acta histochemica 113 (2011) 363–368

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acta histochemica

0065-12

doi:10.1

n Corr

Autonom

E-m

journal homepage: www.elsevier.de/acthis

Cell death causes relocalization of photosensitizing fluorescent probes

Marco Alvarez a, Angeles Villanueva b, Pilar Acedo b, Magdalena Canete b, Juan C. Stockert b,c,n

a Anatomical Institute ‘‘Jose Izquierdo’’, Faculty of Medicine, Central University of Venezuela, 1050 Caracas, Venezuelab Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Cantoblanco, 28049 Madrid, Spainc Center for Biological Research, High Council of Scientific Research, 28040 Madrid, Spain

a r t i c l e i n f o

Article history:

Received 7 January 2010

Received in revised form

17 January 2010

Accepted 18 January 2010

Keywords:

Photosensitizers

Fluorescent probes

Cell cultures

Organelle labeling

Relocalization

Cell death

Necrosis

81/$ - see front matter & 2010 Elsevier Gmb

016/j.acthis.2010.01.008

esponding author at: Department of Bio

ous University of Madrid, Cantoblanco, 280

ail address: juancarlos.stockert@uam.es (J.C. S

a b s t r a c t

When cultured cells are treated with fluorescent organelle probes or photosensitizer agents, a

characteristic redistribution of fluorescence in cell structures occurs frequently after light irradiation. It

is currently assumed that such changes, referred to as relocalizations of the fluorescent compounds,

represent an important aspect of the photodynamic process, which is based on the excitation of

photosensitizers by light in the presence of oxygen. As cell damage and death result from the oxidative

stress induced by photodynamic treatments, we have studied here the redistribution of acridine orange

(AO) and 3,30-dimethyl-oxacarbocyanine (DiOC1(3)) fluorescence after incubation of HeLa cell cultures

with these compounds followed by blue light irradiation to achieve lethal effects. The relocalization of

dyes from their original labeling sites (AO: lysosomes, DiOC1(3): mitochondria) to nucleic acid-

containing structures (cytoplasm, nuclei and nucleoli) appeared clearly associated with cell death.

Therefore, the relocalization phenomenon simply reflects fluorescence changes due to the different

affinity of these dyes for living and damaged or dead cells. As fluorescent probes are often

photosensitizers, prolonged light exposures using fluorescence microscopy will produce lethal

photodynamic effects with relocalization of the fluorescent signal and changes in the cell morphology.

& 2010 Elsevier GmbH. All rights reserved.

Introduction

It is widely known that during light irradiation, numerousfluorescent probes (FPs) used for selective organelle labeling ofcultured cells are also capable of inducing severe photodamagefollowed by cell death. In these cases, the FP acts as aphotosensitizer (PS), which transfers the energy of its excitedsinglet state to the triplet state, and then to molecular oxygen.Highly reactive oxygen species generated by this process produceoxidative stress and lethal effects on tumor cells, and thisbiological response is increasingly applied in photodynamictherapy (PDT) of cancer (Villanueva et al., 2003; Stockert et al.,2004; Canete et al., 2009).

There is a striking difference in the fluorescence of cells whenFPs and PSs are used as labeling or staining agents before or afterfixation (Stockert, 1974; Horobin et al., 2006). Living cells treatedwith pyronin Y as FP only show mitochondrial fluorescence, butafter continuous microscopic excitation or in cells with damagedplasma membrane, the emission is found in basophilic cytoplasm,nuclei and nucleoli (Cowden and Curtis, 1983), which agrees withthe binding affinity of this dye for nucleic acids in fixed cells(Armas-Portela and Stockert, 1979).

H. All rights reserved.

logy, Faculty of Sciences,

49 Madrid, Spain.

tockert).

In this context, when PSs are used in photosensitizationprotocols on cell cultures, a characteristic redistribution offluorescence often occurs after light irradiation, which has beenreferred to as ‘‘relocalization’’ of the PS. It is currently suggestedthat this phenomenon represents an important process in cellphotosensitization. Upon light irradiation, the PS dye methyleneblue was seen to dramatically relocalize from the lysosomes oftreated cells to the cytoplasm and then the nucleus (Ruck et al.,1992; Walker et al., 2004). In the case of macrocyclic PSs, rapidfluorescence relocalization (sometimes with changes in emissionyield) was found during or following light exposure of cellstreated with porphyrins (Woodburn et al., 1991, 1992; Ruck et al.,1992; Patito et al., 2001; Kessel, 2002; You et al., 2003; Sibrian-Marquez et al., 2007), porphycenes (Kessel and Luo, 1998; Kessel,2004; Kessel et al., 2005; Mak et al., 2003) and phthalocyanines(Wood et al., 1997; Malik et al., 1997; Ball et al., 1999; Qualls andThompson, 2001).

Interestingly, this redistribution only occurs after a period ofillumination, so it should be considered as a photoinducedrelocalization due to the release of PSs from their initial organelletargets following photodamage. In this context, conspicuousmorphological changes occur in photosensitized cells showingrelocalized PSs. Taking into account the differences betweenlabeling and staining pattern of living and fixed cells, respectively,we have studied here the fluorescence redistribution of the wellknown photosensitizing dyes acridine orange and 3,30-dimethyl-oxacarbocyanine after light irradiation at lethal doses.

M. Alvarez et al. / acta histochemica 113 (2011) 363–368364

Our findings show that the relocalization of these PSs results fromthe damage and cell death just induced by photosensitization.

Material and methods

HeLa cells were grown on 22 mm square coverslips placed into35 mm culture dishes using Dulbecco’s modified Eagle’s medium(DMEM) containing 10% (v/v) fetal bovine serum (FBS), 50 U/mlpenicillin, 50 mg/ml streptomycin and 1% (v/v) 0.2 M L-glutamine(complete DMEM; all products from Gibco, Paisley, Scotland, UK).Cell cultures were carried out at 37 1C in a humidified atmospherecontaining 5% CO2, and the medium was changed daily.

The photosensitizing and fluorescent probes acridine orange(AO) and 3,30-dimethyl-oxacarbocyanine (DiOC1(3)) (both fromSigma-Aldrich, St. Louis, MO, USA) were used for labeling oflysosomes and mitochondria, respectively. A small volume(0.1 ml) of 10�3 M dye solutions in distilled water was added to10 ml of complete DMEM (final dye concentration: 10�5 M). Cellcultures were incubated in 2 ml of final dye solutions for 1 min(DiOC1(3)) or 2 min (AO). Light irradiation of cell cultures inDMEM was performed after washing with phosphate bufferedsaline (PBS) or directly (without dye washing) for 25 min using aReflecta slide projector equipped with a 150 W lamp. The lightwas filtered through a blue filter (360olo460 nm), and 3-cmwater layer to absorb heat. The light intensity at the cell cultureposition was 7 mW/cm2, as measured with an M8 SpectrumPower energy meter, corresponding to a light dose of 10.5 J/cm2.After dye treatment, either alone or followed by light irradiation,cells were washed with PBS, mounted in DMEM and observedimmediately. In some cases, cells were subjected to formaldehydevapor fixation to allow permanent mounting (Canete et al., 2001).

Fig. 1. Fluorescence micrographs of HeLa cells subjected to AO labeling. (A) Shows cells

followed by blue light irradiation, with membranous bubbles arrowed. The correspondi

signal from the original pictures, conversion to gray images, and displayed with the 16

Bars=20 mm.

Microscopic observations and photography were performedwith an inverted CKX31 microscope and a BX-UCB epifluores-cence microscope (both from Olympus, Tokyo, Japan) equippedwith an HBO 100 W mercury lamp and the filter sets for blue(460–490 nm, DiOC1(3)) and blue plus green (460–490 nm+510–550 nm, AO) exciting light. Photographs were obtainedwith a digital camera (Olympus DP70) over a time period notmore prolonged than 10 min. They were processed usingAdobe Photoshop CS2 software (Adobe Systems, San Jose, CA).Image processing and analysis (IPA) procedures were performedwith the public domain ImageJ 1.42 software (http://rsb.info.nih.gov/ij/). Pseudocolor options were used to break the continuousbright gradient of photomicrographs. The isolated red or greensignals were converted to gray images and then visualized using aselected option from the lookup table of the IPA software.Conversion of original images to equidensity areas showingseveral levels in the black–white scale was also made usingthe Adobe Photoshop software. Convolved images (ImageJ)were obtained by means of the corresponding convolve filter(a matrix of 25 points (5 columns and 5 rows), the central pointwith a[+25] value and the remaining peripheral points witha[�1] value).

Thiazolyl blue (MTT, Sigma-Aldrich) was used for the assess-ment of cell survival 24 h after photosensitization treatments.A stock solution (1 mg/ml) in PBS was prepared immediatelyprior to use. Solutions (100 ml) were added to each culture dish(containing 2 ml of complete medium; final MTT concentration:47.6 mg/ml). After 3 h incubation, formazan precipitates weredissolved in 1.5 ml DMSO and the absorbance was measured at540 nm with a plate reader (SpectraFluor, Tecan, Mannedorf,Switzerland). Cell survival was expressed as the percentageof absorption of treated cells in comparison with that of

exposed to AO with no subsequent irradiation. (B) Shows the results of AO labeling

ng pseudocolor images (C, D, respectively) were generated after isolation of the red

colors option from the lookup table of the ImageJ software (inset: gray-color code).

M. Alvarez et al. / acta histochemica 113 (2011) 363–368 365

control cells. Results were the mean value and standard deviationfrom at least three different experiments.

Results

Normal fluorescent labeling

Cells treated with AO for lysosome labeling showed an orange-red fluorescence of these granular organelles. The remainingcytoplasm and nuclei revealed a pale green emission, whereasnucleoli appeared with a stronger green signal. These morpholo-gical features were clearly observed both in original fluorescencepictures and in IPA images (Fig. 1A, C). Using DiOC1(3) labeling,filamentous mitochondria revealed a bright green emission, andalmost no fluorescence was found in nuclei (Fig. 2A, C, E). Afterobservation and photography of the normal pattern of organellelabeling, other AO- and DiOC1(3)-treated cell cultures weresubjected to blue light irradiation and observed immediately, inorder to analyze the relation between cell photodamage andrelocalization of dyes.

Morphological necrotic changes

The observation of AO- and DiOC1(3)-treated cells immediatelyafter light irradiation revealed a rapid and generalized inductionof necrotic cell death as well as conspicuous changes in thefluorescence pattern (Figs. 1B, D and 2B, D, F). The amount of celldamage was somewhat higher when cultures were irradiated inthe presence of PSs. Cell death by necrosis was recognized on the

Fig. 2. Fluorescence micrographs of HeLa cells subjected to DiOC1(3) labeling. (A) Sho

labeling followed by blue light irradiation. Membranous bubbles are arrowed. The lower

breaking of the gray gradient of cells into 8 levels (C, D); and image filtered by the con

basis of a typical sequence of morphological alterations affectingfundamentally the plasma membrane (Villanueva et al., 2003;Stockert et al., 2004; Rello et al., 2005). The necrotic processstarted with an immediate and massive production of smallsurface evaginations (bubbles) clearly visible by both fluorescencemicroscopy (Fig. 1B) and phase contrast optics (Fig. 3). Plasmamembrane deformations developed rapidly into a small numberof large ‘‘membranous’’ bubbles which finally detached fromthe cell, leaving the cell body still remaining firmly attached tothe substrate. The nuclear morphology was not greatly affectedimmediately after lethal treatments, but several hours later nucleiappeared pyknotic (with highly and homogeneously condensedchromatin) and the cytoplasm became unstained and severelydisorganized or lost, with a refringent and coarsely granularstructure of cytoplasmic remnants (not shown).

In the case of cells subjected to lethal photosensitization withAO, bright green (orthochromatic) emission was always foundin nuclei, whereas the cytoplasm showed variable colors, fromyellow-green to orange-red (metachromatic) fluorescence(Fig. 1B). In addition to the visual recognition of different emissioncolors, IPA procedures also allowed to confirm and analyze inmore detail the morphological characteristics of AO-labeled livingand dead cells. When the red (or green) signal was isolated fromoriginal images, converted into a gray scale, and processed to apseudocolor equidensity map, the changes in cell structures wereobserved more precisely (Fig. 1C, D).

Likewise, the DiOC1(3) labeling pattern of mitochondria incontrol cells (Fig. 2A) was lost and changed to a strong greenfluorescence of nuclei and nucleoli in dead cells (Fig. 2B). Thesefeatures were also observed in the equidensity (Fig. 2C, D) andconvolved images (Fig. 2E, F) obtained by means of IPA

ws cells exposed to DiOC1(3) with no subsequent irradiation. (B) Shows DiOC1(3)

panels correspond to cells from the rectangles marked in (A) and (B), and represent

volve command (E, F). Bars=20 mm.

Fig. 3. Phase contrast micrographs of HeLa cells after labeling with AO (A, B) and DiOC1(3) (C, D), followed by irradiation with blue light and immediate observation. Most

cells show a necrotic morphology, with a massive occurrence of refringent membranous bubbles with different sizes at the cell surface (arrows). Bars=20 mm.

M. Alvarez et al. / acta histochemica 113 (2011) 363–368366

procedures. On the other hand, binary images using differentthresholds were obtained from living and dead cells. With athreshold of 160, a clear separation of cell structures wasachieved, allowing a highly selective visualization of mitochon-dria and nuclei from living and dead cells, respectively (notshown).

To confirm the microscopical results and to support themorphological criteria for cell death, the MTT assay for cellviability was used after AO and DiOC1(3) labeling and bluelight irradiation in the presence of the PSs. Fig. 4 shows thesurvival of control cells, those exposed to light or PS alone,and photosensitized cells. In agreement with microscopicobservations, almost complete cell lethality was observed in thecase of cultures subjected to photodynamic treatments with AOand DiOC1(3).

Discussion

The relocalization of PSs is a poorly understood process thathowever has become of increasing concern in PDT research (Rucket al., 1992; Wood et al., 1997; Kessel, 2002). As the damagingphotodynamic action only occurs during light irradiation, thepractical relevance of PS relocalization is small unless when theirradiation is long or repeated. But although this process couldgenerate additional adverse effects, relocalization as such iscaused merely by photodamage to a target organelle followed

by PS release. Therefore, it is unlikely that relocalization occurs innormal living cells, and it is likely that most PS redistributionprocesses only occur in severely damaged or dead cells.

In living cells the cationic fluorescent probes and photosensi-tizing dyes AO and DiOC1(3) selectively label lysosomes andmitochondria, respectively (Johnson et al., 1981; Canete et al.,2001), which is in agreement with QSAR studies on thelocalization of FPs and PSs (see Rashid et al., 1991; Horobin,1999; Stockert et al., 2004; Horobin et al., 2006). In this context,the nucleosomal organization of chromatin in living nuclei doesnot allow a massive DNA intercalation of compounds such as AO(Delic et al., 1991). DiOC1(3) and similar flexible carbocyaninedyes bind to the DNA minor groove (Seifert et al., 1999; Mikheikinet al., 2000) and induce nuclear fluorescence only in fixed cells(Stockert, 2000; Stockert et al., 2005).

However, exposure to these acridine and carbocyanine dyes,with subsequent significant light irradiation, induces photo-damage and cell death (Zdolsek et al., 1990; Lee et al., 1995).Consequently this process should result in both significantmorphological changes and in a clear redistribution of dyefluorescence in dead cells. It is well known that both lysosomesand mitochondria are key target organelles for cell damageinduced by photosensitization (Zdolsek et al., 1990; Geze et al.,1993; Lee et al., 1995; Kessel and Luo, 1998; Villanueva et al.,2003; Canete et al., 2009). A novel experimental condition wasemployed in the present work, consisting in the irradiation of cellcultures in the presence of the PS to induce greater photodamage

Fig. 4. Dark toxicity (A) and photosensitization response (B) of the cells to the PS

dyes used. After AO and DiOC1(3) treatments (without PSs washing), blue light

irradiation induced a lethal effect. Evaluation of the cell viability of untreated

controls (C), light alone, PS alone and photodynamic protocol (PS+light) was

performed 24 h after treatments by using the MTT method. Data correspond to

mean values7standard deviation from at least three different experiments.

M. Alvarez et al. / acta histochemica 113 (2011) 363–368 367

and higher staining reactions as a consequence of the more severephotodynamic treatment.

Interestingly, results from several authors summarized belowsupport strongly our observations and also our interpretationsabout PS relocalization being due to cell damage followed by celldeath.

(a)

The lysosomal localization of AO in living cells changes to anuclear fluorescence when cells are exposed to prolongedmicroscopical exciting light, which induces cell death (Delicet al., 1991).

(b)

After irradiation, relocalization of TPPS4 from lysosomes intothe cytoplasm and finally into the nucleus was observedsimultaneously with lethal cell damage (Strauss et al., 1995).

(c)

Photosensitizing ruthenium complexes such as Ru(bipyri-dine)3 and Ru(phenanthroline)3 do not enter into culturedcells (Dobrucki, 2001) but they induce strong fluorescencein the chromatin of fixed cells (Bertolesi et al., 1995). Culturedmacrophages can internalize these Ru PSs, and after cellphotodamage of plasma membrane and endosomes, theluminescent Ru complexes just appear in cell nuclei(Dobrucki, 2001).

(d)

The localization of anionic and cationic porphyrins changesfrom lysosomes to nuclei not only after light irradiation but

following cell damage by oxidative stress induced with H2O2

(Patito et al., 2001).

(e) After cell damaging by photosensitization with a cationic

porphyrin, the fluorescence of the mitochondrial FP rhoda-mine 123 changes to a nuclear localization (Hatz et al., 2007).

In keeping with results from the present study using AO andDiOC1(3), all the above examples clearly illustrate the correlationbetween relocalization of PSs and cell damage or death inducedby photosensitization or other lethal treatments. It can beconcluded that the fluorescence relocalization simply reflectsthe different affinity of FPs and PSs for living and dead cells. Asfluorescent dyes are often photosensitizers, prolonged lightexcitation using fluorescence microscopy will produce photo-dynamic lethal effects with the relocalization of the fluorescentsignal and changes in the cell morphology. A practical implicationis that prolonged microscopic excitation must be avoided whenFP- or PS-labeled organelles from living cells are studied.

Acknowledgements

We thank the Consejo de Desarrollo Cientıfico y Humanıstico(CDCH), Venezuela, for support (Group Project 09-00-6879-2007).This work was also supported by a grant from the Ministerio deCiencia e Innovacion (CTQ2007-67763-C03/BQU), Spain.

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