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
to PF-PDT (30), the radiation-inducedRIF-1 fibrosarcoma
cells exposedto Pc 4-PDTin vitro(37), andCV-1cellsirradi-
ated aftera shortincubationwith PF allowingaccumulation
of the photosensitizerprimarilyin the plasma membrane
(31). With ALA-PDT, Noodt et al. (32) found apoptosisin
ChinesehamsterV79 cells andnecrosisin WiDrhumanade-
nocarcinomacells. The particularmode of cell death in
responseto PDT dependson experimentalconditions,such
as the dose of PDT (34,38) andthe subcellularlocalizationof
the photosensitizers(39, 40). It shouldbe kept in mindthat
the definitionof apoptosisis itselfnot firmlyestablished,and
someof the exceptionsmayturnout to be a formof apoptosis
in whichthe nucleareventsarenot observed.
The sensitivityof cells to PDT can varybetween closely
related cell lines as a functionof the expressionof defined
genes that have a role in controllingapoptosis.Thus HL60
cells expressingwild-type p53 were more sensitive to cell
killingby PDT, with eitherPF or SnET2as the photosensi-
tizer, than were HL60 cells in which the p53 genes were
deleted or inactive. All of these cell lines underwentapo-
ptosis rapidly in response to PDT (35). Chinese hamster
ovary cells expressinga humanBCL2 gene were partially
resistant to apoptosis and to overall cell death after PDT
sensitizedby the phthalocyaninePc 4 (36).
TARGETS
Assumingan adequateoxygensupplyand lightintensity,
the site of photodamage depends on the location of the
photosensitizerat the time of irradiationwith visible light.
Secondarily,it can depend upon movement of the photo-
sensitizerto a secondarysite duringcontinuingirradiation.
Nearly all of the photosensitizerscurrentlybeing studied
are hydrophobicmoleculesthat tend to bind to membranes
and are excludedfromthe nucleus.
Photodynamictherapyof cells resultsin extensivedam-
age to cell structures.Depending on the photosensitizer,
overall dose and experimentalprotocol, damage hasbeen
observedin microtubules,membranous organelles,especially
lysosomes and mitochondria,plasma membrane and the
nucleus.Muchof the earlywork in this area was done with
PF or one of its precursors,but since these are complex
mixtures,it is impossibleto distinguishthe location of the
active comparedto the inactive components.More recent
studies have focused on chemicallypure photosensitizers,
identifyingsites of bindingby fluorescencemicroscopyin
comparison to probes of specific organelles. Studies of
some of these targetswill be summarizedbelow.
Cytoskeletal Elements
Berg and Moan (41) have suggested that nonpolymer-
ized tubulin may be a target for cytotoxicity induced by
photodynamicaction.Some of the photosensitizersbind to
tubulin, and after illumination its polymerizationis pre-
vented. Tubulinis affected by low doses of photodynamic
action similarly to treatment with inhibitors of microtubulin
S149
function. Thus photodynamic action and the inhibitors
inducethe formationof micronucleiand giantcells and the
accumulationof cells in mitosis(reviewedin ref. 41). Sporn
and Foster (42) have shownthat depolymerizationof tubu-
lin results 15 min after illuminationand is reversibleafter
low fluencesbut irreversibleafterhighfluences.The authors
suggestthat the depolymerizationis causedby an increase
in intracellularcalcium. Actin filaments remained unaf-
fected. At higherPDT doses, however,Wiemanet al. (43)
reportedmicrofilamentdisruption.
Lysosomes
Although a variety of photosensitizers localize in the
lysosomes, it appears that lysosomal disruptionmay not
always lead to cell killing. Lin and colleagues have
reported that the degree of lysosomal damage caused by
PDT was not correlatedwith the photocytotoxicity(44, 45).
Some cells survivepartiallysosomal disruption,probably
because lysosomal enzymes are inactivated by the treat-
ment, as well as by cytosolicinhibitors(46).
Mitochondria
There is now considerableevidence that mitochondria
are important targets in the cytotoxicity of PDT. Early
studies with PF showed a promptpost-PDT inhibition of
respiration,measuredboth in intact cells and in isolated
mitochondria,inhibitionof the actionof electrontransport
components,suchas succinatedehydrogenase andcytochrome
c-oxidase,and disruptionof the mitochondrialelectrochem-
ical gradient,all of which occurredearlierthan loss of the
abilityof the cells to exclude trypanblue (47-49). Because
PPIX is producedin mitochondria,mitochondrialdamage
is prominentwhen cells are irradiatedat earlytimes (-4 h)
after introducingthe metabolicprecursorALA; however,
becausePPIX can diffuseout of the mitochondria,damage
is foundat othersites as well (17, 50-52). Cationiclipophilic
compounds,such as the krytocyaninedye EDKC and rho-
damine-123,accumulatein mitochondriaas a result of the
highly negative electrochemical potential of the active
inner mitochondrialmembrane(53). However,some por-
phyrin-relatedphotosensitizersthat are negativelycharged,
suchas PF, or neutral,suchas Pc 4, also accumulatein mito-
chondria2(49), possiblybecauseof bindingto specificmito-
chondrialconstituents.Ratcliffe and Matthews(52) found
that the mitochondrialeffects of photoactivatedPPIXwere
inhibitedby ligandsof the peripheralbenzodiazepine receptor
(PBR), a mitochondrialpore-formingprotein. Porphyrins
are endogenousligandsfor this receptor(54), and Morgan
and Oseroff3have recentlydemonstratedthat the binding
of a specificPBR ligand(PK11195)could be competitively
inhibitedby pyropheophorbidephotosensitizersthat local-
ize to mitochondria.Wilson et al. (55) demonstratedthe
2N. L. Oleinick, A. L. Nieminen and N. Trivedi, unpublishedobservation.
3j. MorganandA. R. Oseroff,unpublishedobservation
S150 OLEINICKAND EVANS
abilityof a cardiolipinprobe, lON-nonylacridineorange,to
competitivelyinhibit the uptake of PF into mitochondria,
indicatingthat some photosensitizersmight bind to cardi-
olipinsof the innermitochondrialmembrane.
The PBR is a complex of several proteins that includes
the 18-kDa receptor in the mitochondrial outer mem-
brane linked to the voltage-dependentanion channel and
the adenine nucleotide translocator that is responsible
for ADP/ATP exchange (56, 57). The latter two compo-
nents of the PBR are also thought to be components of
the membranepermeabilitytransition (MPT), a nonspe-
cific ion channel that brings the outer and inner mito-
chondrial membranes into contact (58, 59). Normally,
passage of ions and molecules across the inner mem-
brane is tightly regulated by the membrane potential.
However, the MPT senses changes in redox state, pH,
etc. and can open to permit depolarization of the mem-
brane, inhibition of oxidative phosphorylationand efflux
of Ca+. The results of a recent study of PDT with
hematoporphyrin on isolated rat liver mitochondria
showed that photodynamic action probably degrades
critical histidines of the MPT, leading to site-selective
inactivationof discrete pore functions (60).
The mitochondrionis a criticalsite for release of factors,
especiallycytochromec, that triggerthe finalstagesof apo-
ptosis (61, 62). It has been proposedthat cytochromec, in
combinationwith the cytoplasmicprotein, apoptosis-acti-
vating factor 1 (APAF1), directlyactivatesthe cascade of
caspases (cysteine proteases acting on asparticacid) that
carryout the final stages of apoptosis (63). Such caspases
are rapidly activated in PDT-treated L5178Y-R, HL60,
P388 and L1210 cells (34, 64, 65). Expression of BCL2, a
proteinof the mitochondrialouter membrane,preventsthe
release of the mitochondrialfactors (66, 67), partiallypro-
tects cells from PDT-inducedapoptosis, and endows cells
with a partialresistanceto PDT (36). Furthermore,Kessel
and Luo have shown that photosensitizers that bind to
mitochondria induce apoptosis upon photoirradiation,
whereas those that bind to the plasmamembraneor lyso-
somes,but not to mitochondria,kill cells less efficientlyand
by a mechanismnot involvingapoptosis(39, 40).
TheNucleus:DNA Alterationsand Mutagenicity
As indicatedabove, the lipophilicPDT photosensitizers
generally localize in membranes, including the nuclear
membrane (68). Although these photosensitizers do not
appearto enter the nucleus, illuminationof cells preincu-
bated with the photosensitizerscauses DNA damage that
subsequentlyresultsin chromosomeaberrationsand muta-
tions. Singlet oxygen, a form of reactiveoxygen generated
by PDT, has a rangeof -0.1 pm and a lifetime of less than
1 s (69), but may reach DNA closely positioned near the
nuclear membrane. In agreement with this possibility,
Evenson and Moan (70) have reporteddata suggestingthat
the chromosomalcentromericand telomericregionswhich
bind to the nuclearmembraneare quite sensitive to PDT- sitizers tested when measured at the F37doses (Table I).
generateddamage.Alternatively,it is possiblethat relocal-
ization of the sensitizers to the nucleus occurs during
illuminationand is responsiblefor the inductionof DNA
damage. Relocalization has been reported for several
photosensitizers(71-75).
Photodynamictherapyof varioustypes of cells resultsin
DNA lesionssuchas single-strand breaksand/oralkali-labile
sites (76-78). In two closelyrelatedL5178Y(LY) cell lines,
the cytotoxicityof PDT did not correspondto the induction
or rejoiningof single-strandbreaks,but did correlatewith
the numberof DNA-protein crosslinksand with the extent
of DNA degradationcausedby the treatment(78). Chromo-
some aberrationsand sisterchromatidexchangeshave also
been observedin CHO andNHIKcellstreatedwithhemato-
porphyrinandredlight(70,77, 79).
The mutagenicity of PDT varies with the photosensi-
tizerand the cell line, as well as the targetgene. Noodt et al.
(80, 81) comparedthe mutagenicityof PF and three closely
related porphyrins at the Hprt locus in V79 cells. PDT
was significantly mutagenic when sensitized by the
lipophilic photosensitizers,PF or 3THPP, but not by the
hydrophilicphotosensitizers, TPPS4 and TmPyPh2.The
differences in mutagenicitywere not correlated with dif-
ferences in the efficiency of induction of DNA strand
breaks, nor could they be explained by the intracellular
distributionof the photosensitizers.No significantmuta-
genicitywas detected at the Hprtor Na+/K+ATPase loci in
Chinese hamsterV79 or CHO cells treated with hemato-
porphyrinand red light (76, 79, 82), nor was morphologi-
cal oncogenictransformation inducedundertheseconditions
in C3H 101/2cells (82).
We havestudiedthe mutagenicityof PF andtwo phthalo-
cyanines(AlPcandPc 4) in murineandhumanlymphoblasts,
all heterozygousfor the autosomalthymidinekinase gene
(Table I). TK6 and WTK1 human lymphoblastsare simi-
larlysensitiveto the cytotoxiceffects of PDT with AlPc or
PF, but the mutabilityof these two cell lines differs:WTK1
cells are significantlymutagenizedby PDT with either sen-
sitizer. However, TK6 cells are poorly mutagenized by
PDT. The mutabilityof TK6 cells is also significantlylower
thanthat of WTK1cells aftertreatmentwithUVC or ioniz-
ing radiation(TableI). TK6cellspossessan activep53 gene,
while p53 is inactive in WTK1 cells (92, 93). TK6 cells
show higherlevels of PDT-inducedapoptosis(83) and are
more deficient in recombination(94) and the rejoiningof
DNA double-strandbreaks than WTK1cells (83, 85, 95).
Any one or a combinationof these characteristicscould
explainthe low mutabilityof TK6cells.
The frequencyof PDT-inducedmutantsis muchhigherin
the murineLY-S1cell line thanin eitherof the humanlym-
phoblasticlines or the LY-R16cell line. Both LY cell lines
also harborinactivep53 genes (96). In the murinecells and
in human WTK1 cells, PDT with PF resulted in a higher
mutant frequency than did AlPc-PDT, while Pc 4-PDT
inducedthe lowestmutantfrequencyof the threephotosen-
 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
kinaselocus comparedto the Hprtand Na+/K+ATPase loci
is most probablycausedby the higherrecoveryof mutants
withintergenicmutationsin the case of the thymidinekinase
autosomallocus. When the target gene is in a hemizygous
target region (i.e. the Hprt gene on the X chromosome),
mutants with intergenic mutations are poorly recovered
because of the inactivationof essentialgenes linked to the
target.In contrast,when the targetgene is on an autosomal
chromosome (such as the thymidine kinase gene), the
homologouschromosomecan usuallysupplyan activecopy
of the linked essential gene. Since multilocus lesions are
induced by PDT (97), the location of the target gene can
explainthe highermutantfrequencyinducedby PDT at the
thymidinekinase comparedto the Hprt locus. Use of the
co-dominantgene for Na+/K+ATPase also does not detect
large or even small deletions,because the sequencecoding
for the ouabain-bindingsite (the mutationof whichis nec-
essary for ouabain resistance) very closely neighbors the
sequencefor the ATP-bindingsite. Thusdeletionsaffecting
the ouabain-bindingsite almostalwaysinactivatethe ATP-
binding site, resulting in inactivation of the resistant
enzyme.
The high mutabilityof the LY-S1 cells comparedto the
humancells maybe causedby a higherfrequencyof regions
of hemizygosityin essentialgenes neighboringthe TK gene
in the WTK1and TK6 humancells (98). Alternatively,the
higher sensitivity of the human cells (and of the LY-R16
cells) to the cytotoxic effects of PDT comparedto LY-S1
cells could also explain the lower mutabilityof the human
and LY-R16cells, assumingthat the TK'- mutantsof these
cell lines are more sensitivethanthe TK'- cells.
Our results show that the mutagenicityof PDT varies
greatly with the target gene and the cell line. The mutant
frequency at a particulartarget gene appears to depend
upon the essentialgenes in its vicinity,as well as the activ-
ity of these target genes on the homologouschromosome,
and thereforewould be expectedto varywith the arrange-
ment and activity of genes on the chromosomes of each
particularcell line. Because of the extensive genetic vari-
ation in the humangenome,it is probablethat humancells
may varygreatlyin theirmutabilityfromindividualto indi-
vidual,from cell type to cell type, and from locus to locus.
Use of PDT for the treatmentof benign conditionsshould
thereforebe undertakenwith caution.
SIGNALTRANSDUCTIONPATHWAYS
ACTIVATEDBY PDT
Photodynamictherapyactivatesmanysignalingreactions
that have been characterizedin responseto other oxidative
stresses or physiological stimuli. Table II lists the major
reactionsthat have been reportedto occurin one or more
PDT-treatedcell lines in vitro,alongwith the cell lines and
photosensitizersused experimentally.It is apparentthat
PDT causes the release of a variety of lipid and inorganic
second messengers,as well as the release of calciumions
fromintracellularstores.Eitherindependentlyor as a result
of stimulationby one or more second messengers,several
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
Althoughthe leadingphotosensitizers do not localizeinitially 17. Q. Peng, K. Berg, J. Moan, M. Kongshaug and J. M. Nesland,
in nuclei,both DNA damageandmutationsare producedby
PDT, the extentof the mutagenicitydependingon the photo- 18. K. R.
sensitizer,the targetcell and the targetgenetic locus. PDT
activatesa varietyof signaltransductionpathwaysthat can
lead to cell deathby apoptosisor to the inductionof cytopro- 19. M. Ochsner, Photophysicaland photobiologicalprocesses in the
tective stress responses. The unique combinationof PDT
at criticalsubcellularsites and activation of death-
damage
promotingsignalingpathwayscollectivelycontributeto the 20. J.
efficientandrapidprogressof cell deathprocesses.
ACKNOWLEDGMENTS
Research on photodynamictherapy in the authors' laboratoriesis
supported by PHS grants P01-CA48735 and P30-CA43703 from the
NationalCancerInstitute,DHHS.
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