ICANCIÃŒRRKSKARCH 58. "MO-946, March I. 19981

Protease Activation and Cleavage of Poly(ADP-ribose) Polymerase: An IntegralPart of Apoptosis in Response to Photodynamic Treatment1

Jin He, Cecilia M. Whitacre, Liang-yan Xue, Nathan A. Berger, and Nancy L. Oleinick2

De/wrlnu-nlx t>fRattii>li>R\/J. H.. L-Y. X.. N. L O.] und Medicine ¡C.M. W., N. A. B.¡and thèCWRU/Irelund Cancer Center ¡N.A. B.. N. L. O.¡.Case Western Reser\-e University

Sciami of Medicine. Cleveland. Ohio 44106-4942

ABSTRACT

Apoptosis induced by numerous cancer chemotherapeutic and othertoxic agents has been shown to proceed through a cascade of proteases,now termed caspases, culminating in cleavage of a set of proteins. Theability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 toactivate cellular caspases has been assessed during the rapid apoptosis inmurine lymphoma L5178Y-R cells. Cells were exposed to combinations ofPc 4 and activating red light that result in a90% cell death, as judged bya clonogenic assay. The rate of entry of cells into apoptosis was dosedependent. For 0.5 /DIMPc 4 and either 2.1 or 3 kj/m2, which kill 90 or

99.9% of the cells, oligonucleosomal fragmentation was visible on agarosegels as early as 60 or 30 min after PDT, respectively. To assess caspaseactivation, cells were harvested at various times after PDT, and cellproteins were subjected to electrophoresis and Western blot analysis,using an antibody to poly(ADP-ribose) polymerase (PARP). The cleavageof the normally MT 116,000 PARP into fragments of Mr -90,000 was

observed at approximately the same time as the earliest DNA fragmentation. An antibody to the polymer, poly(ADP-ribose), did not recognize theMr —¿�90,000PARP cleavage products, in contrast to the parent enzyme.This analysis also revealed that levels of a poly(ADP-ribosylated) U,

100,000 protein, tentatively identified as topoisomerase I, were maintainedin cells after PARP was fully cleaved. Caspase-3 (and/or caspase-7) activity, as measured in cell lysates with the fluorogenic substrate DEVD-AMC, was elevated almost immediately after PDT. The cell-permeable,irreversible caspase inhibitor, benzyloxycarbonyl-Val-AIa-Asp(0-methyD-fluoro-methylketone, inhibited PDT-induced apoptosis andPARP cleavage, whereas the inactive peptide analogue, benzyloxycar-bonyl-Phe-Ala-fluoromethyl ketone, was without effect. The results indicate that PDT-induced apoptosis is mediated by activation of caspase-3

and/or other similar caspases.

INTRODUCTION

Photodynamic therapy, a newly approved cancer treatment, uses aphotosensitizer and visible light to produce singlet oxygen and otherreactive oxygen species (1, 2), leading to lipid peroxidation anddamage to membranes, DNA, cytoskeleton. and other sites (3), andeventual cell death and tumor ablation (4). PDT3 of mammalian cells

in culture induces programmed cell death or apoptosis in some celllines (5-8). In mouse L5178Y-R lymphoma cells, PDT induces rapid

cell death by apoptosis that appears to be dependent upon the activation of phospholipases C and A2 but not upon new gene expression(9). Evidence for the importance of protein serine/threonine phos-phatases and protein kinase C in the progress of apoptosis in HL-60

Received 11/27/97; accepted 1/6/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by USPHS Grants POI CA48735. POI CASI 183. P30

CA63200, and P30 CA43703 from the National Cancer Institute.2 To whom requests for reprints should be addressed, at Division of Radiation Biology.

School of Medicine (BRB-324). Case Western Reserve University. 10900 Euclid Avenue.Cleveland, OH 44106-4942. Phone: (216)368-1117; Fax: (216)368-1142; E-mail:nlo@po.cwru.edu.

' The abbreviations used are: PDT. phouxlynamic treatment; PARP. poly( ADP-ribose)

polymerase; ICE. ¡nterleukin-1/3 converting en/,yme: TdT. terminal deoxynucleotidyltransfera.se; AMC. 7-amino-4-methyl-coumarin: caspase. cysteine-dependent aspartate-directed protease; ¿FA-fmk. benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone; ¿VAD-fmk. ben/yloxycarbonyl-Val-Ala-Asp(CMnethyl)-f1uoromethylketone.

cells has been obtained with the aid of the inhibitors calyculin andstaurosporine. respectively (10).

Apoptosis is a programmed event characterized by chromatin condensation, cell membrane blebbing. and DNA cleavage (11-13),

which are the most commonly used indicators of apoptosis. Theendonucleolytic cleavage of DNA proceeds in two phases, first generating large (50-300 kb) chromosomal fragments and subsequently,in many but not all situations, oligonucleosome-sized (multiples of

180 bp) fragments.In recent years, it has become apparent that a cascade of proteases

with properties resembling those of ICE is also activated duringapoptosis (14-17). Some of the earliest indications of the regulation

of cell death by proteases were described for CTLs and natural killercells, which induce apoptosis upon contact of their cytoplasmic granules with target cells. The proteins that are released from the granulesand are responsible for the induction of cell death are perform as wellas a series of serine proteases, including granzyme B/fragmentin-2,

which cleaves its substrates at Asp residues (18). Further evidence ofthe involvement of proteases in the induction of programmed celldeath came from studies of the nematode Caenorhahditis elegans, inwhich the cell death protein Ced-3 was recognized to be a proteasewith cysteine at its active site: Ced-3 is a homologue of ICE (19),

having 29% overall sequence identity, 43% in the most highly conserved region, and complete identity in the pentapeptide QACRG thatcontains the active site cysteine (20).

A family of ICE-like cysteine proteases has been described in

mammalian cells based on the varying degrees of sequence homologyamong the family members and on their preferred cleavage sites (16,17). All members of this family of proteases have in common anessential cysteine residue at the active site and an absolute requirement for aspartic acid at the substrate cleavage site. A systematicnomenclature, as proposed by Alnemri et al. (21), established the termcaspases for cysteine proteases acting on aspartic acid, and the properties of the caspases and their known substrates have been reviewedrecently (17). Among the caspases are: Nedd-2/Ich-l/caspase-2 (22,23); CPP32/YAMA/Apopain/caspase 3 (24. 25): TX/Ich-2/caspase-4(26, 27): and Mch-2/caspase-6 (28), Mch3/ICE-LAP3/caspase-7 (29,30), Mch4/caspase-10 (31). Mch5/FLICE/caspase-8 (31, 32), andMch6/ICE-LAP6/caspase-9 (33, 34). Overexpression of the caspases

in cultured cells has been shown to induce apoptosis, and it is inferredthat physiological levels participate in apoptosis after appropriatestimuli. Among the most commonly observed final substrates thatbecome cleaved during apoptosis are a set of nuclear proteins, including lamin Bl (35, 36), topoisomerases I and II and histone HI (37,38). PARP ( 14. 39). and others (17). The cleavage of one or more ofthese proteins is an important hallmark of apoptosis.

During apoptosis. PARP most often undergoes proteolytic cleavagebetween Asp 216 and Gly 217. This reaction cleaves the Mr 116.000native enzyme into a Mr —¿�90,000fragment, which contains theCOOH-terminal catalytic domain, and a MT—¿�26.000fragment, whichcontains a truncated NH,-terminal DNA-binding domain (14, 39).Asp 216 is the preferred cleavage site for caspase-3 and other closely

related proteases (14, 17. 20).When apoptosis is induced by PDT. hallmarks such as chromatin

940

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from

PARP CLEAVAGE IN RESPONSE TO PC 4-PDT

condensation (5), DNA fragmentation (5-8), and inhibition by Bcl-2

(40) have been shown to be components of the process. Until recently,caspase activation was not characterized during the rapid apoptosisproduced by PDT. After submission of this report, caspase activationand PARP cleavage were reported in HL60 cells treated with PDTsensitized by benzoporphyrin derivative (41 ), and PARP cleavage wasbriefly demonstrated in PDT-treated L1210 and P388 cells with

aluminum phthalocyanine (42). The purpose of the present study wasto determine the ability of PDT to activate caspases, as assessed byPARP cleavage, and to better define the relationship to the extent andtiming of PDT-induced cell death.

MATERIALS AND METHODS

Photosensitizer, Protease Inhibitors, and Substrate. The phthalocyaninephotosensitizer Pc 4 (43) was provided by Drs. Ying-syi Li and Malcolm E.

Kenney. Department of Chemistry, Case Western Reserve University, Cleveland, OH. Stock solutions (0.5 mM in dimethyl formamide) were stored at0-4°C. zVAD-fmk and zFA-fmk were purchased from Enzyme Systems

Products (Dublin. CA). Stock solutions (50 mM in DMSO) were stored at-70°C. DEVD-AMC was from BIOMOL Research Laboratory, Inc. (Plym

outh Meeting, PA). A stock solution (10 mM in DMSO) was stored at -20°C.

Cell Culture. Mouse lymphoma L5178Y-R (LY-R) cells are designated Rbecause of their relative resistance to X-irradiation in comparison to the closelyrelated strain L5178Y-S but have been shown to be relatively sensitive to UVC

radiation, hydrogen peroxide, and PDT in comparison to LY-S cells (44).LY-R cells were grown in suspension culture in Fischer's medium containing

0.1% pluronic F68, 2 mM sodium pyruvate. and 10% heat-inactivated horseserum in a humidified atmosphere at 37°Cwith 5% CO, (44). For most

experiments, the cultures were grown in T-25 flasks; in the studies withprotease inhibitors, 96-well plates were used.

Photodynamic Treatment of Cell Cultures. Cells were loaded with Pc 4by the addition of an aliquot of the stock solution to the culture medium —¿�18h before irradiation. When present, zVAD-fmk or zFA-fmk was added l hprior to irradiation. The light source was a tungsten-halogen lamp with a600-nm long-pass filter. The fluence rate was 75 mW/m2. Unless otherwise

indicated, irradiations were performed at room temperature. The temperatureduring irradiation did not exceed 34°C.

Gel Electrophoresis of DNA. Cells were collected by centrifugaron atvarious times after irradiation. DNA isolation and gel electrophoresis wereperformed as described previously (5). Briefly, cell pellets were resuspended in500 jul of a solution containing IX SSC and 10 mM EDTA, then 1% (w/v)sodium lauryl sarkosinate and 0.1 mg/ml Proteinase K were added, and themixture was incubated at 50°Cfor at least 2 h. The DNA was precipitated by

the addition of 2 volumes of absolute ethanol, resuspended in 200 /nl of TE,Tris-EDTA, and briefly treated with 1 mg/ml RNase before loading onto a

1.5% agarose gel. After electrophoresis, the gel was stained with 0.5 /ng/mlethidium bromide and photographed under UV light.

Origin of Antibodies. Monoclonal mouse anti-PARP antibody was developed to purified human PARP and isolated by protein A-Sepharose chroma-tography in our laboratories as described previously (45). The antibody 4C10-5is a mouse IgG I Kthat shows specificity for the NAD-binding domain of PARPand reacts with a M, 90.000 degradation product in mitogen-stimulated humanlymphocytes (45). Monoclonal anti-poly(ADP-ribose) antibody 10H (46) was

purchased from BIOMOL Research Laboratory, Inc. This antibody recognizesthe linear structure of poly(ADP-ribose) with ribose-ribose linkages and bindsalmost all molecular sizes of the polymer (46). Polyclonal anti-topoisomerase

I antibody was from TopoGen, Inc. (Columbus. OH), and the monoclonalanti-mouse actin and peroxidase-linked sheep anti-mouse immunoglobulin

were from Amersham (Arlington Heights. IL).Protease Activity. Various times after PDT, 4 x IO6cells were collected

by centrifugation. and the washed cell pellet was resuspended in 250 fi\ of lysisbuffer containing 100 mM HEPES, 10% sucrose, 0.1% 3-[(3-cholamidopropy-l)dimethylammoniol]-l-propanesulfonic acid. 0.25 MEDTA. 5 mM DTT, 1 mM

phenylmethylsulfonyl fluoride, 2 /ng/ml pepstatin, and 2 ng/ml leupeptin.Aliquots containing 50 jug of cellular protein [estimated by the Bradfordmethod (47)] were incubated with 50 JU.MDEVD-AMC in 250 u,l of lysisbuffer at 37°Cfor 30 min. Fluorescence was measured in a Perkin-Elmer LS50

fluorometer (Acxc.360 nm: Aem.460 nm). Cellular protein served as the blank.Results were compared with a standard curve constructed with 0-3000 pmol

of AMC.Gel Electrophoresis of Proteins and Western Blot Analysis. Following

PDT. cultures were returned to the 37°Cincubator until harvest. Cells were

lysed and sonicated in a solution comprised of 0.5% sodium deoxycholate.0.2% SDS, 1% Triton X-100. 5 mM EDTA. 10 fig/ml leupeptin, 10 /xg/ml

aprotinin, and 1 mM phenylmethylsulfonyl fluoride in PBS. An equal volumeof SDS sample buffer [50 mM Tris (pH 6.8). 1% SDS. 1% mercaptoethanol.0.1 M DTT, and 5% sucrose] was added to the cell lysate. Equivalent amountsof protein were loaded onto an 8% polyacrylamide gel. subjected to electrophoresis, transferred to a polyvinylidene difluoride membrane, and incubatedwith either antibody 4C10-5 (45), anti-poly(ADP-ribose). or anti-topoisomerase I antibody overnight at 4°C.The immune complexes were detected by the

ECL system (Amersham). The images of the Western blots were scanned, andthe intensity of each band was determined using SigmaGel (Jandel Scientific.San Rafael, CA). In most cases, gels were also probed with anti-actin to correct

for unequal loading of protein.Because the 4C10-5 antibody recognizes the M, 116,000 native PARP band

and the Mr —¿�90,000PARP cleavage fragments, the percentage of the total

PARP in each lane that is present as fragments was calculated as follows:

PARP cleaved =Intensity of M, 90,000 PARP bands

Intensity of M, 90,000 PARP bands

+ intensity of M, 116,000 PARP band

- X 100

The intensity of the bands for the native and cleaved PARP deviatedfrom a linear dependence on amount of protein loaded. However, intest samples, the % PARP cleaved was independent of protein loadingfor control (0% cleaved) and fully cleaved (100%) samples andfluctuated <15% about the average value over a 10-fold range of

protein loaded for a partially cleaved sample. Therefore, no furthercorrection was made for nonlinear band intensity.

Flow Cytometry. Cell fixation and staining were performed based on theAPO-DIRECT kit (Phoenix Flow Systems. Inc.; Ref. 48). In brief, cells werefixed in 1% paraformaldehyde for 15 min and stored in 70% ethanol at -20°C

overnight. Fixed cells were then washed with PBS. resuspended in 50 p.1 ofTdT reaction buffer containing: 10 /il of 5X concentrated buffer solution [1 Mcacodylic acid, 125 mM Tris-HCl (pH 6.6). and 1.26 mg/ml BSA], 2.5 mMcobalt chloride, 6 units TdT, and 0.25 nM FITC-dUTP and incubated at 37°C

for 1 h. The cells were rinsed twice and incubated in 1 ml of a solutioncontaining 2.5 jug/ml propidium iodide and 0.1% DNase-free RNase A in the

dark at room temperature for 30 min. Analysis was carried out in the FlowCytometry Facility of the Case Western Reserve University/Ireland CancerCenter. Fluorescence measurements were made on an EPICS ESP (low cy-

tometer (Coulter Corp.); fluorescence was activated at 488 nm, and fluorescence emission was monitored at 520 nm (fluorescein) and 623 nm (propidiumiodide).

Fluorescence Microscopy. The cell pellets from control or PDT-treatedcultures were resuspended at 1X107 cells/ml in fresh medium, and a 25-/¿l

aliquot was mixed with 1 /¿Iof a solution of acridine orange and ethidiumbromide ( 100 /¿g/ml).The mixture was immediately placed on the stage of anOlympus fluorescence microscope. The samples were excited at 330-380 nm.

and the image was observed and photographed under a combination of a400-nm dichroic mirror and the 420-nm high-pass filter. Live, apoptotic. and

necrotic cells were scored under the microscope. Cells with a green, condensedchromatin pattern were considered apoptotic, whereas those with orange nucleibut without nuclear condensation were considered necrotic.

RESULTS

Fig. 1«presents a Western blot analysis of the time course of PARPcleavage after each of three doses of PDT. Cells not treated with PDTrevealed only a single protein band reactive with the anti-PARP

antibody at M, 116.000. the expected size of unmodified, intact PARP.When cells were treated with 0.5 ¡JLMPc 4 and 2.1 kJ/m2 of red light,

a PDT dose resulting in 90% loss of colony-forming ability (LDl)0;

941

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from

PARP CLEAVAGE IN RESPONSE TO PC 4-PDT

Fig. 1. Kinetics of PARP cleavage as a functionof PDT dose to LY-R cells. LY-R cells wereexposed to 0.5 /¿MPc 4 and either 2.1, 3, or 10kJ/itr of red light. At the indicated times thereafter, cells were collected, subjected to SDS-PAGE.transferred, and reacted with the 4C1Ü-5antibody,

a representative immunoblot analysis of PARPcleavage. C, untreated control cells, h, quantitationof PARP cleavage. The relative intensity of eachband in experiments such as a was measured usingSigma Gel (Jandel Scientific), and the percentageof PARP cleaved was calculated as described in"Materials and Methods." Data represent the

means from at least three independent experiments, except for data from cells treated with 10U/m~, which were from a single experiment: hurs.

SE.

a

116kDa-

90 kDa -

0.5 pM Pc 4

2.1 kJ/m2 3 kJ/m2 10kJ/m2

C 10 30 60 12024048010 30 60 120 240480 10 30 60 120 min post-PDT

100 -

o>O)ro

_O£L[T

20 40 60 80 100

Minutes post-PDT

120230 240

Ref. 49), a new immunoreactive protein band migrating at A/r-90,000 was visible by l h after PDT, and the cleavage of PARP was

nearly complete by 4 h. Treatment of cells with 0.5 /MMPc 4 and 3kJ/m2 of red light, a PDT dose resulting in 99.9% loss of colony-

forming ability, revealed a faster time course of PARP cleavage, withthe cleavage fragments visible by 30 min and all PARP appearing inthe cleaved form by 1 h. After an even higher dose. 0.5 /MMPc 4 and10 kJ/m2 of red light, which results in more than a four-log loss of

colony-forming ability, PARP cleavage was essentially complete by10 min after PDT. The marked reduction in anti-PARP-reactive ma

terial in the last two lanes of Fig. la is due to underloading of thelanes, because cell destruction was extensive and protein recovery waspoor after the highly supralethal dose.

The percentage of PARP in the combined cleaved form has beenestimated by densitometry. and the data from several experiments arecombined in Fig. \b. Because the density of the bands was not linearlyrelated to the amount of protein throughout the entire range, the dataare most useful for determining the time period over which PARPcleavage occurs. On average, approximately 50% cleavage of PARPrequired 80, 30, or <10 min after PDT with 2.1, 3, or 10 kJ/nr redlight, respectively. A comparison of the time course of PARP cleavage to that of other hallmarks of apoptosis was made after a singlePDT dose (0.5 ¿AMPc 4 and 3 kJ/nr; Fig. 2).

In LY-R cells, PDT induces apoptosis that is monitored by oligo-nucleosomal DNA fragmentation, observable as DNA "ladders" upon

static-field agarose gel electrophoresis (Ref. 5; Fig. 2«).Quantifica

tion of the number of cells in apoptosis was accomplished by flowcytometry after marking cells with FITC-labeled dUTP catalyzed byTdT. To compare the kinetics of PARP cleavage (Fig. 2b). oligonu-cleosomal DNA fragmentation (Fig. 2a), and the appearance of FITC-

positive apoptotic cells (Fig. 2c), cultures were treated with PDT, andaliquots were taken at various times after treatment were analyzed forall three features. In the examples shown in Fig. 2, the earliestevidence of increases in each of the three measures was at 20 min afterPDT. By 45 min after PDT, PARP cleavage was completed, whereasonly 30% of the cells appeared to be in apoptosis, as estimated by flowcytometry, and much of the cellular DNA remained too large to enterthe gel. Although the progress of apoptosis in PDT-treated LY-R cells

is rapid, an increase in caspase activity appears to occur prior toendonuclease activity.

To investigate the role of the caspase(s) responsible for the cleavage of PARP during PDT-induced apoptosis. we first directly assayedfor caspase-3 activity by incubating cell lysates with DEVD-AMC

and measuring the release of AMC. As shown in Fig. 3, peptidecleavage activity was increased above control levels as early as 10min and reached a maximum level by 45 min after PDT. We thenexposed the cells to PDT in the presence of zVAD-fmk, a cell-permeable peptide inhibitor of ICE-like proteases, or zFA-fmk, an

inactive peptide analogue (50). These experiments were carried out in96-well plates (Fig. 4«).PARP cleavage started at —¿�30min and was

complete at 60 min. No cleavage of PARP was found at any time upto 4 h after PDT when 200 /MMzVAD-fmk was present, whereas the

inhibitor did not affect the size of PARP in the absence of PDT. Asshown in Fig. 4b, zVAD-fmk was less effective in inhibiting PARPcleavage at concentrations of 50 or 100 /MM,and zFA-fmk was

ineffective at all concentrations between 50 and 200 /MM.Following alower dose of PDT (0.5 /MMPc 4 and 2.1 kJ/nr of red light), PARPcleavage was partial by 2 h and nearly complete by 4 h; in this caseas well, 200 /MMzVAD-fmk provided complete inhibition of PARP

cleavage (data not shown).The above results indicate that lethal doses of PDT activate pro

teases capable of the specific cleavage of PARP in LY-R cells.Because zVAD-fmk inhibits the cleavage, we next asked whetherzVAD-fmk prevented PDT-induced apoptosis. The extent of apoptosis was estimated by fluorescence microscopy of acridine orange- andethidium bromide-stained cells. Representative fields are shown in

Fig. 5a, and quantification of live, apoptotic, and necrotic cells isrecorded in Fig. 5b. No apoptotic cells were found in any of thecultures that were not exposed to PDT, although in cultures treatedwith zFA-fmk alone, about 18% of the cells appeared to be necrotic

(ethidium bromide positive but lacking condensed chromatin). AfterPDT, —¿�20%of the cells were scored as necrotic, and this percentagewas unaffected by either zVAD-fmk or zFA-fmk. The majority of thePDT-treated cells (65%) were morphologically apoptotic, and thepresence of 200 JU.MzVAD-fmk, but not 200 /MMzFA-fmk, prevented

the appearance of apoptotic cells.942

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from

PARP CLEAVAGE IN RESPONSE TO PC 4-PDT

a 0.5 uM Pc 4 + 3 kJ/m2

C Pc 4 5 120 min post-PDT

0.5 jjM Pc 4 +•3 kJ/m2

C Pc 4 5 10 20 30 45 60 120 min post-PDT

116kDa-90kDa-

- Actin

R100CO

ä 80'S 60

£ 40

CL 20 JillC Pc 4 5 10 20 30 45 60 120 min post-PDT

min post-PDT

0.5 MM 3kJ/m2

Fig. 2. Kinetics of apoptotic events and PARP cleavage in PDT-treated LY-R cells.Cell.s were treated with 0.5 (¿MPc 4 and 3 kJ/m2 red light and collected at various times

a.s indicated, a, cell lysates were processed for agarose electrophoresis of DNA. b.immunoblot analysis, upper panel, PARP; middle panel, actin; lower panel, quantitationof PARP cleavage, c. TÚNEL assay and flow cytometry to determine the percentage ofapoptotic cells. Similar results were obtained in three independent experiments.

During the course of these studies, the fragmented PARP occasionally appeared as a double band migrating at Mr ~85,000 and Mr—¿�90,000(e.g., Fig. la, Lanes 5 and 6; Fig. 2b, Lane 6; Fig. 4a, Lane

3). Because PARP contains only one DEVD j G site, we considered

the possibility that the two apparent PARP fragments may differ withrespect to the extent of poly(ADP-ribosylation). The presence ofpoly(ADP-ribose) on various proteins was assessed with an anti-poly(ADP-ribose) antibody. As shown in the top panel of Fig. 6, theintact form of PARP was poly(ADP-ribosylated) in the control and in

treated samples collected up to 30 min after PDT. The antibody didnot recognize the Mr 116,000 PARP from samples taken 45 min orlater after PDT, when intact PARP was completely cleaved (Figs, laand 2b). No PARP fragments were detected with this antibody at anytime during the 2-h post-PDT period. However, a strong poly(ADP-ribosylated) protein band was observed at A/r ~ 100.000. Western

blotting indicated that topoisomerase I is one of the proteins migratingat this position (Fig. 6, middle panel). PDT does not appear to modify

either the poly(ADP-ribosylation) of the M, 100,000 protein or theamount of topoisomerase I protein until 45-120 min after PDT.

DISCUSSION

The present study provides direct evidence for the involvement ofcaspases in PDT-induced apoptosis. Three lines of evidence supportthis conclusion for apoptosis in LY-R cells:

(a) The nuclear protein PARP is cleaved in response to PDT (Fig.1-2, 4, 6). Although all of the specific proteases that are activated by

PDT have not been identified, the data of Fig. 3 suggest that one ormore caspases that cleave at the sequence of DEVD J, AMC, which isalso the cleavage site for PARP (DEVD j G) (14), are activated afterPDT. In HL-60 cells exposed to PDT sensitized by benzoporphyrinderivative, evidence was provided for the activation of caspase-3, butcaspase-1 activation was not detected (41). Caspase-3 and caspase-7

preferentially cleave PARP at that sequence into a Mr 90.000 fragment(51). Caspase-6. although cutting at the same sequence, has a muchlower activity toward PARP but readily cleaves lamin A (51). More-

4000

£•'£1000

•¿�^3Q-Ãœ O)TO E

C/5 FTO ECDlñ

CL Ee

100 r

20 40 60 80

min post-PDT100

Fig. 3. Detection of protease activity in PDT-treated LY-R cell.s. LY-R cells werecollected and lysed at the indicated times after PDT. Aliquots (50 /xg of protein) wereincubated with DEVD-AMC at 37°Cfor 30 min. The amount of fluorochrome released

was determined by comparison to an AMC standard curve in lysis buffer. Data representthe means from at least three independent experiments; hars, SE. Where the error bars arenot shown, they were smaller than the symbol. All means were significantly different fromthe control at O min (**,/>< 0.05).

a.0.5 uM Pc 4 + 3 kJ/m2

C 10

0.5 \iM Pc 4 + 3 kJ/m'

. .7 + +++ + + 200 uM zVAD-fmk60 120240240 10 30 60 120 240 min post-PDT

b.I

0.5 pM Pc 4 + 3 kJ/m2

zvAP.fmk zFA-fmk

C 200 200 0 50 100 200 50 100 200 uM

90 kDa •¿�

Fig. 4. Effects of zVAD-fmk and zFA-fmk on PDT-induced PARP cleavage in LY-Rcells. Cells were grown in 96-well plates and treated with 0.5 /AMPc 4 and 3 kJ/m2. The

protease inhibitors were added I h before light exposure and every 2 h thereafter at theindicated concentrations, a, extent of PARP cleavage in cells at various times after PDTwith or without 200 U.M zVAD-fmk. b, concentration dependence of the effects ofzVAD-fmk and zFA-fmk on PARP cleavage. Cells were collected 2 h after PDT. The celllysates were processed for immunoblot analysis with anti-PARP antibody.

943

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from

PARP CLEAVAGE IN RESPONSE TO PC 4-PDT

Control zVAD-fmk zFA-fmk

Fig. 5. Morphological evidence for the effects ofzVAD-fmk and zFA-fmk on PDT-induced apopto-sis in LY-R cells. Cells were grown in 96-well

plates and treated as described in Fig. 4. Cells werecollected 2 h after PDT, double-stained with acri-

dine orange and ethidium bromide, and viewed andphotographed in a fluorescence microscope. Theresults are shown as representative fluorescence images (a) and quantification of apoptotic cells andnecrotic cells (b). Similar data were obtained fromtwo additional experiments.

Pc 4 + 3 kJ/m2, 2 h

b 80

<DO

60

40

20

Apoptotic cells

Necrotic cells

Control zVAD-fmk zFA-fmk PDT zVAD-fmk zFA-fmk

PDT PDT

over, studies from Srinivasula et al. (34) have demonstrated thatcaspase-9 and caspase-6 are downstream proteases activated bycaspase-3. Martins et al. (52) also showed activation of multiplecaspases in etoposide-induced apoptosis in HL-60 cells. Thus, our

results are in agreement with others, suggesting that apoptosis, especially PDT-induced apoptosis, is accompanied by the activation of a

cascade of multiple caspases (17). The mechanism of activation of thecascade by PDT is presently unclear, and further studies are required.

By Western blot analysis with anti-poly(ADP-ribose), the Mr116,000 PARP appears to be poly(ADP-ribosylated) (Fig. 6). The

antibody, which recognizes all size classes of the polymer (46), didnot react with the PARP cleavage fragments (Fig. 3), arguing thatremoval of poly(ADP-ribose) from PARP precedes or accompanies

cleavage, or that only free, inactive PARP is cleaved. However,modifications of PARP that would not be detected by this antibodyand that could remain in the cleavage products include phosphoryla-tion (53, 54) and mono(ADP-ribosylation) (55, 56).

(b) PARP cleavage has a PDT dose and time dependence (Figs. 1,2, and 4) that is approximately the same as that for the appearance ofother markers of apoptosis, i.e., DNA fragmentation and TdT responsiveness, as measured by flow cytometric identification of cells thathave incorporated fluorescently labeled nucleotides (Fig. 2). Based on

the induction of apoptosis that follows the overexpression of proteasesin cultured cells and the demonstration that exogenous proteases canproduce morphological apoptosis in isolated nuclei, protease action

has been considered a precursor to endonuclease action and chromatincondensation (16). After treatment of a series of tumor cell lines withthe chemotherapeutic agent topotecan, an excellent correlation wasobserved between the extent of PARP cleavage and the appearance ofapoptotic cells, as detected by staining with acridine orange (57). Inthe case of PDT-treated LY-R cells, the very rapid induction and

progress of apoptosis has precluded a clear demonstration of temporalorder of the measured events. In fact, in each experiment wherecomparison of the kinetics was sought, the initiation of PARP cleavage and of DNA fragmentation was nearly simultaneous, althoughPARP cleavage seemed to be completed earlier than the entry of thefinal cells into apoptosis, when measured by flow cytometry. However, activation of the cascade of proteases that are ultimately responsible for cleavage of PARP and other proteins must have preceded theappearance of PARP fragments. As estimated by the cleavage of thefluorogenic peptide substrate DEVD-AMC (Fig. 3), caspase-3-like

activity began to increase almost immediately after PDT and was2-3-fold elevated in 10 min and 100-fold elevated in 30 min. Because

944

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from

PARP CLEAVAGE IN RESPONSE TO PC 4-PDT

0.5 MM Pc 4 + 3 kJ/m2

C 5 10 20 30 45 60 120 Pc 4 min post-PDT

Fig. 6. Western blotting of poly(ADP-ribosy-lated) proteins in PDT-treated LY-R cells. Toppanel PDT-treated cell lysates were subjected toimmunoblolting against poly(ADP-ribose) antibody; middle panel, observation of topoi some rase Ias one of the poly( ADP-ribose(-modified proteins;bottom panel, actin control. The appearance ofmultiple bands in the middle panel results fromoverexposure of the blots to compensate for thepoor reactivity of anti-human topoisomerase I with

the mouse enzyme.

97kDa66kDa

PARP100kDa

97kDa Topo I

_ - «—Actin

it is not known what level of activity increase is needed to initiate thenext steps in apoptosis, the small early elevation may be sufficient.

It is interesting that very rapid and complete PARP cleavage wasobserved for the highest PDT dose tested (Fig. 1), a dose that killsLY-R cells without producing oligonucleosomal DNA fragmentation

(58). Perhaps the excess oxidative damage produced by a markedlysupralethal PDT dose allows apoptosis to be initiated but interfereswith the late steps of the process. A similar proposal has been madeby Dellinger (59), based on studies of the morphology of cellsundergoing apoptosis in response to PDT sensitized by Photofrin.However, Luo and Kessel (42) found that PARP cleavage and oligonucleosomal DNA fragmentation were blocked in P388 cells treatedwith a high dose of PDT sensitized by aluminum phthalocyanine.

(c) An inhibitor of ICE-like cysteine proteases, zVAD-fmk, blocks

both PARP cleavage and the appearance of morphologically apoptoticcells, whereas an inactive peptide analogue, zFA-fmk. blocked neitherevent (Figs. 4 and 5). zVAD-fmk has broad specificity for cysteineproteases (50), and its ability to block PDT-induced apoptosis is

strong evidence that one or more of such proteases are critical forapoptosis after PDT. However, whether the cleavage of PARP per seis important cannot be discerned from these results. PARP is one of aset of nuclear proteins that has been demonstrated to be proteolyticallycleaved during apoptosis. Which, if any, of these proteins is criticalfor initiating the later stages of apoptosis is not known.

The demonstration that proteases are important in PDT-induced

apoptosis has implications for the treatment of tumors by this modality. PDT produces rapid tumor ablation in experimental animals, withcomplete loss of visible tumor often occurring within 1-2 days (4).

Apoptosis is frequently observed at early times during tumor ablation(60-62). If protease action and/or the cleavage of PARP is important

in tumor response, PARP cleavage may be a useful marker of tumorcell death and overall response of PDT-treated tumors.

REFERENCES

1. Weishaupt, K. R.. Corner, C. J., and Dougherty, T. J. Identification of singlet oxygenas the cytotoxic agent in photoinactivation of a murine tumor. Cancer Res., 36:2326-2329. 1976.

2. Agarwal. R., Athar, M., Urban, S. A., Bickers, D. R.. and Mukhtar, H. Involvementof singlet oxygen in chloroaluminum phthalocyanine tetrasulfonate-mediated pho-

toenhancement of lipid peroxidation in rat epidermal microsomes. Cancer Lett., 56:125-129, 1991.

3. Moan. J., and Berg. K. Photochemotherapy of cancer: experimental research. Photo-chem. Photobiol.. 55: 931-948. 1992.

4. Henderson. B. W.. and Dougherty, T. J. How does photodynamic therapy work?Photochem. Pholobiol., 55: 145-157, 1992.

5. Agarwal, M. L.. Clay. M. E.. Harvey, E. J., Evans. H. H., Antunez. A. R.. andOleinick, N. L. Photodynamic therapy induces rapid cell death by apoptosis inL5178Y mouse lymphoma cells. Cancer Res., 5/: 5993-5996, 1991.

6. He. X. Y., Sikes, R. A., Thomsen. S.. Chung. L. W. K.. and Jacques, S. L.Photodynamic therapy with Photofrin 11induces programmed cell death in carcinomacell lines. Photochem. Photobiol., 59: 468-473, 1994.

7. Laukka. M. A.. Wang. K. K.. and Bonner, J. A. Apoptosis occurs in lymphoma cellsbut not in hepatoma cells following ionizing radiation and photodynamic therapy.Dig. Dis. Sci., 39: 2467-2475. 1994.

8. Luo. Y., Chang. C. K., and Kessel. D. Rapid initiation of apoptosis by photodynamictherapy. Photochem. Photobiol.. 63: 528-534. 1996.

9. Agarwal, M. L., Larkin, H. E., Zaidi, S. I. A.. Mukhtar, H.. and Oleinick, N. L.Phospholipase activation triggers apoptosis in photosensiti/ed mouse lymphomacells. Cancer Res.. S3: 5897-5902. 1993.

10. Luo. Y., and Kessel, D. The phosphatase inhibitor calyculin aniagoni/es the rapidinitiation of apoptosis by photodynamic therapy. Biochem. Biophys. Res. Commun.,221: 72-76. 1996.

11. Kerr, J. F. R.. Wyllie. A. H.. and Currie. A. R. Apoptosis: a basic biologicalphenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer, 26:239-257, 1972.

12. Wyllie. A. H.. Kerr, J. F. R., and Currie. A. R. Cell death: the significance ofapoptosis. Int. Rev. Cytol.. 6Ä:251-306, 1980.

13. Ellis. R. E.. Yuang. J. Y.. and Horvit/. H. R. Mechanisms and functions of cell death.Ann. Rev. Cell Biol., 7: 663-698. 1991.

14. Lazebnik, Y. A.. Kaufmann. S. H., Desnoyers. S.. Poirier. G. G., and Earn.shaw, W. C.Cleavage of poly(ADP-ribose) polymerase by a proleinase with properties like ICE.Nature (Lond.). 371: 346-347, 1994.

15. Stewart. B. W. Mechanisms of apoptosis: integration of genetic, biochemical, andcellular indicators. J. Nail. Cancer Inst.. 86: 1286-1296, 1994.

16. Hale A. J.. Smith. C. A.. Sutherland. L. C., Stoneman. V. E. A., Longthome, V. L..Culhane. A. C.. and Williams. G. T. Apoptosis: molecular regulation of cell death.Eur. J. Biochem.. 236: 1-26. 1996.

17. Cohen. G. M.. Caspases: the executioners of apoptosis. Biochem. J.. 326: 1-16, 1997.

18. Shi. L.. Kam, C. M., Powers. J. C.. Aebersold. R.. and Greenberg. A. Purification ofthree cytotoxic lymphocyte granule serine proteases that induce apoplosis throughdistinct subslrate and target cell interactions. J. Exp. Med., 176: 1521-1529, 1992.

19. Yuan. J.. Shaham. S.. Ledoux. S.. Ellis, H. M.. and Horviiz. H. R. The C. elegans celldeath gene ced-3 encodes a protein similar to mammalian interlcukin-lß-convertingenzyme. Cell. 75: 641-652. 1993.

20. Nicholson. D. W., Ali, A.. Thomberry. N. A.. Vaillancourt, J. P., Ding. C. K.. Gallant,M.. Gareau. Y.. Griffin. P. R.. Labelle, M.. Lazebnik, Y. A.. Munday, N. A.. Raju,S. M.. Smulson. M. E.. Yamin. T-T.. Yu. V. L.. and Miller. D. K. Identification andinhibition of the 1CE/CED-3 protease necessary for mammalian apoptosis. Nature(Lond.), 376: 37-43. 1995.

21. Alnemri, E. S., Livingston. D. J.. Nicholson. D. W., Salvesen. G.. Thornberry, N. A.,Wong, W. W.. and Yuan. J. Human ICE/CED3 protease nomenclature. Cell. «7:171,1996.

22. Kumar, S., Kinoshita. M.. Noda. M.. Copeland. N. G.. and Jenkins, N. A. Inductionof apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the productof the Caenorhabditis eleguns cell death gene ced-3 and the mammalian IL-lß-converting en/yme. Genes Dev., A: 1613-1626, 1994.

23. Wang. L., Miura. M., Bergeron, L.. Zhu. H.. and Yuan, J. Ich-1. an lCE/ceil-3 related

gene, encodes both positive and negative regulators of programmed cell death. Cell,78: 739-750. 1994.

24. Fernandes-Alnemri, T.. Litwack, G., and Alnemri, E. S. CPP32, a novel humanapoptotic protein with homology to Caenorhabdilis elegans cell death protein ced-3and mammalian interleukin-lß-converting enzyme. J. Biol. Chem.. 269: 30761-

30764, 1994.

945

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from

PARP CLEAVAGE IN RESPONSE TO PC 4-PDT

25. Tewari. M., Quan, L. T., O'Rourke, K., Desnoyers. S., Zeng. Z., Beidler, D. R.,

Poirier, G. G., Salvesen. G. S., and Dixit, V. M. Yama/CPP32/3, a mammalianhomolog of CED-3. is a Crm-A-inhibitable protease that cleaves the death substratepoly(ADP-ribose) polymerase. Cell, 81: 801-809. 1995.

26. Enari, M.. Hug, H., and Nagata, S. Involvement of an ICE-like protease in Fas-mediated apoplosis. Nature (Lond.). 375: 78-81. 1995.

27. Kamens. J., Paskind, M.. Hugunin. M.. Talanian. R. V.. Allen. H.. Banach. D.. Bump.N., Hackett. M., Johnston. C. G.. Li, P., Mankovich, J. A.. Terranova. M., andGhayur. T. Identification and characterization of ICH-2. a novel member of theinlerleukin-1-converting enzyme family of cysteine proteases. J. Biol. Chem., 270:15250-15256, 1995.

28. Fernandes-Alnemri, T., Litwack. G.. and Alnemri. E. S. Mch2. a new member of theapoptotic Ced-3/Ice cysteine protease gene family. Cancer Res., 55: 2737-2742,

1995.29. Fernandes-Alnemri. T.. Takahashi. A.. Armstrong, R.. Krebs, J., Fritz, L., Tornaseli!,

K. J.. Wang, L., Yu, Z., Croce, C. M., Salvesen, G., Eamshaw, E. C., Litwack, G., andAlnemri. E. S. Mch3, a novel human apoptotic cysteine protease highly related toCPP32. Cancer Res., 55: 6045-6052, 1995.

30. Duan, H., Chinnaiyan. A. M., Hudson, P. L., Wing. J. P.. He. W. W.. and Dixit. V. M.ICE-LAP3, a novel mammalian homologue of the Caenurhiibditis elegans cell deathprotein Ced-3, is activated during Fas- and tumor necrosis factor-induced apoptosis.J. Biol. Chem.. 271: 1621-1625, 1996.

31. Fernandes-Alnemri, T., Armstrong, R. C.. Krebs. J.. Srinivasula. S. M.. Wang. L..Bullrich. F.. Fritz, L. C., Trapani. J. A., Tornaseli!. K. J.. Litwack, G., and Alnemri.E. S. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptoticcysteine protease containing two FADD-like domains. Proc. Nati. Acad. Sci. USA,9.?.-7464-7469, 1996.

32. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke. K., Shevchenko, A., Ni,

J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M.. Krammer, P. H., Peter.M. E., and Dixit, V. M. FLICE, a novel FADD-homologous ICE/CED-3 like protease,is recruited to the CD95 (Fas/APO-1) death-inducing signalling complex. Cell, 85:817-827, 1996.

33. Duan, H., Orth, K., Chinnaiyan A. M.. Poirier, G. G., Froelich. C. J., He, W. W., andDixit. V. M. 1CE-LAP6. a novel member of the lCE-Ced-3 gene family, is activatedby the cytotoxic T cell protease granzyme B. J. Biol. Chem.. 271: 16720-16724,

1996.34. Srinivasula, S. M.. Femandes-Alnemri. T.. Zangrill. J., Robertson. N., Armstrong,

R. C.. Wang. L.. Trapani. J. A.. Tornaseli!. K. J.. Litwack. G., and Alnemri, E. S. Theced-3/interleukin 1/3 convening enzyme-like homolog Mch6 and the lamin cleavingenzyme Mch2a are substrates for the apoptotic mediator CPP32. J. Biol. Chem., 271:27099-27106. 1996.

35. Neamati. N.. Fernandez, A., Wright. S.. Kiefer, J., and McConkey, D. J. Degradationof lamin B, precedes oligonucleosomal DNA fragmentation in apoptotic thymocytesand isolated thymocyte nuclei. J. Immunol., 154: 3788-3795, 1995.

36. Lazebnik, Y. A., Takahashi, A., Moir. R. D., Goldman. R. D., Poirier, G. G.,Kaufmann. S. H., and Earnshaw. W. C. Studies of the lamin protease reveal multipleparallel biochemical pathways during apoptolic execution. Proc. Nati. Acad. Sci.USA, 92: 9042-9046. 1995.

37. Kaufmann, S. H. Induction of endonucleolytic DNA cleavage in human acute my-

elogenous leukemia cells by etoposide, camptothecin. and other cytotoxic anticancerdrugs. Cancer Res., W: 5870-5878. 1989.

38. Voelkek-Johnson. C.. Entingh. A. J.. Wold. W. S. M.. Gooding, L. R., and Laster,S. M. Activation of intracellular proteases is an early event in TNF-induced apoptosis.J. Immunol.. 154: 1707-1716. 1995.

39. Kaufmann. S. H.. Desnoyers. S.. Ottaviano, Y.. Davidson, N. E.. and Poirier. G. G.Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker ofchemotherapy-induced apoptosis. Cancer Res., 53: 3976-3985. 1993.

40. He. J.. Agarwal, M. L., Larkin, H. E.. Friedman, L. R., Xue, L. Y., and Oleinick, N. L.The induction of partial resistance to photodynamic therapy by the protooncogeneBCL-2. Photochem. Photobiol.. 64: 845-852, 1996.

41. Granville. D. J.. Levy. J. G.. and Hunt. D. W. C. Photodynamic therapy inducescaspase-3 activation in HL-60 cells. Cell Death Differ.. 4: 623-628. 1997.

42. Luo, L.. and Kessel, D. Initiation of apoptosis versus necrosis by photodynamictherapy with chloroaluminum phthalocyanine. Photochem. Photobiol.. 66: 479-483.

1997.43. Oleinick. N. L., Antunez, A. R., Clay. M. E., Rihter. B. D.. and Kenney. M. E. New

phthalocyanine photosensitizers for photodynamic therapy. Photochem. Photobiol..57: 242-247, 1993.

44. Ramakrishnan. N.. Oleinick, N. L., Clay, M. E., Homg, M. F.. Antunez, A. R., andEvans, H. H. DNA lesions and DNA degradation in mouse lymphoma L5178Y cellsafter photodynamic treatment sensitized by chloroaluminum phthalocyanine. Photochem. Photobiol., 50: 373-378. 1989.

45. Ranjit. G. B., Cheng, M-F.. Mackay. W.. Whitacre, C. M.. Berger, J. S., and Berger,

N. A. PolyUdenosine diphosphoribose) polymerase in peripheral blood leukocytesfrom normal donors and patients with malignancies. Clin. Cancer Res., /: 223-234,

1995.46. Kawamitsu. H.. Hoshino, H.. Okada. H.. Miwa. M.. Momoi. H.. and Sugimura. T.

Monoclonal antibodies to polyladenosine diphosphate ribose) recognize differentstructures. Biochemistry, 23: 3771-3777, 1984.

47. Bradford. M. M. A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. Anal. Biochem..72: 248-254. 1976.

48. Li. X., Tráganos, F., Melamed, M. R., and Darzynkiewicz, Z. Single step procedurefor labeling DNA strand breaks with fluorescein- or BODlPY-conjugated de-oxynucleotides: detection of apoptosis and bromodeoxyuridine incorporation. Cytom-etry, 20: 172-180. 1995.

49. He. J.. Larkin. H. E.. Li. Y. S.. Rihter, B. D.. Zaidi. S. I. A.. Rodgers. M. A. J..Mukhlar. H.. Kenney. M. E.. and Oleinick, N. L. The synthesis, photophysical andphotobiological properties, and in vitro structure-activity relationships of a set ofsilicon phthalocyanine PDT photosensitizers. Photochem. Photobiol., 65: 581-586,

1997.50. Jacobson, M. D., Weil. M.. and Raff, M. C. Role of Ced-3/ICE-family proteases in

staurosporine-induced programmed cell death. J. Cell Biol.. 133: 1041-1051,1996.51. Takahashi. A.. Alnemri. E. S.. Lazebnik. Y. A.. Fernandes-Alnemri, T. Litwack. G..

Moir. R. D.. Poirier, G. G., Kaufmann, S. H., and Earnshaw. W. C. Cleavage of laminA by Mch2a but not CPP32: multiple ¡nterleukin Ißconverting enzyme-related

proteases with distinct substrate recognition properties are active in apoptosis. Proc.Nati. Acad. Sci. USA, 93: 8395-8400. 1996.

52. Martins. L. M.. Kottke, T.. Mesner. P. W., Basi, G. S.. Sinha, S., Frigon, N., Jr., Tatar,E., Tung, J. S., Bryant, K.. Takahashi. A., Svingen, P. A., Madden. B. J.. McCormick.D. J., Earnshaw, W. C., and Kaufmann, S. H. Activation of multiple interleukin-lßconverting enzyme homologues in cytosol and nuclei of HL-60 cells during etopo-side-induced apoptosis. J. Biol. Chem.. 272: 7421-7430, 1997.

53. Bauer. P. I.. Farkas. G.. Mihalik. R., Kopper, L.. Kun. E., and Farago. A. Phospho-rylation of poly(ADP-ribose) polymerase protein in human peripheral lymphocytesstimulated with phytohemagglutinin. Biochim. Biophys. Acta. /22.ÃŽ:234-239, 1994.

54. Bauer. P. 1., Farkas. G.. Buday. L., Mikala, G., Meszaros, G., Kun, E., and Farago, A.Inhibition of DNA binding by the phosphorylation of poly(ADP-ribose) polymerase

protein catalysed by protein kinase C. Biochem. Biophys. Res. Commun.. 187:730-736, 1992.

55. Alvarez-Gonzalez, R., and Mendoza-Alvarez. H. Dissection of ADP-ribose polymer

synthesis into individual steps of initiation, elongation, and branching. Biochimie, 77:403-407, 1995.

56. Bauer. P. I.. Hakam, A., and Kun. E. Mechanisms of poly(ADP-ribose) polymerasecatalysis: mono-ADP-ribosylation of poly(ADP-ribose) polymerase at nanomolarconcentrations of NAD. FEES Lett., 195: 331-338, 1986.

57. Whitacre. C. M.. and Berger. N. A. Factors affecting topolecan-induced programmedcell death: adhesion protects cells from apoptosis and impairs cleavage of polyl ADP-ribose Ipolymerase. Cancer Res.. 57: 2157-2163. 1997.

58. He, J., and. Oleinick, N. L. Cell death mechanisms vary with photodynamic therapydose and photosensitizer. Proc. SPIE Int. Soc. Opt. Eng.. 2371: 92-96, 1995.

59. Dellinger. M. Apoptosis or necrosis following Photofrin® photo-sensitization: influence of the incubation protocol. Photochem. Photobiol., 64: 182-187, 1996.

60. Zaidi, S. I. A., Oleinick, N. L., Zaim, M. T.. and Mukhtar, H. Apoptosis duringphotodynamic therapy-induced ablation of RIF-I tumors in C3H mice: electron

microscopic, histopathologic. and biochemical evidence. Photochem. Photobiol., 58:771-776. 1993.

61. Agarwal. R.. Korman, N. J.. Mohan. R. R., Feyes, D. K., Jawed, S., Zaim, M. T., andMukhtar. H. Apoptosis is an early event during phthalocyanine photodynamic therapy-induced ablation of chemically induced squamous papillomas in mouse skin.Photochem. Pholobiol., 63: 547-552, 1996.

62. Zhou. G.. Shunji. C.. Jinsheng. D.. Junlin. L.. Jori. G., and Milanesi. C. Apoptosis ofmouse MS-2 fibrosarcoma cells induced by photodynamic therapy with Zn(II)-phthalocyanine. J. Photochem. Photobiol. B Biol., 33: 219-223, 1996.

946

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from

1998;58:940-946. Cancer Res   Jin He, Cecilia M. Whitacre, Liang-yan Xue, et al.   Photodynamic TreatmentPolymerase: An Integral Part of Apoptosis in Response to Protease Activation and Cleavage of Poly(ADP-ribose)

  Updated version

  http://cancerres.aacrjournals.org/content/58/5/940

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/58/5/940To request permission to re-use all or part of this article, use this link

Research. on January 31, 2021. © 1998 American Association for Cancercancerres.aacrjournals.org Downloaded from