Escherichia colimazEF Toxin-Antitoxin System as a Tool to Target Cell Ablation in Plants.

BACKGROUND/AIMS: The Escherichia coli MazF is an endoribonuclease that cleaves mRNA at ACA sequences, thereby triggering inhibition of protein synthesis. The aim of this study is to evaluate the efficiency of the mazEF toxin-antitoxin system in plants to develop biotechnological tools for targeted cell ablation. METHODS: A double transformation strategy, combining expression of the mazE antitoxin gene under the control of the CaMV 35S promoter, reported to drive expression in all plant cells except within the tapetum, together with the expression of the mazF gene under the control of the TA29 tapetum-specific promoter in transgenic tobacco, was applied. RESULTS: No transgenic TA29-mazF line could be regenerated, suggesting that the TA29 promoter is not strictly tapetum specific and that MazF is toxic for plant cells. The regenerated 35S-mazE/TA29-mazF double-transformed lines gave a unique phenotype where the tapetal cell layer was necrosed resulting in the absence of pollen. CONCLUSION: These results show that the E. colimazEF system can be used to induce death of specific plant cell types and can provide a new tool to plant cell ablation.

Ribonuclease binase decreases destructive changes of the liver and restores its regeneration potential in mouse lung carcinoma model.

The successful application of exogenous ribonucleases of different origin to suppress tumor growth allows one to consider them as perspective therapeutics for treatment of oncological diseases. An important aspect of the success of an anti-cancer drug is low hepatotoxicity, which will reduce, if not eliminate entirely the undesirable side effects of treatment. Previously we have shown that ribonuclease from Bacillus intermedius (binase) exhibits high antitumor and antimetastatic activity in tumor models of different histological origin. In this work we studied hepatotoxic action of binase using mouse tumor model of Lewis lung carcinoma. Binase at doses of 0.1-1 mg/kg which produced effective suppression of tumor growth and metastasis, showed positive effect on the liver of tumor-bearing mice expressed in a significant reduction in the volume of destructive changes in the liver parenchyma and return to the normal level of the liver regenerative potential impaired due to endogenous intoxication and tumor burden.

Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells.

Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS 40 and melanoma B-16. We show for the first time that intraperitoneal injection of binase at a dose range 0.1-5 mg/kg results in retardation of primary tumor growth up to 45% in LLC and RLS 40 and inhibits metastasis up to 50% in LLC and RLS 40 and up to 70% in B-16 melanoma. Binase does not exhibit overall toxic effect and displays a general systemic and immunomodulatory effects. Treatment of RLS 40-bearing animals with binase together with polychemotherapy revealed that binase decreases the hepatotoxicity of polychemotherapy while maintaining its antitumor effect. It was demonstrated that the cytotoxic effect of binase is realized via the induction of the intrinsic and extrinsic apoptotic pathways. Activation of intrinsic apoptotic pathway is manifested by a drop of mitochondrial potential, increase in calcium concentration and inhibition of respiratory activity. Subsequent synthesis of TNF-α in the cells under the action of binase triggers extrinsic apoptotic pathway through the binding of TNF with cell-death receptors and activation of caspase 8. Thus binase is a potential anticancer therapeutics inducing apoptosis in cancer cells.

Sensitivity of acute myeloid leukemia Kasumi-1 cells to binase toxic action depends on the expression of KIT and АML1-ETO oncogenes.

Some RNases selectively attack malignant cells, triggering an apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Here we studied the effects of Bacillus intermedius RNase (binase) on murine myeloid progenitor cells FDC-P1; transduced FDC-P1 cells ectopically expressing mutated human KIT N822K oncogene and/or human AML1-ETO oncogene; and human leukemia Kasumi-1 cells expressing both of these oncogenes. Expression of both KIT and AML1-ETO oncogenes makes FDC-P1 cells sensitive to the toxic effects of binase. Kasumi-1 cells were the most responsive to the toxic actions of binase among the cell lines used in this work with an IC50 value of 0.56 µM. Either blocking the functional activity of the KIT protein with imatinib or knocking-down oncogene expression using lentiviral vectors producing shRNA against AML1-ETO or KIT eliminated the sensitivity of Kasumi-1 cells to binase toxic action and promoted their survival, even in the absence of KIT-dependent proliferation and antiapoptotic pathways. Here we provide evidence that the cooperative effect of the expression of mutated KIT and AML1-ETO oncogenes is crucial for selective toxic action of binase on malignant cells. These findings can facilitate clinical applications of binase providing a useful screen based on the presence of the corresponding target oncogenes in malignant cells.

Structure-cytotoxicity relationships in bovine seminal ribonuclease: new insights from heat and chemical denaturation studies on variants.

Bovine seminal ribonuclease (BS-RNase), a homodimeric protein displaying selective cytotoxicity towards tumor cells, is isolated as a mixture of two isoforms, a dimeric form in which the chains swap their N-termini, and an unswapped dimer. In the cytosolic reducing environment, the dimeric form in which the chains swap their N-termini is converted into a noncovalent dimer (termed NCD), in which the monomers remain intertwined through their N-terminal ends. The quaternary structure renders the reduced protein resistant to the ribonuclease inhibitor, a protein that binds most ribonucleases with very high affinity. On the other hand, upon selective reduction, the unswapped dimer is converted in two monomers, which are readily bound and inactivated by the ribonuclease inhibitor. On the basis of these considerations, it has been proposed that the cytotoxic activity of BS-RNase relies on the 3D structure and stability of its NCD derivative. Here, we report a comparison of the thermodynamic and chemical stability of the NCD form of BS-RNase with that of the monomeric derivative, together with an investigation of the thermal dissociation mechanism revealing the presence of a dimeric intermediate. In addition, we report that the replacement of of Arg80 by Ser significantly decreases the cytotoxic activity of BS-RNase and the stability of the NCD form with respect to the parent protein, but does not affect the ribonucleolytic activity or the dissociation mechanism. The data show the importance of Arg80 for the cytotoxicity of BS-RNase, and also support the hypothesis that the reduced derivative of BS-RNase is responsible for its cytotoxic activity.

An overview on nucleases (DNase, RNase, and phosphodiesterase) in snake venoms.

In this review, we have compiled the data on pharmacological activities associated with endogenous purine release related enzymes-nucleases (DNases, RNases, and phosphodiesterases). The results of studies on toxic effects of these enzymes, emphasizing the future directions in this field, are summarized. One of the major problems facing toxicologists is the identification and characterization of specific venom nucleases since they share similar substrate specificities and biochemical properties. In this review, we have attempted to clarify some of the discrepancies about these enzymes. Further, we have tried to correlate the existence of nuclease enzymes in relation to endogenous release of purines, a multitoxin, during snake envenomation, and we also discuss the possible actions of purines. We hope that this review will stimulate renewed interest among toxicologists to biologically characterize these enzymes and elucidate their role in envenomation.

The buried diversity of bovine seminal ribonuclease: shape and cytotoxicity of the swapped non-covalent form of the enzyme.

Bovine seminal ribonuclease exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer. The molecular envelope and the exposed surface of the two isomers are practically indistinguishable and their diversity is almost completely buried in the interior of the protein. Surprisingly, the cytotoxic and antitumor activity of the enzyme is a peculiar property of the swapped dimer. This buried diversity comes into light in the reducing environment of the cytosol, where the unswapped dimer dissociates into monomers, whereas the swapped one generates a metastable dimeric form (NCD-BS) with a quaternary assembly that allows the molecule to escape the protein inhibitor of ribonucleases. The stability of this quaternary shape was mainly attributed to the combined presence of Pro19 and Leu28. We have prepared and fully characterized by X-ray diffraction the double mutant P19A/L28Q (PALQ) of the seminal enzyme. While the swapped and unswapped forms of the mutant have structures very similar to that of the corresponding wild-type forms, the non-covalent form (NCD-PALQ) adopts an opened quaternary structure, different from that of NCD-BS. Moreover, model building clearly indicates that NCD-PALQ can be easily sequestered by the protein inhibitor. In agreement with these results, cytotoxic assays have revealed that PALQ has limited activity, whereas the single mutants P19A and L28Q display cytotoxic activity against malignant cells almost as large as the wild-type enzyme. The significant increase in the antitumor activity, brought about by the substitution of just two residues in going from the double mutant to the wild-type enzyme, suggests a new strategy to improve this important biological property by strengthening the interface that stabilizes the quaternary structure of NCD-BS.

Synergism of Rana catesbeiana ribonuclease and IFN-gamma triggers distinct death machineries in different human cancer cells.

Rana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-gamma. However, it is unclear how RC-RNase and RC-RNase/IFN-gamma induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-gamma-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-gamma induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-gamma can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity.

Comprehensive comparison of the cytotoxic activities of onconase and bovine seminal ribonuclease.

Onconase (ONC) and bovine seminal ribonuclease (BS-RNase) are homologs of bovine pancreatic ribonuclease (RNase A). Unlike RNase A, ONC and BS-RNase can evade the cytosolic ribonuclease inhibitor protein and are potent cytotoxins. Here, the endogenous cytotoxic activities of ONC and BS-RNase are compared in a wide variety of assays. Injections of ONC into one or both testes of mice and rats evokes a stronger aspermatogenic activity than does the injection of BS-RNase. Epididymides exposed to ONC lose mass and all sperm. Testicular tissue is gradually colonized by immunite and fibrocytic cells. Yet, Leydig cells are always present and functional in the ligamented parts of testicles injected with ONC or BS-RNase. ONC is likewise more toxic to mouse embryos than is BS-RNase, both in vitro and in vivo. The antiproliferative effect of ONC on human tumor cell line ML-2 and lymphocytes in a mixed lymphocyte culture is also more pronounced than is that of BS-RNase. The number of granulocyte-macrophage colony-forming units is repressed almost completely by ONC, whereas a five-fold higher dose of BS-RNase does not cause substantial inhibition. In mice, ONC is less immunogenic than BS-RNase but more immunogenic than RNase A. Together, these data indicate that ONC is a pluripotent cytotoxin, and serves as the benchmark with which to gauge the cytotoxicity of other ribonucleases.

Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.

Polymer conjugated bovine seminal ribonuclease inhibits growth of solid tumors and development of metastases in mice.

Bovine seminal ribonuclease (BS-RNase) exerts a potent cytotoxic activity when administered intratumorally (i.t.) to the nude mice bearing human tumors. The ineffective treatment with intravenous (i.v.) or intraperitoneal (i.p.) administration led us to the synthesis of polymeric conjugates with BS-RNase to prevent it from degradation in the blood vessel. Hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification and a PHPMA-BS-RNase conjugates were prepared. Classic conjugate (P-BS) with BS-RNase bound to the polymer by its oligopeptide site chains was prepared by aminolytic reaction of the polymer precursor bearing reactive ester groups situated in the side chains of polymer, while star-like conjugate (S-BS) was synthesized by the reaction of PHPMA containing end-chain reactive group with BS-RNase in aqueous buffer solution at pH 8. In contrast to the total ineffectiveness of free BS-RNase administered i.v. at a daily dose 10 mg/kg, application of P-BS and S-BS conjugates at doses 2 mg/kg and 0.5 mg/kg caused significant inhibition of the growth of human melanoma in nude mice. On the base of these results the effect of i.v. administered S-BS on the metastatic process and the survival of C57Bl/6 inbred mice inoculated with B16 melanoma cells was investigated. Sixty per cent of mice treated with S-BS (0.5 mg/kg/day) survived 100 days without metastatic foci when the experiment terminated. The average survival time of the treated groups was 75.5 days compared to 32.7 days in the control group. BS-RNase conjugated to water soluble polymers appears to be the first BS RNase preparation which exerts anticancer and antimetastatic activity following its intravenous administration.

Involvement of the amino-terminal beta-hairpin of the Aspergillus ribotoxins on the interaction with membranes and nonspecific ribonuclease activity.

Ribotoxins are a family of potent cytotoxic proteins from Aspergillus whose members display a high sequence identity (85% for about 150 amino acid residues). The three-dimensional structures of two of these proteins, alpha-sarcin and restrictocin, are known. They interact with phospholipid bilayers, according to their ability to enter cells, and cleave a specific phosphodiester bond in the large subunit of ribosome thus inhibiting protein biosynthesis. Two nonconservative sequence changes between these proteins are located at the amino-terminal beta-hairpin of alpha-sarcin, a characteristic structure that is absent in other nontoxic structurally related microbial RNases. These two residues of alpha-sarcin, Lys 11 and Thr 20, have been substituted with the equivalent amino acids in restrictocin. The single mutants (K11L and T20D) and the corresponding K11L/T20D double mutant have been produced in Escherichia coli and purified to homogeneity. The spectroscopic characterization of the purified proteins reveals that the overall native structure is preserved. The ribonuclease and lipid-perturbing activities of the three mutants and restrictocin have been evaluated and compared with those of alpha-sarcin. These proteins exhibit the same ability to specifically inactivate ribosomes, although they show different activity against nonspecific substrate analogs such as poly(A). The mutant variant K11L and restrictocin display a lower phospholipid-interacting ability correlated with a decreased cytotoxicity. The results obtained are interpreted in terms of the involvement of the amino-terminal beta-hairpin in the interaction with both membranes and polyadenylic acid.

Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor.

Bovine seminal RNase (BS-RNase) is a homodimeric enzyme with a cytotoxic activity selective for tumor cells. In this study, the relationships of its cytotoxic activity to its dimeric structure and its resistance to the cytosolic RNase inhibitor (cRI) are investigated systematically by site-directed mutagenesis. The results show that (1) the dimericity of BS-RNase is essential for its full cytotoxic action; (2) the role of the dimeric structure in the antitumor activity is that of making the enzyme insensitive to the cytosolic RNase inhibitor; (3) a RNase may not be completely insensitive to cRI to exploit a full cytotoxic potential.

Bovine seminal ribonuclease exerts selective cytotoxicity toward neuroblastoma cells both sensitive and resistant to chemotherapeutic drugs.

BACKGROUND: Bovine seminal ribonuclease (BS-RNase) exerts selective cytotoxicity toward different types of tumor cells. In the present study, we tested the effects of BS-RNase on cultured neuroblastoma (NB) cells resistant to chemotherapeutic agents. The selectivity of the antitumoral activity of BS-RNase was evaluated using cultures of CD34+ hematopoietic stem cells. MATERIALS AND METHODS: Human NB cell lines including IMR-32, UKF-NB-2 and UKF-NB-3 were selected for resistance against vincristine, doxorubicin or cisplatin by exposure to increasing concentrations of the respective drug. The cytotoxicity of the drugs to NB cells was evaluated using a clonogenic assay in a methylcellulose medium. Peripheral blood progenitor cells were obtained from adult healthy donors by positive selection using specific anti-CD34+ antibodies. The toxicity of BS-RNase to CD34+ cells was assessed in the direct clonogenic assay using methylcellulose medium or in ex vivo expansion culture supplemented with hematopoietic growth factors. RESULTS: In the clonogenic assay it was shown that BS-RNase completely inhibits growth of both parental NB cells and their sublines resistant to chemotherapeutic drugs at concentrations (up to 50 micrograms/ml) which have no significant influence on the growth of colony-forming units, granulocyte macrophage and erythroid burst-forming units. Moreover, BS-RNase had no effect on the ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors. CONCLUSIONS: BS-RNase is a highly efficient agent against NB cells resistant to chemotherapeutic drugs. The lack of toxicity to hematopoietic progenitor cells suggests that BS-RNase is also likely to have tolerable hematopoietic toxicity.

The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease. Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5'- and 3'-RACE technique. The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.