glucose regulated protein induction and cellular ... · glucose regulated protein induction and...

[CANCER RESEARCH 51. 6574-6579, December 15. 199I|

Glucose Regulated Protein Induction and Cellular Resistance to Oxidative StressMediated by Porphyrin Photosensitization1

Charles J. Corner,2 Angela Ferrano, Natalie Rucker, Sam Wong, and Amy S. Lee

Clayton Ocular Oncology Center, Childrens Hospital of Los Angeles [C. J. G., A. F. , N. R., S. W.]; Kenneth Norris Jr. Comprehensive Cancer Center [C. J. G., A. S. LJ,and Departments of Pediatrics [C. J. G.], Radiation Oncology [C. J. G.], Molecular Pharmacology and Toxicology Â¡C.J. G.]; and Biochemistry [A. S. L.], University ofSouthern California School of Medicine, Los Angeles, California 90027

ABSTRACT

Photodynamic therapy (PDT) utilizes a tumor localizing porphyrinphotosensitizer in the clinical treatment of cancer. At a mechanistic level,porphyrin photosensitization generates reactive oxygen species whichinitiate oxidative damage to a wide spectrum of biomolecules. Cellularstress proteins are also increased following oxidative stress treatments.In the current study, we examined porphyrin photosensitizing parametersassociated with induction of the glucose regulated family of stress proteins. Elevated levels of mRNA encoding glucose regulated proteins(GRPs) as well as increases in GRP protein synthesis were observed formouse radiation induced fibrosarcoma cells exposed to an extended (16-h) porphyrin incubation prior to light exposure. However, a short (1-h)porphyrin incubation prior to light treatment (designed to produce comparable phototoxicity as PDT using the 16-h porphyrin incubation protocol) was associated with only minimal increases in GRP mRNA levelsor GRP protein synthesis. The relationship between GRP levels andPDT sensitivity was examined in radiation induced fibrosarcoma cellspretreated with the calcium ionophore A-23187 in order to overexpressGRPs prior to photosensitization. Resistance to PDT was observed incells overexpressing GRPs only under photosensitizing conditions associated with the extended porphyrin incubation protocol, and this responsewas not due to changes in cellular porphyrin uptake. In separate experiments, a transient elevation of GRP mRNA levels was observed intransplanted mouse mammary carcinomas following in vivo PDT treatments. Our results indicate that specific targets of oxidative damage(modulated by porphyrin incubation conditions) instead of generalizedcellular exposure to reactive oxygen species are correlated with PDTmediated GRP induction. In this regard, GRP induction may be a usefulin vivo biochemical marker of PDT mediated injury. These results alsosupport the hypothesis that GRPs may play a role in modulating sensitivity to cellular stresses including certain types of oxidative injury.

INTRODUCTION

PDT1 is the treatment of solid tumors with visible light

following the systemic administration of the photosensitizerPhotofrin II (1, 2). Photofrin II accumulates in malignant tissueand the photosensitizing action of this hematoporphyrin derivative generates reactive oxygen species such as singlet oxygen(3). Lasers produce the light used in clinical PDT and fiberopticcables deliver the light to the various treatment sites (1, 2).Currently, five Phase III clinical trials are evaluating the efficacyof Photofrin II mediated PDT for tumors of the bronchus,esophagus, and bladder (3). PDT induces oxidative damage to

Received 2/22/91; accepted 10/7/91.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was performed in conjunction with the Clayton Foundation forResearch and was supported in part by USPHS Grants R37-CA-31230 to C. J.G. and R37-CA-27607 to A. S. L. from the National Cancer Institute.

2To whom requests for reprints should be addressed, at Clayton OcularOncology Center. Mail Stop 67, Childrens Hospital of Los Angeles, 4650 SunsetBoulevard. Los Angeles, CA 90027.

3The abbreviations used are: PDT, photodynamic therapy; GRP, glucoseregulated protein; HSP, heat shock protein; RIF. radiation induced fibrosarcoma;FCS. fetal calf serum; TCA, trichloroacetic acid; SDS. sodium dodecyl sulfate;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; cDNA,complementary DNA.

numerous lipids, proteins, and nucleic acids (2, 3). Subcellulartargets associated with porphyrin photosensitization includethe plasma membrane, cytoplasmic organdÃ-es, enzymes, andDNA (4). In vivo injury in tumor tissue involves both directtumor cell killing and vascular disruption (5).

Oxidative stress associated with PDT induces the synthesisof stress proteins of both the HSP and GRP families (2, 3, 6).Heat shock proteins are grouped into molecular weight familiesof 20,000-30,000, 70,000-73,000, 90,000, and 104,000-110,000 (7). Circumstantial evidence suggests that increasedexpression of heat shock proteins correlates with the development of thermotolerance (7-11). HSPs therefore play an integral role in the development of cellular resistance to heat inaddition to their essential functions in cytosolic translocationof proteins and transient binding to nascent proteins and poly-peptides (12, 13). Cellular sensitivity to PDT is not altered inheat resistant cell lines that constitutively overexpress HSP-70when compared to parental control cells (14).

Glucose regulated proteins with molecular weights of 78,000and 94,000 share sequence homology with HSPs but are neitherheat inducible nor associated with thermotolerance (7, 15). TheGRP family is coordinately induced under conditions of glucosestarvation, anoxia, alterations in intracellular calcium, and exposure to inhibitors of glycosylation (7, 15-18). The M, 78,000GRP is identical in sequence to the immunoglobulin heavy-chain binding protein, and both GRP-78 and GRP-94 arelocalized in the endoplasmic reticulum (19). GRP-78 bindstransiently to nascent, secretory, and transmembrane proteinsand binds permanently to abnormally folded or processed proteins which accumulate in the endoplasmic reticulum (13, 17,20). GRP-78 may therefore have a protective function duringand after cellular stress when protein processing in the endoplasmic reticulum is perturbed. Conditions which lead to theinduction of GRPs (such as glucose starvation or incubationwith A23187 or 2-deoxyglucose) are associated with cellularresistance to doxorubicin, etoposide, tumor necrosis factor, andimmunological attack by cytotoxic T-cells (21-23).

The current study was performed to define in vitro porphyrinphotosensitization parameters associated with increased levelsof GRPs. Additional experiments were performed to examinewhether exposure to A23187 (which induces the overexpressionof GRPs) could modify the sensitivity of cells to PDT and todetermine whether GRP induction could be observed followingin vivo PDT.

MATERIALS AND METHODS

Drugs. Photofrin II (the derivative of hematoporphyrin currentlyused in clinical PDT) was provided by Quadra Logics Technology, Inc.,Vancouver, British Columbia, Canada. The drug was supplied as asterile aqueous solution at a concentration of 2.5 mg/ml. The calciumionophore A23187 was obtained from Calbiochem, La Jolla, CA, anda stock solution was made up in ethanol at a concentration of 1 mg/ml.

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PORPHYRIN PHOTOSENSmZATlON AND STRESS PROTEIN INDUCTION

Cells and Cell Culture Conditions. Mouse radiation induced fibrosarcoma cells (RIF-1) in passages 4-7 were used in all experiments (14).The cells were grown in RPMI 1640 supplemented with 15% PCS andantibiotics.

Photosensitization Treatment Conditions. Appropriate numbers ofRIF-1 cells were plated into 60-mm plastic Petri dishes (for clonogenicsurvival determinations) or into T-75 plastic flasks (for protein ormRNA assays) and incubated at 37Â°Cfor 24 h to allow for attachment.

The growth medium was removed and the cells were then incubated inthe dark with Photofrin II (25 Mg/ml) for 16 h (extended incubation)in medium supplemented with 5% PCS or for l h (short incubation) inmedium supplemented with 1% PCS (24). Following porphyrin incubation cells were either immediately rinsed with fresh growth mediumwithout serum (after the 1-h incubation) or were rinsed for 30 min infresh growth medium supplemented with 10% PCS (after the 16-hincubation). Medium was then removed and the dishes or flasks wereexposed to visible red light at a fluence rate of 0.35 mW/cm2 generatedby a parallel series of 30-W fluorescent bulbs filtered with clear Plexiglass and a ruby Mylar film (24). The emission spectrum of the lightsource ranged from 590 to 650 nm as measured with a scanningmonochromator (American ISA, Metuchen, NJ). After light exposure,cells were refed with complete growth medium and returned to a 37Â°C

incubator. Cell survival was determined by a standard clonogenic assayand three dishes were evaluated at each dose point in experiments thatwere repeated at least twice (24). Treatments with A23187 were performed by incubating RIF-1 cells with 7 ^M A23187 in complete growthmedium for 16 h. The 16-h A23187 incubation was performed eitherimmediately before the l -h porphyrin incubation protocol or concurrentwith the 16-h porphyrin incubation protocol.

Cellular Porphyrin Uptake. Intracellular porphyrin concentrationswere determined by absorption spectroscopy (25). Photofrin II wasextracted from cells by sonication in 0.1 N NaOH. Porphyrin levelswere then determined from a calibration curve using absorption ratiomeasurements at 390 and 470 nm. Protein concentrations were determined using the Bio-Rad protein assay and cellular Photofrin II wasthen calculated on the basis of ^g porphyrin/mg protein.

mRNA Analysis. Total RNA was extracted from exponentially growing cultures of RIF-1 cells or from in vivo mouse mammary tumorsusing the guanidine isothiocyanate method (26). The RNA was eitherblotted directly onto a nylon filter under vacuum using a slot blotapparatus or size separated by denaturing electrophoresis on a 1%agarose gel containing 6.6% formaldehyde and then transferred to anylon filter. The p3C5 hamster cDNA encoding GRP-78 (27) and theaT human cDNA encoding a-tubulin (14) were labeled with 32P viarandom-primed DNA labeling (28). The filters were prehybridized for3 h at 42Â°Cin hybridization solution (10% dextran sulfate-1 M NaCl-

1% sodium dodecyl sulfate-50% formamide-100 ^g/ml salmon spermDNA). The filters were then incubated overnight at 42"C in hybridization solution containing the "P-labeled p3C5 probe. After hybridization, the filters were washed first at room temperature and then at 42Â°C

in a solution containing 0.1 x standard saline citrate (0.15 M NaCI,0.015 M Na3 citrate) and 1% SDS. The filters were then exposed toKodak XAR-5 X-ray film. Radioactivity was removed from the filtersby rinsing in water at 95Â°Cand the filters were rehybridized under

identical conditions using the a-tubulin probe. The slot blot autoradi-ograms were quantified by densitometry to obtain relative levels ofGRP-78 mRNA as a function of time after PDT.

Analysis of Stress Protein Synthesis. Cells were labeled for l h with30 Ã-iCi/ml/raÂ«Ã--['5S]methioninein methionine free RPMI 1640 at

selected time intervals after PDT treatments described above. Cellswere dissolved in SDS loading buffer and lysates containing equalamounts of radioactivity were size separated by electrophoresis using a10% polyacrylamide gel (29). Gels were dried and exposed to KodakXAR-5 X-ray film and the resulting autoradiograms were quantified bydensitometery (30).

Protein Glycosylation Measurement. Uptake of ['HJfucose into TCA

precipitable protein was determined in control and PDT treated RIF-1cells as described previously (17). Cells were pulse labeled for l h withRPMI 1640 containing ['Hjfucose (4 Â¿iCi/ml)starting either 0, 2, 4, or

6 h after treatment. Cells were rinsed twice with phosphate bufferedsaline and then placed in a lysing buffer (0.1 M Tris-HCI-10% glycerol-2% SDS). Total radioactivity was determined by counting a sample ofthe cell lysate. TCA precipitable counts were obtained by adding TCAat a final concentration of 10% to equal volumes of the cell lysate. Theprecipitate was collected on filters, dried, and then counted.

In Vivo PDT. Female C3H/HeJ mice (8-12 weeks old) were transplanted s.c. with a BA mammary carcinoma to the hind flank (31).Treatment of the tumors with PDT began when the lesions were 7-9mm in diameter. Mice received an i.p. injection of Photofrin II at adose of 5 mg/kg 24 h prior to light exposure. An argon pumped dyelaser tuned to 630 nm was used as the in vivo PDT light source and apower density of 75 mW/cm2 was used in order to eliminate thermalinteractions. A 200-J/cm2 light dose was delivered to all mice. This

PDT dose produces an initial cytotoxic response in all tumors andresults in approximately 50% long term cures (31). Animals weresacrificed at various time intervals following PDT treatment and RNAwas extracted from the tumors as described above. Levels of GRP-78and rt-tubulin mRNA were then determined by slot blot and densito-metric analysis.

RESULTS

GRP Induction by PDT Is Affected by Porphyrin IncubationConditions. Fig. 1 shows the levels of GRP-78 and Â«-tubulinmRNA in RIF-1 cells as a function of time following PhotofrinII mediated photosensitization using either the short (1-h) orextended (16-h) porphyrin incubation procedures, respectively.The filters were first hybridized to 12P-labeled probes for GRP-

78 and then to Â«-tubulin in order to evaluate RNA loadingprofiles. Comparable levels of PDT mediated cytotoxicity (corresponding to 20-40% survival levels) were elevated for boththe short and extended porphyrin incubation protocols. Controlcells, Photofrin II alone, and light alone conditions were notassociated with increased expression of GRP-78. In addition,photosensitization in which the short (1-h) porphyrin incubation was used (Fig. \A) did not result in altered GRP-78 mRNAlevels when compared to control conditions. However, photosensitization in which the extended (16-h) porphyrin incubationwas used (Fig. \B) produced significant increases in the levelsof GRP-78 mRNA. Maximum increases in GRP-78 mRNAwere observed 10-12 h following PDT. Similar kinetics andlevels of GRP-94 mRNA, evaluated with the p4A3 cDNA (16),were also observed following PDT treatments (data not shown).

Fig. 2 shows a SDS-PAGE analysis of the major stressproteins induced by heat, A23187, or PDT using an extended(16-h) porphyrin incubation. Treatment with A23187 inducedthe Mr 78,000 and 94,000 GRPs. Heat exposure induced M,70,000 and 90,000 HSPs. Increased temporal synthesis of bothHSPs and GRPs were observed following PDT using the extended (16-h) porphyrin incubation. The M, 32,000 proteininduced by PDT has recently been identified by Western im-munoblot and Northern analysis to be the microsomal enzymeheme oxygenase (30).

The kinetics of GRP synthesis following Photofrin II mediated PDT is shown in Fig. 3. Densitometric ratios of the A/r78,000 and 94,000 GRPs versus actin were obtained by scanningautoradiographs resulting from SDS-PAGE of fraÂ«i-[-"S]me-

thionine labeled cell lysates. Ratios are plotted as a function oftime following PDT treatment. Increased synthesis of GRPswas observed for RIF-1 cells subjected to the extended (16-h)porphyrin incubation prior to light exposure as shown inFig. 3. However, only minimal increases in GRP synthesiswere seen when cells received a 1-h porphyrin incubationprior to light exposure which produced comparable levels ofcytotoxicity.

6575



PORPHYRIN PHOTOSENSmZATION AND STRESS PROTEIN INDUCTION

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O .2" X OU _l Q O.

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alpha TUBULIN

tGRP-78B

GRP Induction Is Correlated with Decreased Cell Sensitivityto PDT. Survival curves for RIF-1 cells treated with PhotofrinII mediated PDT using the short (1-h) or extended (16-h)porphyrin incubation protocol are shown in Fig. 4. In additionto PDT treatment alone, cells were also subjected to pretreatment incubation with A23187 in order to increase intracellularlevels of GRPs at the time of PDT. The A23187 incubationproduced a 6-8-fold increase in GRP protein synthesis whenanalyzed by SDS-PAGE and densitometry. Fig. 4A shows thatexposure to A23187 prior to PDT (using the short porphyrinincubation protocol) had no effect on the sensitivity of cells toPDT. However, Fig. 4B shows that preincubation with A23187prior to PDT using the extended (16-h) porphyrin incubationresulted in a 50- to 100-fold decrease in cellular sensitivity atthe highest light treatment doses when compared to cells treatedwith either PDT alone or PDT plus the ethanol solvent used todissolve the A23187. Intracellular levels of Photofrin II forcells incubated for the extended (16-h) time period with orwithout A23187 were 3.72 Â±0.65 (SD) and 4.10 Â±1.02 ngporphyrin/mg protein, respectively. The uptake of Photofrin II

5 -COTT UJÂ» T Ãœ â„¢¿� ~

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GRP-78

Fig. 1. Northern analysis of GRP-78 and n-tubulin mRNA levels in RIF-1cells exposed to stress conditions. Cells were exposed to: no external treatment(CONTROL), 420 J/m2 light treatment alone (LIGHT), 25 tig/mi Photofrin IIfor either l h (DHE-I h) or 16 h (DHE-16 h) without light, 7 Â»MA23187 for 16h (A23187), 0.25% /3-mercaptoethanol for 12 h (B-ME), 20-min exposure at 45Â°C(HEAT), or a combination of Photofrin II plus light which yields a 20-40%survival (PDT). Cells exposed to heat were analyzed 4 h after treatment and cellsexposed to PDT were analyzed 0-18 h after treatment. After the various treatments total RNA was isolated, size separated on a denaturing gel, and transferredto nylon membranes. The membranes were sequentially hybridized to "P-labeledcDNA probes for GRP-78 and n-tubulin. A, mRNA levels as a function of timeafter PDT using the short (1-h) porphyrin incubation; B, mRNA levels as afunction of time after PDT using the extended (16-h) porphyrin incubation. Forcomparison. mRNA isolated 10 h after PDT using the 16-h incubation protocolis shown in both A and B.

OC OC OC OCOCEEOCOCXXX Ixxxxx--p>o>Â¡i;T-rjmr-oÂ»"-Â»-.- CM

H' UIH

Protein Glycosylation Is Not Altered following PDT. Table 1documents the levels of glycosylation for control and PDTtreated cells. RIF-1 cells were analyzed following both 1-h and16-h porphyrin incubation protocols. Incorporation of TCAprecipitable [3H]fucose was used as a quantitative measurementof protein glycosylation (17, 33-35). Overall uptake of ['H]

fucose in RIF-1 cells was slightly decreased during the first 5 hafter PDT when the short (1-h) porphyrin incubation protocol

was used but the level of glycosylation (percentage of TCAprecipitable uptake) was comparable to that observed in controlcells. PDT treatments which utilized the extended (16-h) porphyrin incubation resulted in a minor decrease in glycosylationat the earliest time period after PDT without any changes incellular fucose uptake.

DHE: 25 UG/ML; 16 HR

HEAT: 20 MIN; 45 C

PDT: 315 JOULES/SQ M

Fig. 2. Protein synthesis patterns in Rill cells exposed to stress conditionsdescribed in Fig. 1. PDT profiles are for cells incubated with Photofrin II for theextended (16-h) time period prior to light exposure. Cells were pulse labeled withfrans-[35S]methionine (30 uCi/ml) for 1 h immediately before obtaining cell

lysates. Samples of lysates containing equal amounts of radioactivity were sizeseparated by electrophoresis on a 10% polyacrylamide gel. An autoradiogram ofthe Coomassie blue stained gel is presented with molecular weights shown on theleft and major stress proteins induced by A23I87, heat and PDT shown on theright. For abbreviations, see legend to Fig. 1.

6576



PORPHYRIN PHOTOSENSITIZATION AND STRESS PROTEIN INDUCTION

was comparable under both conditions; therefore the resistanceto PDT documented in Fig. 4B cannot be explained on thebasis of differential cellular uptake of porphyrin.

GRP niRNA Levels Are Increased in Tumor Tissue Treatedwith PDT. In vivo induction of GRP-78 was evaluated by slotblot analysis of RNA extracted from tumors treated with Pho-tofrin II mediated PDT. Table 2 shows the ratios of GRP-78versus a-tubulin for control and PDT treated tumor samples.Drug alone and light alone samples were comparable to thebasal levels observed in control tumor tissue. Slight increasesin the GRP-78 versus a-tubulin ratios were seen at 3 and 5 hfollowing in vivo PDT. The largest increase in GRP-78 mRNAwas observed at 8 h following PDT and then ratios returned tobackground levels at 12 and 18 h.

DISCUSSION

Increased levels of GRPs were documented following PDTmediated oxidative stress in cultured RIF cells and in tumor

o s 10 is 20

HOURS AFTER PHOTOSENSITIZATION

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10 15 2 0

HOURS AFTER PHOTOSENSITIZATIONFig. 3. Kinetics of GRP protein synthesis following PDT using short (I-h) or

extended (16-h) porphyrin incubation conditions. The PDT treatments resultedin 20-40% survival. Densitometer readings were obtained by scanning autoradi-ograms resulting from SDS-PAGE of [frans-"S|methionine labeled cell lysates.A, ratios of GRP-78 versas actin; B, ratios of GRP-94 versus actin. Time zerocorresponds to control conditions prior to light treatment. Points representdensitometry ratios calculated from individual SDS-PAGE experiments. Ph-ll,Photofrin II.

Table 1 Effect of Photofrin II incubation time prior to light treatment on proteinglycosylation in KIF-1 cells

Background counts were subtracted from the dpm values shown. The resultsrepresent the mean Â±SD. PDT treatments resulted in survival levels rangingfrom 25 to 50%.

Uptake and incorporation of |JH]fucose

" Total uptake TCA ppt. uptake _ .(dpm) (dpm) "'"â€¢'"'

ControlconditionsNolight, no PhotofrinIIPhotofrin

11alone (1hincubation)Photofrin

II alone (16hincubation)Light

alone (420 J/m2)43388045

Â±10727547Â±9649904

Â±15748300

Â±16671044

Â±186858Â±86866

Â±147865

Â±23912.9711.368.7410.42

Photosensitization with 1-h Photofrin II incubation (25 Â»jg/ml,367.5 J/m2)Ihpost-PDT 5 6220 Â±3944 476 Â±125 7.653hpost-PDT 5 4428 Â±714 461 Â±170 10.415hpost-PDT 5 5984 Â±816 540 Â±177 9.027hpost-PDT 5 9729 Â±2549 1294 Â±305 13.30

Photosensitization with 16-h Photofrin II incubation (25 Â»jg/ml,280 J/m1)Ihpost-PDT 5 7507 Â±1286 383+154 5.103hpost-PDT 5 9147 Â±1208 763 Â±108 8.34Shpost-PDT 5 8903 Â±1787 973 + 325 10.927hpost-PDT 5 9772 Â±1936 II59 Â±256 11.86

tissue. In vivo samples of the BA mammary carcinoma contained a mixed population of normal and tumor cells while thein vitro RIF tumor cells were homogeneous. Therefore, quantitative comparisons of stress protein induction for the in vitroand in vivo data cannot be made. However, induction kineticsof both GRP-78 and GRP-94 mRNA and protein were similar

in RIF cells exposed to porphyrin mediated photosensitization.The coordinate induction of GRPs by PDT is comparable tothat seen following exposure to A23187 (15). Interestingly,GRP induction following photosensitization was observed onlywhen extended (16-h) porphyrin incubation protocols wereused. A short (1-h) porphyrin incubation followed by light

treatment, which leads primarily to plasma membrane damagein LI210 cells (4, 32), was not associated with increases inGRPs. Extended porphyrin incubation prior to light exposurecauses injury to subcellular organdÃ-es (mitochondria and lyso-somes) as well as decreased protein synthesis and inactivationof numerous cellular enzymes in LI210 cells (4), and thisprotocol resulted in a large increase in GRP levels in RIF cells.Therefore, the induction of GRPs by porphyrin mediated PDTappears to be related to specific sites or types of oxidativedamage as opposed to only the generation of reactive singletoxygen.

The rapid and preferential synthesis of stress proteins is anubiquitous characteristic for cells exposed to various environmental insults (7, 10,13). Hyperthermia leads to elevated levelsof heat shock proteins which are associated with transientresistance to subsequent heat treatments (9). It is unclearwhether increased levels of individual members of the HSPfamily are sufficient to produce thermotolerance or whethercoinduction of all HSPs is required (8, 11). Porphyrin mediatedPDT induces increased synthesis of HSPs (2, 6) but increasesin cellular HSP-70 are not correlated with any change in PDTsensitivity (14). Interestingly, conditions which induce GRPs(such as exposure to A23187, 2-deoxyglucose, chronic anoxia,or glucose starvation) are associated with transient resistanceto Adriamycin, etoposide, tumor necrosis factor, and cytotoxicT-cells (21-23). In vitro experiments evaluating PDT mediatedGRP induction with Adriamycin sensitivity show similar re-

6577




1001

PH-II AlÃ³n*

PH-II + ETOH

PH-II Â»A23187

200 400 600 800 1 000

LIGHT DOSE (Joules/sq.m.)

1001

OC

i

tuOCCÃ¼iO. .001

.0001200 400 600 800 1000

LIGHT DOSE (Joules/sq.m.)Fig. 4. Survival curves for RIF-l cells exposed to porphyrin photosensitization.

A. percentage of survival plotted versus light dose for cells incubated for l h withPhotofrin II (Ph II) (25 (Â¿g/ml)in medium supplemented with l% PCS prior tolight exposure. Increased intracellular levels of GRP-78 were induced by a 16-hexposure to A23187 (7 M) immediately prior to Photofrin II incubation. B,survival curves for RIF-l cells exposed to porphyrin photosensitization underconditions in which cells were incubated concurrently for 16 h with Photofrin II(25 fjg/ml) and A23187 (7 /Â¿M)in medium supplemented with 5% FCS. PDTtreatments after a 16-h incubation with the solvent used to dissolve A23187[0.35% ethanol (ETOH)\ are also shown. Individual curves are shown for cellsexposed to light after incubation with Photofrin II alone, Photofrin 11 plusethanol. or Photofrin II plus A23187. Points, average of 2 experiments: bars, SE.

suits.4 In all cases, the cellular resistance is correlated with

stress induced increases in GRPs but biochemical mechanismsassociated with the resistance have not been identified. In thecurrent study, A23187 induced overexpression of GRPs wascorrelated with resistance to porphyrin mediated PDT onlyunder photosensitizing conditions involving an extended porphyrin incubation. Exposure to A23187 can alter normal phys-

4 A. M. R. Fisher and C. J. Corner, unpublished data.

Table 2 Slot blot analysis of GRP-78 and n-tubulin levels in BA mammarycarcinoma following in vim PDT

C3H/HeJ mice with BA mammary carcinomas were treated with PDT (5 mg/kg Photofrin II administered via i.p. injection 24 h prior to a 200-J/cm2 lighttreatment). GRP-78 and Â«-tubulinmRNA levels were determined by densitometerfollowing slot blot analysis. One tumor sample was obtained per treated mouse.Mean Â±SD.

Samp,e Sample no. Ratio (GRP-78 vs. a-tubulin)

ControlPhotofrin11aloneLight

alonePDTPDTPDTPDTPDT3581218533565130.709

Â±0.030.657Â±0.020.685

Â±0.150.763Â±0.070.937Â±0.171.615+0.280.7270.523

Â±0.04

iological parameters in cells in addition to inducing GRP synthesis (36). Therefore, PDT resistance associated with A23187may involve actions unrelated to stress proteins. For example,inhibition of general protein synthesis, associated with iono-phore incubation, could deplete nascent protein targets requiredfor PDT mediated cytotoxicity. Cellular porphyrin content wasnot altered by A23187 incubation; therefore the observed resistance was not due to changes in cellular photosensitizerlevels. Overexpression of intracellular GRPs using stably trans-fected expression vectors could provide definitive informationregarding the role of GRPs in PDT sensitivity. Likewise, useof a reporter gene fused to normal and deleted 5' untranslatedregions of the GRP-78 promoter (36) could provide novelinformation regarding sequences and/or regions associated withGRP induction by PDT.

Our observation that GRP-78 mRNA levels are increased intumor tissue following PDT implies that gene induction responses occurring after in vitro PDT (using an extended porphyrin incubation) are similar to photosensitizing reactionsoccurring after standard in vivo PDT. These results suggest thatin situ hybridization or immunohistochemical studies, usingGRP-78 oligonucleotide probes or antibodies, could be appliedin identifying initial targets and sites of in vivo PDT damage.Pathological and physiological data indicate that both tumorcells and tumor vasculature are injured following PDT (3, 5).Unfortunately, the sequence of in vivo events and the primarytargets associated with PDT mediated tumoricidal action havenot been defined. Therefore, the induction of stress proteinscould serve as specific molecular markers for identifying theinitial cells, tissue regions, and/or tumor depths responding toPDT mediated damage.

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1991;51:6574-6579. Cancer Res Charles J. Gomer, Angela Ferrario, Natalie Rucker, et al. Oxidative Stress Mediated by Porphyrin PhotosensitizationGlucose Regulated Protein Induction and Cellular Resistance to

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