C cleavage by caspase-6 activation is crucial for apoptotic induction by photodynamic therapy with hexaminolevulinate in human B-cell lymphoma cells

Cancer Letters 339 (2013) 25–32

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Lamin A/C cleavage by caspase-6 activation is crucial for apoptotic induction by photodynamic therapy with hexaminolevulinate in human B-cell lymphoma cells Susan Shahzidi a, Andreas Brech b, Mouldy Sioud c, Xiaoran Li a, Zhenhe Suo a, Jahn M. Nesland a, Qian Peng a,⇑ a

Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital and Medical Faculty, University of Oslo, Oslo, Norway Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway c Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway b

a r t i c l e

i n f o

Article history: Received 5 July 2013 Accepted 24 July 2013

Keywords: Photodynamic therapy Hexaminolevulinate Lymphoma Apoptosis Lamin A/C Caspase-6

a b s t r a c t Photodynamic therapy (PDT) with a light-activated drug is an approved modality for cancer treatment. Hexaminolevulinate (HAL), a hexylester of 5-aminolevulinic acid as the photosensitising protoporphyrin IX (PpIX) precursor, is clinically used for both PDT and photodetection. Our previous studies have shown that HAL–PDT can effectively induce apoptosis in several human blood malignant cell lines. However, the mechanisms involved in the apoptotic induction are still not fully elucidated. In this study we have focused on the role of cellular lamin A/C in the apoptotic induction. HAL–PDT-mediated apoptosis was confirmed by various techniques including fluorescence microscopy and electron microscopy in both human B-cell lymphoma Ramos and Daudi cell lines. The lamin A/C, together with caspases-6 and -3, was cleaved during the apoptosis. Western blots, immunocytochemistry, fluorescence microscopy and electron microscopy demonstrated that the specific caspase-6inhibitor abrogated the HAL-PDT-mediated cleavages of both caspase-6 and lamin A/C and subsequent apoptosis in these two cell lines, suggesting that the cleavage of lamin A/C by the caspase-6 activation is crucial for such apoptotic induction. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Photodynamic therapy (PDT), a modality using a light-activated drug, is based on the energy transfer from the photosensitiser to oxygen to generate cytotoxic singlet oxygen. PDT with protoporphyrin IX (PpIX), endogenously induced from 5-aminolevulinic acid (ALA) or its esters via heme biosynthetic pathway, has shown to be an effective and safe treatment for several premalignant and malignant diseases [1–3]. However, the mechanisms of action on cell-killing are still not fully elucidated. Apoptosis is well defined with morphological and biochemical changes of the cell nucleus including chromatin condensation (pyknosis), nucleosomal-sized DNA fragmentation (DNA laddering) and nuclear fragmentation (karyorrhexis) [4]. Active cytosolic

Abbreviations: ALA, 5-aminolevulinic acid; HAL, hexaminolevulinate; PDT, photodynamic therapy; PpIX, protoporphyrin IX; TdT, terminal deoxynucleotid transferase. ⇑ Corresponding author. Address: Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Montebello, 0310 Oslo, Norway. Tel.: +47 22935553; fax: +47 22934832. E-mail address: [email protected] (Q. Peng). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.07.026

aspartate-specific (cysteine) proteases, named caspases, are primarily responsible for such characteristic features of the nucleus. Although more than 14 caspases have been identified [5], due to their structural homology only caspases-3, -6 and -7 are classified as executioner caspases responsible for cleaving downstream target polypeptides causing apoptotic cell death [6–8]. The role of caspase-3 in apoptotic execution has widely been investigated, involving the cleavage of poly(ADP-ribose) polymerase [9] as well as the release of the active DNA fragmentation factor [10]. However, the specific role of caspase-6 is poorly understood with conflicting information. In some reports the caspase-6 activation depends upon the caspase-3 activation [11,12]; while in others does not [13] or takes place before the caspase-3 activation [14]. The caspase-6 has been reported to exclusively cleave the lamin A/C responsible for typical apoptotic nuclear morphology [15]. Nuclear lamina is a dense network of lamin filaments and lamin-associated proteins, both of which are physically associated with the inner nuclear membrane and peripheral chromatin. Besides regulating important cellular events such as DNA replication, the nuclear lamina also provides mechanical support and determines chromatin organization [16]. Lamins are classified as types

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A/C and B. The two types differ in their biochemical properties [17]. The type A/C has been shown to form functional association with polynucleosomes, histones and DNA [18]. In the present report we have investigated the effect of caspase6 activation on the lamin A/C cleavage during apoptosis in the human Ramos and Daudi B-cell lymphoma cell lines after PDT with hexaminolevulinate (hexylester of ALA), a PpIX precursor used for both clinical PDT and photodetection [19,20]. 2. Materials and methods 2.1. Chemicals Hexaminolevulinate (HAL) was kindly provided by Photocure ASA (Oslo, Norway). Stock solution of 12 mM was freshly prepared for each experiment. The pan-caspase inhibitor (InSolution™ Q-VD-OPh, Non-O-methylated) and caspase-6 inhibitor-I were from CalbiochemÒ (USA). The anti-cytochrome C antibody was from Santa Cruze Biotechnology (Santa Cruz, CA) and the secondary antibody conjugated with DyLightÒ549 from AbD seroTec (Oxford, UK). All other antibodies used in this work were from Cell Signaling (Beverly, MA). 2.2. Cell culture Human B-cell lymphoma cell lines, Ramos and Daudi, were subcultured every second day in RPMI-1640 medium (PAA Laboratories GmbH, Fisher Scientific, Norway) containing 10% fetal bovine serum (FBS) (Saveen & Werner, Oslo, Norway), 100 units/ml penicillin, 100 lg/ml streptomycin, and 1% glutamine (Gibco, Invitrogen, Norway) at 37 °C in a 5% CO2 humidified incubator. All experiments were carried out at a cell density of 8  105/ml. 2.3. PDT with HAL Ramos and Daudi cells were incubated for 4 h in the dark in serum-free RPMI 1640 medium containing 5 or 20 lM of HAL. The cells were then exposed to the light from a bank of four fluorescent tubes (model 3026, applied Photophysics, London, UK) emitting light mainly in the region of 410–500 nm with a maximum around 440 nm. The fluence rate of the light reaching the cells was 12 mW/cm2. The light doses were 0.18 J/cm2 for Ramos cells and 0.78 J/cm2 for Daudi cells, respectively. After irradiation the medium was immediately replaced with HAL-free medium containing 10% FBS. In some experiments the inhibitor of pan-caspase or of caspase-6 (20 lM) was added to the samples before and after irradiation. 2.4. Assessment of apoptotic cells Typical nuclear morphology of apoptotic cells such as chromatin condensation, chromatin margination and nuclear fragmentation was assessed by fluorescence microscopy after staining cells with 4 lg/ml Hoechst 33342 (H342) (Sigma, USA) at 37 °C for 10 min. The percentage of apoptotic cells was calculated by counting at least 200 cells in each sample and each experiment was always done in triplicate. Such counting followed the apoptotic verification by electron microscopy as described below. Furthermore, DNA fragmentation was determined in control and treated samples by using the Apoptotic DNA Ladder Kit (Life technologies, USA) according to the manufacture’s instructions. An equivalent amount of DNA from samples at various times after HAL–PDT were resolved on 1.5% agarose gel containing 0.5 lg/ml ethidium bromide in Tris–borate ethylenediaminetetraacetic acid (EDTA) (1xTBS). The bands had been visualised under an UV Tran-illuminator before the photographs were taken.

centrifuged to precipitate cellular debris. The amount of protein in the lysates was determined. 150 lg of the lysate proteins from each sample were incubated at 37 °C for 3 h in a reaction buffer containing caspase-3 substrate (N-acetyl-Asp-Glu-val-Asp(DEVD)-p-nitroanilide) according to the manufacture’s instruction. Absorbance at the 405 nm was read on a microtiter plate reader (Multiskan Ex, Labsystems, Finland). 2.7. Western blot Control and PDT-treated cells (8  108) at various times after HAL-PDT were collected by centrifugation. After being washed once with cold PBS, the pellets were resuspended in 200 ll lysis buffer (Sigma, St. Louis, MO), followed by incubation on ice for 30 min and sonication for 15 s to obtain whole-cell extracts. For the cytosolic fraction the cell pellets were resuspended in 200 ml HEPES medium containing digitonin (20 mg/ml) (Sigma, St. Louis, MO), incubated for 5 min at room temperature and then 30 min on ice to permeabilize the cells to leak their cytosolic proteins into the medium. The cells were finally centrifuged at 14,000 rpm for 10 min at 4 °C and the supernatant was collected as the cytosolic fraction. The whole-cell extracts and cytosolic fractions were kept at 70 °C before use. Total proteins were quantified by the Bradford method using the bovine serum albumin (BSA) protein assay kit (Pierce, Inc., Rockford, IL) according to the protocol. An equal amount of proteins (20 lg per lane) from the whole-cell lysates and cytosolic fractions was electrophoresed on SDS–polyacrylamide gels (10–15%) and the gel-separated proteins were transferred to polyvinylidene difluoride membranes by a semi-wet transfer apparatus (Bio-Rad, CA). The membranes had been washed once (5 min) with 0.1% TBSTween, blocked with 5% non-fatty milk in the TBS-Tween for 1 h at room temperature before they were probed overnight at 4 °C with primary antibodies. The membranes were then washed three times and incubated for 1 h at room temperature with an anti-mouse or anti-rabbit secondary antibody. Finally, they were visualized using a chemiluminescence detection kit, ECL-PLUS (Amersham Biosciences, Piscataway, NJ). The recognition of b-actin with its antibody (1:5000 dilution) allowed reconfirmation of the total amount of proteins loaded on the gels. 2.8. siRNA and transfection Two siRNA sequences targeting lamin A/C, (NM_005572) (50 -CAGGCAGTCTGCTGAGAGGAA-30 and 50 -CCCACCAAAGTTCACCCTGAA-30 ) and one irrelevant siRNA (control) sequence (50 -UUGAUGUGUUUAGUCGCUA-30 ) were purchased from Qiagen (USA). Transfections of both cell lines with siRNAs were performed using electroporation technique as described previously [21]. Briefly, 2.5  106 cells were suspended in 0.5 ml serum-free RPMI-1640 medium containing 2 lg siRNA using the BTX electroporation apparatus in a 4-mm BTX cuvette and placed on ice for 5 min. The cells were then pulsed at 500 V for 2 ms. After transfection the cells were diluted in 2.5 ml of pre-warmed medium and incubated at 37° in 5% CO2 for 48 h. After that the protein extracts were prepared and separated as described at the Section 2.7; and then probed with the anti-lamin A/C monoclonal antibody. After densitometric analyses of the signals and normalisation with b-actin signals the percentages of the lamin A/C inhibition by siRNAs were calculated. 2.9. Cell Death Detection ELISAPLUS The kit of Cell Death Detection ELISAPLUS (Roche Applied Science, Mannheim, Germany) was used to determine the cytosolic histone-associated DNA fragments (mono- and oligonucleosomes) in the lamin A/C-targetting siRNA transfected and untransfected cells according to the manufacture’s instructions. The ELISA signals representing the cytoplasmic histone-associated DNA fragments released from the nuclei after apoptotic cell death were quantified by measuring the absorbance at 405 nm using an ASYS UVM340 96-well plate reader at 48 h after transfection.

2.5. Electron microscopy

2.10. Immunocytochemistry of lamin A/C

Control and PDT-treated cells were washed with phosphate buffered saline (PBS), fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 2% OsO4 and 1.5% KFeCN, followed by the staining with 1% uranyl acetate. The cells were then dehydrated through a graded ethanol series and embedded in Epon/Araldite mixture. Semithin sections were cut with glass knives, mounted on glass slides and stained with toluidine blue; and observed under a light microscope for the orientation. Ultrathin sections were cut with diamond knives, floated onto a 100-mesh copper grid, stained with uranyl acetate and lead citrate, and finally examined by transmission electron microscopy (TEM) (JEOL-JEM 1230 at 80 kV). The TEM images were recorded on a digital camera (Morada, Olympus, Tokyo, Japan) and further processed using the Adobe Photoshop software.

Controlled and PDT-treated cells (5  104) were washed once with PBS containing 1% FBS before being cytospun on specially coated slides and then airdried overnight. The cells were permeabilized with 0.1% Saponin for 5 min, blocked with 10% sheep serum in PBS for 30 min, incubated with the primary antibody for 90 min at room temperature; and subsequently with the secondary sheep anti-rabbit IgG antibody conjugated with DyLightÒ549 for 45 min. The fluorescence images of the lamin A/C were obtained by a fluorescence microscope (Nikon Eclipse E800, Nikon) equipped with a highly light-sensitive thermo-electrically cooled charge-coupled device camera (ORCA11-ER, Hamamatsu, Japan). 2.11. Isolation of cell nuclei

2.6. Caspase-3 activity The activity of caspase-3 was measured with a colorimetric assay kit (Chemicon, Temecula, CA). Subsequent to PDT (4 h for Ramos and 10 h for Daudi; respectively) the cells were collected and suspended in lysis buffer at 0 °C for 10 min. Lysates were

To address if there was a direct effect of HAL-PDT on the cleavage of the lamin A/C, the Nuclei EZ Prep Kit (Sigma, St. Louis, MO) was used to isolate the nuclei of the Ramos and Daudi cell lines according to the manufacture’s instructions. The nuclei of the cells were immediately incubated with PpIX at a concentration of 0.1 or

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Fig. 1. HAL-PDT-induced apoptosis of the Ramos and Daudi cells. (A) Fluorescence images of the control (Ctrl, before HAL-PDT) and apoptotic cells (20 h after HAL-PDT) in the Ramos and Daudi cell lines. The magnification of the images is the same with the bar (in the Ramos Ctrl image) of 10 lm. (B) Percentage of apoptotic cells induced by HAL-PDT with or without the pan-caspase inhibitor. The bars are SD. (C) Immunoblots of cytosolic translocation of mitochondrial cytochrome C (Cyt.C) and cleavage of caspase-3 at various times after HAL-PDT. ‘C’ indicates control samples; while ‘0’ for the samples taken immediately after PDT. (D) Inhibitory effect of the pan-caspase inhibitor on the caspase-3 activity at 4 h for Ramos cells and 10 h for Daudi cells after HAL-PDT. The bars are SD.

0.5 lg/ml for 1 or 2 h before light irradiation at the same doses as used for HAL-PDT of the whole cells. Four hours later, the treated nuclear samples were stained with the Hoechst 33342 and examined by fluorescence microscopy.

2.12. Statistical analysis The rank sum (Mann–Whitney) test was used to statistically analyse data and all tests were done at the 0.05 significance level.

3. Results 3.1. HAL-PDT induced apoptotic cell death The apoptosis was induced during the period of 4–20 h after HAL-PDT in both cell lines. After 20 h most apoptotic cells became necrotic assessed by the double staining of H342 for apoptosis and

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Fig. 2. A, Inhibition of the HAL-PDT-mediated apoptotic DNA fragmentation by pan-caspase inhibitor and caspase-6 inhibitor in both Ramos and Daudi cell lines. The samples were taken 20 h after HAL-PDT. (B) Immunoblots of PDT-mediated cleavages of caspase-6 and lamin A/C in both cell lines. (C) Immunoblots of inhibition of PDT-mediated caspase-6 cleavage by pan-caspase inhibitor, but not by caspase-3 inhibitor in both cell lines. (D and E) Correlation between the HAL-PDT-mediated destruction of lamin A/C and apoptotic induction in the Ramos (D) and Daudi (E) cell lines. The caspase-6 inhibitor blocks both PDT-mediated cleavage of lamin A/C and subsequent apoptosis. Such correlations are shown by immunocytochemistry (small arrow for lost lamin A/C; big arrows for intact lamin A/C), Hoechst 33342-based fluorescence microscopy and electron microscopy (EM, Bars: 3 lm).

propidium iodide for necrosis (data not shown). The time course and extent of the apoptotic induction are cell type-dependent. The Ramos cells were more sensitive than Daudi cells to the PDT-mediated apoptosis and a lower light dose for PDT was thus

applied. The typical nuclear alterations of apoptotic cells were found in both cell lines with fluorescence microscopy using the H342 for nuclear staining 20 h after HAL-PDT (Fig. 1A). This finding was further confirmed by electron microscopy (Fig. 2D and 2E) and

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flow cytometry with the apoptotic TdT staining (data not shown). Quantitatively, about 80% of apoptotic cells could be induced by PDT based on the fluorescent microscopic counting (Fig. 1B) (P < 0.05 as compared to controls) in both cell lines. This apoptosis was effectively inhibited by the pan-caspase inhibitor, InSolution™ Q-VD-OPh, Non-O-methylated (Fig. 1B) (P < 0.05 as compared to those without inhibitor), suggesting that the apoptotic induction

was through the caspase pathway. These results were supported by the Western blots on the cytosolic fractions of treated cells, which showed the release of cytochrome c from the mitochondria and cleavage of caspase-3 (Fig. 1C), together with the measurements of caspase-3 activity (Fig. 1D, P < 0.05 as compared to the cells with the pan-caspase inhibitor). The DNA gels also confirm the apoptotic laddering of DNA fragmentation (Fig. 2A).

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3.2. Effect of caspase-6 activation on lamin A/C cleavage Having shown the typical morphological change of apoptosis, we investigated the effect of caspase-6 activation on lamin A/C cleavage. Fig. 2A shows that the caspase-6 inhibitor blocks the HAL-PDT-based apoptotic laddering of DNA fragmentation in both cell lines, suggesting the involvement of caspase-6 in such apoptotic induction. Western blots demonstrate that HAL-PDT mediates not only the cleavage of caspase-6, but also the lamin A/C (Fig. 2B). The use of the pan-caspase inhibitor or specific caspase6 inhibitor could completely stop the PDT-induced cleavages of both caspase-6 and lamin A/C (Fig. 2B), but not by the use of the specific caspase-3 inhibitor (Fig. 2C), indicating clearly the cleavage of lamin A/C by the PDT-mediated caspase-6 activation. 3.3. Effect of lamin A/C cleavage on apoptotic induction Next, we have studied the effect of lamin A/C cleavage on apoptotic induction. Immunocytochemistry revealed the disappearance of the lamin A/C rings in the nuclei of those PDT-mediated damaged cells in both cell lines (Figs. 2D and E, small arrow). Such destruction of the lamin A/C was accompanied by the apoptotic induction confirmed by both H342-based fluorescence microscopy and electron microscopy (Figs. 2D and E). This effect of cleaved lamin A/C on the apoptotic induction was totally abolished by the use of the specific caspase-6 inhibitor (Figs. 2D and E). Furthermore, transfection with the lamin A/C siRNA sequence-1 resulted in 72% and 61% inhibition of lamin A/C expression in the Ramos and Daudi cell lines, respectively; while 77% and 66% inhibition with the siRNA sequence-2 (Fig. 3A). These results are consistent with the finding that the partial knockdown of lamin A/C by the two siRNA sequences led to increased amounts of cytoplasmic his-

A

tone-associated DNA fragments (mono- and oligonucleosomes) released from the nuclei of apoptotic cells in the two cell lines as detected by the Cell Death Detection ELISAPLUS kit (Fig. 3B; P < 0.05 as compared to control samples). In addition, a direct effect of PDT with exogenous PpIX on the isolated nuclei of these two cell lines did not induce nuclear apoptotic changes (data not shown), suggesting a requirement of caspase-6 activation for the degradation of lamin A/C. Overall, these data strongly indicate the relationship between PDT-mediated degradation of lamin A/C via the caspase-6 activation and its subsequentapoptotic induction.

4. Discussion PDT is an approved treatment modality for various cancers [1]. PDT with exogenous photosensitisers can induce apoptotic cell death [22]. Our previous studies have demonstrated that PDT with PpIX endogenously derived from HAL, can effectively induce apoptotic cell death in several human blood malignant cell lines [23– 25]. The mechanisms involved in apoptotic induction are different in various cell lines [22–25]. In the present study, HAL-PDT induced about 80% of apoptotic cell death in both human B-cell lymphoma Ramos and Daudi cell lines (Figs. 1A and B). The mitochondrion-initiated caspase-dependent pathway is one of the few well-characterised pathways in the apoptotic induction. This includes the release of a caspase activator, cytochrome c, from the mitochondria into the cytosol, which works together with apoptotic protease activating factor-1 and procaspase-9 in the presence of dATP to produce caspase-9, which further activates caspase-3. This pathway appears also to be involved in the apoptotic induction in the Ramos and Daudi cell lines after HAL-PDT (Figs. 1C and D). However, the use of a specific caspase-6 inhibitor

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Fig. 3. (A) Silencing effect of lamin A/C small interfering RNAs (siRNAs) on the Ramos and Daudi cell lines using the BTX electroporation method. The down-regulation of lamin A/C by siRNAs was determined by western blots. Ctrl (controls), only eletroporated without siRNAs; Se-1, siRNA sequence-1; Se-2, siRNA sequence-2; Se-ctrl (siRNA control), irrelevant siRNA sequence. (B) Cytoplasmic histone-associated DNA fragments (mono- and oligo-nuclesomes) released from the nuclei of apoptotic cells after downregulation of lamin A/C by siRNAs, measured by the Cell Death Detection ELISAPLUS kit. The bars are SD.

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could inhibit the cleavages of both caspase-6 and lamin A/C (Fig. 2B) and also stop the apoptotic induction (Figs. 2A, D, E); while the caspase-3 inhibitor could not inhibit the cleavage of caspase-6 (Fig. 2C), neither stop the apoptosis (data not shown), suggesting that caspase-6 might act upstream of caspase-3 and its activation does not depend on the caspase-3 activation. This finding is in good agreement with previous reports [14,15,26,27] demonstrating that caspase-6 is required for the completion of chromatin condensation and formation of apoptotic bodies during apoptosis. Lamins, the major components of nuclear lamina, are implicated in various nuclear functions including the maintenance of chromatin organisation, apoptosis and replication [28,29]. The lamins are known as substrates of proteases and the caspase-6mediated proteolysis of the lamins, lamin A/C in particular, is crucial for nuclear apoptotic events such as shrinkage, disassembly of nuclear membrane and formation of apoptotic bodies [15,30,31]. This is consistent with the results of the present study, showing the dependence of lamin A/C cleavage on the activation of caspase-6 and its subsequent apoptotic alterations in both Ramos and Daudi cell lines after HAL-PDT (Figs. 2B, D, and E). Inhibition of such lamin A/C cleavage by the caspase-6 inhibitor gave rise to an entire blockage of theHAL-PDT-mediated apoptotic induction (Figs. 2A, D, and E). In addition, the results that show no apoptotic alterations of isolated nuclei after PDT with exogenous PpIX in both cell lines (data not shown) suggest a requirement of the cytosolic caspase-6 rather than a direct effect of PDT on the cell nuclei in the HAL-PDT-mediated apoptosis. The presence of lamin A/C in the nuclear interior as well as in nuclear periphery [32,33] and its physically close association with chromatin [34,35] have been shown to play a pivotal role for the structural and functional properties of chromatin. In the current study the suppression of lamin A/C by siRNA resulted in an increased release of the histone-associated DNA fragments from the nuclei to the cytosol in both Ramos and Daudi cell lines (Figs. 3A and B). This finding further highlights the significance of lamin A/C cleavage in the abrogation of lamin-chromatin organization and of its contribution to disassembling of nuclear shape and architecture in apoptotic cell death mediated by HAL-PDT. In conclusion, HAL-PDT can induce apoptotic cell death in both human B-cell lymphoma Ramos and Daudi cell lines. The cleavage of lamin A/C by the caspase-6 activation appears to play a crucial role in such apoptotic induction, as the specific caspase-6 inhibitor can completely block such induction. Conflicts of interest None. References [1] J. Dougherty, C.J. Gomer, B.W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan, Q. Peng, Review: photodynamic therapy, J. Natl. Cancer Inst. 90 (1998) 889–905. [2] Q. Peng, T. Warloe, K. Berg, J. Moan, M. Kongshaug, K.-E. Giercksky, J.M. Nesland, 5-Aminolevulinic acid-based photodynamic therapy. Clinical research and future challenges, Cancer 79 (1997) 2282–2308. [3] J.M. Gaullier, K. Berg, Q. Peng, H. Anholt, P.K. Selbo, L.W. Ma, J. Moan, Use of 5aminolevulinic acid ester to improve photodynamic therapy on the cells in culture, Cancer Res. 57 (1997) 1481–1486. [4] G. Kroemer, L. Galluxxi, P. Vandenabeele, et al., Classification of cell death: recommendations of the nomenclature committee on cell death, Cell Death Differ. 16 (2009) 3–11. [5] N.A. Thornberry, Y. Lazebnik, Caspases: enemies within, Science 281 (1998) 1312–1316. [6] G.S. Salvesen, V.M. Dixit, Caspase activation: the induced-proximity model, Proc. Natl. Acad. Sci. USA 96 (1999) 10964–10967.

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