Onconase, an anti-tumor ribonuclease suppresses intracellular oxidative stress.

Onconase (ONC), an antitumor ribonuclease from oocytes of a frog Rana pipiens, capable of inducing apoptosis in many cell lines is synergistic with several other anticancer drugs. Since cytotoxic effects of numerous drugs are modulated by reactive oxygen intermediates (ROI), we have studied effects of ONC on the intracellular level of oxidants in several normal cell types as well as tumor cell lines. It is demonstrated for the first time that ONC substantially decreases the content of ROI in all cell lines studied. This effect depends on the ribonucleolytic activity of the enzyme and is due to both, decreased rate of ROI generation and accelerated rate of their degradation. Onconase decreases the mitochondrial transmembrane potential and consequently, generation of ATP. Simultaneously the enzyme decreases the expression of an antiapoptotic protein Bcl-2, and upregulates the proapoptotic Bax protein. These finding are consistent with the enzyme propensity to induce apoptosis. The observed antioxidant activity of ONC may be an important element of its cytotoxicity towards cancer cells. The enzyme seems to exert its biological activities by interfering with the redox system of cellular regulation.

Enhancement of vincristine cytotoxicity in drug-resistant cells by simultaneous treatment with onconase, an antitumor ribonuclease.

BACKGROUND: Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. PURPOSE: We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. METHODS: Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT-29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. RESULTS: The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001). CONCLUSION: Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene.

Physical parameters and chemical composition of porcine pancreatic elastase II.

Some molecular properties of the elastase II preparation, homogenous in ultracentrifugation, have been determined. The molecular weight is 25 000, the sedimentation coefficient and the diffusion coefficient are 3.69-10(-13) s(-1) and 12.09-10(-7) cm2/s, respectively. The partial specific volume was 0.716 g/cm3, and the axial ratio is 1.95. Elastase II exhibited a considerably lower content of arginine, tyrosine, and valine, and a higher content of proline, serine and conjugated carbohydrates than elastase I. The N-terminal amino acid of the enzyme is leucine, and its isoelectric point was 10.7.

Neutral elastolytic proteinase from canine leucocytes. Purification and characterization.

1. A neutral proteinase (EC 3.4.-.-) with elastolytic activity was isolated from canine bloodstream leucocytes, and purified to apparent homogeneity by a two-step procedure consisting of DEAE-Sephadex chromatography and molecular sieving on Sephadex G-75. 2. The molecular weight of the enzyme was 23 500, and the absorbance (A1%1cm) at 282 nm was 6.1. Amino acid analysis showed high content of glycine, aspartic acid, and valine, and low proportion of methionine, lysine and histidine as well as the absence of tyrosine in the enzyme molecule. 3. The proteinase was active against several protein substrates as well as towards N-t-butyloxycarbonyl-L-alanine p-nitrophenyl ester, N-acetyl-L-alanyl-tyrosine ethyl ester. 4. The enzyme was inactivated by diisopropylfluorophosphate, N-acetyl-L-alanyl-L-alanyl-L-alanine chloromethyl ketone, and N-p-tosyl-L-phenylalanine chloromethyl ketone. Inhibition by some natural proteinase inhibitors was also noted.

Effect of single amino acid replacements on the thermodynamics of the reactive site peptide bond hydrolysis in ovomucoid third domain.

We have measured equilibrium constants, Khyd, at pN 6 for the hydrolysis of the reactive site peptide bond (bond between residues 18 and 19) in 42 sequenced variants (39 natural, 3 semisynthetic) of avian ovomucoid third domains. The values range from 0.4 to approximately 35. In 35 cases the effect of a single amino acid replacement on Khyd could be calculated, 13 are without effect and 22 range from a factor of 1.25 to 5.5. Several, but not all, of the effects can be rationalized in terms of residue-residue interactions that are affected by the reactive site hydrolysis. As the measurements are very precise it appears that additional measurements on designed rather than natural variants should allow for the precise measurement of side-chain--side-chain interaction energies.

Refined 1.7 A X-ray crystallographic structure of P-30 protein, an amphibian ribonuclease with anti-tumor activity.

The X-ray crystallographic structure of P-30 protein (Onconase) has been solved by multiple isomorphous replacement and the structure has been refined at 1.7 A resolution to a conventional R-factor of 0.178. The molecular model comprises all 826 non-hydrogen protein atoms, 96 solvent molecules and a sulfate anion that is bound at the active site. The molecular structure is similar to that of ribonuclease A. The active site cleft is located at the junction of two three-stranded beta-sheets and the N-terminal helix. A sulfate anion is non-covalently bound by Lys9, His10, His97, Phe98 and an intermolecular contact involving Lys55' from a neighboring molecule. The N-terminal pyroglutamyl (Pyr) residue is part of the active site and its O epsilon 1 atom forms a hydrogen bond with the Lys9 N zeta. The previously constructed comparative molecular model of P-30 based on ribonuclease A correctly predicted the overall fold of P-30 and the conformation of its active site residues. The model failed to predict the conformation of Pyr1 and the conformation of the two loops following helix alpha 3 and strand beta 3.

Refined X-ray crystal structures of the reactive site modified ovomucoid inhibitor third domains from silver pheasant (OMSVP3*) and from Japanese quail (OMJPQ3*).

Tetragonal and triclinic crystals of two ovomucoid inhibitor third domains from silver pheasant and Japanese quail, modified at their reactive site bonds Met18-Glu19 (OMSVP3*) and Lys18-Asp19 (OMJPQ3*), respectively, were obtained. Their molecular and crystal structures were solved using X-ray data to 2.5 A and 1.55 A by means of Patterson search methods using truncated models of the intact (virgin) inhibitors as search models. Both structures were crystallographically refined to R-values of 0.185 and 0.192, respectively, applying an energy restraint reciprocal space refinement procedure. Both modified inhibitors show large deviations from the intact derivatives only in the proteinase binding loops (Pro14 to Arg21) and in the amino-terminal segments (Leu1 to Val6). In the modified inhibitors the residues immediately adjacent to the cleavage site (in particular P2, P1, P1') are mobile and able to adapt to varying crystal environments. The charged end-groups, i.e. Met18 COO- and Glu19 NH3+ in OMSVP3*, and Lys18 COO- and Asp19 NH3+ in OMJPQ3*, do not form ion pairs with one another. The hydrogen bond connecting the side-chains of Thr17 and Glu19 (i.e. residues on either side of the scissile peptide bond) in OMSVP3 is broken in the modified form, and the hydrogen-bond interactions observed in the intact molecules between the Asn33 side-chain and the carbonyl groups of loop residues P2 and P1' are absent or weak in the modified inhibitors. The reactive site cleavage, however, has little effect on specific interactions within the protein scaffold such as the side-chain hydrogen bond between Asp27 and Tyr31 or the side-chain stacking of Tyr20 and Pro22. The conformational differences in the amino-terminal segment Leu1 to Val6 are explained by their ability to move freely, either to associate with segments of symmetry-related molecules under formation of a four-stranded beta-barrel (OMSVP3* and OMJPQ3) or to bind to surrounding molecules. Together with the results given in the accompanying paper, these findings probably explain why Khyd of small protein inhibitors of serine proteinases is generally found to be so small.

Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity.

A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily. Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals. To explore the structural basis of these differences among members in the RNAse family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coli with final yields of 10 to 50 mg per liter of culture after purification. A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity. Cleavage of the recombinant onconase Met-1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity. Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated. Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C). Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity. The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex.

Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution.

The molecular structure of the complex between bovine pancreatic alpha-chymotrypsin (EC 3.4.4.5) and the third domain of the Kazal-type ovomucoid from Turkey (OMTKY3) has been determined crystallographically by the molecular replacement method. Restrained-parameter least-squares refinement of the molecular model of the complex has led to a conventional agreement factor R of 0.168 for the 19,466 reflections in the 1.8 A (1 A = 0.1 nm) resolution shell [I greater than or equal to sigma (I)]. The reactive site loop of OMTKY3, from Lys13I to Arg21I (I indicates inhibitor), is highly complementary to the surface of alpha-chymotrypsin in the complex. A total of 13 residues on the inhibitor make 113 contacts of less than 4.0 A with 21 residues of the enzyme. A short contact (2.95 A) from O gamma of Ser195 to the carbonyl-carbon atom of the scissile bond between Leu18I and Glu19I is present; in spite of it, this peptide remains planar and undistorted. Analysis of the interactions of the inhibitor with chymotrypsin explains the enhanced specificity that chymotrypsin has for P'3 arginine residues. There is a water-mediated ion pair between the guanidinium group on this residue and the carboxylate of Asp64. Comparison of the structure of the alpha-chymotrypsin portion of this complex with the several structures of alpha and gamma-chymotrypsin in the uncomplexed form shows a high degree of structural equivalence (root-mean-square deviation of the 234 common alpha-carbon atoms averages 0.38 A). Significant differences occur mainly in two regions Lys36 to Phe39 and Ser75 to Lys79. Among the 21 residues that are in contact with the ovomucoid domain, only Phe39 and Tyr146 change their conformations significantly as a result of forming the complex. Comparison of the structure of the OMTKY3 domain in this complex to that of the same inhibitor bound to a serine proteinase from Streptomyces griseus (SGPB) shows a central core of 44 amino acids (the central alpha-helix and flanking small 3-stranded beta-sheet) that have alpha-carbon atoms fitting to within 1.0 A (root-mean-square deviation of 0.45 A) whereas the residues of the reactive-site loop differ in position by up to 1.9 A (C alpha of Leu18I). The ovomucoid domain has a built-in conformational flexibility that allows it to adapt to the active sites of different enzymes. A comparison of the SGPB and alpha-chymotrypsin molecules is made and the water molecules bound at the inhibitor-enzyme interface in both complexes are analysed for similarities and differences.

G1 arrest of U937 cells by onconase is associated with suppression of cyclin D3 expression, induction of p16INK4A, p21WAF1/CIP1 and p27KIP and decreased pRb phosphorylation.

Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.

Potentiation of tumor necrosis factor induced apoptosis by onconase.

Onconase (ONC) a ribonuclease from amphibian oocytes is cytostatic and cytotoxic to many human tumor lines, shows in vivo antitumor activity in mouse tumor models and is in Phase III clinical trials. The mechanism of antitumor activity of ONC is presumed to be due to its internalization, degradation of intracellular RNA and suppression of protein synthesis. Since apoptosis triggered by TNF-alpha is known to be potentiated by inhibitors of protein synthesis, we have hypothesized that it also may be potentiated by ONC. Indeed, preincubation of U-937 or HL-60 leukemic cells with 0.17 microM ONC rendered them more sensitive to induction of apoptosis by TNF-alpha or antibody to CD95 (Fas). The mechanism by which ONC amplifies the effect of TNF-alpha may involve suppression of induction of the survival genes whose expression is triggered by activation of NFkB by this factor.

Enhancement of activation-induced apoptosis of lymphocytes by the cytotoxic ribonuclease onconase (Ranpirnase).

Onconase (Onc) is an amphibian ribonuclease of the pancreatic RNase family that is cytostatic and cytotoxic to several tumor lines. It also shows anti-tumor activity in mouse tumor models and is currently in phase III clinical trials. In animal tests and clinical trials Onc shows lesser toxicity and fewer side effects compared to most chemotherapeutic drugs. Intriguingly, repeated infusions of this protein do not cause apparent immunological reactions in patients. The aim of the present study was to investigate sensitivity to Onc of human lymphocytes during their mitogenic stimulation in response to the polyvalent mitogen phytohemagglutinin (PHA), and in mixed allogeneic lymphocyte cultures. Unexpectedly, we observed that frequency of cells undergoing activation-induced apoptosis was markedly increased in all cultures containing Onc. Apoptosis was measured by flow cytometry using markers that detect activation of caspases, the in situ presence of DNA strand breaks, and loss of fragmented DNA ('sub-G1' cell subpopulation). The enhancement of frequency of activation-induced apoptosis (up to 244%) was observed at 4.2-83 nM Onc concentration, which is at least an order magnitude lower than its minimal concentration reported to affect proliferation or induce apoptosis of leukemic and solid tumor cell lines. The cell cycle progression of lymphocytes that responded to PHA mitogenically was not affected at 8.3 or 83 nM Onc concentration. Because activation-induced apoptosis is the key mechanism regulating several in vivo immunological functions including induction of tolerance, the observed effects of Onc may explain the apparent lack of immune reactions to this protein in treated patients. The propensity of Onc to potentiate the activation-induced apoptosis suggests that this drug may have clinical utility as immunomodulating agent, e.g., to suppress transplant rejection or treat autoimmune diseases.

Thermodynamics and kinetics of the hydrolysis and resynthesis of the reactive site peptide bond in turkey ovomucoid third domain by aspergillopeptidase B.

1. Aspergillopeptidase B rapidly hydrolyses the -Leu18-Glu19-reactive site peptide bond in turkey ovomucoid third domain (OMTKY3) within the pH-range of 4.0-8.4. The reaction proceeds to equilibrium between OMTKY3 and its modified form with the reactive site peptide bond cleaved (OMTKY3). 2. The dependence of the equilibrium constant (Khyd) on pH indicates that hydrolysis of the reactive site peptide bond apparently does not perturb the pK-values of any preexistent ionizable groups in OMTKY3. 3. The obtained Khyd0 value indicates that free energies of OMTKY3 and OMTKY3 are essentially the same. 4. Hydrolysis of the reactive site peptide bond by aspergillopeptidase B at neutral pH is about 60 times faster than the same reaction catalyzed by subtilisin (Carlsberg), the enzyme strongly inhibited by OMTKY3. 5. Resynthesis of the reactive site peptide bond at neutral pH catalyzed by aspergillopeptidase B (reverse reaction) is almost four orders of magnitude faster than the forward reaction.

Specificity of elastases: degradation of the oxidized beta-chain of insulin by porcine pancreatic elastase II and dog leucocyte elastase.

Porcine elastase II (EC 3.4.21.-), a pancreatic proteinase with elastolytic activity, hydrolyses the oxidized beta-chain of insulin with major cleavages occurring at Leu17-Val18, Phe24-Phe25, Phe25-Tyr26 and Tyr26-Thr27. Canine leucocytic elastase splits the same substrate with major sites at Val12-Glu13 and Val18-Cys19 O3H. This indicates similarity of elastase II to chymotrypsins (EC 3.4.21.1 or 3.4.21.2) and of dog leucocyte enzyme to human granulocyte elastase and porcine pancreatic elastase I (EC 3.4.21.11).