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Verocytotoxin-1 induces apoptosis in vero cells
Journal of Infection(1995) 3o, 213-218
Ver0cytotoxin-1 Induces Apoptosis in Vero Cells C. D. Inward, J. Williams, I. Chant 1, J. Crocker 2, D. V. Milford, P. E. Rose 1 and C. M. Taylor Renal Research Laboratory, Queen Elizabeth Hospital, Birmingham, 1Department of Haematology, Warwick Hospital and 2Department of Pathology, Heartlands Hospital, Birmingham, U.K. Accepted for publication 7 December 1994
Introduction Epidemiological studies have firmly linked infection with verocytotoxin-producing Escherichia coli (VTEC) with both haemorrhagic colitis and childhood haemolytic uraemic syndrome (HUS). Although details of the pathogenesis of HUS remains unknown, it seems likely that the exotoxins verocytotoxin I and 2 (VT1 and VT2), also known as shiga-like toxins 1 and 2, play a role. The holotoxins consist of one A-subunit and five B-subunits. The latter bind to the terminal di-galactose moiety of the cell membrane glycolipid globotriaosylceramide, Gb3 (also known as CD77 or blood group Pk). There are preliminary data to show that these receptors are expressed in the glomeruli of young humans, x although Gb3 is not found in the renal vasculature of most laboratory animals. Once bound the toxin is internalised by an energy dependent process and the A-subunit released. This has enzymatic activity and cleaves adenosine from ribosomal RNA at a point where aminoacyl transfer RNA is assembled, thus effectively truncating protein synthesis. The cytopathic effect on eukaryotic cells by these toxins has been thought to be the consequence of protein synthesis inhibition. Cell death may take place through two descriptive processes. In necrosis there is a chaotic breakdown of cell integrity, often with cell swelling and membrane rupture. By contrast eukaryotic cells in both physiological and pathological circumstances, may undergo gene-controlled programmed cell death; apoptosis. 2-4 In this case crucial events in the dismantling of the cell include a characteristic cleavage of DNA into 200 base pair fragments, contraction of the cytoplasm and the packaging of nuclear remnants into membrane bound vesicles. Apoptotic cells are rapidly recognised and phagocytosed Address correspondenceto: C. M. Taylor,Department of Nephrology, Children's Hospital, Ladywood Middleway, Ladywood, Birmingham, B16 8ET,U.K.
0163-4453/95/030213 +06 $08.00/0
by their neighbours. We have looked further into the mode of cell death using vero cells, which are exquisitely sensitive to the toxin. The following experiments show that apoptosis is the principal mode of cell killing by VT1.
Materials and Methods Giemsa, diphenylamine (DPA), sodium lauryl sarkosinate, proteinase K, RNase and ¢I)X 174 Hae III digest DNA marker were obtained from Sigma. Agarose was provided by Pharmacia. Verocytotoxin 1 and anti-VT1 monoclonal antibody were generously provided by Dr Mohammed Karmali. Anti-CD 7 7 antibody was obtained from Serotec. Veto cells originating from green African monkey kidney tissue were obtained from the European collection of animal cell culture, Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K. and grown in Dulbecco's modified essential medium, supplemented with 10% foetal calf serum, L-glutamine, penicillin and streptomycin at 5% CO2, at 3 7°C. Vero cells were seeded in 8 well Permanox slides and a minimum of 500 cells per well were counted under phase contrast microscopy after treatment with VT1. Cells were scored as either apoptotic or normal. Alternatively, cells were stained with Giemsa and photographed.
Gel electrophoresis DNA was prepared for gel electrophoresis as previously described. S Vero cells were subjected to centrifugation at 750 x g for 5 rain and the pellets washed with ice-cold TBS. Cells were lysed with 50 mM Tris, 10 mM EDTA, 0.5% sodium lauryl sarkosinate and 0 . 5 m g / m l proteinase K and incubated at 50°C for 1 h. RNase (0.5 rag/ ml) was added and samples incubated for a further I h © 1995 The British Societyfor the Study of Infection
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Verocytotoxin Induces Apoptosis
Figure 1. Giemsa stained vero cells. (a) Untreated cells, (b) cells treated with 10 pg/ml VT1 for 24 hours showing nuclear and cytoplasmic condensation, (c) cells treated with 100 pg/ml VT1 for 24 hours showing characteristic blebbing of the plasma membrane; apoptotic bodies. Magnification x 200.
C. D. Inward et aL
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at 50°C. Loading buffer (10 mM EDTA, pH 8.0, 1% low gelling temperature agarose, 40% sucrose and 0.25% bromophenol blue) was added to each sample. Electrophoresis was performed on a 2 % agarose gel containing 0.1 ~tg/ml ethidium bromide at 40V for 2-3 h with a TBE running buffer. DNA was visualised under ultraviolet light and photographed.
acid was added to a final concentration of 0.5 M, to both supernatant and pellet, followed by 2 volumes of diphenylamine solution (0.088 M diphenylamine, 98% v/v glacial acetic acid, 1.5% v/v concentrated sulphuric acid and 0.5 % v/v of 1.6% acetaldehyde solution. Samples were stored at 4°C for 48 h after which they were quantiffed spectrophotometrically at OD575 nm. 6 DNA fragmentation was expressed as a percentage using the equation:
Quantification of DNA fragmentation
OD Supernatant/(OD Supernatant + OD Pellet) x 100 = % DNA fragmentation
Cell pellets were prepared by centrifugation at 750 x g for 5 min and lysed with 400 gl of lysis buffer (0.2% Triton X-IO0, 10 mM Tris and i mM EDTA, pH 8.0). Lysates were subjected to centrifugation at 13,000 x g for 10 rain. The supernatant, containing small DNA fragments, was removed from the pellet of uncut DNA. The pellet was resuspended in 2 O0 ~tl of lysis buffer. Perchloric
Results
Oualitative data The addition of VT1 to vero cells caused changes in cell structure seen by light microscopy. These consisted of condensation of cell cytoplasm and nuclear chromatin with nuclear material appearing in membrane bound vesicles; apoptotic bodies (Fig. 1). No necrosis was observed. VT1 induced degradation of DNA in vero cells giving rise to the typical ladder pattern on gel electrophoresis (Fig. 2). This indicated endonuclease activity.
Quantitative data Under phase contrast microscopy apoptotic cells could be clearly identified by their smaller, rounded and opalescent appearance, often being detached form the surface of the well. The 24 hour dose dependent counts of apoptotic cells are given in Fig. 3. Similar results were obtained using the diphenylamine (DPA) assay. DPA binds to deoxyribose replacing a purine base. DNA thus labelled, can be quantified because of the characteristic light absorbance of DPA. After centrifugation of the sample, small cleaved fragments of DNA are measured in the supernatant. Using this assay increasing fragmentation induced by VT1, could be seen in a dose dependent manner (Fig. 4). Fragmentation at lOpg/ml of VT1 could be completely blocked by monoclonal antibody to VT1, and partial blocking could be achieved by preincubation with anti CD 77 monoclonal antibody for an hour at 37°C before the toxin was applied (data not shown). The time course of VT1 action is given in Fig. 5, the earliest fragmentation appeared at 6-8 h and continued up to 24 h. Figure 2. DNA degradation s h o w n by gel electrophoresis in vero cells treated with VT1. 5 x 106 vero cells were incubated with or or without VT1 for 24 hours. Lane 1, untreated cells; lane 2, cells treated with 100 pg/ml VT1, lane 3, molecular size markers 7 2 - 1 3 5 3 base pair fragments following DNA digestion with ~ X 1 7 4 Hae III.
Discussion In these experiments we demonstrated both DNA fragmentation and the cytopathic changes characteristic of
Verocytotoxin Induces Apoptosis
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Figure 3. Effectof VT1 on the percentage of apoptoticvero cells observed by phase contrast microscopyat 24 h. (a) Untreated cells; (b) 10 pg/ ml VT]; (c) 100 pg/ml VT1; (d) 10 ng/ml VT1. Results axe mean and S.E.M. for five experiments.
apoptosis; findings which have not been previously demonstrated in vero cells. The gel electrophoresis (Fig. 2) provides qualitative information on the fragmentation of DNA, whereas the diphenylamine reaction allows quantification of the same process. With the latter we were able to show that DNA fragmentation was apparent some 6-8 h after incubation with the toxin and continued to increase to 24 h, the end point of these experiments. In 2 4 h dose response assays, there was concordance between the percentage of DNA fragmented and the proportion of cells appearing apoptotic by phase contrast microscopy. Giemsa staining provided further qualitative information and confirmed the typical pyknotic appearance and nuclear packaging of apoptotic ceils. From the magnitude of the apoptotic response at 24 h and
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Figure 4. Effect of VT1 on the percentage of DNA fragmentation. Vero cells were treated for 24 h with VT1 and percentage of DNA fragmentation estimated using DPA reaction. (a) Untreated cells; (b) 1 pg/ml VT1; (c) 5 pg/ml VT3_;(d) 10 pg/ml VT1; (e) 100 pg/ml VT1. Results are mean and s.E.u, for three experiments.
comparing this with the overall cytocidal effect of verotoxin on vero ceils, we conclude that programmed cell death is the principal mode of killing. This result is not surprising. Pyknotic changes were reported in ileal enterocytes in rabbits infected with VTEC 0 1 5 7 : H 7 or exposed to Shiga toxin alone. 7'8 More importantly other bacterial and plant toxins have similar protein inhibitory and cytopathic effects? Diphtheria and pseudomonas A toxins catalyse ADP ribosylation of a residue on elongation factor 2 (EF2), inactivating it and effectively blocking protein synthesis. 1° The A-chain of the plant lectin ricin operates in an identical way to that of the Shiga/verocytotoxin family in cleaving the Nglycosidic bond of adenosine at position 4 3 2 4 from the 5' end of 28S ribosomal RNA of the 60S ribosomal snbunit.11.12 Diphtheria, pseudomonas A and ricin toxins
C. D. Inward et aL
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changes in the number of toxin receptors expressed on the cell surface. 16 However, Chang et al., ~7 experimenting with other h u m a n cell lines found that whereas diphtheria toxin induced DNA fragmentation and cytolysis, cells in which comparable protein synthesis inhibition was obtained with cyclohexamide did not. A further separation between overall protein synthesis inhibition and apoptosis was found in experiments by Khan and 30 Waring on murine peritoneal macrophages38 Apoptosis is an important physiological process in macrophages which serves to curtail the inflammatory response. Ricin precipitated this in a dose dependent manner. However, once macrophages had become adherent, ricin no longer induced DNA fragmentation, even though protein syn20 thesis was inhibited. An additional pathway by which verocytotoxin might induce apoptosis was suggested by Mangeney et al. 19 They showed that in Burkitt's l y m p h o m a cells, which bear the CD77 receptor, the B-subunit of the toxin alone could induce the characteristic DNA fragmentation and histological changes of apoptosis. The B-subunit is 10 thought not to have any enzymatic activity of its own. It thus appears that this is a novel signal transduction pathway unrelated to protein synthesis inhibition. Many of the experiments outlined above used transformed cell lines which m a y enter apoptosis differently from their parent tissues. Extrapolation from in vitro 0 observations to the action of the toxin in vivo m a y be 0 5 10 15 20 25 misleading. In part this objection was removed in a subtle hours experiment by Buchman et al.,2° using porcine endothelial Figure 5. Timecourse of DNA fragmentation estimated by DPAreaction cells. These authors showed that pre-treatment of endoin vero cells treated with VT1. Open symbols: untreated cells. Solid thelium with bacterial endotoxin (lipopolysaccharide, symbols:cells treated with 100 pg/ml VT1. Results are mean and S.E.M. LPS), could prime cells so that a second signal consisting for four experiments. of either a heat shock or protein synthesis inhibition (cycloheximide) would induce apoptosis. Independently the LPS, heat shock or cycloheximide treatment itself all induce apoptosis in eukaryotic cells. ~°'~3 Sandvig and was sub-lethal and reversing the order of the experiment van Deurs 14 showed that Shiga toxin and ricin induce did not give rise to cell death. The concept that two DNA fragmentation and the ultrastructural changes of different signals in a specific sequence can induce apopapoptosis in vero and MDCK cells, although these authors tosis is interesting and m a y relate to disease states. We used cell lysis to quantify cell death. note that in HUS associated with Shigellosis there is Is apoptosis related to the protein synthesis inhibitory properties held in c o m m o n by these toxins? Certain evidence that LPS enters the circulation accompanied h u m a n cell lines, e.g. HL-60, undergo apoptosis w h e n by a leukaemoid reaction. 2° In VTEC 0 1 5 7 infection treated with inhibitors of transcription (actinomycin- endotoxaemia is suggested indirectly by neutrophilia D) or translation (cycloheximide)35 It has been noted which occurs in those who proceed to HUS, 22 and depreviously that rapidly dividing cells appear more sens- rangement of LPS-binding proteins in plasma. 23 It would itive to the action of these toxins. 13 Cell vulnerability to be reasonable to find out whether LPS can operate as protein synthesis inhibitors is in part related to their the first, and verocytotoxin as the second signal to cause position in the cell reproductive cycle, being maximal at apoptosis in h u m a n endothelinm. If so this might explain the G 1-S interphase. Vero cells which replicate vigorously the nature and distribution of the vascular lesion of in culture, have been shown to be increasingly sensitive haemorrhagic colitis and the haemolytic uraemic synto verotoxin at this point, a p h e n o m e n o n divorced from drome.
40 .~
Verocytotoxin Induces Apoptosis
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Acknowledgement The authors are indebted to Dr M. A. Karmali for the generous gift of VT1 and anti-VT1 monoclonal antibody. CDI was supported by the Renal Research Fund of the Children's Hospital, Birmingham, IC by the Paul O'Gorman Foundation.
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References 1 Lingwood CA. Verotoxin-binding in human renal sections. Nephron 1994; 66: 21-28. 2 Kerr JFR, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kinetics. Br ] Cancer 1972; 26: 239-257. 3 Alison MR, Sarraf CE. Apoptosis: a gene-directed programme of cell death. ] Roy Coil Phys London 1992; 26: 25-35. 4 Wyllie AH. Apoptosis (The 1992 Frank Rose Memorial Lecture). Br ] Cancer 1993; 67: 205-208. 5 Smith GA, Williams GT, Kingston R, Jenkinson EJ, Owen IIT. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 1989; 337:181-183. 6 Burton K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Chem ] 1956; 52: 315-323. 7 Pal CH, Kelly JK, Myers GL. Experimental infection of infant rabbits with verotoxin-producing Escherichia coll. Infect Immunol 1986; 51: 16-23. 8 Keenan KP, Sharpnack DD, Collins H, Formal SB, O'Brien AD. Morphologic evaluation of the effects of Shiga toxins and E. coli Shiga-like toxin in the rabbit intestine. Am ] Pathol 1986; 125: 69-80. 9 Kochi SK, Collier RJ. DNA fragmentation and cytolysis in the U937 ceils treated with diphtheria toxin or other inhibitors of protein synthesis. Exp Cell Res 1993; 208: 296-302. 10 Morimoto H, Bonavida B. Diphtheria toxin- and pseudomonas A toxin-mediated apoptosis. ] Immunol 1982; 149: 2089-2094. 11 Endo Y, Tsurgi K. RNA N-glycosidase activity of ricin A-chain,
14 15
16 17 18 19 20
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22
23
Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem 1987; 262: 812-813. Furutani M, Kashiwagi K, Ito K, Endo Y, Igarashi K. Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin and of sarcin. Arch Bioehem Biophys 1982; 293: 140-146. Griffiths GD, Leek MD, Gee DJ. The toxic plant proteins ricin and abrin induce apoptotic changes in mammalian lymphoid tissues and intestine. ] Pathol 1987; 151: 221-229. Sandvig K, van Deurs B. Toxin-induced cell lysis: Protection by 3-methyladenine and cyclohexamide. Exp Cell Res 1992; 200: 253-262. Martin SJ, Lennon SV, Bonham AM, Cotter TG. Induction of apoptosis (programmed cell death) in human leukaemic HL-60 cells by inhibition of RNA or protein synthesis. J Immunol 1990; 145: 1859-1867. Pudymaitis A, Lingwood CA. Susceptibility to verotoxin as a function of the cell cycle. J Cell Physiol 1992; 150: 632-639. Chang MP, Bramhall J, Graves S, Bonavida B, Wisnieski BJ. Internucleosomal DNA cleavage precedes diphtheria toxin-induced cytolysis. ] Biol Chem 1989; 264: 15261-15267. Khan T, Waxing P. Macrophage adherence prevents apoptosis induced by ricin. Eur ] Cell Biol 1993; 62: 4 0 6 4 1 4 . Mangeney M, Lingwood CA, Taga S, Caillou B, Tursz T, Wiels J. Apoptosis induced in Burkitt's lymphoma cells via GB3/CD77, a glycolipid antigen. Cancer Res 1993; 53: 5314-5319. Buchman TG, Abello PA, Smith EH, Bulkley GB. Induction of heat shock response leads to apoptosis in endothelial ceils previously exposed to endotoxin. Am ] Physiol 1993; 265: H165-170. Koster K, Levin J, Walker L, Tung KS, Gilman RH, Rahaman MM, Majid MA, Islam S, Williams RC. Hemolytic uremic syndrome after shigellosis, Relation to endotoxemia and circulating immune complexes. N Engl J IVied 1978; 298: 927-933, Pavia AT, Nichols CR, Green DP, Tauxe RV, Mottice 8, Greene K, Wells JG, Siegler RL, Brewer El), Harmon D, Blake PA. Hemolytic uremic syndrome during an outbreak of Escherichia coli O157:H7 infection in institutions for mentally retarded persons: clinical and epidemologic observations. ] Pediatr 1990; 116: 544-551. Heyderman RS, Fitzpatrick MM, Barclay GR. Haemolytic-uraemic syndrome. Lancet 1994; 343: 1042.
Ver0cytotoxin-1 Induces Apoptosis in Vero Cells C. D. Inward, J. Williams, I. Chant 1, J. Crocker 2, D. V. Milford, P. E. Rose 1 and C. M. Taylor Renal Research Laboratory, Queen Elizabeth Hospital, Birmingham, 1Department of Haematology, Warwick Hospital and 2Department of Pathology, Heartlands Hospital, Birmingham, U.K. Accepted for publication 7 December 1994
Introduction Epidemiological studies have firmly linked infection with verocytotoxin-producing Escherichia coli (VTEC) with both haemorrhagic colitis and childhood haemolytic uraemic syndrome (HUS). Although details of the pathogenesis of HUS remains unknown, it seems likely that the exotoxins verocytotoxin I and 2 (VT1 and VT2), also known as shiga-like toxins 1 and 2, play a role. The holotoxins consist of one A-subunit and five B-subunits. The latter bind to the terminal di-galactose moiety of the cell membrane glycolipid globotriaosylceramide, Gb3 (also known as CD77 or blood group Pk). There are preliminary data to show that these receptors are expressed in the glomeruli of young humans, x although Gb3 is not found in the renal vasculature of most laboratory animals. Once bound the toxin is internalised by an energy dependent process and the A-subunit released. This has enzymatic activity and cleaves adenosine from ribosomal RNA at a point where aminoacyl transfer RNA is assembled, thus effectively truncating protein synthesis. The cytopathic effect on eukaryotic cells by these toxins has been thought to be the consequence of protein synthesis inhibition. Cell death may take place through two descriptive processes. In necrosis there is a chaotic breakdown of cell integrity, often with cell swelling and membrane rupture. By contrast eukaryotic cells in both physiological and pathological circumstances, may undergo gene-controlled programmed cell death; apoptosis. 2-4 In this case crucial events in the dismantling of the cell include a characteristic cleavage of DNA into 200 base pair fragments, contraction of the cytoplasm and the packaging of nuclear remnants into membrane bound vesicles. Apoptotic cells are rapidly recognised and phagocytosed Address correspondenceto: C. M. Taylor,Department of Nephrology, Children's Hospital, Ladywood Middleway, Ladywood, Birmingham, B16 8ET,U.K.
0163-4453/95/030213 +06 $08.00/0
by their neighbours. We have looked further into the mode of cell death using vero cells, which are exquisitely sensitive to the toxin. The following experiments show that apoptosis is the principal mode of cell killing by VT1.
Materials and Methods Giemsa, diphenylamine (DPA), sodium lauryl sarkosinate, proteinase K, RNase and ¢I)X 174 Hae III digest DNA marker were obtained from Sigma. Agarose was provided by Pharmacia. Verocytotoxin 1 and anti-VT1 monoclonal antibody were generously provided by Dr Mohammed Karmali. Anti-CD 7 7 antibody was obtained from Serotec. Veto cells originating from green African monkey kidney tissue were obtained from the European collection of animal cell culture, Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K. and grown in Dulbecco's modified essential medium, supplemented with 10% foetal calf serum, L-glutamine, penicillin and streptomycin at 5% CO2, at 3 7°C. Vero cells were seeded in 8 well Permanox slides and a minimum of 500 cells per well were counted under phase contrast microscopy after treatment with VT1. Cells were scored as either apoptotic or normal. Alternatively, cells were stained with Giemsa and photographed.
Gel electrophoresis DNA was prepared for gel electrophoresis as previously described. S Vero cells were subjected to centrifugation at 750 x g for 5 rain and the pellets washed with ice-cold TBS. Cells were lysed with 50 mM Tris, 10 mM EDTA, 0.5% sodium lauryl sarkosinate and 0 . 5 m g / m l proteinase K and incubated at 50°C for 1 h. RNase (0.5 rag/ ml) was added and samples incubated for a further I h © 1995 The British Societyfor the Study of Infection
214
Verocytotoxin Induces Apoptosis
Figure 1. Giemsa stained vero cells. (a) Untreated cells, (b) cells treated with 10 pg/ml VT1 for 24 hours showing nuclear and cytoplasmic condensation, (c) cells treated with 100 pg/ml VT1 for 24 hours showing characteristic blebbing of the plasma membrane; apoptotic bodies. Magnification x 200.
C. D. Inward et aL
215
at 50°C. Loading buffer (10 mM EDTA, pH 8.0, 1% low gelling temperature agarose, 40% sucrose and 0.25% bromophenol blue) was added to each sample. Electrophoresis was performed on a 2 % agarose gel containing 0.1 ~tg/ml ethidium bromide at 40V for 2-3 h with a TBE running buffer. DNA was visualised under ultraviolet light and photographed.
acid was added to a final concentration of 0.5 M, to both supernatant and pellet, followed by 2 volumes of diphenylamine solution (0.088 M diphenylamine, 98% v/v glacial acetic acid, 1.5% v/v concentrated sulphuric acid and 0.5 % v/v of 1.6% acetaldehyde solution. Samples were stored at 4°C for 48 h after which they were quantiffed spectrophotometrically at OD575 nm. 6 DNA fragmentation was expressed as a percentage using the equation:
Quantification of DNA fragmentation
OD Supernatant/(OD Supernatant + OD Pellet) x 100 = % DNA fragmentation
Cell pellets were prepared by centrifugation at 750 x g for 5 min and lysed with 400 gl of lysis buffer (0.2% Triton X-IO0, 10 mM Tris and i mM EDTA, pH 8.0). Lysates were subjected to centrifugation at 13,000 x g for 10 rain. The supernatant, containing small DNA fragments, was removed from the pellet of uncut DNA. The pellet was resuspended in 2 O0 ~tl of lysis buffer. Perchloric
Results
Oualitative data The addition of VT1 to vero cells caused changes in cell structure seen by light microscopy. These consisted of condensation of cell cytoplasm and nuclear chromatin with nuclear material appearing in membrane bound vesicles; apoptotic bodies (Fig. 1). No necrosis was observed. VT1 induced degradation of DNA in vero cells giving rise to the typical ladder pattern on gel electrophoresis (Fig. 2). This indicated endonuclease activity.
Quantitative data Under phase contrast microscopy apoptotic cells could be clearly identified by their smaller, rounded and opalescent appearance, often being detached form the surface of the well. The 24 hour dose dependent counts of apoptotic cells are given in Fig. 3. Similar results were obtained using the diphenylamine (DPA) assay. DPA binds to deoxyribose replacing a purine base. DNA thus labelled, can be quantified because of the characteristic light absorbance of DPA. After centrifugation of the sample, small cleaved fragments of DNA are measured in the supernatant. Using this assay increasing fragmentation induced by VT1, could be seen in a dose dependent manner (Fig. 4). Fragmentation at lOpg/ml of VT1 could be completely blocked by monoclonal antibody to VT1, and partial blocking could be achieved by preincubation with anti CD 77 monoclonal antibody for an hour at 37°C before the toxin was applied (data not shown). The time course of VT1 action is given in Fig. 5, the earliest fragmentation appeared at 6-8 h and continued up to 24 h. Figure 2. DNA degradation s h o w n by gel electrophoresis in vero cells treated with VT1. 5 x 106 vero cells were incubated with or or without VT1 for 24 hours. Lane 1, untreated cells; lane 2, cells treated with 100 pg/ml VT1, lane 3, molecular size markers 7 2 - 1 3 5 3 base pair fragments following DNA digestion with ~ X 1 7 4 Hae III.
Discussion In these experiments we demonstrated both DNA fragmentation and the cytopathic changes characteristic of
Verocytotoxin Induces Apoptosis
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Figure 3. Effectof VT1 on the percentage of apoptoticvero cells observed by phase contrast microscopyat 24 h. (a) Untreated cells; (b) 10 pg/ ml VT]; (c) 100 pg/ml VT1; (d) 10 ng/ml VT1. Results axe mean and S.E.M. for five experiments.
apoptosis; findings which have not been previously demonstrated in vero cells. The gel electrophoresis (Fig. 2) provides qualitative information on the fragmentation of DNA, whereas the diphenylamine reaction allows quantification of the same process. With the latter we were able to show that DNA fragmentation was apparent some 6-8 h after incubation with the toxin and continued to increase to 24 h, the end point of these experiments. In 2 4 h dose response assays, there was concordance between the percentage of DNA fragmented and the proportion of cells appearing apoptotic by phase contrast microscopy. Giemsa staining provided further qualitative information and confirmed the typical pyknotic appearance and nuclear packaging of apoptotic ceils. From the magnitude of the apoptotic response at 24 h and
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Figure 4. Effect of VT1 on the percentage of DNA fragmentation. Vero cells were treated for 24 h with VT1 and percentage of DNA fragmentation estimated using DPA reaction. (a) Untreated cells; (b) 1 pg/ml VT1; (c) 5 pg/ml VT3_;(d) 10 pg/ml VT1; (e) 100 pg/ml VT1. Results are mean and s.E.u, for three experiments.
comparing this with the overall cytocidal effect of verotoxin on vero ceils, we conclude that programmed cell death is the principal mode of killing. This result is not surprising. Pyknotic changes were reported in ileal enterocytes in rabbits infected with VTEC 0 1 5 7 : H 7 or exposed to Shiga toxin alone. 7'8 More importantly other bacterial and plant toxins have similar protein inhibitory and cytopathic effects? Diphtheria and pseudomonas A toxins catalyse ADP ribosylation of a residue on elongation factor 2 (EF2), inactivating it and effectively blocking protein synthesis. 1° The A-chain of the plant lectin ricin operates in an identical way to that of the Shiga/verocytotoxin family in cleaving the Nglycosidic bond of adenosine at position 4 3 2 4 from the 5' end of 28S ribosomal RNA of the 60S ribosomal snbunit.11.12 Diphtheria, pseudomonas A and ricin toxins
C. D. Inward et aL
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changes in the number of toxin receptors expressed on the cell surface. 16 However, Chang et al., ~7 experimenting with other h u m a n cell lines found that whereas diphtheria toxin induced DNA fragmentation and cytolysis, cells in which comparable protein synthesis inhibition was obtained with cyclohexamide did not. A further separation between overall protein synthesis inhibition and apoptosis was found in experiments by Khan and 30 Waring on murine peritoneal macrophages38 Apoptosis is an important physiological process in macrophages which serves to curtail the inflammatory response. Ricin precipitated this in a dose dependent manner. However, once macrophages had become adherent, ricin no longer induced DNA fragmentation, even though protein syn20 thesis was inhibited. An additional pathway by which verocytotoxin might induce apoptosis was suggested by Mangeney et al. 19 They showed that in Burkitt's l y m p h o m a cells, which bear the CD77 receptor, the B-subunit of the toxin alone could induce the characteristic DNA fragmentation and histological changes of apoptosis. The B-subunit is 10 thought not to have any enzymatic activity of its own. It thus appears that this is a novel signal transduction pathway unrelated to protein synthesis inhibition. Many of the experiments outlined above used transformed cell lines which m a y enter apoptosis differently from their parent tissues. Extrapolation from in vitro 0 observations to the action of the toxin in vivo m a y be 0 5 10 15 20 25 misleading. In part this objection was removed in a subtle hours experiment by Buchman et al.,2° using porcine endothelial Figure 5. Timecourse of DNA fragmentation estimated by DPAreaction cells. These authors showed that pre-treatment of endoin vero cells treated with VT1. Open symbols: untreated cells. Solid thelium with bacterial endotoxin (lipopolysaccharide, symbols:cells treated with 100 pg/ml VT1. Results are mean and S.E.M. LPS), could prime cells so that a second signal consisting for four experiments. of either a heat shock or protein synthesis inhibition (cycloheximide) would induce apoptosis. Independently the LPS, heat shock or cycloheximide treatment itself all induce apoptosis in eukaryotic cells. ~°'~3 Sandvig and was sub-lethal and reversing the order of the experiment van Deurs 14 showed that Shiga toxin and ricin induce did not give rise to cell death. The concept that two DNA fragmentation and the ultrastructural changes of different signals in a specific sequence can induce apopapoptosis in vero and MDCK cells, although these authors tosis is interesting and m a y relate to disease states. We used cell lysis to quantify cell death. note that in HUS associated with Shigellosis there is Is apoptosis related to the protein synthesis inhibitory properties held in c o m m o n by these toxins? Certain evidence that LPS enters the circulation accompanied h u m a n cell lines, e.g. HL-60, undergo apoptosis w h e n by a leukaemoid reaction. 2° In VTEC 0 1 5 7 infection treated with inhibitors of transcription (actinomycin- endotoxaemia is suggested indirectly by neutrophilia D) or translation (cycloheximide)35 It has been noted which occurs in those who proceed to HUS, 22 and depreviously that rapidly dividing cells appear more sens- rangement of LPS-binding proteins in plasma. 23 It would itive to the action of these toxins. 13 Cell vulnerability to be reasonable to find out whether LPS can operate as protein synthesis inhibitors is in part related to their the first, and verocytotoxin as the second signal to cause position in the cell reproductive cycle, being maximal at apoptosis in h u m a n endothelinm. If so this might explain the G 1-S interphase. Vero cells which replicate vigorously the nature and distribution of the vascular lesion of in culture, have been shown to be increasingly sensitive haemorrhagic colitis and the haemolytic uraemic synto verotoxin at this point, a p h e n o m e n o n divorced from drome.
40 .~
Verocytotoxin Induces Apoptosis
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Acknowledgement The authors are indebted to Dr M. A. Karmali for the generous gift of VT1 and anti-VT1 monoclonal antibody. CDI was supported by the Renal Research Fund of the Children's Hospital, Birmingham, IC by the Paul O'Gorman Foundation.
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