Olga N. Ilinskaya

Cytotoxic ribonucleases: molecular weapons and their targets

Many ribonucleases (RNases) are highly cytotoxic. In some cases, they attack selectively malignant cells, triggering apoptotic response, and therefore are considered as alternative chemotherapeutic drugs. Factors that determine the cytotoxicity of RNases, primarily of those of microbial origin, are reviewed here. These factors include catalytic activity, ability to escape natural inhibitors, stability, and efficiency of internalization. The latter is, in turn, determined by positive charge on the molecule and interaction with cell membrane. Cellular targets and molecular determinants of RNases decisive for their cytotoxic action are characterized.

Bacteria and archaea involved in anaerobic digestion of distillers grains with solubles

Cereal distillers grains, a by-product from bioethanol industry, proved to be a suitable feedstock for biogas production in laboratory scale anaerobic digesters. Five continuously stirred tank reactors were run under constant conditions and monitored for biogas production and composition along with other process parameters. Iron additives for sulfide precipitation significantly improved the process stability and efficiency, whereas aerobic pretreatment of the grains had no effect. The microbial communities in the reactors were investigated for their phylogenetic composition by terminal restriction fragment length polymorphism analysis and sequencing of 16S rRNA genes. The bacterial subcommunities were highly diverse, and their composition did not show any correlation with reactor performance. The dominant phylotypes were affiliated to the Bacteroidetes. The archaeal subcommunities were less diverse and correlated with the reactor performance. The well-performing reactors operated at lower organic loading rates and amended with iron chloride were dominated by aceticlastic methanogens of the genus Methanosaeta. The well-performing reactor operated at a high organic loading rate and supplemented with iron hydroxide was dominated by Methanosarcina ssp. The reactor without iron additives was characterized by propionate and acetate accumulation and high hydrogen sulfide content and was dominated by hydrogenotrophic methanogens of the genus Methanoculleus.

Binase and other microbial RNases as potential anticancer agents

Some RNases possess preferential cytotoxicity against malignant cells. The best known of these RNases, onconase, was isolated from frog oocytes and is in clinical trials as anticancer therapy. Here we propose an alternative platform for anticancer therapy based on T1 RNases of microbial origin, in particular binase from Bacillus intermedius and RNase Sa from Streptomyces aureofaciens. We discuss their advantages and the most promising directions of research for their potential clinical applications.

Changing the net charge from negative to positive makes ribonuclease Sa cytotoxic.

Ribonuclease Sa (pI = 3.5) from Streptomyces aureofaciens and its 3K (D1K, D17K, E41K) (pI = 6.4) and 5K (3K + D25K, E74K) (pI = 10.2) mutants were tested for cytotoxicity. The 5K mutant was cytotoxic to normal and v‐ras‐transformed NIH3T3 mouse fibroblasts, but RNase Sa and 3K were not. The structure, stability, and activity of the three proteins are comparable, but the net charge at pH 7 increases from −7 for RNase Sa to −1 for 3K and to +3 for 5K. These results suggest that a net positive charge is a key determinant of ribonuclease cytotoxicity. The cytotoxic 5K mutant preferentially attacks v‐ras‐NIH3T3 fibroblasts, suggesting that mammalian cells expressing the ras‐oncogene are potential targets for ribonuclease‐based drugs.

Barnase and binase: twins with distinct fates.

RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant cells triggering an apoptotic response. A number of eukaryotic and bacterial RNase‐based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. This review focuses on the properties of the extracellular RNases from Bacillus amyloliquefaciens (barnase) and Bacillus intermedius (binase), the characteristics of their biosynthesis regulation and their physiological role, with an emphasis on the similarities and differences. Barnase and binase can be regarded as molecular twins according to their highly similar structure, physical–chemical and catalytic properties. Nevertheless, the ‘life paths’ of these enzymes are not the same, as their expression in bacteria is controlled by diverse signals. Binase is predominantly synthesized under phosphate starvation, whereas barnase production is strictly dependent on the multifunctional Spo0A regulator controlling sporulation, biofilm formation and cannibalism. Barnase and binase also have some distinctions in practical applications. Barnase was initially suggested to be useful in research and biotechnology as a tool for studying protein–protein interactions, for RNA elimination from biological samples, for affinity purification of RNase fusion proteins, for the development of cloning vectors and for sterility acquisition by transgenic plants. Binase, as later barnase, was tested for antiviral, antitumour and immunogenic effects. Both RNases have found their own niche in cancer research as a result of success in targeted delivery and selectivity towards tumour cells.

A Direct Technique for Magnetic Functionalization of Living Human Cells

Functionalized living cells are regarded as effective tools in directed cell delivery and tissue engineering. Here we report the facile functionalization of viable isolated HeLa cells with superparamagnetic cationic nanoparticles via a single-step biocompatible process. Nanoparticles are localized on the cellular membranes and do not penetrate into the cytoplasm. The magnetically responsive cells are viable and able to colonize and grow on substrates. Magnetically facilitated microorganization of functionalized cells into viable living clusters is demonstrated. We believe that the technique described here may find a number of potential applications in cell-based therapies and in development of whole-cell biosensors.

Bacillus intermedius ribonuclease as inhibitor of cell proliferation and membrane current.

The antiproliferative action of the guanine-specific ribonuclease secreted by Bacillus intermedius (binase) was studied in different chicken and mouse cell lines. The proliferation rate of chicken embryo fibroblasts, either normal or Rous sarcoma virus-transformed, was significantly reduced by binase treatment. Among mouse fibroblasts, v-ras-transformed NIH3T3 cells were sensitive to binase, whereas the growth of non-transformed, v-src-transformed or v-fms-transformed NIH3T3 cells was not affected. A 48 h treatment with binase inhibited the Ca2+-dependent K+ current of v-ras-transformed NIH3T3 cells but had no effect on this membrane current in non-transformed and in v-src- or v-fms-transformed NIH3T3 cells. Our results suggest that mammalian cells expressing the ras-oncogene are a potential target for the antiproliferative action of binase.

SOS-inducing ability of native and mutant microbial ribonucleases

The results of genotoxicity testing of microbial ribonucleases from Bacillus species with different catalytic activity obtained by site-directed mutagenesis in SOS chromotest are reported. At the concentrations 0.1-1 mg/ml, the induction factor for wild-type bacillar binase, barnase and mutant Arg58Lys binase with 100% activity was found to be significantly higher than 1.5 (1.8-2.8). Mutant RNases having decreased catalytic activity (binases with replacements Lys26Ala, Arg61Gln, His101Glu) or through natural inhibitor barstar inactivated wild-type RNase exhibited no SOS-inducing potency. The ability of native bacillar RNases and mutant enzymes possessing high catalytic activity comparable with the activity of wild-type RNase to cause the SOS response indicates that genotoxicity is mediated through the probable cleavage of cellular RNA.

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.

Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca(2+) and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.