Josef Matoušek

Antitumor Activity and Other Biological Actions of Oligomers of Ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.

Immunosuppressive activity of angiogenin in comparison with bovine seminal ribonuclease and pancreatic ribonuclease.

Angiogenin, a member of the pancreatic-like ribonuclease family with a special biological action (RISBAses), is a basic protein that induces blood vessel formation. Another member of these special ribonucleases, bovine seminal ribonuclease (BS RNase), displays biological properties, including aspermatogenic, embryotoxic, antitumor and immunosuppressive activities. The effects of two angiogenin preparations tested on the biological activities mentioned above are reported and compared with those of BS RNase and RNase A. In contrast to RNase A, which was ineffective in all biological activities tested, angiogenin suppressed significantly the proliferation of human lymphocytes stimulated by phytohemagglutinin or concanavalin A or by allogenic human lymphocytes (mixed lymphocyte culture). However, angiogenin did not affect the growth of human tumor cell lines, development of cow and mouse embryos and spermatogenicity in mice. On the basis of these results, angiogenin is the first monomeric ribonuclease described so far that displays immunosuppressive activity similar to that of the dimeric BS RNase. The immunosuppressive activity of angiogenin might synergize with the effect on neovascularization of tumor tissues and thus contribute to the development of tumor.

PEG chains increase aspermatogenic and antitumor activity of RNase A and BS-RNase enzymes.

RNase A (bovine pancreatic ribonuclease) and BS-RNase (bovine seminal ribonuclease) are monomeric and dimeric enzymes, respectively, with aspermatogenic and antitumor activities. While the aspermatogenic and, in some experimental situations, the antitumor effects of the RNase A are only minor, the activity of BS-RNase in these phenomena is very significant. These differences can be annulled by means of conjugation of the enzymes with PEG (polyethylene glycol) chains. Aspermatogenic activity was studied histologically following subcutaneous injections of RNase A and BS-RNase conjugates in ICR mice, and the antitumor activity in athymic nude mice with growing human melanoma with i.p. injection of these conjugated ribonucleases. The experiments proved that RNase A, when conjugated to PEG, produced identical aspermatogenic and antitumour effects as BS-RNase conjugated to this polymer. Immunogenicity of RNase A and BS-RNase did not change substantially after the conjugation with PEG polymers. Binding of produced antibodies to both ribonucleases attached to PEG, however, was substantially reduced.

Immunosuppressive Activity of Bovine Seminal Ribonuclease and its Mode of Action.

Two preparations of dimeric BS RNase-native and recombinant proteins caused identical immunosuppressive effects on MLC-stimulated human lymphocytes. The monomers of RNase A and BS RNase were ten times less active. The inhibitory effect on MLC-stimmulation was followed by 90% inhibition of cell-mediated lympholysis (CML) caused by BS RNase (10 micrograms/ml). This effect indicated that BS RNase suppressed the recognition phase of the cytotoxic reaction, resulting in inhibition of generation of cytotoxic effector cells. BS RNase exerted a similar effect on generation of cytotoxic LAK cells. Cytotoxic activity of LAK cells or CTLs against K562 target cells was abrogated only when BS RNase was added at the beginning of the sensitizing phase, but the cytotoxicity of effector cells in the destruction phase was not influenced. The effect of RNase A on the generation of cytotoxic cells was much less pronounced. To get more information about the site of action, the effect of BS RNase on early lymphocyte stimulation by PHA was investigated by using fluorescein cell probes. BS RNase (100 micrograms/ml) prevented a shift in fluorescein emission occurring within one hour of activation using fluorescein diacetate as a marker for changes in the cytoplasmic matrix. On the contrary, it did not block the shift in fluorescence emission when tested with diphenylhexatrien as a marker for changes in membrane fluidity. Furthermore the effect of BS RNase on expression of membrane antigens expressed on activated human lymphocytes was estimated. BS RNase significantly inhibited the expression of CD25, CD38 and CD71 antigens on PHA-, Con A- and MLC-stimulated human T and B lymphocytes. No substantial change in expression of these antigens was observed on IL-2-stimulated cells, but DNA synthesis was totally abrogated. These results indicate that the mode of action of BS RNase on activated T and B lymphocytes is based mainly on the suppressed expression of receptors for interleukin-2-alpha-chain and transferrin.

The cytosolic ribonuclease inhibitor contributes to intracellular redox homeostasis

The hypothesis that the cytosolic RNase inhibitor (cRI) has a role in the protection of the cellular redox homeostasis was investigated testing the effects of oxidants and anti‐oxidants on normal, primary endothelial HUVE cells, and malignant HeLa cells, before and after their engineering into cRI‐deprived cells. We found that cRI plays an important, possibly a key, physiological role in the protection of cells from redox stress, as demonstrated by decreased GSH levels as well as increased oxidant‐induced DNA damage in cRI deprived cells.

Bovine seminal ribonuclease attached to nanoparticles made of polylactic acid kills leukemia and lymphoma cell lines in vitro.

Bovine seminal ribonuclease (BS-RNase) is a protein with a number of biological effects. It shows antitumoral, aspermatogenic, antiembryonic, immunosuppressive and antiviral properties. The cytotoxic effects appear to be specific for tumor cells as non-malignant cells seem to be unaffected in vitro. Unfortunately, the in vivo application of BS-RNase so far was successful only when it was administered intratumorally. Therefore, the objective of the present investigation was to improve the properties of BS-RNase by attachment to nanoparticles made of polylactic acid (PLA-NP) using an adsorption method. This preparation was tested in vitro against leukemia (MOLT-4) and lymphoma (H9) cell lines sensitive and resistant to cytarabine. No difference between the nanoparticle preparation and pure BS-RNase was found in these tests. To examine the in vivo effects, the preparations were tested for their aspermatogenic and antiembryonal efficacy compared to the pure BS-RNase as a rapid test for antitumoral activity. The aspermatogenic and antiembryonal effects were enhanced by the nanoparticle preparation. Consequently, BS-RNase loaded adsorptively to PLA-NP holds promise for the in vivo use as an antitumoral agent. Further research will investigate the efficacy of this preparations in an in vivo tumor model.

Coupling of the antitumoral enzyme bovine seminal ribonuclease to polyethylene glycol chains increases its systemic efficacy in mice.

Bovine seminal ribonuclease (BS-RNase) is an antitumoral active enzyme exhibiting specific antitumoral action against a number of different cancer cell lines. However, its systemic use is limited by its pharmacokinetic properties and antigenicity. Therefore, it was conjugated to polyethylene glycol (PEG) chains to overcome these problems. Measurement of aspermatogenic effects of the preparation after s.c. injection and injection into the scrotum was chosen as a model for the distribution of the enzyme in the body mediated by the linkage to PEG chains. Additionally, the antigenicity of BS-RNase coupled to PEG chains (BS-RNase–PEG) was compared to that of free BS-RNase, as antigenicity is known to be one of the main obstacles in the use of protein-based drugs. BS-RNase–PEG caused aspermatogenic effects after systemic administration to mice in very low concentrations at which free BS-RNase is not effective. Moreover, BS-RNase possessed a very low antigenicity as long as it was coupled to the PEG chains. In order to investigate the antitumoral efficacy of BS-RNase–PEG in vivo, preliminary experiments on the effect of the conjugate on neuroblastoma growth in mice were performed in a UKF-NB-3 xeno-transplantate model, demonstrating a drastically increased anti-tumoral activity of the conjugate compared to the free enzyme.

Antitumor effects and cytotoxicity of recombinant plant nucleases.

Recombinant plant nucleases R-TBN1 and R-HBN1 were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant nucleases were as effective against melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about 10 times lower concentrations compared to the use of bovine seminal RNase (BS-RNase). Because the recombinant nucleases R-HBN1 and R-TBN1 share only 67.4% amino acid identity and showed only partial immunochemical cross-reactivity, their similar anticancerogenic effects can be mainly explained by their catalytical similarity. Both recombinant nucleases showed lower degree of aspermatogenesis compared to BS-RNAse and PN nuclease. Unlike BS-RNase, aspermatogenesis induced by both recombinant nucleases could not be prevented by the homologous antibody complexes. Owing to relatively low cytotoxicity on the one hand, and high efficiency at low protein levels on the other, recombinant plant nucleases R-HBN1 and R-TBN1 appear to be stable biochemical agents that can be targeted as potential antitumor cytostatics.

Ribonucleases Endowed with Specific Toxicity for Spermatogenic Layers

Bovine seminal ribonuclease (BS-RNase) is a dimer in which the subunits are cross-linked by disulfide bonds between Cys31 of one subunit and Cys32 of the other. Dimeric BS-RNase is resistant to ribonuclease inhibitor (RI), a protein endogenous to mammalian cells, and is toxic to a variety of cell types. Monomeric BS-RNase (like its homolog, RNase A) is bound tightly by RI and is not cytotoxic. The three-dimensional structure of the RI·RNase A complex suggests that carboxymethylation of C32S BS-RNase (to give MCM31) or C31S BS-RNase (MCM32) could diminish affinity for RI. We find that MCM31 and MCM32 are not only resistant to RI, but are also aspermatogenic to mice. In contrast to the aspermatogenic activity of dimeric BS-RNase, that of MCM31 and MCM32 is directed only at spermatogenic layers. Intratesticular injection of MCM31 or MCM32 affects neither the diameter of seminiferous tubules nor the weight of testes. Also in contrast to wild-type BS-RNase, MCM31 and MCM32 are not toxic to other cell types. Direct immunofluorescence reveals that MCM31 and MCM32 bind only to spermatogonia and primary spermatocytes. This cell specificity makes MCM31 and MCM32 of potential use in seminoma therapy and contraception.

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation.

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.