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FEBS Letters 579 (2005) 1324–1331 FEBS 29295

Minireview Ribosome inactivating proteins and apoptosis

Sriram Narayanana,1, Kalpana Surendranathb, Namrata Borab, Avadhesha Suroliaa, Anjali A. Karandeb,* a Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India b Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

Received 6 December 2004; revised 13 January 2005; accepted 18 January 2005

Available online 29 January 2005

Edited by Vladimir Skulachev

the 28 S rRNA. This activity prevents the formation of a crit- Abstract Ribosome inactivating proteins (RIPs) are protein that are of plant or microbial origin that inhibit protein ical stem–loop configuration, to which the elongation factor synthesis by inactivating ribosomes. Recent studies suggest that (EF) is known to bind during the translocation step of transla- RIPs are also capable of inducing cell death by apoptosis. tion. The end result of this activity is complete inhibition of Though many reports are available on cell death induced by cellular translation. The role of the B-chain is to help in the RIPs, the mechanism involved is not well studied. Comparison transport of RIPs into the cells by binding to specific sugar res- of pathways of apoptosis and cellular events induced by various idues of glycoproteins or glycolipids on the plasma membrane RIPs suggests a central role played by mitochondria, probably and internalization by endocytosis. Many bacteria and fungi acting as an integrator of cellular stress and cell death. The pur- are also known to produce RIPs that are capable of bringing pose of this review is to compare the various apoptotic pathways about inhibition of protein synthesis, albeit by a mechanism that may be involved and propose a general pathway in RIP-in- that is different from that of plant RIPs. Examples of bacterial duced cell death. Ó 2005 Federation of European Biochemical Societies. Published and fungal RIPs include diphtheria (DT), pseudomonas by Elsevier B.V. All rights reserved. (PE), (ST), a-sarcin, restrictocin, etc. DT and PE are known to ADP-ribosylate the EF-2 and thus lead- Keywords: Ribosome inactivating protein; Protein synthesis ing to its inactivation. A notable exception among the bacterial inhibition; Ribosome inactivation; Ribotoxic stress response; toxins is ST, whose mechanism of ribosome inactivation is Mitochondrial stress; Apoptosis identical to that of plant RIPs. Ribosome inactivating proteins have, for quite some time, been known for their selective killing of tumor cells compared to normal cells. In fact, the interest in RIPs was rekindled as 1. Introduction a result of their potential for cancer immunotherapy using immunotoxins. Many studies report that RIPs and immuno- Ribosome inactivating proteins (RIPs) are a family of highly toxins made using the toxic A-chain of RIPs are able to bring potent protein toxins that bring about inhibition of protein about cell death by inducing apoptosis [4–8]. Most of these synthesis by directly interacting with and inactivating the ribo- studies show that RIPs are capable of inducing apoptosis in a somes or by modification of factors involved in translation, wide variety of cells and cell lines, while few of them have tried usually the elongation step. These toxins are known to be pro- to delineate the apoptotic pathway and propose a mechanism duced by a wide variety of plants. Examples of plant RIPs in- for cell death induction by these toxins. However, it should clude abrin, , gelonin, momordin, mistletoe lectin (ML), be kept in mind that these pathways need not necessarily be etc. (for reviews on plant RIPs, refer [1–3]). Type I RIPs are mutually exclusive and more investigation in this area is needed made up of the toxic subunit (A-chain) alone, while type II to determine the factor that triggers the RIP-induced signaling RIPs consist of a toxic A-chain and a lectin like subunit leading to cell death. The purpose of this review is to compare (B-chain) linked together by a disulfide bond. The toxicity of various events and mechanisms that have been proposed for the A-chain is due to its RNA-N-glycosidase activity, by which mode of apoptosis induction by RIPs from reports available in it brings about depurination of adenine at position 4324 in the literature as well as from studies that have been carried out in our laboratory. Apart from plant RIPs. many bacterial tox-

* ins like DT [9,5,6],PE[5,10] and STs [11–13] have also been Corresponding author. Fax: +91 80 2360 0814. found to induce cell death by apoptosis. However, this review E-mail address: [email protected] (A.A. Karande). will focus on the cell death induced by plant RIPs and STs 1 S.N. is a senior research fellow under the Special Research alone because though ST is of bacterial origin its mechanism Programme of The Indian Institute of Science, Bangalore. of ribosome inactivation is identical to that of plant RIPs.

Abbreviations: RIP, ribosome inactivating protein; TNF, tumor necro- sis factor; AOP, anti-oxidant protein; MMP, mitochondrial membrane 2. Ribosome inactivating protein-induced apoptosis potential (Dwm); ML, mistletoe lectin; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; ST, Shiga toxin; PE, Pseudomonas exotoxin; CHX, cycloheximide; PNAG, Polynucleotide It was thought that cells treated with RIPs or RIP contain- N-glycosidase ing immunotoxins died simply because of arrest of protein

0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.01.038 S. Narayanan et al. / FEBS Letters 579 (2005) 1324–1331 1325 synthesis and consequently necrosis. However, it was found pyrimidine nucleotide antibiotic blasticidin S are known to in- that the morphology of cells treated with RIPs resembled that hibit the peptidyl activity of ribosomes. These anti- of cells undergoing death by apoptosis. Soon after the report biotics also resulted in activation of SAPK/JNK1 further by Griffiths et al. [14] on ricin- and abrin-induced apoptosis, confirming the above mentioned hypothesis. Antibiotics that many other bacterial as well as plant toxins were also found inhibit protein synthesis at stages other than translocation to induce apoptosis in mammalian cells [4–9]. Though many did not induce phosphorylation of SAPK/JNK1 upon treat- reports are available on toxin-induced apoptosis, very little ment with anisomycin [25]. progress has been made on elucidating the mechanism and Shiga toxin 1 has also recently been shown to trigger a sim- pathways of RIP-induced apoptosis. Most of the work that ilar ribotoxic stress response resulting in the activation of p38 has been published is on ST-induced apoptosis. MAPK and JNK, culminating in apoptosis of HCT cells. The One of the earliest reports that RIPs are capable of inducing finding that mutant ST (E167D) lacking RNA-N-glycosidase cell death came from the studies carried out by Griffiths et al. activity was not able to induce phosphorylation of p38 MAPK in 1987 [14]. They showed that intramuscular injection of ricin clearly indicates that the enzymatic activity of the A-chain is and abrin into rats resulted in death of cells in rapidly dividing essential for apoptosis signaling. Further, blocking of p38 tissues, where these toxins were known to concentrate. It was MAPK as well as JNK using the inhibitor SB202190 was able observed that the morphology of cells in para-aortic lymph to rescue the cells from ST-induced apoptosis [13]. Langer nodes and PeyerÕs patches of ricin or abrin injected rats resem- et al. [19,20,26] and others have found that ML are capable bled that of cells undergoing apoptosis characterized by the of inducing apoptosis in many cell lines. Site specific mutation formation of apoptotic bodies. It was also found that abrin of E166Q and R169Q in recombinant ML resulted in the loss and ricin treatment resulted in exhibition of apoptotic mor- of protein synthesis inhibitory activity as well as apoptosis phology, in addition to heterochromatin condensation and inducing capacity of the toxin [20]. Additionally, in the fungal DNA laddering in bovine pulmonary endothelial cells [15] ribotoxin a-sarcin, mutation of His137 to Gln also led to loss and also in U937, a human macrophage cell line [6]. In HeLa of apoptosis induction [27]. This suggests that the glycosidase cells, ricin was found to induce the classical apoptotic morpho- activity of RIPs is essential for their toxicity. It is known that logical changes and also activation of caspase-3. Ricin also JNK is involved in stress-induced apoptosis triggered via the brought about an arrest of cell cycle at G2/M phase, but no ef- mitochondria [28]. fect on the G0/G1 phase [16]. In addition to ricin and abrin, In the case of ricin, it has recently been shown to induce several other RIPs like ST, ML, saporin and gelonin are also apoptotic cell death in the macrophage cell line RAW 264.7 reported to induce apoptosis [11,17–21]. [29]. It was observed that in this case, treatment of RAW 264.7 cells with ricin led to increased secretion of TNF-a by these cells and consequently apoptosis. Ricin B-chain alone 3. Apoptosis induction by ribotoxic stress response did not induce apoptosis and pretreatment with the p38 MAPK inhibitor, SB202190, resulted in appreciable drop in It has been reported in the literature that many chemical the secreted TNF-a levels and also cell death. Similar results agents that disrupt macromolecular synthesis in cells also in- were obtained with another RIP modeccin and the antibiotic duce cell death. The list includes cycloheximide (CHX) and anisomycin [29]. However, studies carried out in our labora- actinomycin D, inhibitors of protein synthesis and RNA tran- tory on abrin-induced cell death in Jurkat cells revealed that scription, respectively. Treatment of HL-60 cells with CHX there was no significant rescue from apoptosis even upon pre- and actinomycin D resulted in large scale apoptosis [22].It treatment of cells with p38 MAPK or JNK inhibitors (Naraya- has also been shown that CHX alone is a poor inducer of nan et al., unpublished data). It is possible that abrin induces apoptosis, but it potentiates tumor necrosis factor (TNF-a)- multiple signaling pathways resulting in cell death and hence induced apoptosis [23]. Anisomycin, a low molecular weight different cell lines may respond differently towards transla- protein synthesis inhibitor, was shown to have better apoptosis tional inhibition with abrin. inducing capacity in U937 cells than CHX and was compara- With the evidences available at hand, it is safe to surmise ble to ricin in inducing apoptosis [24]. This suggested that the that one of the pathways induced by RIPs could be the ribo- continuous synthesis of certain regulatory proteins is required toxic stress response through their RNA-N-glycosidase activ- to keep the apoptotic machinery in check in some fast dividing ity, resulting in the activation of SAPK/JNK and p38 cell populations. MAPK leading to apoptosis [30]. However, the exact mecha- Earlier, it was shown by Iordanov et al. [25] that ricin, a-sar- nism or the pathway by which signals are transmitted from cin and anisomycin were able to activate the SAP kinases or damaged 28S rRNA to the MAPK pathway needs to be inves- JNK1 in Rat-1 cells. Damage to 28S rRNA by RIPs resulted tigated further. in a novel pathway of kinase activation called ribotoxic stress response, resulting in the activation of SAPK/JNK and its acti- vator, SEK1/MKK4. This study also revealed that activation 4. Apoptosis induction via the stress-induced mitochondrial of SAPK/JNK1 is not just due to protein synthesis inhibition, pathway but also due to signaling from 28S rRNA affected by RIPs. Further, not all inhibitors of protein synthesis were able to eli- Studies on the apoptosis induced by RIPs resulted in the cit the ribotoxic stress response. Hence, it was proposed that finding that mitochondria are involved in many cases, though ribotoxic stress response is specific for inhibitors that either in- the proposed modes of induction of this pathway vary. Most duce damage to the a-sarcin/ricin loop of 28S rRNA or RIPs of the studies reported a loss in mitochondrial membrane po- that ADP-ribosylate the EF-2/EF-G, thus arresting translation tential, MMP (Dwm), rapid release of cytochrome c, activation at the translocation step. Anisomycin and the aminohexose of caspase-9 and a marked increase in the production of reac- 1326 S. Narayanan et al. / FEBS Letters 579 (2005) 1324–1331 tive oxygen species (ROS) in cells. It is known that any or all of apoptosis [34]. Also, the connection between calcium mobiliza- these events can lead to cell death. tion from ER to mitochondria is one of the hot topics of re- In our laboratory, abrin was found to induce apoptosis in search in cell death. That the toxin-induced intrinsic or Jurkat cells and that it occurs via the mitochondrial pathway mitochondrial pathway of cell death is a complex phenomenon of apoptosis. We also determined the kinetics of various apop- with multiple levels of regulation is reflected by the above men- totic events induced by abrin and found that the loss of MMP tioned reports. However, the bottom line of these data is that was the earliest event to occur after protein synthesis inhibi- RIPs are capable of inducing stress-induced apoptosis via the tion. This was followed by caspase-3 activation and DNA frag- mitochondrial pathway. mentation. Unlike some of the lectins that are known to activate death receptors (DR), abrin induced cell death was independent of caspase-8 and hence DR. Overexpression of 5. Apoptosis induction by downregulation of anti-apoptotic Bcl-2 was able to inhibit apoptosis induced by abrin, but these factors cells were found to die by necrosis albeit after a prolonged exposure [4]. This suggested that the abrin-induced apoptosis The fate of a cell, to survive or to die, is decided by the del- follows the intrinsic mitochondrial pathway of cell death. icate balance between the levels of anti-apoptotic and pro- In the case of ST, it was observed that treatment of HeLa apoptotic proteins in the cell. Many pro-apoptotic factors cells with ST resulted in activation of several caspases, includ- are activated during extrinsic as well as intrinsic signaling ing caspase-3, -6, -8 and -9 and apoptosis as measured by of apoptosis. During ST-induced cell death in endothelial DNA fragmentation. It was also reported that enzymatically cells, it was demonstrated that both ST-1 and ST-2 were able active ST alone could bring about cell death. Further, the acti- to bring about a decrease in the cellular level of the Bcl-2 vation of caspase-9 suggested the involvement of mitochon- family protein, Mcl-1, while the levels of Bcl-2 and Bcl-XL dria, and indeed there was a loss of MMP (Dwm) in HeLa were unaltered [35]. Mcl-1 is structurally similar to Bcl-2 ex- cells upon treatment with ST [17]. Though caspase-8 activity cept that it harbors two PEST sequences that target the pro- was found to increase, it could be due to activation by cas- tein for degradation by proteasome. The involvement of pase-9 at late stages of apoptosis. Further, the fact that brefel- caspases in the degradation of Mcl-1 was ruled out as z- din A treatment resulted in inhibition of ST induced apoptosis VAD-FMK, a pancaspase inhibitor was not able to block suggests that the DR pathways might not be involved. It is also Mcl-1 degradation. However, specific inhibitors of protea- of importance to note that ST-induced apoptosis was inhibited some, lactacystin and its derivative b-lactone, were able to by overexpression of Bcl-2, an anti-apoptotic protein acting at block the degradation of Mcl-1, thus rescuing cells from the mitochondrial level [18]. ST-induced apoptosis. It is possible that Mcl-1 is continu- An alternate pathway of apoptosis induction by abrin has ously synthesized and degraded by proteasome and the con- been proposed by Shih et al. [31]. They found that the apopto- tinuous presence of Mcl-1 is probably required to prevent sis signaling by abrin is independent of its glycosidase activity the cells from undergoing death. Protein synthesis inhibition and is due to its direct interaction with a mitochondrial protein by ST leads to a decreased level of Mcl-1, consequently called anti-oxidant protein-1 (AOP-1). They were able to show resulting in apoptosis. Another study by Jones et al. [18] re- that abrin colocalized with the AOP-1 on the mitochondria, in veals that treatment of epithelial cells with ST led to an in- addition to showing that AOP-1 interacts with an active site crease in the levels of the pro-apoptotic protein, Bax. This mutant A-chain using yeast two-hybrid system. Also, it was mechanism of action of RIP toxins might be cell line depen- found that abrin was able to bring about a decrease in the dent as it is known that different cell lines express differing anti-oxidant function of AOP-1 and consequently an increase levels of pro- and anti-apoptotic factors and needs to be stud- in ROS production, and cytochrome c release into the cytosol. ied further. This study suggested that abrin is able to induce apoptosis by production of ROS, loss of MMP and release of cytochrome c into the cytosol. Shih et al. propose that the apoptogenic activity of abrin A-chain could be independent of its RNA- 6. Apoptosis induction due to NAD+ depletion by PARP N-glycosidase activity. In addition to inducing loss of MMP, hyperactivation abrin was also found to induce elevation in ROS levels due to mitochondrial damage [4]. There are also reports available on RIP-induced apoptosis A third mode of action of these toxins is supposedly by ROS that involves alternative pathways of apoptosis. It was re- production in response to stress and by increase in intracellular ported that treatment of U937 cells with ricin led to a marked calcium levels. Ricin induced a rapid elevation of cellular cal- decrease in the intracellular levels of NAD+ and ATP before cium level in hepatoma cells [32]. Trichosanthin, a type I RIP, inducing apoptosis [36]. Recent studies by Izyumov et al. [37] has been shown to induce; apoptosis in many cells. Trichosan- showed that decrease in intracellular ATP levels by a combina- thin induces high levels of ROS production in human chorio- tion of inhibitors of glycolysis and oxidative phosphorylation carcinoma cells [33]. This event was shown to be dependent on resulted in the mitochondrial pathway of cell death. Poly- extracellular as well as intracellular calcium levels and ROS (ADP-ribose) polymerase (PARP) was also implicated to play production paralleled calcium elevation, suggesting that ROS a role in this apoptotic pathway as 3-amino benzamide, a production might be a consequence of calcium signaling. The PARP inhibitor, was able to block loss of NAD+ and thus anti-oxidant Trolox and ROS scavengers catalase and manni- apoptosis to some extent. Indeed, some recent reports conclu- tol were able to rescue the cells from trichosanthin-induced cell sively show that hyperactivation of PARP-1 can result in death [33]. Recently, many studies have established a role for depletion of NAD+ levels and hence can induce mitochondrial cellular calcium signaling in the mitochondrial pathway of damage and apoptosis [38,39]. S. Narayanan et al. / FEBS Letters 579 (2005) 1324–1331 1327

Barbieri et al. [40] have shown that the RIPs saporin L2, saporin S6, gelonin and momordin have transforming activity that involves auto-ADP-ribosylation of PARP. They also show that RIPs directly depurinate auto-modified PARP releasing adenine from the ADP-ribosyl group. It is known that the auto-modification of PARP is required for the base excision repair mechanism of DNA repair pathway. They pro- pose that depurination of auto-modified PARP could result in the inhibition of DNA repair pathway as well as availability of PARP for further ADP-ribosylation, leading to depletion of intracellular levels of NAD+ thus inducing apoptosis. How- ever, similar studies carried out by our group showed that PARP inhibitor was not able to rescue Jurkat cells from Fig. 1. Confocal microscopy for localization of abrin-FITC in SIHA cells. SIHA cells grown for 12 h on teflon coated slides were treated abrin-induced cell death [4]. It is possible that this pathway with FITC-conjugated abrin (1 lg/ml) for 3 and 6 h. The cells were is relevant only in certain types of cell lines that are sensitive then stained with DAPI for staining the nucleus. Abrin localization is to alterations in cellular NAD+ levels. indicated by FITC fluorescence (green) and the nucleus is stained blue. Note the absence of green fluorescence in the nuclei of cells.

7. Apoptosis induction by DNA damage due to nuclease activity whether RIPs can enter the nucleus to induce DNA damage by nuclease activity and trigger apoptosis. Another mode of apoptosis induction by RIPs is by their non-specific nuclease activity, by which they bring about DNA damage and hence apoptosis. RIPs have been shown 8. Discussion to be active in inducing depurination of naked DNA as well as RNA in vitro and are known to have nuclease activity at Table 1 shows the toxicity with respect to apoptosis induc- high concentrations. The nuclease activity has been proposed tion by various RIPs and the low molecular weight protein to be due to the cleavage of DNA backbone at the apurinic/ synthesis inhibitor, CHX. The table includes unpublished apyrimidinic sites [41]. The RIPs dianthin 30, saporin 6 and data gathered from studies carried out in our laboratory on gelonin have been shown to have DNase activity, while ricin various adherent and non-adherent cell lines with abrin and did not show any such activity under the conditions used for observations from the literature. It can be seen from the table the assay [42]. Contradicting this is the report where ricin and that though there is not much difference in the IC50 for pro- ST have been shown to induce DNA damage by their nucle- tein synthesis inhibition in Jurkat cells and U937 cells, the ex- ase activity in human umbilical vein endothelial cells (HU- tent of apoptosis is different. Also, the cell lines Raji, U937 VECs) [12]. Similarly, trichosanthin has been shown to cut and U266B1 did not show much apoptosis even up to 72 h, supercoiled double stranded DNA into linear and relaxed but instead exhibited necrotic cell death (Bora et al., unpub- forms in vitro. This activity has also been observed with lished observation). It is known that the B-cell line Raji over- other RIPs like gelonin, saporin and dianthin. The nuclease expresses the anti-apoptotic protein, Bcl-2. Studies have activity of RIPs is still not very clear because the conditions shown that Bcl-2 overexpression in Jurkat cells results in under which the assay was performed and the concentrations necrosis rather than apoptosis upon treatment with abrin of RIPs used vary considerably from one report to another. [4]. Though the table indicates that RIPs induce cell death At least in two cases, contaminating nucleases have been by apoptosis, there are considerable differences in the kinetics found along with the RIP preparations. Further, it was re- as well as extent of apoptosis observed for a given concentra- ported that nuclease free preparations of gelonin, momordin tion of toxin. 1, PAP-S and saporin S6 have been found to harbor only the Though all RIPs induce protein synthesis inhibition, it is polynucleotide N-glycosidase (PNAG) activity and no DNA possible that the pathway of apoptosis induced by RIPs de- nicking or nuclease activity [43]. In spite of all these reports pends upon the cells as well as the RIP used. Studies carried the question whether RIPs are capable of entering nucleus out by Brinkmann et al. [44] in genetic screens for cDNAs to bring about depurination or nicking of DNA in vivo re- that confer resistance to PE-induced apoptosis resulted in mains to be answered. With FITC-conjugated abrin, confocal the identification of a gene that encoded an antisense frag- microscopy studies were carried out in our laboratory to find ment of the cellular apoptosis susceptibility (CAS). a gene if abrin could be localized in the nuclei of various cell lines. that is involved in nuclear import and chromosome segrega- The outcome of the study indicated no nuclear localization tion. While downregulation of CAS gene was able to confer of abrin even after it has entered the cytosol. Fig. 1 shows resistance to PE and DT induced apoptosis, it was not, able the confocal microscopy of SIHA cells after treating with to inhibit cell death induced by ricin. This suggests that the abrin-FITC for 3 and 6 h. From the figure, it is evident that mechanism of apoptosis induction might depend on the abrin does not localize to the nuclei of SIHA cells and hence mechanism of the action of the toxin rather than protein cannot act on the nuclear DNA. Similar results were ob- synthesis inhibition, the end result of ribosome inactivation. served in MCF-7 and HeLa cells (data not shown). This is Further, the picture is made more complex by contradictory in contradiction with the earlier studies by Dodson et al. reports in the literature with respect to the involvement [41], Roncuzzi et al. [42] and Brigotti et al. [12]. It is evident of p38 MAPK and JNK 1/2 in CHX-induced apoptosis that this aspect needs to be studied in depth to determine [45,46]. 1328 S. Narayanan et al. / FEBS Letters 579 (2005) 1324–1331

Table 1 Effects of type I and type II RIPs and the low molecular weight inhibitor, CHX, on protein synthesis and cell death induction in various cell lines

Cell line IC50 for PSI Concentration Time (h) % apoptosis observed Reference Abrin Jurkat 0.2 ng/ml 10 ng/ml 12 30 [4] U937 0.75 ng/ml 1 lg/ml 72 20a Bora et al.b Raji 35 ng/ml 1 lg/ml 72 8c Bora et al.b U266B1 3.0 ng/ml 1 lg/ml 48 20a Bora et al.b BeWo 7.5 ng/ml 1 ng/ml 48 55 Bora et al.b KB 10 ng/ml 100 ng/ml 48 40 Bora et al.b MCF-7 0.4 ng/ml 10 ng/ml 36 25 Surendranath et al.d T47D 0.4 ng/ml 10 ng/ml 36 36 Surendranath et al.d Ovcar-3 0.3 ng/ml 10 ng/ml 36 35 Surendranath et al.d U937 NAe 0.1 nM 6 40 [6]

Ricin U937 NA 1 nM 6 18 [6]

Shiga toxin HeLa NA 10 ng/ml 4 64 [17] HEp-2 NA 12.5 ng/ml 24 20 [18] THP-1 NA 1 ng/ml 5 48.2 [11]

Mistletoe lectin MOLT-4 NA 100 pg/ml 24 22 [20] Lymphocytes NA 50 ng/ml 72 40 [19] HUVEC NA 50 lg/ml 48 25 [21]

Trichosanthin JAR NA 50 ng/ml 24 30 [33]

Immunotoxin RFB4(dsFv)PE38 CA 46 0.6 ng/ml 1 ng/ml 24 16 [8] Daudi 20 ng/ml 1 ng/ml 24 84 [8] JD38 0.3 ng/ml 1 ng/ml 24 16 [8] Namalwa 1.5 ng/ml 1 ng/ml 24 27 [8] Raji 0.4 ng/ml 1 ng/ml 24 27 [8]

Cycloheximide MRC-5 NA 50 lg/ml 72 45.2 [22] HL-60 NA 50 lg/ml 48 48 [22] aNecrosis was observed. bUnpublished observation by Namrata Bora. cExtensive necrosis was observed at 72 h. dUnpublished observation by Kalpana Surendranath. eNA: data not available.

Fig. 2 summarizes various signaling cascades and pathways namely ribotoxic stress in cells, as it is the origin of intrinsic that are triggered by RIPs to induce cell death. It is quite apoptotic signaling cascade. clear from the picture that most of the pathways are funneled into the mitochondria. It is well known that mitochondria play a major role in several stress induced cell death path- 9. Future prospects ways and damage to mitochondria with subsequent loss of mitochondrial membrane potential has been known to be Since the potential application of RIP toxins lies in their tar- the point of no return in apoptotic cascades. It is logical to geted delivery in cancer immunotherapy, it is imperative that a assume that RIPs do not activate the cellular apoptosis systematic study be carried out on various cancer cells and cell machinery directly as the primary activity of these toxins is lines to determine their efficacy in inducing apoptosis. Such a to inactivate ribosomes and stop protein synthesis in cells. study will throw light on the sensitivity of cancer cells to apop- Cells are known to respond to several stress conditions like tosis induced by RIPs. It is desirable that toxin mediated ther- heat shock, infection by viruses or loss of cellular ATP. Cells apy of cancers induces apoptotic cell death and not necrosis, depending upon the extent of stress they are exposed to, since necrosis results in the spillage of cellular content to extra- either try to overcome the stress by activating various stress cellular milieu, thereby triggering inflammatory response, response genes or undergo cell death. But how the cells de- which can be deleterious. Also, knowledge about the mecha- cide when to activate the stress negating pathway and when nism of RIP-induced cell death pathways will also enable us to induce apoptosis is still not known. Mitochondrion is a to tackle the problem of development of resistance to these good candidate for sensing the stress induced by RIPs, toxins by cancer cells. S. Narayanan et al. / FEBS Letters 579 (2005) 1324–1331 1329

Fig. 2. Various pathways of RIP-induced apoptosis. The possible mechanisms and pathways by which RIPs can induce cell death are shown.

At present, the molecular link between protein synthesis Acknowledgments: We gratefully acknowledge Prof. S.K. Podder for inhibition and the triggering of apoptotic cell death is unclear. helpful scientific discussions. The Council of Scientific and Industrial Is it the direct damage to the 28S rRNA that is induced by the Research, Government of India and the Indian Institute of Science, Bangalore are acknowledged for their financial support. RNA-N-glycosidase activity of RIPs or ADP-ribosylation of EF-2/EF-G in the case of DT and PE that triggers apoptosis? Is it the loss of protein synthesis in the cells that acts as the crit- ical factor in the signaling of apoptosis? Do RIPs interact with References mitochondria and affect them directly? These are some of the [1] Stirpe, F. (2004) Ribosome-inactivating proteins. Toxicon 44 (4), questions that need to be addressed in order to understand 371–383. the mechanism of translation inhibition induced cell death. [2] Hartley, M.R. and Lord, J.M. (2004) Cytotoxic ribosome- In addition to plant tissues, proteins that bring about ade- inactivating lectins from plants. Biochim. Biophys. Acta 1701 nine glycosylase activity under certain specific cellular stress (1–2), 1–14. conditions have been reported to be found in mammalian tis- [3] Barbieri, L., Battelli, M.G. and Stirpe, F. (1993) Ribosome- inactivating proteins from plants. Biochim. Biophys. Acta 1154 sues also. This activity is very similar to that of many plant (3–4), 237–282. RIPs and has been observed in extracts of cells or tissues under [4] Narayanan, S., Surolia, A. and Karande, A.A. (2004) Ribosome- stress or infection [47]. It is, therefore, probable that a mecha- inactivating protein and apoptosis: abrin causes cell death via nism of cell death induction that is triggered during cellular mitochondrial pathway in Jurkat cells. Biochem. J. 377 (1), 233– 240. stress responses like protein synthesis inhibition exists in cells. [5] Morimoto, H. and Bonavida, B. (1992) - and RIPs might activate this pathway to trigger cell death upon Pseudomonas A toxin-mediated apoptosis. ADP ribosylation of translational inhibition. elongation factor-2 is required for DNA fragmentation and cell 1330 S. Narayanan et al. / FEBS Letters 579 (2005) 1324–1331

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