Staurosporine induced poly (ADP-ribose) polymerase independent cell death in Dictyostelium discoideum

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1 Indian Journal of Experimental Biology Vol.50, January 2012, pp Staurosporine induced poly (ADP-ribose) polymerase independent cell death in Dictyostelium discoideum Hina Mir, Jyotika Rajawat & Rasheedunnisa Begum* Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara , India Received 20 May 2011; revised 9 August 2011 In the present study D. discoideum has been used as a model organism to understand the role of poly (ADP-ribose) polymerase (PARP) in caspase independent paraptotic cell death pathways. D. discoideum lacks caspases and Bcl-2 family proteins; nevertheless it has 9 potential genes for PARP. PARP has been known to get activated in various cell death associated diseases. In this study kinetics of cell death induced by staurosporine (STS), a bacterial alkaloid, was established to unravel the role of PARP. It was found that STS induced cell death in D. discoideum did not involve PARP activation, however it involved cathepsin D. Results indicated that an alternative mechanism may be existing in D. discoideum that lacks Bcl-2 family proteins for STS induced cell death that evades Bax involvement. Keywords: Apoptosis inducing factor, Benzamide, Cathepsin, Pepstatin, Staurosporine Staurosporine (STS), a bacterial alkaloid obtained from Streptomyces staurosporeus, possesses inhibitory activity against fungi and yeast. It is a cell permeable protein kinase inhibitor and induces cell death in many mammalian cell types by both caspase dependent and caspase independent apoptotic pathways. STS induced caspase independent cell death mimics mitochondrial death pathway in which Bax and Bak play key roles. STS causes limited lysosomal destabilization and thereby releases several cathepsins - B, D and L into cytosol. Amongst these, cathepsin D an aspartate protease acts at neutral ph because the active conformation of cathepsin D is stabilised by binding with target cytosolic proteins. Cytosolic cathepsin D via Bax activation causes the release of Apoptosis inducing factor (AIF) from mitochondria. Once released in to cytosol, AIF translocates to nucleus where it brings about large scale DNA fragmentation and ultimately paraptotic cell death 1. In the present study, Dictyostelium discoideum, an organism that lacks caspases 2,3 has been used as the model organism. Besides various developmental studies 4 D. discoideum is also a good model organism to study evolutionary aspects of cell This article was presented in the Conference on Molecular Medicine (MOLMED-2011) at CHARUSAT, Changa, India. *Correspondent author Telephone: rasheedunnisab@yahoo.co.in death and has been extensively used to study effect of oxidative stress 5-8 and UV-C stress 9 on vegetative cell death and development. However, in this study D. discoideum has been used to explore STS induced caspase independent cell death which might be occurring in absence of Bax. STS induced cell death is well understood to be mediated by AIF, however the role of poly (ADPribose) polymerase (PARP) and its contribution in AIF mediated cell death has not been well explored. PARP is a nuclear enzyme found in eukaryotes that gets activated due to DNA damage 10 and catalyses transfer of long, linear or branched chains of poly (ADP-ribose) (PAR) onto a wide range of proteins using NAD + as its substrate. PARP activation is a dynamic and flexible metabolic pathway that can be targeted to stu``dy its specific response in higher eukaryotic cells to DNA damage 11. PARP activation has been shown to participate in carcinogenesis, DNA repair and cell death. Early and rapid PARP activation in cells exposed to high levels of DNA damage by alkylating agents has been associated with subsequent apoptotic or necrotic death 12,13. Thus, under conditions of basal DNA damage where PARP contributes to cell homeostasis, however during moderate/severe cellular stress PARP contributes to cell death. Hence, under various pathological conditions PARP inhibition seems to be beneficial 7,14,15 to the organism.

2 MIR et al.: STAUROSPORINE INDUCED CELL DEATH IN DICTYOSTELIUM 81 Cell death pathways initiated by activation of PARP are known to be mediated by AIF. PARP activators, including DNA-alkylating agent N-methyl- N -nitro-n-nitrosoguanidine, hydrogen peroxide (H 2 O 2 ) and NMDA induce the translocation of AIF to nucleus, along with other mitochondrial changes and ultimately cell death 16,17. The present study attempts to explore the role of PARP in STS induced AIF mediated cell death. Materials and Methods Induction of cell death using staurosporine Log phase cells at a density of ~ cells/ml were exposed to different doses of STS (0, 100, 250, 400 and 500 nm) in HL-5 medium at 22 C in a sterile flask. Cell death was monitored using trypan blue at intervals of 2 h for 8 h when the percent cell death started declining. Assessment of cell death by AnnexinV-FITC/PI dual staining To differentiate between apoptotic and necrotic cell death, dual staining with Annexin V-FITC/PI was performed using apoptosis detection kit (Molecular Probes). ~ D. discoideum cells were pelleted and washed twice with 1X Sorenson s buffer (SB). Cells were then suspended in binding buffer (ph 7.2) provided in the kit and incubated with Annexin V for 10 min and then with PI for 5 min in dark at 22 C. PARP activity assay 18 Cells were centrifuged and washed with PBS, fixed in 70% chilled methanol for 10 min at -20 C, washed with blocking solution (1.5% BSA with 0.05% Tween-20 in PBS), and then incubated for 1 h with antibody against PAR raised in mouse at a concentration of 0.5 ug/ml. Cells were washed twice or thrice with blocking solution, and incubated for 1 h with FITC-conjugated anti-mouse IgG as secondary antibody, used at a dilution of 1:200. Cells were then again washed twice or thrice with PBS, and fluorescence was observed at 60 magnification using a Nikon (Tokyo, Japan) fluorescence microscope with a charge-coupled device camera. Data were analyzed by image proplus software to calculate the mean density of fluorescence from different fields, and ~50 cells were examined for each dose. NAD + estimation Intracellular levels of NAD + in D. discoideum were determined by enzymatic recycling method employing alcohol dehydrogenase to reduce NAD + to NADH 19. In brief, cells were pelleted and washed twice with ice cold PBS and NAD + was extracted with perchloric acid and then neutralized with KOH. NAD + levels were estimated following protein normalization with modified Lowry method 20. Evaluation of mitochondrial membrane potential Potential sensitive dye DiOC 6 (3, 3 -dihexyloxacarbocyanine iodide) (Sigma) was used to evaluate changes in mitochondrial membrane potential (MMP). ~ cells were pelleted and washed twice with 1X SB. Cells were stained with DiOC 6 (400 nm) for 15 min in dark and then washed once with 1X SB and monitored for the fluorescence (Nikon Eclipse TE2000S, Japan). Monitoring AIF release by immunofluorescence 1 AIF translocation to nucleus was monitored by immunofluorescence. D. discoideum cells were pelleted and washed once with phosphate buffered saline (PBS) ph 7.4, fixed in 70% chilled ethanol for 10 min at -20 C and then washed with blocking solution (1.5% BSA with 0.05% Tween 20 in PBS) followed by incubation for 1 h in primary antibody using rabbit anti-aif polyclonal antibodies raised against amino acids of human AIF (Cayman Chemical) at 1:1000 dilution and then anti rabbit IgG (whole molecule) TRITC conjugate (Sigma) at 1:400 dilution. Nuclear counterstaining with DAPI (1 ug/ml) for 5 min was performed after the removal of excess secondary antibody and observed for the fluorescence. DNA fragmentation TUNEL assay (Terminal deoxynucleotidyl transferase dutp nick end labeling) was performed according to the procedure supplied by the manufacturer (ApoBrdu DNA fragmentation kit; Sigma). This assay is based on labeling of 3ʹOH ends of fragmented DNA with Br-dUTP catalyzed by terminal transferase 21. Fluorescence was monitored by using Anti Brdu-FITC antibody. Protocol was followed according to the instructions given along with ApoBrdu DNA fragmentation detection kit supplied by Sigma. Results Induction of cell death by staurosporine and PARP activity Dose dependent effect of staurosporine (STS) on cell death was studied. It can be observed (Fig. 1) that as the concentration of STS increases from 100 to 500 nm percentage of cell death increases. This trend continues till 8 h with considerable decrease in number of live cells starting at 2 h with STS (500 nm).

82 INDIAN J EXP BIOL, JANUARY 2012 In STS (400 nm) treated cells, PS exposure was obtained at 3 h while PI staining at 6 h (Fig. 2). Thus, for further experiments a lower dose of 400 nm was selected.

In STS (500 nm) treated cells PS-PI positive fluorescence was seen simultaneously at 3 h.

PARP activity was also assayed at later time points (data not shown) to see if PARP was activated late after STS stress.

Consequent to PARP activation cellular NAD + pools deplete within an hour hence NAD + levels were assayed 2 h after STS treatment to find out PARP activation immediately after STS treatment.

3 82 INDIAN J EXP BIOL, JANUARY 2012 In STS (400 nm) treated cells, PS exposure was obtained at 3 h while PI staining at 6 h (Fig. 2). Thus, for further experiments a lower dose of 400 nm was selected. Benzamide pretreated cells also showed PS and PI staining at 3 and 6 h, respectively, after STS (400 nm) treatment. In STS (500 nm) treated cells PS-PI positive fluorescence was seen simultaneously at 3 h. To study the kinetics of PARP activation, PARP activity was assayed at various time points (5, 10 and 15 min) post STS treatment and the results are shown in Fig. 3 a and b. PARP activity was also assayed at later time points (data not shown) to see if PARP was activated late after STS stress. Increase in PARP activity could not be observed after STS treatment as compared to control cells. Consequent to PARP activation cellular NAD + pools deplete within an hour hence NAD + levels were assayed 2 h after STS treatment to find out PARP activation immediately after STS treatment. NAD + levels in STS treated cells (86 ± 9.47) were not significantly changed compared to control cells (94 ± 8.21). These results support the PARP assay results. Fig. 1 Dose and time dependent cell death induced by STS (100, 250, 400, 500 nm) in D. discoideum cells as monitored by trypan blue exclusion method [Values are mean ± SE of 5 independent experiments]. Fig. 3 (a)-parp activity assay by indirect immunofluorescence. Under control conditions basal PARP activity was observed. No significant change in PAR immunoreactivity was seen under STS stress [Photographs were taken at 60 objective]; (b)-densitometric analysis of PARP activity. PARP activity was measured at different time points post STS stress [Values are mean ± SE of 5 independent experiments]. Fig. 2 Annexin V staining of STS treated D. discoideum cells. PS exposure was seen at 3 h while PI staining at 6 h with STS (400 nm). STS (500 nm) was found to be necrotic as both AnnexinV-FITC and PI staining were observed at 3 h. Benzamide did not affect STS induced paraptotic cell death. Pepstatin A delayed PS exposure and PI staining to 6 h with 400 nm STS. While with 500 nm dose the cell death was converted to paraptotic dose [Data represents value of 3 independent experiments. Photographs were taken at 60 objective].

MIR et al.: STAUROSPORINE INDUCED CELL DEATH IN DICTYOSTELIUM 83 Mitochondrial and nuclear changes induced by STS Time dependent changes in MMP were monitored with staurosporine.

Cells treated with 500 nm STS exhibited increased MMP 1 h after stress which later declined by 3 h (Fig. 4a,b). Benzamide had no effect (Fig. 4a and c) on STS induced MMP changes.

4 MIR et al.: STAUROSPORINE INDUCED CELL DEATH IN DICTYOSTELIUM 83 Mitochondrial and nuclear changes induced by STS Time dependent changes in MMP were monitored with staurosporine. 400 nm STS led to significant changes in MMP indicated by less accumulation of the dye at 3 h (Fig. 4a,b). Cells treated with 500 nm STS exhibited increased MMP 1 h after stress which later declined by 3 h (Fig. 4a,b). Benzamide had no effect (Fig. 4a and c) on STS induced MMP changes. These effects of STS and benzamide on MMP changes were also reflected in their effect on AIF release. During STS induced cell death AIF release was observed at different time points with paraptotic and necrotic doses. With 400 nm STS translocation was seen at 4 h (Fig. 5). Inhibition of PARP activity by benzamide did not intercept this AIF release during paraptosis. DNA fragmentation could be detected by TUNEL assay and a significant increase in TUNEL positive cells was seen (data not shown) at 6 h after 400 nm STS treatment suggesting that D. discoideum cells undergo DNA fragmentation. Effect of cathepsin D inhibition on STS induced cell death As STS induced cell death is known to be mediated by cathepsin D 1 in other systems experiments were done using cathepsin D inhibitor. Cathepsin D inhibition delayed the cell death as seen by PS-PI dual staining (Fig. 2). Pepstatin A pretreatment could prevent the changes in MMP induced by both doses of STS (Figs. 4a,c). Also pepstatin A prevented nuclear translocation of AIF 4 h after STS (400 nm) treatment (Fig. 5). Indicating that cathepsin D activity mediates STS induced cell death in D. discoideum. Fig. 4 (a)-mitochondrial membrane potential changes induced by STS. Live cells accumulate the dye in mitochondria hence fluorescence intensity was found to change with STS stress as compared to control. Benzamide did not affect STS induced MMP changes. Pepstatin A partially prevented STS induced MMP changes; (b)-densitometric analysis of mitochondrial membrane potential changes induced by STS. STS (400 nm) leads to significant reduction in MMP at 3 h whereas STS (500 nm) results in significant changes at 1 and 3 h after stress [Values are mean of 3 independent experiments. Photographs were taken at 60 objective]; (c)-densitometric analysis of mitochondrial membrane potential changes induced by STS with benzamide and pepstatin A pretreatment. MMP change with STS (400 nm) at 3 h and STS (500 nm) at 1 h were not affected by benzamide. Pepstatin A pre-treatment partially rescued the MMP change induced by STS (400 nm) at 3 h and STS (500 nm ) at 1 h [Values are mean ± SE from 3 independent experiments. a P value <0.05 compared to respective dose of STS without pepstatin A pre-treatment].

84 INDIAN J EXP BIOL, JANUARY 2012 Fig. 5 Fluorescence microscopy for the mitochondrial-nuclear translocation of AIF at different time points after STS treatment.

AIF translocation to nucleus was observed at 4 h of STS treatment as seen by cells showing pink fluorescence in nuclei of due to overlapping of two fluorescence signal.

5 84 INDIAN J EXP BIOL, JANUARY 2012 Fig. 5 Fluorescence microscopy for the mitochondrial-nuclear translocation of AIF at different time points after STS treatment. Nucleus of control cells stained blue due to DAPI indicating AIF is localized in mitochondria. AIF translocation to nucleus was observed at 4 h of STS treatment as seen by cells showing pink fluorescence in nuclei of due to overlapping of two fluorescence signal. Pretreatment with pepstatin A prevented translocation of AIF to nucleus while benzamide failed to do so [Values represents mean of 3 independent experiments. Photographs were taken at 60 objective]. Fig. 6 Diagrammatic representation of mechanism of staurosporine induced cell death in D. discoideum. Based on the results a pathway for kinetics of STS induced cell death (Fig. 6) is proposed. Discussion Staurosporine is widely used to induce apoptosis in many mammalian cell types 22. It induces caspase dependent as well as caspase independent types of cell death 23. Caspase independent cell death is executed with the help of lysosomal destabilisation causing release of cathepsin D in to cytosol. The key player in this cascade is apoptosis inducing factor (AIF) which is released due to limited permeablization of outer mitochondrial membrane. Events following AIF release include chromatin condensation and large scale DNA fragmentation leading to caspase independent paraptotic cell death 1. Staurosporine induced cell death in D. discoideum cells is mediated via AIF Human corneal cells show PI staining after 12 h of STS treatment 24. In this study D. discoideum cells showed PS exposure at 3 h and PI staining at 6 h post 400 nm STS treatment. These cells underwent MMP changes at 3 h, while exhibiting AIF release 4 h post STS treatment similar to melanoma cells that showed MMP changes at 6 h and AIF release at 16 h post STS treatment 23. Absence of oligonucleosomal ladder formation (data not shown) and positive TUNEL assay suggested large scale DNA fragmentation in

6 MIR et al.: STAUROSPORINE INDUCED CELL DEATH IN DICTYOSTELIUM 85 D. discoideum following AIF release at 6 h after 400 nm STS treatment which is in accordance with already published report 1. STS (500 nm) treated D. discoideum cells exhibited both PS-PI simultaneously at 3 h. MMP changes with this necrotic dose commence within 1 h of STS treatment. These cells exhibited a smeared DNA on agarose gel (data not shown) further indicating the cell death type to be of necrosis. Staurosporine induced cell death is independent of PARP activity Reports suggest that PARP is activated when DNA is damaged 25,14. PARP activation occurs within minutes of DNA damage induction by UV-C 11. Similarly, HeLa cells when subjected to MNNG stress, a DNA alkylating agent, also showed peak PARP activity at ~5 min 26. Activation of PARP within minutes of oxidative and UV-C stress induction in D. discoideum has been reported 5,7-9. STS and PARP activation both are known to initiate a caspase independent cell death pathway which is mainly dependent on AIF release and consequent large-scale DNA fragmentation in various cell types including neuroblastoma cells 27. However, till date the role of PARP in the cascade of events led by STS is not known. In the present study, effort has been made to deal with this aspect of STS induced cell death. PARP assay suggested that PARP did not get activated immediately after STS and also inhibition by benzamide did not rescue the mitochondrial changes induced by STS. Unchanged NAD + levels in STS treated cells further support PARP independent cell death. These results point out that PARP might not be involved in STS induced cell death although PARP activation might occur after the DNA fragmentation that was led by AIF in the nucleus. However this activation could not be of significance as benzamide did not affect STS induced cell death. Staurosporine induced cell death in D. discoideum cells involved cathepsin D Pepstatin A is an inhibitor of acid proteases (aspartyl peptidases) viz., cathepsin D. It functions as a transition state analogue. It binds to the active site of the enzyme and prevents its binding with cytosolic proteins like Bax. Caspase independent cell death induced by STS is known to be inhibited by pepstatin A 1,28. D. discoideum cells preincubated with pepstatin A were also rescued from STS induced cell death as seen with PS-PI dual staining. Cathepsin D is reported to mediate its effect in STS induced cell death via Bax activation. Activated Bax leads to limited permeabilisation of the outer mitochondrial membrane. This in turn results in the release of AIF from the inter-membrane space of mitochondria to the cytosol 1. Hence, inhibition of cathepsin D delays cell death. In the present study, the rescuing effect of cathepsin D inhibition on the mitochondria changed in terms of MMP and AIF release underlaid the delaying effect of pepstatin A on STS induced cell death. Interestingly, Lam et al. 29 have reported the absence of Bcl-2 family proteins in D. discoideum by in silico investigations. These facts made the above stated results more intriguing raising the possibility of an alternative mechanism for the execution of STS induced cell death in D. discoideum that involved cathepsin D. The present study showed that like mammalian cell types, cathepsin D and AIF played central role in STS induced cell death in D. discoideum as well, albeit via a mechanism that circumvented the involvement of Bcl-2 family proteins. However, PARP did not involve in this type of cell death. In other words, this study emphasised that PARP was dispensable for paraptotic cell death which was mediated via AIF. Acknowledgment Thanks are due to the Department of Science and Technology, New Delhi for research support to RB (No.SR/SO/BB-03/2010); the Department of Biotechnology, New Delhi for research support to RB (BT/PR9496/BRB/10/562/2007) and Council of Scientific and Industrial Research (New Delhi) for awarding SRF to the author (JR). References 1 Bidère N, Lorenzo H K, Carmona S, Laforge M, Harper F, Dumont C & Senik A, Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis, J Biol Chem, 278 (2003) Olie R A, Durrieu F, Cornillon S, Loughran G, Gross J, Earnshaw W C & Golstein P, Apparent caspase independence of programmed cell death in Dictyostelium, Curr Biol, 8 (1998) Katoch B, Sebastians S, Sahdev S, Padh H, Hasnain S E & Begum R, Programmed cell death and its clinical implications, Indian J Exp Biol, 40 (2002) Mir H A, Rajawat J, Pradhan S & Begum R, Signaling molecules involved in the transition of growth to development of Dictyostelium discoideum, Indian J Exp Biol, 45 (2007) 223.

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The Annexin V Apoptosis Assay

The Annexin V Apoptosis Assay Development of the Annexin V Apoptosis Assay: 1990 Andree at al. found that a protein, Vascular Anticoagulant α, bound to phospholipid bilayers in a calcium dependent manner.