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• W2098753502 abstract "Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay. Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay. The crystal (Cry) 3The abbreviations used are: Cry, crystal; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDI, protein-disulfide isomerase; LDH, lactate dehydrogenase; PI, propidium iodide; TEM, transmission electron microscopy; PEG, polyethylene glycol; PBS, phosphate-buffered saline; FCS, fetal calf serum; DMEM, Dulbecco's modified essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; (DiBAC4(3), bis(1,3-dibarbituric acid)-trimethine oxonol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; DAPI, 4′,6′-diamidino-2-phenylindole; ER, endoplasmic reticulum.3The abbreviations used are: Cry, crystal; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PDI, protein-disulfide isomerase; LDH, lactate dehydrogenase; PI, propidium iodide; TEM, transmission electron microscopy; PEG, polyethylene glycol; PBS, phosphate-buffered saline; FCS, fetal calf serum; DMEM, Dulbecco's modified essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; (DiBAC4(3), bis(1,3-dibarbituric acid)-trimethine oxonol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; DAPI, 4′,6′-diamidino-2-phenylindole; ER, endoplasmic reticulum. proteins produced in Bacillus thuringiensis, a Gram-positive bacterium, are known to show high cytotoxicity against insects (1Höfte H. Whiteley H.R. Microbiol. Rev. 1989; 53: 242-255Crossref PubMed Google Scholar). Once an organism has ingested a parasporal Cry protein in B. thuringiensis, the protein is solubilized under alkaline conditions in the midgut and processed to an active toxin by digestive system proteases. The activated toxin then binds to a specific receptor on the membrane surface of epithelial gut cells, leading to permeable pore formation and finally the death of the insect (2Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar, 3de Maagd R.A. Bravo A. Crickmore N. Trends Genet. 2001; 17: 193-199Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Because of their nonpathogenicity toward vertebrate organisms and species-specific toxicities toward insects, Cry proteins have been applied world-wide as biopesticides, and some Cry protein genes are now used in transgenic crops to control insect pests (4Cannon R.J.C. Biol. Rev. 1996; 71: 561-636Crossref Scopus (39) Google Scholar). On the other hand, these toxins are not only important tools for organic farming but have also made important contributions to the control of insect-mediated diseases, such as African river blindness. Recently, Cry proteins have also been shown to target nematodes, including the intestinal parasite Nippostrongylus brasiliensis (5Wei J.Z. Hale K. Carta L. Platzer E. Wong C. Fang S.C. Aroian R.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2760-2765Crossref PubMed Scopus (325) Google Scholar), and to kill pathogenic protozoan parasites, including Trichomonas vaginalis (6Kondo S. Mizuki E. Akao T. Ohba M. Parasitol. Res. 2002; 88: 1090-1092Crossref PubMed Scopus (22) Google Scholar). Therefore, elucidating the molecular actions of antiparasitic Cry proteins may be useful for controlling parasites in medical fields.The genes for the B. thuringiensis Cry proteins appear to reside on plasmids, often as a part of composite structures that include a variety of transportable elements (3de Maagd R.A. Bravo A. Crickmore N. Trends Genet. 2001; 17: 193-199Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 7Mahillon J. Rezsohazy R. Hallet B. Delcour J. Genetica (Dordr.). 1994; 93: 13-26Crossref PubMed Scopus (84) Google Scholar). This high degree of genetic plasticity results in a remarkable diversity of B. thuringiensis strains and Cry proteins, and a growing number of these strains and toxin proteins are being isolated and cloned (2Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar, 8Crickmore N. Zeigler D.R. Feitelson J. Schnepf E. Van Rie J. Lereclus D. Baum J. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 807-813Crossref PubMed Google Scholar). Although a number of B. thuringiensis strains producing insecticidal toxins have been identified, many other B. thuringiensis strains containing noninsecticidal inclusion proteins have also been ubiquitously discovered in natural environments and are rather more widely distributed than the insecticidal strains (9Ohba M. Aizawa K. J. Invertebr. Pathol. 1986; 47: 12-20Crossref Scopus (119) Google Scholar, 10Meadows M.P. Ellis D.J. Butt J. Jarrett P. Burges H.D. Appl. Environ. Microbiol. 1992; 58: 1344-1350Crossref PubMed Google Scholar). Through a wide screening of noninsecticidal Cry protein cytotoxicities toward several human cell lines, we have identified novel B. thuringiensis toxins, the parasporins, that possess cytotoxic and nonhemolytic activities against a wide range of human cells (11Mizuki E. Ohba M. Akao T. Yamashita S. Saitoh H. Park Y.S. J. Appl. Microbiol. 1999; 86: 477-486Crossref PubMed Scopus (136) Google Scholar, 12Mizuki E. Park Y.S. Saitoh H. Yamashita S. Akao T. Higuchi K. Ohba M. Clin. Diagn. Lab. Immunol. 2000; 7: 625-634Crossref PubMed Scopus (121) Google Scholar). The parasporins are heterogeneous in their cytotoxicity spectra, because some are active on human cells, whereas others kill a few specific cells.A potent toxin was discovered in the noninsecticidal and nonhemolytic B. thuringiensis strain A1547, which produces agglutinative Cry proteins with cytocidal activity against MOLT-4 human leukemic T cells (13Kim H.S. Yamashita S. Akao T. Saitoh H. Higuchi K. Park Y.S. Mizuki E. Ohba M. J. Appl. Microbiol. 2000; 89: 16-23Crossref PubMed Scopus (27) Google Scholar). In a previous study, we obtained the gene encoding the purified new toxin protein, which was named parasporin-2 (or Cry31Aa, as designated by the B. thuringiensis δ-endotoxin nomenclature committee) (14Katayama H. Yokota H. Akao T. Nakamura O. Ohba M. Mekada E. Mizuki E. J. Biochem. (Tokyo). 2005; 137: 17-25Crossref PubMed Scopus (50) Google Scholar), and we examined the cytotoxic activities of a recombinant parasporin-2 against a variety of cultured human cells. Parasporin-2 was found to have strong cytocidal activities against various human cells with markedly divergent target specificities. For example, it was highly cytotoxic toward human hepatocyte cancer cells (HepG2 cells) and less cytotoxic toward normal hepatocyte cells (HC cells) (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In slices of liver and colon cancer tissues, it was surprisingly found that parasporin-2 preferentially killed the cancer cells, while leaving the normal cells unaffected (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).Because parasporin-2 possesses highly selective cytotoxicity toward human cells, and especially has the potential to recognize and kill some classes of cancer cells, the possibility of its application to medical and biological fields has been anticipated (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, the actions of parasporin-2 have hardly been characterized at the molecular and cellular levels. For instance, the active form of parasporin-2 and its exact mechanism for inducing cell death are currently unknown. Through our present analyses of its proteolytic activation and cytocidal effects, we show the following: (i) that parasporin-2 is highly activated through processing of both its N- and C-terminal propeptides; (ii) that it specifically binds to the plasma membrane of hepatocyte cancer cells; (iii) that it rapidly increases the membrane permeability; and (iv) that it dramatically alters the cytoskeleton and organelle morphologies. Thus, parasporin-2 is a cell-discriminating, membrane-targeting, and pore-inducing toxin that subsequently causes irreversible intracellular decay in cancer cells.EXPERIMENTAL PROCEDURESCell Culture and Materials—HepG2 and COS-7 cells were cultured in Dulbecco's modified essential medium (DMEM; Nissui) containing 10% fetal calf serum (FCS; Biological Industries), whereas HeLa cells were cultured in minimal essential medium (Nissui) containing 10% FCS, under 5% CO2 at 37 °C. The polyclonal antibody against parasporin-2 was raised in rabbits against the purified protein. The monoclonal antibodies against α-tubulin and cadherin were purchased from ICN Biomedicals and Sigma, respectively. The anti-GAPDH and anticytochrome c monoclonal antibodies were obtained from HyTest and Zymed Laboratories Inc., respectively. The rabbit polyclonal antibodies against PDI and actin were purchased from StressGen Biotechnologies Corp. and Sigma, respectively. Tom40 and cytochrome P450 reductase polyclonal antibodies were gifts from Dr. K. Mihara (Kyushu University) and Dr. T. Ogishima (Kyushu University), respectively. Horseradish peroxidase-conjugated secondary antibodies were obtained from BIOSOURCE.Purification of Recombinant Parasporin-2—Escherichia coli BL21 (DE3) cells transformed with the pET-37k plasmid, containing a gene for full-length parasporin-2 and a C-terminal hexahistidine tag, were cultured and lysed as described previously (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Following centrifugation, the material in the pellet was solubilized in 50 mm Na2CO3 at 37 °C for 1 h and then centrifuged for 10 min at 15,000 × g. The resultant supernatant was loaded onto a nickel-chelating column (Amersham Biosciences) equilibrated with an alkaline solution (20 mm Tris-HCl, pH 8.0, 50 mm Na2CO3) and eluted with 50 mm Na2CO3 and 500 mm imidazole. The purified protein was digested with 0.1 mg/ml proteinase K at 37 °C for 30 min and then 1 mm phenylmethylsulfonyl fluoride was added to stop the proteolysis. The protease-treated protein was applied to a Q-Sepharose Fast Flow column (Amersham Biosciences) equilibrated with the alkaline solution, and the protease-resistant 30-kDa parasporin-2 toxin was eluted with 500 mm NaCl. The 31-kDa toxin, a truncated protein lacking the N-terminal 51 residues, with an N-terminal initial methionine and a C-terminal hexahistidine tag was produced and purified as described previously (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).MALDI-TOF Mass Spectrometry—Proteinase K-treated parasporin-2 was mixed with a matrix solution of sinapinic acid as described previously (16Okumura S. Saitoh H. Ishikawa T. Wasano N. Yamashita S. Kusumoto K. Akao T. Mizuki E. Ohba M. Inouye K. J. Agric. Food Chem. 2005; 53: 6313-6318Crossref PubMed Scopus (26) Google Scholar), and the mixture was analyzed using an Autoflex mass spectrometer (Bruker Daltonics). The spectrometer was calibrated using ubiquitin, myoglobin, trypsinogen, and bovine serum albumin as molecular weight standards.Determination of Cell Viability—Cells were plated in 96-well plates at a density of 2 × 104 cells/well and cultured overnight, before parasporin-2 was added to each well. To determine the LD50 (LD50) of the toxin for each cell type, the viable cells were measured by the MTT assay using a Cell Titer 96™ nonradioactive cell proliferation assay kit (Promega) after intoxication at 37 °C for 24 h as described previously (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). When a kinetic analysis was performed for the cell death, we determined the cell viability by quantification of ATP, which indicates the presence of metabolically active cells, using a CellTiter-Glo™ luminescent cell viability assay kit (Promega). The chemiluminescence signals were captured with a cooled CCD camera system (Cool Saver; ATTO), and their intensities were quantified using image analysis software (CSAnalyzer; ATTO).Protein Efflux and PI Influx Measurements—Cells were plated in 96-well plates at a density of 2 × 104 cells/well and cultured overnight. After two washes with phosphate-buffered saline (PBS), parasporin-2 was added to the cells in DMEM without FCS. For determination of LDH efflux from the cells, the medium was centrifuged to remove floating cells. Next, the resultant supernatant was mixed with the solution of the LDH cytotoxicity detection kit (Takara), and the optical densities at 490 nm were measured with a microplate reader model 550 (Bio-Rad). To inhibit the LDH efflux, 30 mm PEG (Wako) in DMEM was added to the cells followed by treatment with parasporin-2 for 8 h. The amounts of leaked LDH were determined and represented as percentages of the LDH activity obtained after treatment of the cells with 1% (w/v) Triton X-100. For PI (Sigma) staining, cells (2 × 104 cells/well) were grown on 96-well plates overnight and washed twice with PBS, before PI (final concentration: 5 mg/ml) in DMEM was added together with parasporin-2. At the indicated times, the uptake of PI into the cells was measured with a FLA-5000 Phosphor-Imager (Fuji Film) with excitation at 510 nm and emission at 665 nm. 100% entry of PI was determined by treatment of the cells with 0.2% Triton X-100.Measurement of the Membrane Potential—HepG2 cells (2 × 104 cells/well) were grown overnight on Optilux 96-well clear-bottom plates (Falcon) precoated with collagen type I (Sigma). The cells were washed twice with a dye solution (Hanks' balanced salt solution containing 20 mm HEPES-NaOH, pH 7.4, and 1 mm bis(1,3-dibarbituric acid)-trimethine oxonol (DiBAC4(3)) (Dojin)), and then incubated in the dye solution at 37 °C for 30 min. The fluorescence intensities of the dye, which depended on the membrane potential, were monitored using a Flex Station (Molecular Devices) with excitation at 488 nm and emission at 520 nm. After the fluorescence intensity had stabilized, parasporin-2 was added. Maximal depolarization was obtained at the end of each experiment by adding valinomycin (final concentration, 10 μm). Single fluorescence traces were expressed as the ratio I(t)/Imax, i.e. fluorescence intensity relative to the maximal fluorescence intensity after the addition of valinomycin.Electron Microscopy—After MOLT-4 cells had been incubated with parasporin-2 at 37 °C for appropriate times, the cells were harvested by centrifugation. For transmission electron microscopy (TEM), ultrathin sections were prepared as described previously (17Lee D. Katayama H. Akao T. Maeda M. Tanaka R. Yamashita S. Saitoh H. Mizuki E. Ohba M. Biochim. Biophys. Acta. 2001; 1547: 57-63Crossref PubMed Scopus (27) Google Scholar) and observed using an electron microscope (model H-7100; Hitachi).Immunofluorescence Microscopy—For immunofluorescence, cells were grown on collagen I-coated chamber slides. The cells were seeded at a density of 2 × 104 cells/chamber and incubated overnight. After washing with PBS, parasporin-2 was added to the cells in DMEM without FCS, and the cells were incubated for appropriate times. Immunofluorescence experiments were performed as described previously (18Mitoma J. Ito A. EMBO J. 1992; 11: 4197-4203Crossref PubMed Scopus (102) Google Scholar). Briefly, the cells were washed with PBS, fixed with 2% paraformaldehyde, 0.1% glutaraldehyde in PBS for 15 min, washed several times with PBS, and treated with 1% Triton X-100 for 2 min to permeabilize the membranes. Excess aldehyde was quenched by incubation with 1 mg/ml NaBH4 for 10 min. The intoxicated, fixed, and permeabilized cells were treated with an anti-parasporin-2 antibody as the primary antibody, and then labeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) as a fluorescent dye-conjugated secondary antibody, in PBS containing 10 mm glycine and 10% bovine serum albumin. For double immunostaining, Alexa Fluor 568-conjugated goat antimouse IgG was also used as a secondary antibody.Immunohistochemistry of Tissue Samples—For immunohistochemical observation of parasporin-2 in liver and colon cancer tissues, cancer specimens were cut into small pieces and incubated in RPMI 1640 medium containing 10% FCS and 100 nm parasporin-2 for 24 h in 37 °C at an atmosphere of 95% air and 5% CO2, before being fixed in 10% formaldehyde and embedded in paraffin (19Sasaguri Y. Komiya S. Sugama K. Suzuki K. Inoue A. Morimatsu M. Nagase H. Am. J. Pathol. 1992; 141: 611-621PubMed Google Scholar). After deparaffinization and rehydration, 5-mm sections were incubated in 3% H2O2 for 10 min to block endogenous peroxidase activity. Next, the sections were rinsed and incubated with a polyclonal anti-parasporin-2 antibody for 1 h. After washing with PBS, secondary antibody/peroxidase-linked polymers were applied, and the sections were incubated with 100 ml of Tris-HCl, pH 7.6, containing 20 mg of 3,3′-diaminobenzidine tetrahydrochloride, 65 mg of sodium azide, and 20 ml of 30% H2O2. After counterstaining with Meyer's hematoxylin, the sections were observed under a light microscope. The diagnosis of each cancer tissue specimen was re-evaluated and confirmed by three pathologists who examined formalin-fixed and paraffin-embedded tissue sections stained with hematoxylin and eosin or appropriate immunohistochemical stains.RESULTSProparasporin-2 Is Cleaved to a 30-kDa Core Fragment by Proteinase K—Digestion of Cry protein precursors by digestive system proteases in the insect midgut or in vitro is essential for toxin activation. The protein inclusion of parasporin-2 can also be activated in vitro by proteolysis under alkaline conditions. We previously reported the possibility that parasporin-2 undergoes N-terminal processing, because of differences between the N-terminal amino acid sequences of the predicted protein from the nucleotide sequence of the gene and the protease-activated native toxin (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The protein sequence of the activated native toxin started with an aspartic acid that represented residue 52 of the full-length toxin (proparasporin-2). To examine the exact molecular form of the active toxin, recombinant proparasporin-2 and a truncated toxin lacking the N-terminal 51 residues, each with a His6 tag at the C terminus, were expressed in E. coli and purified on a nickel-chelating column. When the purified proteins were separated by SDS-PAGE and stained, almost homogeneous polypeptides for proparasporin-2 (37 kDa) and the N-terminal truncate (31 kDa) were detected (Fig. 1A). After treating the recombinant parasporin-2 with proteinase K, the molecular sizes of the processed proteins were essentially the same as that of native parasporin-2 in the gel (Fig. 1B). The migration of the N-terminally truncated parasporin-2 was slower than the proteinase K-activated parasporin-2 (30 kDa) obtained from parasporal inclusion bodies of B. thuringiensis strain A1547 or the recombinant protein (Fig. 1B, compare lane 3 with lanes 2 and 6). The observed differences in the protein sizes were not because of the C-terminal His6 tag addition, because the N-terminally truncated parasporin-2 without a His6 tag was also larger than the mature 30-kDa toxin (Fig. 1C, upper panel). No His6 tag epitope was detected in the 30-kDa toxin using a monoclonal antibody against polyhistidine (Fig. 1C, lower panel). Therefore, the differences appear to result from cleavage at the C-terminal region. MALDI-TOF mass spectrometry analysis of the molecular masses of the 30-kDa proteins obtained from native and recombinant sources revealed that the proteins were essentially the same size, specifically 27,869.9 and 27,873.3 Da for the native and recombinant proteins, respectively. The N-terminal protein sequence of the recombinant 30-kDa toxin was DVIRE, which was the same sequence obtained in our previous report using the activated native toxin (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These mass values and protein sequences are consistent with the value of 27,855.5 Da calculated for the toxin sequence corresponding to amino acid residues 52-306. Thus, parasporin-2 is cleaved by proteinase K not only at the N terminus but also at the C terminus, and the resultant product is a 30-kDa core toxin, as illustrated in Fig. 1D.C-terminal Processing Enables Parasporin-2 to Convert to the Potent Toxin—To elucidate the effect of the C-terminal cleavage on the cytocidal activity, the N- and C-terminally processed parasporin-2 (30 kDa) was tested for its cytotoxicity toward various human cells. When the cytotoxicities of various concentrations of parasporin-2 against cultured cells were monitored using the MTT assay and the LD50 values at 24 h after administration were determined, the cytotoxicity was found to vary from cell type to cell type (Table 1). The toxin proteins were evenly cytotoxic toward MOLT-4, Jurkat, HL-60, and HepG2 cells, and the LD50 values against these cells were all around 20 ng/ml. HeLa cells were moderately susceptible to the 30-kDa form of parasporin-2. Next, the cytocidal activities for cell lines from other mammals were determined. The toxin was found to be inefficient toward monkey cells but toxic toward rodent cell lines (Table 2). It was of interest that the C-terminally truncated toxin killed Sawano and CACO-2 cells much more efficiently than the nontruncated 31-kDa parasporin-2. A marked difference between the cytocidal activities of the C-terminally processed and nonprocessed toxins was observed in kinetic analyses of their cytotoxicities. The 30-kDa toxin reduced HepG2 cell viability in a comparatively rapid manner, with LD50 times of 1.2 and 5 h for 0.1 mg/ml of the 30- and 31-kDa toxins, respectively, whereas the purified 37-kDa proparasporin-2 showed little toxicity toward the cells (Fig. 1E). The immediate intoxication appeared to be a general characteristic of the 30-kDa parasporin-2, because it was also observed for MOLT-4 cells (Fig. 1F). Because MOLT-4 cells suffered slower cytocidal effects than HepG2 cells, there may be a difference in the mode of the toxin action between these two cell types. Investigation for processing of the 31-kDa toxin by HepG2 cells revealed that the toxin was hardly cleaved, indicating that the 31-kDa form was not activated by cellular proteases and by itself (Fig. 1G). In any case, these results indicate that the C-terminal processing is involved in the efficient and rapid cytotoxicity of parasporin-2, in addition to its N-terminal cleavage. Subsequently, we used the highly toxic form to elucidate the cytocidal action of parasporin-2 in the experiments described below.TABLE 1Cytocidal activities of the 30-kDa and 31-kDa fragments of parasporin-2 toward various cultured human cellsCell nameCharacteristicsLD50 (μg/ml)aCell viabilities at 20 h of intoxication were determined based on the metabolically active cells using the MTT assay as described under “Experimental Procedures.” The LD50 (50% lethal dose) values were calculated from the cell viability data for each dose of toxin30 kDa31 kDabPreviously reported values are presented in this table (15)MOLT-4Leukemic T cells0.0220.044JurkatLeukemic T cells0.0180.015HL-60Leukemic T cells0.0190.066T cellNormal T cellsNDcND indicates not determined0.148HCNormal hepatocytes1.1>10HepG2Hepatocyte cancer0.0190.023HeLaUterine (cervical) cancer2.5>10SawanoUterine cancer0.0020.041TCSUterine (cervical) cancer7.8>10UtSMCNormal uterus2.59.28MRC-5Normal embryonic lung fibroblasts0.47.15A549Lung cancer0.34>10CACO-2Colon cancer0.0134.86a Cell viabilities at 20 h of intoxication were determined based on the metabolically active cells using the MTT assay as described under “Experimental Procedures.” The LD50 (50% lethal dose) values were calculated from the cell viability data for each dose of toxinb Previously reported values are presented in this table (15Ito A. Sasaguri Y. Kitada S. Kusaka Y. Kuwano K. Masutomi K. Mizuki E. Akao T. Ohba M. J. Biol. Chem. 2004; 279: 21282-21286Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar)c ND indicates not determined Open table in a new tab TABLE 2Cytocidal activities of the 30- and 31-kDa fragments of parasporin-2 toward various cultured mammalian cellsCell nameCell type and originLD50 (μg/ml)aCell viabilities at 20 h of intoxication were determined as described under “Experimental Procedures.” The LD50 values were calculated are described for Table 130 kDa31 kDaVeroKidney (monkey)>10>10COS-7Kidney (monkey)>10>10NIH3T3Fibroblast (mouse)0.0090.038CHO-K1Ovary (Chinese hamster)1.24.2a Cell viabilities at 20 h of intoxication were determined as described under “Experimental Procedures.” The LD50 values were calculated are described for Table 1 Open table in a new tab Parasporin-2 Causes Marked Morphological Alterations in the Cells—HepG2 cells showed marked morphological alterations during incubation with the highly toxic form of parasporin-2. A number of balloon-like shapes suddenly emerged one after another on the cell periphery in the presence of 0.1 mg/ml of the toxin (Fig. 2A, panel b). One or several blebs surrounded each cell, gradually enlarged over a few minutes (Fig. 2B), and finally became detached from the cells (Fig. 2A, panel b, see arrowheads). The number of cells carrying blebs increased in a dose- and time-dependent manner for the range of toxin concentrations investigated (0.1-10 mg/ml; data not shown). NIH-3T3 cells showed similar morphological alterations to HepG2 cells after treatment with the toxin, whereas intoxicated MOLT-4 cells seemed to differ from the blebbing morphology, in that the cells simply swelled and contained some small vacuolated structures (Fig. 2A, panels f and g). Video documentation revealed the sequence of the morphological changes in MOLT-4 cells, which consisted of intracellular vacuolations, cell swelling, and finally cell bursting (data not shown). When the toxin-treated MOLT-4 cells were examined by TEM, many vacuolar structures were observed in the cytoplasmic space during the early stage of the toxin action (Fig. 2C, panel b), and the cytosolic volume was decreased with nuclear deformation at the late stage (Fig. 2C, panel c). Parasporin-2 hardly induced any morphological changes to HeLa cells (Fig. 2A, panel d) or COS-7 cells (data not shown), which showed low sensitivities to the toxin. Thus," @default.
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• W2098753502 date "2006-09-01" @default.
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• W2098753502 title "Cytocidal Actions of Parasporin-2, an Anti-tumor Crystal Toxin from Bacillus thuringiensis" @default.
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