Studying arsenic trioxide-induced apoptosis of Colo-16 cells with two-photon and confocal laser scanning microscopy

With two-photon and confocal laser scanning microscopy in combination with fluorescent probes Hoechst 33342, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and Fluo 3-AM, we simultaneously observed arsenic trioxide (As2O3)-induced changes in nuclear morphology, reactive oxygen species (ROS) and intracellular calcium concentration [Ca 2+ ]i within human skin squamous carcinoma cells (Colo-16 cells). Our results indicated that As2O3 induced [Ca 2+ ]i elevation and ROS production within Colo-16 cells, and both [Ca 2+ ]i elevation and ROS production were involved in the apoptosis of Colo-16 cells. These results suggested that two-photon and confocal laser scanning microscopy might provide a real-time, sensitive and noninvasive method for simultaneously multi-parameter observation of As2O3induced apoptosis at the single cell level.


INTRODUCTION
Although arsenic is poisonous and chronic arsenic exposure from industrial or natural sources can cause serious toxicity, arsenic has been used therapeutically for more than 2,400 years (Klaassen, 1996); in traditional Chinese medicine, arsenic trioxide (As 2 O 3 ) is used to treat syphilis, rheumatosis, and psoriasis (Shen et al., 1997).Recent researches demonstrate that As2O3 induces partial cytodifferentiation and triggers apoptosis of the leukemia cells, leading to high clinical and molecular remission rates in patients with relapsed acute promyelocytic leukemia (APL) (Soignet et al, 1998;Muto et al, 2001;Zhu et al, 1997), and As2O3 have been approved by Food and Drug Administration of U.S.A. for use in the treatment of relapsed/refractory APL.Fluorescence microscopy is an essential tool of modern biology and has emerged as a novel and noninvasive method for visualization of living cells and biological tissue (Gustafsson, 1999).The effective sensitivity of fluorescence microscopy measurements is often limited by out-of-focus flare.This limitation is greatly reduced in a confocal laser scanning microscopy, where the out-offocus background is rejected by a confocal pinhole to produce thin ( 1 m) and unblerred optical sections from within thick samples (White et al., 1987).The two-photon laser scanning microscopy is a new alternative to confocal microscopy and is also a promising technique to observe biological specimens because of its inherent advantage such as three-dimensional resolution without a confocal pinhole, less photobleaching above and below an observing plane and long depth penetration with nearinfrared light excitation (Denk et al., 1990(Denk et al., , 1995)).In twophoton laser scanning microscopy, the fluorophore is excited by a focus beam of light of approximately twice the wavelength of the normal excitation light.For example, a fluorophore that normally absorbs ultraviolet light (-350 nm) can be excited by two red photons (-700 nm) if they reach the fluorophore at the same time (-10 -18 seconds).Therefore, two-photon laser scanning microscopy in combination with confocal laser scanning microscopy might provide the possibility for simultaneously measurement of multi-labeling biological specimens based on the difference between excitation or emission wavelengths of the fluorescent probes.
Although some Chinese medicines used to cure skin cancer contain As 2 O 3 and show good therapeutic efficacy (Gilman et al., 1996), its accurate mechanism is still unknown.In this paper, two-photon and confocal laser scanning microscopy were used to study As 2 O 3 -induced changes in nuclear morphology, reactive oxygen species (ROS) and intracellular calcium concentration (Ca 2+ ) I within human skin squamous carcinoma cells (Colo-16 cells).Our results indicated that As 2 O 3 induced concentration-dependent apoptosis of Colo-16 cells and both As 2 O 3 -induced (Ca 2+ ) i elevation and ROS production involved in the apoptosis of Colo-16 cells, suggesting that two-photon and confocal laser scanning microscopy might throw new insight into the anti-skin-cancer mechanism of As 2 O 3 .

Cell culture
The human skin squamous carcinoma cell lines (Colo-16 cells) were provided by the Department of Biology, Beijing Normal University.All cells were cultured in RPMI 1640 containing 10% fetal bovine serum in an incubator containing 5% CO2.Exponentially growing cells were used in all the experiments.

Cell viability assays
100 µl of cell suspension (1×10 4 Colo-16 cells) was seeded into 96well tissue culture plates containing different-concentration of As2O3.After treatment for 12, 24, 36, 48 and 60 h separately, 10 µl of MTT [5 mg/ml in phosphate-buffered saline (PBS)] was added into each well.After 4 h of incubation at 37°C, the formazan crystals produced in viable cells were dissolved with 100 µl of 10% SDS.OD540 was measured on a spectrophotometer to determine the relative cell viability.

Detection of DNA fragmentation
Colo-16 cells at a density of 10 6 cells/ml were treated with differentconcentration of As2O3 for 24 h and then were collected by centrifugation at 2500 g for 5 min.The resultant cell pellets were resuspended in phosphate-buffered saline (PBS) buffer containing 5 mM MgCl2 and lysed in 500 l of TE buffer containing 0.1% SDS and 1.5 mg/ml proteinase K overnight at 37°C.After two successive extractions with phenol-chloroform, the aqueous layer was transferred to a new centrifuge tube.The DNA was precipitated with ethanol, resuspended in 100 l water and treated with 400 g/ml RNase A for 2 h at 37°C.Electrophoresis was performed in 1% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA).After electrophoresis, the gel was stained with 1 mg/ml ethidium bromide, watched and photographed under UV light.

Measurement of (Ca2 + )i with confocal laser scanning microscopy
Cells were preloaded with 5 M Fluo 3-AM in RMPI 1640 for 1 h at 37°C.After washing with HBSS three times, cells were treated with 10 M As2O3 and images of (Ca 2+ )i were recorded every 20 s for up to 10 min with confocal laser scanning microscopy at 488 nm excitation and 525 nm emission.
Fluorescence from one field of cells per cover slip was typically recorded under different experimental conditions.After acquiring the images of baseline fluorescence levels, 10 M As2O3 was applied and 6 additional images were obtained every 5 min.

Cell viability assays
When Colo-16 cells were cultured for 24 to 60 h with As 2 O 3 (1 to 12 μM), cell viability were analyzed by MTT and the viable cell number was compared with the untreated control.Figure 1 shows that the viable cell number decreased with both treatment-time and As 2 O 3concentration increased suggesting that the cytotoxic effect of As 2 O 3 on Colo-16 cells was both time and concentration dependent.

Detection of DNA fragmentation with electrophoresis and nuclear morphological observation with twophoton laser scanning microscopy
Cells die either following necrosis or apoptosis.Apoptosis (programmed cell death) is an important feature of normal development as well as various disease states, particularly cancer (Kerr et al., 1994).Apoptosis is distinguished from necrosis by characteristic morphological and biochemical changes, including compaction and fragmentation of the chromatin, cell shrinkage and fragmentation into membrane-bound apoptotic bodies, the activation of cysteine proteases such as caspases and internucleosomal fragmentation of DNA by selectively activated DNases (Kerr et al., 1994).
To assess whether Colo-16 cells underwent apoptosis after exposure up to 10 M As 2 O 3 , we examined the DNA nucleosomal fragmentation via agarose gel electrophoresis (Figure 2) and nuclear morphology via twophoton laser scanning microscopy (Figure 3).The biochemical hallmark of apoptosis is the degradation of DNA by endogenous DNases, which cut the internucleosomal regions into double-stranded DNA fragments of 180 to 200 bp (characteristic DNA ladder in gel electrophoresis) (Kerr et al., 1994).The results (Figure 2) showed that no DNA ladder appeared with As 2 O 3 concentration of less than 8 M, a faint DNA ladder emerged with 8 M As 2 O 3 and distinct DNA ladder appeared with 10 M As 2 O 3 .The result demonstrated that As 2 O 3 induced the apoptosis of Colo-16 cells in a concentration-dependent manner.
As shown in Figure 3, the nuclear morphological observation showed that all the examined cells without As 2 O 3 treatment had the normal nuclear morphology (Figure 3a).After treatment with 4 M As 2 O 3 for 24 h, some of the examined cells had characteristic chromatin condensation and apoptotic body formation (Figure 3b). Figure 3c shows that after treatment with 10 M As 2 O 3 for 24 h, most of the examined cells had apoptotic body formation.These results further confirmed that As 2 O 3 induced apoptosis of Colo-16 cells in a concentration-

Measurement of (Ca2 + ) i with confocal laser scanning microscopy
It had been demonstrated that increase in (Ca 2+ ) i played an important role in the induction of Ca 2+ -dependent endonucleases and in initiation of apoptosis (McConkey et al., 1989(McConkey et al., , 1988)).Simultaneously, observation of nuclear morphological changes via two-photon laser scanning microscopy and (Ca 2+ ) i changes via confocal laser scanning microscopy revealed that the apoptotic cells showed high Fluo-3 fluorescence after 10 M As 2 O 3 treatment for 24 h (Figure 4), and this indicated (Ca 2+ ) i elevation in the apoptotic cells.To confirm whether the (Ca 2+ ) i elevation was induced by As 2 O 3 stimulation, we studied As 2 O 3 -induced changes in (Ca 2+ ) i in a real time with confocal laser scanning microscopy.The results showed that As 2 O 3 rapidly increased (Ca 2+ ) i over the treatment time (Figure 5).Both Figures 4 and 5 suggested that As 2 O 3 -induced (Ca 2+ ) i elevation was involved in the apoptosis of Colo-16 cells.

Measurement of reactive oxygen species (ROS) with confocal laser scanning microscopy
Accumulating evidence pointed to ROS as being second messengers in the activation of enzymes, transcription factors, growth, differentiation and apoptosis (Suzuki et al., 1997;Finkel, 1998;Zhang et al., 2001).In this study, fluorescent probe H 2 DCF-DA was used to study As 2 O 3induced ROS production.H 2 DCF-DA was not sensitive to the oxidizing agents; when H 2 DCF-DA was added to the cells, it diffused across the cell membrane and was hydrolyzed by intracellular esterases to H 2 DCF; upon oxidation, H 2 DCF yielded highly fluorescent DCF (Zhu et al., 1994).To assess whether ROS was involved in the As 2 O 3 -induced apoptosis of Colo-16 cells, we simultaneously observed the nuclear morphological changes via two-photon laser scanning microscopy and ROS production via confocal laser scanning microscopy.The results showed that DCF fluorescence increased in the apoptotic cells with chromatin condensation and apoptotic body formation (Figure 6), indicating ROS production in the apoptotic cells.To confirm whether the ROS formation was induced by As 2 O 3 stimulation, we  was loaded without As 2 O 3 addition (Figure 7).Both Figures 6 and 7 suggested that As 2 O 3 -induced ROS was involved in the apoptosis of Colo-16 cells.

DISCUSSION
Arsenic trioxide (As 2 O 3 ) has been widely used in the clinic treatment of relapsed/refractory acute promyelocytic leukemia (APL); however, exactly how As 2 O 3 mediates its clinical efficacy is not fully understood.Two main mechanisms of action of As 2 O 3 have been identified from both in vivo and in vitro studies: promotion of APL cell differentiation (observed at low levels of As 2 O 3 ) and induction of apoptosis (observed at high levels of As 2 O 3 ) (Chen et al., 1997;Shao et al., 1998).The therapeutic potential of As 2 O 3 might be extended to malignant diseases beyond APL.As 2 O 3 has been reported to have direct cytotoxic effects on different cancer cell lines including leukemic, lymhoma and a variety of solid tumor cell lines in high concentrations (Lee and Ho, 1994).This study showed that As 2 O 3 had direct cytotoxic effect on human skin squamous carcinoma cells (Colo-16 cells) in both concentration-and time-dependent manner (Figure 1).Further study with both agarose gel electrophoresis (Figure 2) and two-photon laser scanning microscopy (Figure 3) demonstrated that As 2 O 3 induced apoptosis of Colo-16 cells at higher concentration (> 8 M), suggesting that apoptosis appeared to be the main phenomenon resulting in significant cell death at higher concentrations of As 2 O 3 .As 2 O 3 -induced apoptosis occurs via a variety of mechanisms (Miller, 2002).It has been reported that superoxide generation was the main mechanism of apoptosis by As 2 O 3 in several experimental models (lee and Ho, 1994;Lynn et al., 2000;Jing and Dai, 1999).Generation of reactive oxygen species can cause loss of mitochondrial membrane potential with subsequent changes in membrane permeability and arsenic trioxide can further inhibit glutathione peroxidase and increase cellular hydrogen peroxide content (Jing and Dai, 1999).Our study with the two-photon and confocal laser scanning microscopy (Figure 4) demonstrated that ROS production increased in the apoptotic cells with chromatin condensation and apoptotic body formation and real-time analysis with confocal microscopy (Figure 5) showed that ROS increased greatly over time in response to As 2 O 3 stimulation, suggesting that As 2 O 3 -induced ROS was involved in apoptosis of Colo-16 cells.The increase in (Ca 2+ ) i played an important role in the induction of Ca 2+dependent endonucleases and in the initiation of apoptosis (McConkey et al., 1989(McConkey et al., , 1988)), although in some cases, apoptosis proceeds in the absence of (Ca 2+ ) i changes (Whyte et al., 1993).It has been reported that intracellular calcium change was involved in As 2 O 3induced apoptosis in three myelocytic leukemia cell lines (Wei et al., 2001).Our study observed with two-photon and confocal laser scanning microscopy (Figure 6) demonstrated that (Ca 2+ ) i was increased in the apoptotic cells with chromatin condensation and apoptotic body formation.Real-time analysis with confocal microscopy (Figure 7) showed that (Ca 2+ ) i increased greatly over time in response to As 2 O 3 stimulation, suggesting that As 2 O 3induced (Ca 2+ ) i elevation was involved in the apoptosis of Colo-16 cells.
Apoptosis occurs in many different cell types and in response to diverse stimuli including DNA damage, intracellular damage, toxins and extracellular signals (Kerr et al., 1994).Apoptosis has become a target subject for understanding cellular mechanisms of many diseases, as well as for developing new drugs that interfere with either proapoptotic or antiapoptotic molecular networks.Consequently, it has become important to develop reliable assays to detect apoptosis.Techniques currently available for apoptosis detection are based on the study of morphology of apoptotic cells (light and fluorescence microscopy coupled to nuclear staining with specific dyes and electron microscopy), DNA fragmentation detected by electrophoretic techniques and terminal transferasemediated dUTP nick-end labeling (TUNEL) and membrane changes detected by annexin V in vivo labeling and on immunological assays using antibodies directed to apoptosis-related proteins (Stadelmann and Lassmann, 2000).However, all the earlier mentioned techniques detect only one specific feature of the process of apoptosis.Two-photon and confocal laser scanning microscopies in combination with specific fluorescent dyes provide the possibility for simultaneously multiparameter observation of apoptosis at the level of single cell.In our study, with combination of two-photon and confocal laser scanning microscopy, we simultaneously observed the changes in nuclear morphology, (Ca 2+ ) i and ROS production during As 2 O 3 -induced apoptosis of Colo-16 cells, suggesting that two-photon and confocal laser scanning microscopy might provide a real-time, sensitive and noninvasive method for simultaneously multiparameter observation of drug-induced apoptosis at the single cell level.

Conclusions
With two-photon and confocal laser scanning microscopy in combination with fluorescent probes Hoechst 33342, DCFH-DA and Fluo 3-AM, we simultaneously observed As 2 O 3 -induced changes in nuclear morphology, ROS and (Ca 2+ ) i within Colo-16 cells.Our results indicated that both As 2 O 3 -induced (Ca 2+ ) i elevation and ROS production were involved in the apoptosis of Colo-16 cells.These results suggested that two-photon and confocal laser scanning microscopy might provide a realtime, sensitive and noninvasive method for simul-taneously multi-parameter observation of drug-induced apoptosis at the single cell level.

Figure 1 .
Figure 1.Cytotoxic effect of As2O3 on Colo-16 cells.Viable cell number was determined by MTT assay as described in materials and methods.Data shown are expressed as percentage of control values (without As2O3 treatment) and represented by the mean  SEM (n  5).

Figure 2 .
Figure 2. DNA electrophoresis of Colo-16 cells after treatment with As2O3 of different concentration for 24 h.

Figure 3 .
Figure 3. Nuclear morphological changes in Colo-16 cells after treatment with As2O3 of different concentration for 24 h.A, 0 M As2O3; B, 4 M As2O3; C, 10 M As2O3.Images of Hoechst 33342 fluorescence was collected by two-photon laser scanning microscopy at 730 nm excitation and 460 nm emission.

Figure 4 .Figure 5 .
Figure 4. As2O3 induced nuclear morphological changes (A) and intracellular calcium changes (B) in Colo-16 cells.Images of Hoechst 33342 fluorescence (A) and Fluo 3 fluorescence (B) were collected simultaneously by two-photon laser scanning microscopy and confocal laser scanning microscopy.

Figure 6 .Figure 7 .
Figure 6.As2O3 -induced nuclear morphological changes (A) and ROS production (B) in Colo-16 cells.Images of Hoechst 33342 fluorescence (A) and DCF fluorescence (B) were collected simultaneously by two-photon laser scanning microscopy and confocal laser scanning microscopy.