
Heme oxygenase V79 PF 120
 PHOTOBIOLOGYOF PDT S153

Althoughthe leadingphotosensitizers do not localizeinitially 17. Q. Peng, K. Berg, J. Moan, M. Kongshaug and J. M. Nesland,
in nuclei,both DNA damageandmutationsare producedby 5-Aminolevulinicacid-basedphotodynamictherapy:Principlesand
experimentalresearch.Photochem.Photobiol.65,235-251 (1997).
PDT, the extentof the mutagenicitydependingon the photo- 18. K. R.
Weishaupt,C. J. GomerandT. J. Dougherty,Identificationof
sensitizer,the targetcell and the targetgenetic locus. PDT singlet oxygen as the cytotoxic agent in photoinactivation of a
activatesa varietyof signaltransductionpathwaysthat can murinetumor.CancerRes.36,2326-2329(1976).
lead to cell deathby apoptosisor to the inductionof cytopro- 19. M. Ochsner, Photophysicaland photobiologicalprocesses in the
tective stress responses. The unique combinationof PDT photodynamic therapy of tumours. J. Photochem. Photobiol. B 39,
at criticalsubcellularsites and activation of death- 1-18 (1997).
damage
Moan and K. Berg, Photochemotherapyof cancer:Experimental
promotingsignalingpathwayscollectivelycontributeto the 20. J. research. Photochem. Photobiol. 55, 931-948 (1992).
efficientandrapidprogressof cell deathprocesses.
21. B. W. Henderson and T. J. Dougherty, How does photodynamic
therapy work? Photochem. Photobiol. 55, 145-157 (1992).
ACKNOWLEDGMENTS 22. T. Hasan and J. A. Parrish,Photodynamictherapy of cancer. In
CancerMedicine,4th ed. (J. F. Holland, E. I. Frei, R. C. J. Bast,
Research on photodynamictherapy in the authors' laboratoriesis D. W. Kufe, D. L. Morton and R. R. Weichselbaum,Eds.), pp.
supported by PHS grants P01-CA48735 and P30-CA43703 from the 739-750.Williams& Wilkins,Baltimore,1997.
NationalCancerInstitute,DHHS. 23. R. W. Boyle and D. Dolphin, Structureand biodistributionrela-
tionshipsof photodynamicsensitizers.Photochem.Photobiol. 64,
469-485 (1996).
REFERENCES
24. D. Kessel, Symposium-in-Print: Subcellularlocalizationof photo-
1. A. Marcacci,Surl'actiondes alcaloidesdansle regnevegetalet ani- sensitizingagents.Photochem.Photobiol.65, 387-388(1997).
mal.Arch.Ital.Biol. 9, 2-4 (1888). 25. S. I. A. Zaidi,N. L. Oleinick,M. T. Zaim andH. Mukhtar,Apopto-
sis duringphotodynamictherapy-inducedablationof RIF-1tumors
2. 0. Raab, Ueber die WirkungfluorescirenderStoffe auf Infusorien. in C3H mice:Electronmicroscopic,histopathologicand biochemi-
Z. Biol. 39, 524-526 (1900). cal evidence.Photochem.Photobiol.58, 771-776 (1993).
3. H. von TappeinerandA. Jesionek,Therapeutische
Versuchemitflu- 26. R. Agarwal, N. J. Korman,R. R. Mohan, D. K. Feyes, S. Jawed,
orescierendenStoffen.Muench.Med.Wochenschr.50,72-76 (1903). M. T. Zaim and H. Mukhtar,Apoptosis is an early event during
4. J. D. Spikes, Photodynamic action: From Parameciumto pho- phthalocyaninephotodynamictherapy-inducedablationof chemi-
tochemotherapy.Photochem.Photobiol.65S, 142S-147S(1997). cally induced squamous papillomas in mouse skin. Photochem.
Photobiol.63, 547-552 (1996).
5. T. J. Dougherty,B. W. Henderson,S. Schwartz,J. W. Winkelman
and R. L. Lipson,Historicalperspective.In PhotodynamicTherapy 27. J. Webber,Y. Luo,R. Crilly,D. FrommandD. Kessel,An apoptotic
(B. W. Henderson and T. J. Dougherty, Eds.), pp. 1-15. Marcel response to photodynamic therapy with endogenous protopor-
Dekker,New York, 1992. phyrin in vivo. J. Photochem. Photobiol. B 35, 209-211 (1996).
28. G. Zhou, C. Shunji,D. Jinsheng,L. Junlin,G. Jori and C. Milanesi,
6. T. Dougherty,D. G. Boyle, K. Weishaupt,C. Gomer,D. Borcicky,
J. Kaufmanand G. Grindey, Phototherapyof human tumors. In Apoptosis of mouse MS-2fibrosarcomacells inducedby photody-
namictherapywith Zn(II)-phthalocyanine. J. Photochem.Photobiol.
Researchin Photobiology(A. Castellani,Ed.), pp. 435-466.Plenum B 33,219-223 (1996).
Press,New York, 1977.
29. M. L. Agarwal, M. E. Clay, E. J. Harvey, H. H. Evans, A. R.
7. T. J. Dougherty,Photodynamictherapy.Photochem.Photobiol.58, Antunez and N. L. Oleinick,Photodynamictherapyinducesrapid
895-900(1993). cell death by apoptosisin L5178Ymouse lymphomacells. Cancer
8. S. A. Khan,T. J. Doughertyand T. S. Mang,An evaluationof pho- Res.51, 5993-5996(1991).
todynamictherapyin the managementof cutaneousmetastasesof 30. X-Y. He, R. A. Sikes,S. Thomsen,L. W. K. ChungandS. L. Jacques,
breastcancer.Eur.J. Cancer29A, 1686-1690(1993). Photodynamictherapywith PhotofrinII inducesprogrammedcell
9. H. Lui and R. Rox Anderson,Photodynamictherapyin dermatol- death in carcinomacell lines. Photochem.Photobiol. 59, 468-473
ogy:Recent developments.Dermatol.Clin.11, 1-13 (1993). (1994).
31. M. Dellinger,Apoptosisor necrosisfollowingPhotofrinphotosensi-
10. H. I. Pass, Photodynamictherapy in oncology: Mechanismsand tization:Influenceof the incubationprotocol. Photochem.Photo-
clinicaluse.J. Natl. CancerInst.85, 443-456(1993).
biol. 64, 182-187(1996).
11. F. Sieber, Phototherapy,photochemotherapyand bone marrow 32. B. B. Noodt, K. Berg, T. Stokke, Q. Peng and J. M. Nesland,Apo-
transplantation. J. Hematother.2, 43-62 (1993). ptosis and necrosisinducedwith light and 5-aminolaevulinicacid-
12. J. G. Levy and M. Obochi,New applicationsin photodynamicther- derivedprotoporphyrin IX. Br.J. Cancer74,22-29 (1996).
apy:Introduction.Photochem.Photobiol.64, 737-739 (1996). 33. E. Ben-Hur,J. OetjenandB. Horowitz,SiliconphthalocyaninePc 4
and red light causes apoptosis in HIV-infected cells. Photochem.
13. K. B. TraunerandT. Hasan,Photodynamictreatmentof rheumatoid Photobiol.65,456-460 (1997).
andinflammatory arthritis.Photochem.Photobiol.64,740-750(1996).
34. Y. Luo and D. Kessel, Initiation of apoptosis versus necrosis by
14. P. Gonschlor, F. Gerheuser, M. Fleuchaus,T. Y. Huehns, A. E.
photodynamictherapywith chloroaluminumphthalocyanine.Pho-
Goetz, U. Welsch,R. Sroka,M. Dellian,H. A. Lehrand B. Hofling, tochem. Photobiol. 66, 479-483 (1997).
Localphotodynamictherapyreducestissuehyperplasiain an experi-
mentalrestenosismodel.Photochem.Photobiol.64,758-763 (1996). 35. A. M. R. Fisher,K. Danenberg,D. Banerjee,J. R. Bertino,P. Danen-
berg and C. J. Gomer, Increasedphotosensitivityin HL60 cells
15. E. Ben-HurandB. Horowitz,Advancesin photochemical approaches expressingwild-typep53.Photochem.Photobiol.66,265-270(1997).
for blood sterilization.Photochem.Photobiol.62, 383-388(1995).
36. J. He, M. L. Agarwal,H. E. Larkin,L. R. Friedman,L. Y. Xue and
16. C. J. Gomer,Preclinicalexaminationof firstand second generation N. L. Oleinick,The inductionof partialresistanceto photodynamic
photosensitizersused in photodynamictherapy.Photochem.Photo- therapyby the protooncogeneBCL-2. Photochem.Photobiol. 64,
biol. 54, 1093-1107(1991). 845-852(1996).
S154 OLEINICKAND EVANS

37. D. Separovic, K. J. Mann and N. L. Oleinick, Association of with the voltage-dependent anion channel and the adenine
ceramideaccumulationwith photodynamictreatment-inducedcell nucleotide carrier. Proc. Natl. Acad. Sci. USA 89, 3170-3174 (1992).
death. Photochem. Photobiol. 68,101-109 (1998).
57. A. Camins,F. X. Sureda,D. Pubill,J. Camarasaand E. Escubedo,
38. J. He and N. L. Oleinick,Cell death mechanismsvarywith photo- Characterizationand differentiationof peripheral-typebenzodi-
dynamic therapy dose and photosensitizer. SPIE 2371, 92-96 azepine receptorsin rat and humanprostate.Life Sci. 54, 759-767
(1995). (1994).
39. D. Kessel,Y. Luo, Y. Deng and C. K. Chang,The role of sub-cellu- 58. M. Zoratiand I. Szabo,The mitochondrialpermeabilitytransition.
lar localizationin initiationof apoptosisby photodynamictherapy. Biochim. Biophys. Acta 1241, 139-176 (1995).
Photochem. Photobiol. 65,422-426 (1997). 59. B. Herman,G. J. Gores, A. Nieminen, T. Kawanishi,A. Harman
40. D. Kessel and Y. Luo, Mitochondrial photodamage and PDT- and J. Lemasters,Calciumand pH in anoxic and toxic injury.Crit.
Rev. Toxicol. 21,127-148 (1990).
induced apoptosis. J. Photochem. Photobiol. B 42, 89-95 (1998).
60. C. Salet, G. Moreno, F. Ricchelli and P. Bernardi,Singlet oxygen
41. K. BergandJ. Moan,Lysosomesandmicrotubules as targetsfor pho-
of cancer.Photochem.Photobiol.65,403-409(1997). producedby photodynamicaction causes inactivationof the mito-
tochemotherapy chondrial permeability transition pore. J. Biol. Chem. 272,
42. L. A. Sporn and T. H. Foster, Photofrin and light induces micro- 21938-21943(1997).
tubule depolymerizationin culturedhumanendothelialcells. Can- 61. G. Kroemer,N. Zamzamiand A. A. Susin,Mitochondrialcontrol
cerRes.52, 3443-3445(1992). of apoptosis.Immunol.Today18,44-51 (1997).
43. T. J. Wieman, K. D. Doak and V. H. Fingar, Effects of PDT on 62. X. Liu, C. N. Kim,J. Yang, R. Jemmersonand X. Wang,Induction
cytoskeletalF-actinstructures.Photochem.Photobiol.53 (Suppl.), of apoptoticprogramin cell-free extracts:Requirementfor dATP
96S (1991).[Abstract] andcytochromec. Cell86, 147-157(1996).
44. C-W. Lin, J. R. Shulok, S. D. Kirley,L. Cincottaand J. W. Foley, 63. H. Zou, W. J. Henzel, X. Liu, A. Lutschigand X. Wang,Apaf-1, a
humanprotein homologous to C. elegans CED-4, participatesin
Lysosomallocalizationand mechanismof uptakeof Nile Blue pho-
tosensitizersin tumorcells. CancerRes.51, 2710-2719(1991) cytochromec-dependentactivationof caspase-3.Cell 90, 405-413
(1997).
45. C-W.Lin, J. R. Shulok,S. D. Kirley,C. M. Bachelder,T. J. Flotte, 64. D. J. Granville,J. G. Levy and D. W. C. Hunt, Photodynamicther-
M. E. Sherwood, L. Cincotta and J. W. Foley, Photodynamic
destructionof lysosomes mediatedby Nile Blue photosensitizers. apy inducescaspase-3activationin HL-60 cells. Cell Death Differ.
Photochem. Photobiol. 58, 81-91 (1993). 4, 623-628(1997).
65. J. He, C. M. Whitacre,L-Y. Xue, N. A. Bergerand N. L. Oleinick,
46. K. Berg and J. Moan, Lysosomes as photochemicaltargets. Int.J. Proteaseactivationand cleavageof poly(ADP-ribose)polymerase:
Cancer59, 814-822(1994). An integralpart of apoptosis in response to photodynamictreat-
47. S. L. Gibsonand R. Hilf, Interdependenceof fluence,drugdose and ment. CancerRes.58, 940-946(1998).
oxygen on hematoporphyrinderivativeinducedphotosensitization 66. J. Yang,K. Bhalla,C. N. Kim,A. M. Ibrado,T. I. Peng,D. P. Jones
of tumormitochondria.Photochem.Photobiol.42,367-373 (1985). andX. Wang,Preventionof apoptosisby Bcl-2:Releaseof cytochrome
c frommitochondria blocked.Science275,1129-1132(1997).
48. A. Atlante, G. Moreno, S. Parsarellaand C. Salet, Hematopor-
phyrinderivative(PF II) photosensitizationof isolated mitochon- 67. R. M. Kluck,E. Bossy-Wetzel,D. R. GreeneandD. W. Newmeyer,
dria:Impairmentof anion translocation.Biochem. Biophys. Res. The release of cytochromec from mitochondria:A primarysite of
Commun.141,584-590 (1986). Bcl-2regulationof apoptosis.Science275, 1132-1136(1997).
49. G. Singh, W. P. Jeeves, B. C. Wilson and D. Jang, Mitochondrial 68. J. Moan,K. Berg, E. Kvam,A. Western,Z. Malik,A. Ruck and H.
photosensitization by Photofrin II. Photochem. Photobiol. 46, Schneckenburger,Intracellularlocalizationof photosensitizers.In
645-649 (1987). Photosensitizing Compounds: Their Chemistry, Biology and Clinical
Use (G. Bock and S. Harnett,Eds.), pp. 95-111. CIBA Foundation
50. K. P. Uberriegler,E. BanieghbalandB. Krammer,Subcellulardam- Symposium146,Wiley,Chichester,1989.
age kineticswithinco-cultivatedWI38andVA13-transformed WI38 69. J. Moan, On the diffusionlength of singlet oxygen in cells and tis-
humanfibroblastsfollowing5-aminolevulinic acid-inducedprotopor- sues. J. Photochem. Photobiol. B 6, 343-344 (1990).
phyrinIX formation.Photochem.Photobiol.62,1052-1057(1995).
70. F. Evensen and J. Moan, Photodynamicaction and chromosomal
51. S. linuma,S. S. Farshi,B. Ortel andT. Hasan,A mechanisticstudy damage:A comparisonof haematoporphyrin derivative(HpD) and
of cellular photodestructionwith 5-aminolaevulinicacid-induced lightwithX-irradiation.Br.J. Cancer45, 456-465(1982).
porphyrin.Br.J. Cancer70, 21-28 (1994). 71. S. R. Wood, J. A. Holroydand S. B. Brown,The subcellularlocal-
52. S. L. Ratcliffe and E. K. Matthews,Modificationof the photody- izationof Zn(II) phthalocyaninesand their redistributionon expo-
namic action of delta-aminolaevulinicacid (ALA) on rat pancre- sureto light.Photochem.Photobiol.65, 397-402(1997).
atoma cells by mitochondrialbenzodiazepinereceptorligands.Br. 72. Z. Malik,I. Amit and C. Rothman,Subcellularlocalizationof sul-
J. Cancer71, 300-305(1995). fonated tetraphenylporphines in colon carcinomacells by spec-
53. A. R. Oseroff, D. Ohuoha, G. Ara, D. McAuliffe, J. Foley and L. trallyresolvedimaging.Photochem.Photobiol.65, 389-396(1997).
Cincotta,Intramitochondrial dyes allowselectivein vitrophotolysis 73. Q. Peng, G. W. Farrants,K. Madslien, J. C. Bommer, J. Moan,
of carcinomacells. Proc.Natl.Acad.Sci. USA83, 9729-9733(1986). H. E. Danielsen and J. M. Nesland,Subcellularlocalization,redis-
tributionand photobleachingof sulfonatedaluminumphthalocya-
54. A. Verma,J. Nye and S. H. Snyder,Porphyrinsare endogenousli-
nines in a human melanoma cell line. Int. J. Cancer49, 290-295
gands for the mitochondrial (peripheral-type) benzodiazepine
(1991).
receptor.Mol. Pharmacol.84, 2256-2260(1987).
74. K. Berg, K. Madslien,J. C. Bommer,R. Oftebro,J. W. Winkelman
55. B. C. Wilson, M. Olivo and G. Singh, Subcellularlocalization of and J. Moan, Light induced relocalization of sulfonated meso-
Photofrinand aminolevulinicacid and photodynamiccross-resis- tetraphenylporphinesin NHIK 3025 cells and effects of dose frac-
tance in vitro in radiation-inducedfibrosarcomacells sensitive or tionation. Photochem. Photobiol. 53, 203-210 (1991).
resistantto Photofrin-mediated photodynamictherapy.Photochem.
Photobiol.65,166-176 (1997). 75. J. Moan, K. Berg, H. Anholt and K. Madslien, Sulfonated alu-
minum phthalocyanines as sensitizers for photochemotherapy.
56. M. W. McEnery,A. M. Snowman,R. R. Trifilettiand S. H. Snyder, Effects of small light doses on localization, dye fluorescence and
Isolationof the mitochondrialbenzodiazepinereceptor:Association photosensitivityin V79 cells. Int.J. Cancer58, 865-870(1994).
 PHOTOBIOLOGYOF PDT S155

76. E. Ben-Hur,T. Fujihara,F. Suzukiand M. M. Elkind,Genetictoxi- 94. F. Xia, S. A. Amundson,J. A. Nickoloffand H. L. Liber,Different
cology of the photosensitization of Chinese hamster cells by capacitiesfor recombinationin closely relatedhumanlymphoblas-
phthalocyanines.Photochem.Photobiol.45, 227-230 (1987). toid cell lines with differentmutationalresponsesto X-irradiation.
Mol. Cell. Biol. 14, 5850-5857 (1994).
77. J. Moan, H. Waksvik and T. Christensen,DNA single-stranded
breaks and sister chromatidexchangesinducedby treatmentwith 95. C. A. Bill, Y. Yongjia,N. R. Miselis,J. B. LittleandJ. A. Nickoloff,
hematoporphyrin and lightor by X-raysin humanNHIK 3025cells. A role for p53 in DNA end rejoiningby humancell extracts.Mutat.
CancerRes.40,2915-2918 (1980). Res. 385, 21-29 (1997).
78. N. Ramakrishnan,N. L. Oleinick, M. E. Clay, M-F. Horng, A. R. 96. R. D. Storer, A. R. Kraynak,T. W. McKelvey, M. C. Elia, T. L.
Antunez and H. H. Evans, DNA lesions and DNA degradationin Goodrow and J. G. DeLuca, The mouse lymphoma L5178Y
mouse lymphomaL5178Ycells afterphotodynamictreatmentsen- (TK'/TK-)cell line is heterozygousfor a codon 170 mutationin the
sitized by chloroaluminumphthalocyanine.Photochem.Photobiol. p53 tumorsuppressorgene. Mutat.Res.373, 157-165(1997).
50, 373-378 (1989). 97. J. T. Deahl, N. L. Oleinick and H. H. Evans, Large mutagenic
79. C. J. Gomer,N. Rucker,A. BanerjeeandW. F. Benedict,Compari- lesions are induced by photodynamictherapyin murine L5178Y
son of mutagenicityand inductionof sister chromatidexchangein lymphoblasts. Photochem. Photobiol. 58,259-264 (1993).
Chinesehamstercells exposed to hematoporphyrin derivativepho- 98. S. A. Amundsonand H. L. Liber,A comparisonof inducedmuta-
toradiation,ionizingradiation,or ultravioletradiation.CancerRes. tion at homologousalleles of the tk locus in humancells. II. Molec-
43, 2622-2627 (1983). ularanalysisof mutants.Mutat.Res.267, 89-95 (1992).
80. B. B. Noodt,E. Kvam,H. B. SteenandJ. Moan,PrimaryDNA dam- 99. M. L.
Agarwal,H. E. Larkin,S. I. A. Zaidi, H. Mukhtarand N. L.
age,HPRTmutationandcell inactivationphoto-inducedwithvarious Oleinick,Phospholipaseactivationtriggersapoptosisin photosensi-
sensitizersin V79 cells.Photochem.Photobiol.58,541-547(1993).
tized mouselymphomacells. CancerRes.53, 5897-5902(1993).
81. B. B. Noodt, J. Moan, E. Kvam and H. B. Steen, No correlation
D. Separovic,J. He and N. L. Oleinick, Ceramidegeneration in
between DNA strandbreaksand HPRT mutationinducedby pho- 100.
tochemicaltreatmentin V79 cells. Mutat.Res.323,75-79 (1994). responseto photodynamictreatmentof L5178Ymouse lymphoma
cells. CancerRes.57, 1717-1721(1997).
82. C. J. Gomer, N. Rucker and A. L. Murphree,Transformationand
101. B. W. Hendersonand J. M. Donovan,Release of prostaglandinE2
mutagenicpotential of porphyrinphotodynamictherapyin mam- from cells by photodynamictreatment in vitro. CancerRes. 49,
maliancells.Int.J. Radiat.Biol. 53, 651-659(1988).
6896-6900(1989).
83. H. H. Evans, M-F. Horng, M. Ricanati, J. T. Deahl and N. L.
Oleinick, Mutagenicityof photodynamictherapyas comparedto
102. E. Ben-Hur, T. M. A. R. Dubbelman and J. Van Steveninck,
UVC and ionizingradiationin humanand murinelymphoblast cell Phthalocyanine-induced photodynamicchangesof cytoplasmicfree
lines. Photochem. Photobiol. 66, 690-696 (1997). calciumin Chinesehamstercells.Photochem.Photobiol.54, 163-166
(1991).
84. S. A. Amundson, F. Xia, K. Wolfson and H. L. Liber, Different
and induced in two human 103. L. C. Penning,M. H. Rasch,E. Ben-Hur,T. M. A. R. Dubbelman,
cytotoxic mutagenicresponses by X-rays
A. C. Havelaar,J. Vander Zee and J. Van Steveninck,A role for
lymphoblastoidcell lines derived from a single donor. Mutat.Res. the transient increase of cytoplasmicfree calcium in cell rescue
286, 233-241(1992).
after photodynamic treatment. Biochim. Biophys. Acta 1107,
85. H. H. Evans, M. Ricanati,M-F. Horng, Q. Jiang,J. Mend and P. 255-260 (1992).
Olive, DNA double-strandbreak rejoiningdeficiencyin TK6 and
other human B-lymphoblastcell lines. Radiat. Res. 134, 307-315 104. S. Gupta,N. Ahmad and H. Mukhtar,Involvementof nitricoxide
(1993). duringphthalocyanine(Pc 4) photodynamictherapy-mediated apo-
ptosis. CancerRes.58, 1785-1788(1998).
86. A. M. Chaudhry,Q. Jiang, M. Ricanati, M-F. Horng and H. H.
Evans, Characterization of multilocus lesions in human cells 105. S. W. Ryterand C. J. Gomer,NuclearfactorkappaB bindingactiv-
exposedto X radiationand radon.Radiat.Res. 145,31-38 (1996). ity in mouse L1210cells followingPhotofrinII-mediatedphotosen-
sitization. Photochem. Photobiol. 58, 753-756 (1993).
87. J. He, M-F. Horng, J. T. Deahl, N. L. Oleinick and H. H. Evans,
Variationof photodynamicefficacyduringthe cellularuptakeof two 106. L-Y. Xue, J. He and N. L. Oleinick, Rapid tyrosine phosphoryla-
phthalocyaninephotosensitizers.Photochem.Photobiol.67, 720-728 tion of HS1 in the responseof mouse lymphomaL5178Y-Rcells to
(1998). photodynamictreatment sensitized by the phthalocyaninePc 4.
Photochem. Photobiol. 66, 105-113 (1997).
88. H. H. Evans,R. M. Rerko,J. Mend, M. E. Clay,A. R. Antunezand
N. L. Oleinick, Cytotoxic and mutagenic effects of the photody- 107. J-S. Tao, J. S. Sanghera,S. L. Pelech, G. Wong and J. G. Levy,
namicactionof chloroaluminumphthalocyanineand visiblelightin Stimulation of stress-activated protein kinase and p38 HOG1
L5178Y cells. Photochem. Photobiol. 49,43-47 (1989). kinase in murine keratinocytes following photodynamictherapy
with benzoporphyrinderivative.J. Biol. Chem.271, 27107-27115
89. R. M. Rerko, M. E. Clay,A. R. Antunez,N. L. Oleinickand H. H.
tk locus (1996).
Evans, PhotofrinII photosensitizationis mutagenicat the
in mouseL5178Ycells. Photochem.Photobiol.55, 75-80 (1992). 108. N. L. Oleinick,J. He, L-Y. Xue and D. Separovic,Stress-activated
90. H. H. Evans, M. Ricanati, C. DiSalvo, M-F. Horng and J. Mend, signallingresponsesleading to apoptosisfollowingphotodynamic
Inductionof multilocuslesionsby UVC-radiationin mouse L5178Y therapy.SPIE 3247,82-88 (1998).
lymphoblasts.Mutat.Res.251, 79-90 (1991). 109. M. C. Luna, S. Wong and C. J. Gomer, Photodynamictherapy
mediated induction of early response genes. Cancer Res. 54,
91. H. H. Evans,J. Mend, M-F. Horng,M. Ricanati,C. Sanchezand J. 1374-1380(1994).
Hozier, Locus specificityin the mutabilityof L5178Ymouse lym-
phoma cells: The role of multilocuslesions. Proc. Natl. Acad. Sci. 110. G. Kick,G. Messer,G. Plewig,P. KindandA. E. Goetz, Strongand
USA 83, 4379-4393(1986). prolongedinductionof c-junand c-fos proto-oncogenesby photo-
92. F. Xia, X. Wang, Y-H. Wang, N-M. Tsang, D. W. Yandell, K. T. dynamictherapy.Br.J. Cancer74, 30-36 (1996).
Kelsey and H. L. Liber,Altered p53 statuscorrelateswith differences 111. S. Evans,W. Matthews,R. Perry,D. Fraker,J. NortonandH. I. Pass,
in sensitivityto radiation-inducedmutation and apoptosis in two Effectof photodynamictherapyon tumornecrosisfactorproduction
closely related human lymphoblastlines. CancerRes. 55, 12-15 by murinemacrophages. J. Natl.CancerInst.82,34-39 (1990).
(1995). 112. C. Anderson,S. Hrabovsky,Y. McKinley,K. Tubesing,H-P. Tang,
93. J. B. Little, H. Nagasawa,P. C. Keng, Y. Yu and C-Y. Li, Absence R. Dunbar,H. Mukhtarand C. A. Elmets, Phthalocyaninephoto-
of radiation-inducedG1 arrestin two closely related humanlym- dynamictherapy:Disparateeffects of pharmacologicinhibitorson
phoblast cell lines that differ in p53 status. J. Biol. Chem. 270, cutaneousphotosensitivityand on tumor regression.Photochem.
11033-11036(1995). Photobiol. 65, 895-901 (1997).
S156 OLEINICKAND EVANS

113. G. Kick,G. Messer,A. Goetz, G. PlewigandP. Kind,Photodynamic mitochondrialtargetingdye VictoriaBlue BO. Photochem.Photo-
therapyinduces expressionof interleukin6 by activationof AP-1 biol. 67, 155-164(1998).
but not NF-KBDNA binding.CancerRes.55, 2373-2379(1995).
119. C. J. Gomer, S. W. Ryter, A. Ferrario, N. Rucker, S. Wong and
114. S. 0. Gollnick,X. Liu,B. Owczarczak, D. A. MusserandB. W. Hen- A. M. R. Fisher,Photodynamictherapy-mediatedoxidativestress
derson,Altered expressionof interleukin6 and interleukin10 as a can induce expression of heat shock proteins. Cancer Res. 56,
result of photodynamictherapyin vivo. CancerRes. 57, 3904-3909 2355-2360(1996).
(1997). 120. C. J. Gomer,M. Luna,A. Ferrarioand N. Rucker,Increasedtran-
115. C. J. Gomer,A. Ferrario,N. Rucker,S. Wong and A. S. Lee, Glu- scription and translationof heme oxygenase in Chinese hamster
cose regulatedproteininductionand cellularresistanceto oxidative fibroblastsfollowing photodynamicstress of PhotofrinII incuba-
stress mediated by porphyrinphotosensitization.CancerRes. 51, tion. Photochem. Photobiol. 53, 275-279 (1991).
6574-6579(1991).
121. C. E. CanmanandM. B. Kastan,Threepathsto stressrelief.Nature
116. P. M. Curryand J. G. Levy, Stress protein expression in murine 384, 213-214 (1996).
tumorcells followingphotodynamictherapywith benzoporphyrin
derivative. Photochem. Photobiol. 58, 374-380 (1993). 122. M. Robinson and M. H. Cobb, Mitogen-activatedprotein kinase
117. L. Y. Xue, M. L. Agarwaland M. E. Varnes,Elevationof GRP-78 pathways. Curr. Opin. Cell Biol. 9, 180-186 (1997).
and loss of HSP-70followingphotodynamictreatmentof V79 cells: 123. J. Yuan, Transducingsignals of life and death. Curr. Opin. Cell
Sensitization by nigericin. Photochem. Photobiol. 62, 135-143 Biol. 9, 247-251 (1997).
(1995). 124. J. C. Lee and P. R. Young, Role of CSBP/p38/RKstress response
118. J. Morgan,J. E. Whitakerand A. R. Oseroff,GRP78inductionby kinase in LPS and cytokine signaling mechanisms.J. Leukocyte
calcium ionophore potentiates photodynamic therapy using the Biol. 59,152-157 (1996).