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to be capable of singlet oxygen generation within the UV, but possibly also the visible range for their phototherapeutic potential. Acknowledgments O u r studies were supported by Grant-In-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (10670801, A M ) , a grant from the Deutsche Forschungsgemeinschaft, SFB 503, Teilproject B2, and a grant from the Alexander-vonHumboldt-Foundation.

[30] S i n g l e t O x y g e n - T r i g g e r e d I m m e d i a t e Preprogrammed Apoptosis By DIANNE E. GODAR Introduction The two distinct morphological forms of cell death are apoptosis and necrosis. These morphological distinctions can be seen using light, fluorescent, or electron microscopy. The salient morphological features of apoptosis include cell shrinkage, plasma membrane "blebbing," vacuolization, and chromatin digestion and condensation along the nuclear membrane. In the final stages, the cell fragments into membrane-bound vesicles called apoptotic bodies. In vivo, these apoptotic cells and bodies are removed by phagocytosis; however, in vitro they are not removed and consequently undergo "secondary necrosis." During secondary necrosis, lysosomes rupture and release hydrolytic enzymes into the cytoplasm, causing further destruction of internal components, including the plasma membrane, which results in cell swelling and lysis. When the term apoptosis was originally coined in 1972,1 it only referred to the morphological changes characteristic of this form of cell death, as the underlying biochemical mechanisms were not yet known. Since then the terms apoptosis and programmed cell death (PCD) 2 have been used synonymously, even though some mechanisms of apoptosis are found to proceed independent of transcription and translation, 3,4 a biochemical mechanism referred to as preprogrammed cell death (prePCD). 4 Thus, the term apoptosis only describes the morphological changes that occur during this mode of cell death, 1 j. F. R. Kerr, A. H. Wyllie, and A. R. Currie, Br. J. Cancer 26, 239 (1972). 2 R. A. Lockshin and C. M. Williams, J. Insect. Physiol. 10, 643 (1964). 3 S. Martin, S. L e n n o n , A. B o n h a m , and T. Cotter, J. Immunol, 145, 1859 (1990). 4 D. E. Godar, Photochem. Photobiol. 63, 825 (1996).

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whereas the terms prePCD and PCD segregate the general underlying biochemical mechanisms that drive these changes. Reactive oxygen species (ROS) cause damage to a variety of cellular components that can initiate different apoptotic mechanisms simultaneously, complicating the interpretation of results. ROS cause damage to mitochondrial membranes, triggering immediate prePCD apoptosis (T <30 min), damage to receptors initiating intermediate prePCD apoptosis (T > 0.5 hr -< 4 hr), and damage to DNA, inducing delayed PCD apoptosis (T >> 4 hr). 5 Both UVA1 (340-400 nm) radiation and photodynamic therapy (PDT; photosensitizer and visible light ->400 nm) mediate primarily singlet oxygen damage to membranes. Singlet oxygen damage to mitochondrial membranes causes immediate depolarization of the inner transmembrane potential and the immediate appearance of apoptotic cells. Unlike most initiators of apoptosis, singlet oxygen triggers the immediate onset of apoptotic morphology because mitochondria are downstream of all initiating signaling events, such as gene activation and the "initiator" caspase cascade (e.g., caspase 8). When apoptosis is triggered at the mitochondria, either apoptosis-initiating factor (AIF) or cytochrome c is released, which activates the "executioner" caspase cascade (e.g., caspase 3) that causes morphological changes. Thus, morphological characteristics associated with apoptosis occur very rapidly (-<1 hr) when triggered by singlet oxygen damage and is consequently referred to as "immediate" apoptosis. 6 For several reasons, it is particularly challenging to detect and accurately quantify apoptotic cells and changes that occur during apoptosis following singlet oxygen damage. In addition to initiating different apoptotic mechanisms simultaneously, singlet oxygen damage causes cells to undergo apoptosis so rapidly that significant percentages of cells can be in the final stages within minutes after exposure (some exposure times are 60 min). In the final stages of apoptosis, the plasma membranes become permeable to dyes such as propidium iodide so that these cells appear necrotic. In addition, secondary necrosis quickly follows singlet oxygen-triggered apoptosis, which further complicates the interpretation of results. Moreover, singlet oxygen damage to plasma membranes renders these cells particularly sensitive to common technical manipulations, such as vortexing, centrifuging, washing, pipetting, fixing, and even vital dye staining methods. Some routine methods cannot be used at all or must be modified to optimize the yield by protecting the cells from further damage and loss during processing. A couple of examples of routine techniques that cannot be used at all are cytospins and vital dye staining. Cytospins cannot be used even if the vessels 5 D. E. Godar, J. Invest. Dermatol. 112, 3 (1999). 6 D. E. Godar, S. A. Miller, and D. P. Thomas, Cell Death Different. 1, 59 (1994).

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and slides are precoated with serum/protein and the centrifugal force and time are decreased as much as possible because singlet oxygen damage to plasma membranes causes them to rupture during this procedure and the remaining cells appear necrotic after fixing and staining (unpublished observations). Simple vital dye stains, such as trypan blue, cannot be relied on to quantify apoptotic cells because they swell and give misleading data. 7 In general, the most reliable techniques include those that have the fewest processing steps or those that can be modified to afford adequate protection of these cells during the entire technical procedure to increase the yield and maintain the true identity of all cell populations. The methods described in this article allow good recovery and separate analysis of both apoptotic and morphologically normal cell populations following singlet oxygen damage by UVA1 radiation or PDT. These methods are divided into three sections. The first section describes general modifications to various common processing techniques that maximize the yield of all cell populations to assure that accurate and reliable results are obtained. It also describes irradiation procedures and postexposure treatments. The second section describes some specific ways to determine if a new system creates singlet oxygen and if this damage causes cells to die by immediate (T < 30 min) prePCD apoptosis or by some other mechanism of apoptosis. The third section describes a flow cytometry procedure that allows separate analysis and quantification of apoptotic and morphologically normal cells and changes that occur to the mitochondria in either population. General Modifications of Methods The delicate nature of singlet oxygen-damaged cells requires some general modifications to all procedures in order to obtain maximum recovery of all cell populations. These modifications will minimize erroneous results created by technical procedures that cause membrane damage before and/ or after exposure.

Centrifuging The time and force of centrifugation must both be decreased, e.g., the time should be reduced to 5-7 min and the rpm should not exceed 12001400 for most centrifuges. For microcentrifuges with a set speed (10,000 rpm), use for only 30-45 sec, unless it is a variable speed model, and then set it no higher than eight (8000 rpm) for no longer than 1 min. 7 j. Z. Beer, K. M. Olvey, S. A. Miller, D. P. Thomas, and D. E. Godar, Photochem. Photobiol. 58, 676 (1993).

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Washing Procedures that require washing the cells, other than annexin V binding to phosphatidylserine residues (because proteins interfere with binding), should contain at least 1% serum or protein (protease-free bovine serum albumin) in all wash buffers. The number of washes should be reduced as much as possible whenever possible (e.g., three washes can usually be reduced to two) and can sometimes be completely eliminated (see Section VI,B).

Suspending Cells To suspend cells after centrifuging and aspirating, either "finger flick" the cell pellet on the bottom of the centrifuge tube or tap the bottom of the tube a few quick times on the countertop. Alternatively, a quick (2-3 sec) vortex at a moderate setting (half maximum speed) may be used, but only on the pelleted cells. Never add buffer or media first and then vortex. For homogeneous suspension of cell pellets, it is important to centrifuge them briefly and lightly as just described. Check for "clumps" of cells and, if present, mix by inversion and "finger flick" the bottom of the tube a few times while upside down. Pipetting the solution gently once or twice (without bubble formation) is better for larger volumes. If clumps of cells are found, the procedure that was used should be modified by decreasing the centrifugal force and/or time and possibly altering the suspending technique.

Vortexing Never vortex singlet oxygen-damaged cells for more than 3 sec, at a high setting, or in solution. Use quick vortex times (-<3 sec) at half the highest setting unless the cells are being fixed (one-third highest setting), and always vortex the cells in pellet form rather than in solution.

Pipetting It is important to pipette without too much force and without forming many bubbles during any processing step, especially when suspending cells after irradiation. Bubbles cause oxidative damage to plasma membranes, increasing the percentage of apoptotic cells, causes loss of cells through lysis, 7 and also increases the percentages of necrotic cells. Irradiation Procedures Irradiation of cells in phosphate-buffered saline (PBS) is preferred to media for three reasons: (1) it does not absorb wavelengths above 200 nm,

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(2) it has no components that can be oxidized like the essential nutrients present in media (nutrient deprivation can cause apoptosis as well); and (3) it cannot produce toxic products. If media must be present during exposure, then a control for the exposed media must be included and added to cells that were not exposed. In addition, the time of irradiation must be increased somewhat to account for the radiation absorbed by the media, if irradiated from above. Note that the time the cells are in PBS should not exceed 2 hr or the cells will start to undergo apoptosis due to nutrient deprivation. Cells in PBS can be irradiated in uncovered cell culture dishes, e.g., 5 ml of 6-10 × 105 suspended cells/ml in 60-mm dishes or 2 ml in 35-mm dishes. Alternatively, flasks (T-25 with 5 ml) or covered dishes can be used to prevent evaporation during irradiation. However, this system should only be used for UVA/UVB and visible light exposures because UVC is absorbed completely by polystyrene and other plastics. If a covered system is chosen, then transmission of the light through the plastic must be assessed and the time of irradiation increased to obtain the correct dose. In addition, dishes no smaller than 35 mm should be used, e.g., 24- or 96-well plates, because edging effects of the light occur and alter the effective dose received by the cells. For ionizing radiation procedures, dishes, flasks, or centrifuge tubes may be used for irradiation of cells in PBS. The dose rate of some UVA-emitting devices, e.g., black light bulbs, is too low to produce a reasonable dose in 2 hr. Although biological affects can be observed using these lower dose-rate bulbs, they are mainly due to the significant amounts of UVA2 (320-340 nm) and UVB (290-320 nm) emitted by these sources. To observe UVAl-triggered immediate apoptosis, the UVA-emitting source should have a dose rate ->25 W/m 2 and emit very little UVA2 and virtually no UVB. The temperature must be controlled and monitored carefully during UVA/visible exposures 8 because almost all high-dose rate UVA/visible light sources (except lasers) emit significant amounts of infrared radiation. The best control of temperature can be achieved with a circulating water bath connected to a black cooling plate (or block). Alternatively, one can use a long shallow dish (to reduce reflected light) with a shallow pool of water (2-3 mm depth), some ice, and possibly salt. Whatever system is chosen, the temperature must be monitored during the entire course of exposure to assure that the highest dose does not create temperatures above 30° for more than a few minutes. For when temperatures exceed 30°, the enzymes involved in apoptosis are more active and higher percentages of 8 D. E. Godar and J. Z. Beer, in "Biological Responses to Ultraviolet A Radiation" (F. Urbach, ed.), p. 65. Valdenmar, Overland, KS, 1992.

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apoptotic cells are obtained immediately after exposure. In addition, cellular damage from IR can also occur and/or heat shock proteins can be induced at higher temperatures, either of which may interfere with the interpretation of results.

Radiation Sources 1. UVA1 Source. UVASUN 2000, 3000, or 5000 sunlamps (Mutzhas, Munich, Germany, and similar high-output UVA sources are available through other companies) emit wavelengths ->340 nm and have an emission peak around 365 nm. The UVA1 irradiance at the sample level (43-71 cm) should be about 200 W/m 2. The emission spectrum of the UVASUN 3000 sunlamp,6 the instrument used for routine dosimetric measurements, and the calibration of the detector have been described previously.9 2. Visible Light Source. UVASUN sunlamps can be equipped with a UF3 cutoff filter (Read Plastics, Rockville, MD) to yield wavelengths ->400 nm. 3. UVAR Source. The UVAR photoactivation chamber (Therakos, West Chester, PA) has an irradiance of about 50 W/m 2 through the plastic top of a T-25 flask at the sample level (16 cm). This source primarily emits UVA, but also emits biologically significant amounts of UVA2 and UVB wavelengths. 4. UVB Source. The FS20 sunlamp (Westinghouse) has an emission peak near 313 nm. An U340 filter (Hoya Optics, Fremont, CA) may be used to eliminate all the UVC and to reduce the visible and UVA wavelengths so that the UVB wavelengths are predominate. Using this filter, UVB irradiance at the sample level (29 cm) is about 0.48 W/m 2 (UVA irradiance is about 0.32 W/m2). 5. X-Ray Source. A 120-kV, constant potential, 3-mm aluminum halfvalue thickness X-ray source can be used at a target-to-source distance of 70 cm. If the dose rate is 0.81 Gy/min, then 12.35 min of exposure will generate 10 Gy of ionizing radiation. A 20-cm-thick lucite sheet should be used to backscatter the beam in air for even dosimetry. Irradiating Suspension Cells Cells, which may be obtained from the American Type Tissue Collection, are grown to 3-5 × 105 cells/ml in complete CO2-independent medium (GIBCO) containing 10% serum, 4 mM glutamine, and sometimes antibiotics. This medium is recommended for all procedures because it does not cause a significant pH change when processing the cells. The cells are 9 D. E. Godar, D. P. Thomas, S. A. Miller, and W. Lee, Photochem. Photobiol. 57, 1018 (1993).

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harvested and concentrated by centrifugation (see Section II), aspirated, suspended by vortexing the pellet, and then diluted in PBS (without serum or protein) using about half the original volume. The final concentration of cells should be between 6 and 10 x 105 cells/ml, about twice the original, but should never exceed 1 x 106 cells/ml to ensure even dosimetry (by reducing shielding effects from "piled-up" cells). Count these cells to be sure they are not too dense and to later determine the recovery after removal from the dishes.

Irradiating Adherent Cells Cells should be grown to 70-80% confluency and no higher to assure even dosimetry. These cells can be irradiated in either the attached or the detached (suspended) state in PBS. If the later is chosen, then the cell concentration should not exceed 106 cells/ml. Postexposure Treatment

Suspension Cells Immediately following exposure, an equal volume of complete medium is added to either the suspension cells or the adherent cells that were irradiated in the unattached state. Cells are then removed by gently pipetting the media over the dish or back of the flask once or twice with minimum bubble production and mild force using a spiraling or back and forth motion, respectively.7 Tip the dish at an angle (30 ° increasing to 45 °) while drawing up the solution to minimize bubble formation. Check the dishes visually for clarity by holding them up to the light under the biological hood and verify that most of the cells have been removed using an inverted microscope. Put these cells in either T-25 flasks or centrifuge tubes for observation at different times postexposure or processing for different end points, respectively. In addition, count the cells to determine the recovery, which should be >-90%. After monitoring the cells over a short time course (<24 hr), more medium may be added for overnight growth so that the concentration does not exceed 2-3 x 105 cells/ml.

Adherent Cells For postexposure times other than immediately after exposure, add an equal volume of medium and incubate at 37°. For the time point immediately after exposure, do not aspirate the PBS off the adherent cells because they detach when undergoing apoptosis and are lost during this process. Instead, remove the PBS containing cells from the dishes or flasks by

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pipetting and place this solution in a centrifuge tube (15 ml). Remove the remaining attached cells using trypsin-EDTA (1 ml for 35-mm dish and 3 ml for 60-mm) at room temperature for 5-10 min with an occasional gentle swirling motion. When detached, add an equal volume or more of complete media and gently collect the cells by pipetting and then pool these cells with the PBS cells already in the centrifuge tube. Harvest the cells by centrifugation and suspend cells in fresh 37° complete media as described in Section II. For dose-response curves and time courses of adherent cell types, a different dish or flask of cells must be used for each dose and postexposure observation time.

Determining which ROS Initiates which General Mechanism of Apoptosis An extensive list of biologically suitable reagents used for ROS studies is given elsewhere in this volume. The few, but most reliable, reagents used in ROS studies in cell culture are given here as a first approach for determining if a given system may involve the production of singlet oxygen. 5 In addition, some alternate approaches are described for obtaining evidence that suggests singlet oxygen is involved and for determining which general apoptotic mechanism a given system initiates. It is important to note that externally added enzymes, such as catalase and superoxide dismutase, can only decrease plasma membrane damage caused by hydroxyl and/or superoxide anion radicals, respectively, because enzymes cannot enter the cell in active form. Furthermore, externally added oxidants, such as HzO2, affect the plasma membrane lipids and proteins to a greater extent than internal components. Immediate apoptosis can be triggered by singlet oxygen- or superoxide anion-mediated damage. 5 In either case, the appearance of a significant number of apoptotic cells can be observed immediately after exposure (real time -<15 rain). Dose-response curves (0-1000 kJ/m z) with clonogenic survival data (platting for reproductive capacity) should be obtained first, and the cells should be monitored over a time course beginning immediately after exposure and extending to at least 48 hr. 4 Cyclosporin A (CsA) is used to distinguish between singlet oxygen- and superoxide anion-triggered immediate apoptotic mechanisms because it only inhibits singlet oxygentriggered immediate apoptosisJ In addition, immediate and intermediate apoptotic mechanisms cannot be inhibited with transcription or translation inhibitors because both immediate apoptosis and intermediate apoptosis are prePCD mechanisms. Only delayed apoptosis is inhibited significantly by transcription or translation inhibitors because D N A damage-induced

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apoptosis is a PCD mechanism that partially relies on the synthesis of protein. 4

In Vitro Evidence for Singlet Oxygen For singlet oxygen studies, one sample of cells is suspended in PBS and maintained in the dark (sham exposed), whereas another sample is exposed and compared to other exposed samples that have added reagents. One sample should be suspended in PBS containing an agent that extends the lifetime of singlet oxygen, such as deuterium oxide (10× PBS may be diluted into D 2 0 , 90%, or powdered PBS may be dissolved into D 2 0 , 100%, either solution must be sterile filtered before use). Another sample should contain an agent that quenches singlet oxygen, such as sodium azide (50-100 mM final concentration in PBS). 5 The control for mitochondrial depolarization and uncoupling of oxidation from phosphorylation is sodium cyanide (1 mM), which does not quench or enhance the lifetime of singlet oxygen. However, these reagents should be removed immediately after exposure for three reasons: (1) they can induce, promote, or inhibit an apoptotic mechanism; (2) they can alter mitochondrial function (e.g., azide, deuterium oxide, and cyanide); and (3) they can interfere with certain cellular processes under study and give misleading results. For example, azide also uncouples oxidation from phosphorylation, which decreases intracellular ATP levels and consequently decreases the activity of energy-dependent enzymes. To remove reagents after irradiation, cells are harvested by centrifugation as described in Section II. Other chemicals, such as water-soluble antioxidant glutathione (10 mM), 5 can also be added to the PBS during irradiation to obtain information as to whether ROS are involved at all. The hydrophobic ROS quencher, vitamin E, can determine if oxidative damage to membranes is important, a° However, because vitamin E (type VI, Sigma) is an oil, it must be emulsified first and then preincubated for 2-24 hr with the cells (with slow rocking) to allow incorporation into the membranes. Furthermore, a variety of agents may be used to test for hydroxyl radicals, such as ethanol, dimethyl sulfoxide, histidine, and mannitol, but care should be exercised because most of these reagents also quench singlet oxygen. A good handbook to consult for designing reactive oxygen studies is "Free Radicals in Biology and Medicine. ''11 Figure 1 shows that UVA1 radiation results in the immediate appearance of apoptotic cells (0 in the figure). The presence of a lifetime extender of singlet oxygen (100% D 2 0 ) increased the percentage of apoptotic cells 10D. E. Godar and A. D. Lucas, Photochem Photobiol. 62, 108 (1995). 11 B. Halliwell and J. M. C. Gutteridge, "Free Radicals in Biology and Medicine." Clarendon Press, Oxford, 1991.

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FIG. 1. UVA1 mediates singlet oxygen damage that results in the immediate appearance of apoptotic cells. The percentages of apoptotic cells in sham (left side) and UVA1 (800 kJ/ m2; right side) samples at 0, 4, and 24 hr after exposure are shown. O, PBS; D20, deuterium oxide (100%); N3, sodium azide (50 mM); CN, sodium cyanide (1 mM). Note that DzO increased the percentages of apoptotic cells, whereas N3 decreased the percentages of apoptotic cells and CN had little affect. Results represent means from three separate experiments and error bars are standard deviations (SD).

significantly, whereas a quencher of singlet oxygen (50 mM N3) decreased the percentage of apoptotic cells significantly. In addition, the control for uncoupling oxidation from phosphorylation, sodium cyanide (1 mM), shows that it has little effect on the percentages of apoptotic cells. These results suggest that UVA1 mediates singlet oxygen damage, which triggers immediate apoptosis. The sham controls are shown on the left-hand side of Fig. 1 and the UVAl-exposed cells are shown on the right-hand side of Fig. 1. In both cases the reagents were removed immediately after exposure. The percentages of apoptotic cells were obtained using a flow cytometry procedure described in Section VI,A, but may also be obtained using other methods of detection (see Section VII).

Systems Known to Generate Singlet Oxygen, Superoxide Anions, and~or Hydroxyl Radicals, and Systems That Do Not Involve ROS in the Initiation of Apoptosis Other systems that are known to generate singlet oxygen, superoxide anions, and/or hydroxyl radicals or do not create any reactive oxygen species may be used to reveal which ROS, if any, and which general mechanism of apoptosis a given system produces. For example, singlet oxygen may be generated by photosensitizers exposed to visible light, i.e., PDT. This can be done using natural precursors, such as 6-aminolevulinic acid (ALA), to increase intracellular levels of a photosensitizer, such as protoporphyrin

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IX, I2 or by adding a photosensitizing dye, such as rose bengal. 13 Superoxide anions may be generated using pyruvate (10 mM) with UVA1 radiation, s vitamin 1(3, or paraquat in the dark. aa Hydroxyl radicals (and superoxide anions) may be generated using high doses of UVB. TMHigh doses of UVB a5 and UVC 16 radiation are also reported to cause cross-linking of the Fas receptor on the plasma membrane. Thus, systems that initiate apoptosis through receptor cross-linking should also be used, such as anti-Fas IgM antibodies, to determine if intermediate apoptosis is initiated. In addition, some systems also cause some D N A damage, such as UVA1 radiation, and others almost exclusively cause DNA damage, such as X-rays, and consequently the delayed apoptotic mechanism. Furthermore, some systems exclusively cause D N A damage without any ROS, such as etoposide. Thus, additional systems should be included that are known to cause DNA damage without producing appreciable ROS, e.g., moderate doses of UVB/ U V C , 10 P U V A , 17 X-rays, 5'18 or etoposide. 19 One approach for generating singlet oxygen inside cells is to increase the intracellular concentration of the photosensitizer protoporphyrin IX using ALA, which is a natural biochemical precusor of protoporphyrin IX, but is not a photosensitizer. Cells are preincubated at 37 °, in complete medium with 0.25-1 mM A L A for 2-4 hr, and no longer, 5 to prevent overproduction and delocalization of protophorpyrin IX to the plasma membrane, which causes excessive damage, resulting in necrosis. The increase in protoporphyrin IX may be monitored by fluorescence emission >650 nm (FL3 channel in flow cytometry). I2 The stock solution of A L A is 1 M in DMSO. To generate singlet oxygen, the cells are processed as described and irradiated with at least 100 kJ/m 2 visible light. A higher dose may be used, but it alone should not induce appreciable apoptosis (<-20%). Two sham-exposed controls must be included: one with only A L A and no light and the other with only visible light and no ALA. Another approach for generating singlet oxygen inside cells uses photosensitizing dyes, such as rose bengal, s'13 Cells are preincubated with 10-100 12 E. A. Hryhorenko, K. Rittenhouse-Diakun, and N. S. Harvey et aL, Photochem. Photobiol. 67, 565 (1998). 13 C. R. Lambert and I. E. Kochevar, J. A m . Chem. Soc. 118, 3297 (1996). 14 A. Gorman, A. MeGowan, and T. G. Cotter, F E B S Lett. 404, 27 (1997). 15y . Aragane, D. Kulms, D. Metze, G. Wilkes, B. Poppelmann, T. A. Luger, and T. Schwarz, J. Cell Biol. 140, 171 (1998). 16 A. Rehemtulla, C. A. Hamilton, A. M. Chinnaiyan, and V. M. Dixit, J. Biol. Chem. 272, 25783 (1997). 17 B. R. Vowels, E. K. Yoo, and F. P. Gasparro, Photochem. Photobiol. 63, 572 (1996). 18j. G. Peak and M. J. Peak, Mutat. Res. 246, 187 (1991). 19 S. Kasibhatla, T. Brunner, L. Genestier, F. Echeveri, A. Mahboubi, and D. R. Green, Mol. Cell. 1, 543 (1998).

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tzM rose bengal (Sigma) for 1-2 hr, or overnight, in complete medium, processed as described, and irradiated with about 100 kJ/m 2 or more of visible light. The stock solution of rose bengal can be 1-10 mM in PBS. Two sham-exposed controls must be included: one with only rose bengal and no light and the other with only the light and no dye. To generate superoxide anions, vitamin K 3 (Sigma) is incubated at different concentrations (10/~M-1 mM) with the cells in complete medium in the dark. 5,n The stock solution is 100 mM in DMSO. To generate hydroxyl radicals, use high doses of UVB (>99.99% clonogenic killing; see Section II). To initiate apoptosis via a mechanism that does not involve ROS, but does involve receptor cross-linking, anti-Fas monoclonal IgM antibody, C H l l (Medical & Biological Laboratories, Nagoya, Japan), can be incubated with the cells at the recommended concentration (100 ng/ml) or higher in fresh complete medium. To generate primarily D N A damage with minimal ROS production, one may use low doses of UVB or UVC radiation (<99% clonogenic killing), X-rays (10 Gy; see Section II,B), chemicals, such as etoposide (5 /~M), or PUVA (see later). To generate DNA damage-induced delayed apoptosis by P U V A , 17 cells are preincubated in complete medium with 100-300 ng/ml (200 ng/ml is shown in Fig. 2) 8-methoxypsoralen (8-MOP) for 15-20 min at 37 ° before processing for irradiation with 10-30 kJ/m 2 (30 kJ/m 2 is shown in Fig. 2) of U V A R from the photoactivation chamber (see Section II). The stock solution of 8-MOP is 200 ~g/ml (or 1000 times higher than that used) in ethanol or DMSO. Two sham-exposed samples must be run: one for 8MOP alone and the other for U V A R alone. The latter will have some immediate apoptosis due to the U V A present in this source, which may be subtracted from the PUVA values to obtain only the effect of PUVA. Alternatively, the U V A R dose may be reduced (e.g., 10 kJ/m 2) and the photosensitizer increased (e.g., 300-400 ng/ml). Figure 2 shows a time course (0-24 hr) after exposure of cells to a variety of different initiators of apoptosis. Note that only singlet oxygengenerating systems, such as protoporphyrin IX (ALA in Fig. 3) and rose bengal, trigger immediate apoptosis, as does UVA1 radiation. Vitamin K and high doses of UVB initiated an intermediate mechanism, whereas moderate dose UVB, PUVA, and X-rays all initiated delayed apoptosis. Collectively, the results in Fig. 1 and 2 show that only single oxygen damage leads to the immediate appearance of apoptotic cells unlike damage produced by other ROS (intermediate and delayed) or damage to D N A that does not involve ROS (delayed).

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lOO

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FIG. 2. Various initiators of apoptosis. Cells were treated as described in Section V,B. All sham-exposed controls were within 5% of the PBS sham, except the U V A R control for PUVA, which had the same value as PUVA immediately and at 4 hr after exposure. Sham, PBS; ALA, 6-aminolevulenic acid (0.5 mM, 2-hr preincubation) and 100 kJ/m 2 visible light, Rose, rose bengal (50 t~M, 1-hr preincubation) and 100 k J / m z of visible light; UVA1, UVA1 radiation (800 kJ/m2; 95% clonogenic killing); Vit K, vitamin K 3 (100/~M) in the dark; UVB Hi, highdose UVB (500 j/m2; >99.99% clonogenic killing); Fas, anti-Fas IgM antibody (CHll, 100 ng/ml); UVB, moderate-dose UVB (100 j/m2; 95% clonogenic killing); PUVA, 200 ng/ml 8-MOP and 30 kJ/m 2 of UVAR; and Xray, X-rays (10 Gy: >99.99% clonogenic killing). Note that only the singlet oxygen-generating systems, ALA and rose bengal, caused immediate apoptosis like UVA1 radiation. Results represent means of three separate experiments and error bars are SD.

Specifically Inhibiting Singlet Oxygen-Triggered Immediate Apoptosis CsA only inhibits singlet oxygen-triggered immediate apoptosis. It blocks the release of apoptosis initiating factor (AIF), a 50-kDa protease, from the mitochondrial megachannel,z° A final concentration of 10-100 ~g/ml (8-80/zM) CsA is preincubated with the cells at 37° in complete medium for 30-45 min, and no longer, because it is only effective for short periods of time (<2 hr). In addition, 10-100/zg/ml of CsA is present during and after exposure. The stock solution is 10 mg/ml in ethanol or DMSO. The sham-exposed cell sample and other samples that did not receive CsA had the same final percentage of solvent (1% here) before, during, and after irradiation because ethanol and DMSO can quench hydroxyl radicals. Furthermore, ethanol leads to increases in NADH levels through the action of alcohol dehydrogenase, which inhibits superoxide anion-triggered immediate apoptosis by UVA1 radiation (400 kJ/m 2) combined with pyruvate (10 mM). 5

20 G. Kroemer, N. Zamzami, and S. A. Susin, Immunol. Today. 18, 44 (1997).

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Figure 3 shows that 100/xg/ml (about 80/zM) CsA only inhibits singlet oxygen (ALA PDT and UVA1 radiation)-triggered immediate apoptosis, whereas it has little effect on superoxide anion-triggered (vitamin K3) or receptor-initiated (anti-Fas antibody) intermediate apoptosis. Although not shown in Fig. 3, CsA also significantly inhibits singlet oxygen-triggered immediate apoptosis generated by rose bengal and visible light. In contrast, it has no inhibitory effect on superoxide anion-triggered immediate apoptosis generated by either UVA1 radiation combined with pyruvate or high concentrations of vitamin K3 (1 mM) in the dark. 5 Furthermore, CsA does not inhibit any intermediate or delayed apoptotic mechanism and can increase the percentages of these cells. Note that concentrations of CsA as low as 8/xM may be used to inhibit singlet oxygen-triggered immediate apoptosis.

Assessing Preprogrammed and Programmed Cell Death Mechanisms of Apoptosis Preprogrammed cell death is an apoptotic mechanism that relies solely on proteins that are synthesized constitutively, 4 whereas programmed cell death is an apoptotic mechanism that also relies on the synthesis of new

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FIG. 3. CsA inhibits only singlet oxygen-triggered immediate apoptosis. All samples had the same final concentration of ethanol present (1%). The ALA and visible light sham-exposed controls were within 5% of that shown as the Sham(-). Sham, sham-exposed PBS cells without ( - ) CsA and with (+) CsA (100/zg/ml); ALA, &aminolevulenic acid (1 mM, 2-hr preincubation) and 100 kJ/m 2 of visible light without ( - ) CsA and with (+) CsA (100/xg/ ml); UVA1, UVA1 radiation (800 kJ/m 2) without ( - ) CsA and with (+) CsA; Vit K, vitamin K3 (100/zM) in the dark without ( - ) CsA and with (+) CsA (100/zg/ml); Fas, anti-Fas IgM antibody (CH11, 100 ng/ml) without ( - ) CsA and with (+) CsA (100 p~g/ml). Note that only singlet oxygen-triggered immediate apoptosis (ALA PDT and UVA1 radiation) is inhibited by CsA, whereas superoxide anion-/hydroxyl radical or receptor-initiated intermediate apoptosis (vitamin K 3 or anti-Fas antibody, respectively) are not affected. Results represent means of three separate experiments and error bars are SD.

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protein. 2 The best way to determine if an apoptotic mechanism is prePCD or PCD is to inhibit translation with cycloheximide (CHX) because it does not inhibit aminopeptidases like puromycin. 2I However, great care must be taken not to inhibit translation much more than 50% or else the cells start to undergo apoptosis (after 4 hr) from the presence of the inhibitor. 4'5 This general concept applies to all other inhibitors as well. Some preliminary testing should be done to find a suitable concentration of CHX that inhibits translation by about 50%, while it does not cause appreciable apoptosis (-<15%) in the controls when present for 48 hr. 4 Low concentrations around 0.1/zg/ml are effective on leukemia and lymphoma cells and are a good starting point for finding the correct concentration for other cell types. The correct concentration should be verified by quantifying the amount of [35S]methionine incorporated into newly synthesized proteins. 4 When the concentration is approximately correct, CHX-treated cells will only incorporate about half the amount of [35S]methionine into trichloroacetic acid-precipitable protein as the controls. In addition, the effect of CHX on translation should be monitored before every experiment by [35S] methionine labeling. Be sure to add some fresh media, with or without CHX, for overnight growth. Note that some adherent cells detach during this procedure, which may be irradiated in the detached state. Alternatively, shorter preincubation (4 hr) with CHX can be used for adherent cell types. Once the correct concentration of CHX has been determined, it is preincubated with the cells for at least 4 hr, or overnight (12-24 hr), for irradiation by UVA1, PDT, or X-rays. However, if PUVA, UVB, or UVC exposures will be used, the CHX should be added immediately after exposure because it absorbs short wavelength UV. Use a UVA1 or PDT dose that gives 30-50% apoptosis at almost zero time postexposure so that significant differences may be observed. Other apoptotic mechanisms will require longer postexposure observation times (e.g., 4-48 hr). In addition, include at least one positive control for intermediate apoptosis, such as high doses of UVB (>99.99% clonogenic death; 500 kJ/m2), anti-Fas IgM antibody (CH 11 at 100 ng/ml), and/or staurosporine (1 tzM), and at least one positive control for delayed apoptosis, such as moderate dose UVB (90-99% clonogenic death; 100 kJ/m2), etoposide (5 /zM), PUVA (see Section IV,B), or X-rays (10 Gy; Section II,B). Figure 4 shows that neither the UVAl-triggered immediate nor the high-dose UVB-initiated intermediate apoptotic mechanisms are inhibited significantly by CHX (0.08-0.1 tzg/ml). However, after 24 hr, a difference is observed, which suggests that both UVA1 radiation and high doses of 21 D. B. Constam, A. R. Tobler, A. Rensing-Ehl, I. Kemler, L. B. Hersh, and A. Fontana, J. Biol. Chem. 270, 26931 (1995).

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Fic. 4. Preprogrammed and programmed cell death mechanisms of apoptosis determined by inhibiting translation with cycloheximide (CHX). Cells were pretreated without ( - ) or with (+) CHX (0.1 /zg/ml) overnight before exposure to UVA1 (800 kJ/m2), Fas (anti-Fas IgM antibodies; C H l l , 100 ng/ml), or Etop (etoposide, 5/,M). CHX was added immediately after UVB exposure (100 and 500 J/m 2) because it absorbs UVB (and UVC) radiation. Similar results for anti-Fas antibodies and etoposide were obtained if the CHX was added immediately after the addition of either reagent. Results are also shown for 48 hr because the UVBinduced delayed apoptotic mechanism, like X-rays, requires more time to display a significant difference. Results represent means of three separate experiments and error bars are SD.

UVB radiation also initiate a delayed apoptotic mechanism by causing D N A damage. Intermediate apoptosis initiated by anti-Fas IgM antibodies is also a prePCD mechanism of apoptosis because it is not inhibited by CHX. Moderate doses of UVB (100 J/m 2) or etoposide (5/zM) cause D N A damage and are positive controls for PCD because they induce a delayed apoptotic mechanism (24-48 hr) that is inhibited by CHX to the same extent as protein synthesis (about 50%). Note that the UVB-induced delayed apoptotic mechanism requires at least 48 hr to display a significant difference. Flow Cytometry Procedures The flow cytometry method described here relies on the differences in size between apoptotic and morphologically normal cells. This procedure is recommended for regularly shaped cells (round), such as most lymphomas and leukemias, and some adherent cell types, such as L929. However, it is difficult to separate apoptotic and morphologically normal cell populations of irregularly shaped cells, such as the human T-helper cell lymphoma, H9, or some large adherent cell types (that must also be analyzed shortly after preparation to minimize "clumping"). In addition, some inducers of apoptosis, such as staurosporine, etoposide, and anti-Fas-initiated

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apoptosis, cause a diverse distribution of shapes and sizes of cells undergoing apoptosis that makes the analysis tricky and less reliable. For these other cell types and inducers of apoptosis, another method for detecting apoptosis, such as Annexin V binding of phosphatidylserine residues expressed on the outer leaflet of the plasma membrane and/or T U N E L labeling of digested DNA, must also be used to confirm these flow cytometry data. These other methods will not be discussed here because kits with detailed instructions are available from a variety of companies.

Analyzing Apoptotic and Morphologically Normal Cells Separately Flow cytometry can be performed using a single beam 488-nm excitation source, e.g., FACscan (Becton-Dickinson, San Jose CA). Apoptotic and morphologically normal cell percentages are determined using a flow cytometric method described previously. 22 At least 10,000 cells are collected and analyzed using suitable software, such as LYSYS II or CellQuest, according to the side light-scattering (SSC) profile (proportional to cellular granularity) versus the forward light-scattering (FSC) profile (proportional to cellular cross-sectional area or size). Depending on the inducer, the apoptotic cells can be more granular, but are always smaller, than the normal cell population. The FSC and SSC settings must be adjusted for the different inducers of apoptosis and cell types to achieve a distinct separation between these two populations of cells. This can be accomplished by first centering (between 400 and 600 on a linear scale) the sham-exposed cells on the FSC (size) and then changing the SSC (granularity) and FSC settings by comparing both sham and exposed populations until adequate separation is achieved between them. The morphologically normal cell population may have to be adjusted up field in some cases (centered around 600), especially for large adherent cells and irregularly shaped cells, such as H9 lymphomas. Once the optimum settings are determined, data are collected in the linear mode for both SSC and FSC. For analysis, the region marker cutoffs between populations are best determined by comparing both sham and exposed (highest dose) populations in adjacent windows. This method can be verified either by sorting these two populations and analyzing the different end points associated with apoptosis or by analyzing different end points, such as Annexin V or TUNEL. An example of a SSC (granularity) versus FSC (size) profile is shown in Fig. 5A as a dot plot with polygon region markers separating apoptotic and morphologically normal cell populations after UVA1 irradiation of Jurkat T-helper cells. 22 C. Dive, C. D. Gregory, D. J. Phipps, D. L. Evans, A. E. Milner, and A. H. Wyllie, Biochim. Biophys. Acta 1133, 275 (1992).

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FIG. 5. UVA1 irradiation of transformed human T-helper cells (Jurkat) immediately (T -< 20 min) reduces the mitochondrial transmembrane potential (A~m) in a dose-dependent manner. The A~Irm w a s monitored using a specific probe, JC-1, as described in Section VI,B. (A) The side (granularity) versus the forward (side) light-scatter profile of a 600-kJ/ m2 UVA1 exposed sample immediately after exposure: the apoptotic cells are smaller (downfield) than the normal population as displayed by the region markers in the dot plot. (B) The Aalfm of only the apoptotic cells in a yellow/orange (FL2) versus green (FL1) fluorescent profile after gating on this population in the side versus the forward light-scatter profile as shown in A. Apoptotic cells display a low/x~ m as indicated in the polygon region marker. (C) Morphologically normal cells, after gating from A, immediately following 600 kJ/m2 UVA1 radiation: these cells show both low and high A~m. (D) Almost all of the morphologically normal sham-exposed cells have a high A~m. (E and F) The A~l~rn of morphologically normal cells immediately after 400 or 800 kJ/m2 of UVA1 radiation, respectively. Similar results were obtained in three separate experiments.

Analysis of Intracellular Organelles: Mitochondria V a r i o u s flow c y t o m e t r y grade fluorescent dyes are n o w available to e x p l o r e changes to different organelles a n d e n d p o i n t s , such as p H , Ca 2+, i n t r a c e l l u l a r G S H , a n d increases in R O S , to m e n t i o n b u t a few ( M o l e c u l a r P r o b e s , E u g e n e , O R ) . F l u o r e s c e n t dyes that e m i t p r i m a r i l y in o n e fluores-

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UVA1-TRIGGERED IMMEDIATE PREPROGRAMMED APOPTOSIS

327

cent channel can be used so that double labeling allows another end point to be monitored simultaneously in another fluorescent channel. Alternatively, confocal microscopy may be used to monitor mitochondrial or other organelle changes. Fluorescent microscope pictures of these cells can be obtained to verify and document the organelle changes. Dramatic changes in the mitochondrial transmembrane potential (A~m) can be measured using a specific fluorescent probe, such as 5,5',6,6'-tetrachloro-l,l',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR). 23 The cells are incubated at 37 ° for about 10 min with JC-1 at a final concentration of 1 ~g/ml. 5 The cells are not washed to remove the unbound dye after incubation because a threshold setting of 52 does not allow the flow cytometer to count the free fluorescent particles as data. The JC-1 stock solution is 1 mg/ml in DMSO, so that 1/zl is added to 1 ml of cells. Stock solutions (10×; 10 mg/ml) may be stored at - 2 0 ° for extended periods of time (1 year or more), and the diluted solution (1 mg/ml), protected from light, is stable for up to a month or more at room temperature. The FACScan settings can vary dramatically between instruments, cell types, and procedures. The following initial settings are recommended for leukemia and lymphoma cells: FL1 (530 --- 15 nm) in log data collection mode with a PMT setting around 425 and FL2 (585 _ 20 nm) in log data collection mode with a PMT setting around 310. Although compensation is not shown in Fig. 5 because it is not necessary for accurate quantification, the different populations may be separated further for better qualitative presentation by adjusting the compensation: FL1-FL2% is recommended at 1.2-1.4 and FL2-FLI% is recommended at 22.4-23.2 for Jurkat T cells. Once suitable settings are obtained, data are collected from 10,000 cells and a separate analysis of each population is achieved by gating on that cell population in the FSC versus SSC profile as shown in Fig. 5A. Each population is then analyzed for mitochondrial changes (A~m) using twodimensional dot plots of the yellow/orange (FL2) versus the green (FL1) fluorescent profiles as shown in Figs. 5B-F. Figure 5B shows a UVA1exposed sample where all the apoptotic cells display a low mitochondrial transmembrane potential (A~m), whereas Fig. 5C shows that morphologically normal cells have both low and high A~m'S. Figure 5D shows a shamexposed sample where almost all of the morphologically normal cells have a high A~m, but as the dose of UVA1 radiation increases, the percentages of cells with a high A~xtm decreases (compare Figs. 5D, 5E, 5C, and 5F). Figure 6 shows that UVA1 radiation causes a dose- and time-dependent decrease of the Aq~min the morphologically normal cell population. Unlike 23 S. Salvioli, A. Ardizzoni, C. Franceschi, and A. Cossarizza, FEBS Lett. 411, 77 (1997).

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80

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FIG. 6. UVAl-mediated singlet oxygen damage to mitochondrial membranes reduces the inner transmembrane potential (A~m)in a dose- and time-dependent manner. UVA1 radiation causes a dose- and time-dependent drop in the Aq~mof morphologically normal cells after gating on this population as shown in Fig. 5A. Results represent means from three separate experiments and error bars are the SD. other inducers of apoptosis, singlet oxygen causes a drop in the m~ttmbefore the cells display typical morphological characteristics of apoptosis. For most other apoptotic mechanisms, the AxI't m drops while the cells are decreasing in size (or after). C o m p a r e mxI-tm changes over a time course using other inducers of apoptosis.

Discussion Unlike other initiators of apoptosis, singlet oxygen causes damage to a variety of cellular components, particularly membranes. Further technical damage to the plasma m e m b r a n e s can give misleading results because m o r e cells undergo apoptosis and some appear necrotic or lyse during processing. To assure that reliable results are obtained, m a n y c o m m o n methods must be modified so that the cells do not acquire m o r e plasma m e m b r a n e damage from these technical procedures. Both U V A 1 radiation 24 and P D T 25 mediate the production of singlet oxygen. 5 Although some reagents, such as deuterium oxide or azide (Fig. 1), and other approaches (Fig. 2) can suggest that singlet oxygen m a y be involved in a given system, only CsA has been found to specifically inhibit singlet oxygen-triggered immediate apoptosis (Fig. 3). 5 I m m e d i a t e apoptosis occurs very rapidly (T -< 30 min) and is a p r e P C D mechanism of apoptosis because it does not rely on the synthesis of new proteins to 24A. Morita, T. Werfel, and H. Stege et al., J. Exp. Med. 186, 1763 (1997). z5D. Kessel and Y. Luo, J. Photobiol. Photochem. 42, 89 (1998).

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proceed (Fig. 4). Singlet oxygen causes damage to a variety of cellular components, but immediate prePCD apoptosis is triggered primarily by mitochondrial membrane damage. This damage causes the mitochondrial transmembrane potential to immediately depolarize (Figs. 5 and 6). Once depolarized, the mitochondrial megachannel opens at the so-called "S", or sulfhydryl sensitive, site 26 and apoptosis-initiating factor (AIF) is released, unless CsA is present. 2° Unlike singlet oxygen-triggered immediate apoptosis, superoxide anion (1 mM vitamin K3)-triggered immediate apoptosis and superoxide anion- or hydroxyl radical-initiated intermediate apoptosis cannot be inhibited with CsA. Moreover, other systems that initiate apoptosis cannot be inhibited by CsA, such as receptor (e.g., antiFas antibody, CH ll)-initiated intermediate apoptosis or D N A damageinduced delayed apoptosis (e.g., etoposide). CsA does not inhibit other mechanisms of apoptosis because they do not rely on the release of AIF, but rather rely on the release of cytochrome c27 after Bax forms a pore 28 at the "P," or pyrimidine nucleotide-sensitive, site of the mitochondrial megachannel. 26 Antioxidants, such as glutathione, 5 or some antioxidant proteins associated with the outer mitochondrial membrane, such as Bcl2, can inhibit UVA1-29 and PDT-mediated 3° immediate apoptosis. Bcl-2, and family members, can also inhibit many other mechanisms of apoptosis. Details of the different apoptotic mechanisms, i.e., immediate prePCD, intermediate prePCD, and delayed PCD are discussed elsewhere. 31 Flow cytometry can be used to determine the order of biochemical events that occur before, during, and/or after the apoptotic morphology occurs, i.e., cell shrinkage. Flow cytometry allows separate analysis of the apoptotic and morphologically normal cells while analyzing various end points. This method relies on the difference in size between apoptotic and morphologically normal cells that is determined by forward (size) versus side (granularity) light-scattering profiles. Unlike other methods that analyze the entire population of cells and average end points associated with apoptosis, this method segregates events that occur before, during, and after cell shrinkage, the salient morphological feature of apoptotic cells. A

26 p. Costantini, B. V. Chernyak, V. Petronilli, and P. Bernardi, J. Biol. Chem. 271, 6746 (1996). 27 E. Bossy-Wetzel, D. D. Newmeyer, and D. R. Green, E M B O J. 17, 37 (1998). 28 j. M. Jurgensmeier, Z. Xie, Q. Deveraux, L. Ellerby, D. Bredesen, and J. C. Reed, Proc. Natl. Acad. Sci. U.S.A. 95, 4997 (1998). 29 C. Pourzand, G. Rossier, O. Reelfs, C. Borner, and R. M. Tyrrell, Cancer Res. 57, 14051 (1997). 30 j. He, M. L. Agarwal, H. E. Larkin, L. R. Friedman, L. Y. Xue, and N. L. Oleinick, Photochem. PhotobioL 64, 845 (1996). 31 D. E. Godar, J. Invest. Dermatol. Symp. Proc. 4, 17 (1999).

330

ULTRAVIOLETA

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variety of flow cytometry methods 32 allow different cellular changes to be monitored, such as external and internal antigens, pH, Ca 2+, and organelles, like mitochondria, while they occur in normal and apoptotic populations. Because singlet oxygen causes damage to mitochondrial membranes resuiting in immediate depolarization of the inner mitochondrial transmembrane potential, measuring mitochondrial transmembrane potential gives a powerful tool to help distinguish singlet oxygen-triggered immediate apoptosis from other apoptotic mechanisms. In addition, the order of signaling events that occur before and after mitochondrial transmembrane depolarization, such as gene induction or activation of the different caspase cascades, can be determined. Thus, biochemical events that initiate the apoptotic morphology can be segregated from those that execute morphological changes. In addition, other flow cytometry methods for detecting apoptosis can help confirm the order of events. For example, Annexin V can determine if an event occurs before or after phosphatidylserine residues are expressed on the outer leaflet of the plasma membrane or TUNEL can determine if an event occurs before or after DNA fragmentation. 33 These methods will help investigators determine which ROS, if any, are responsible for initiating which apoptotic mechanism, i.e., immediate prePCD, intermediate prePCD, or delayed PCD. In addition, the different biochemical changes, such as gene activation, signal transduction, and caspase activation, may be analyzed separately in apoptotic and morphologically normal cells to help determine the order of events. This is particularly important for singlet oxygen-triggered immediate apoptosis because it occurs so rapidly, especially to cancer cells. Since the therapeutic relevance of UVAl-mediated singlet oxygen-triggered immediate apoptosis has recently been noted, e4 understanding how this mechanism works may help improve future clinical applications of UVA1 phototherapy.

32 Z. Darzynkiewicz and H. A. Crissman (eds.), in "Methods in Cell Biology," Vol. 33. Academic Press, New York, 1990. 33 T. G. Cotter and S. J. Martin (eds.), "Techniques in Apoptosis: A Users Guide." Portland Press, London, 1996.