Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells.
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PMID:Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases. 1004 64

Onconase, a homolog of ribonuclease A (RNase A) with low ribonucleolytic activity, is cytotoxic and has efficacy as a cancer chemotherapeutic. Here variants of RNase A were used to probe the interplay between ribonucleolytic activity and evasion of the cytosolic ribonuclease inhibitor protein (RI) in the cytotoxicity of ribonucleases. K41R/G88R RNase A is a less active catalyst than G88R RNase A but, surprisingly, is more cytotoxic. Like Onconase, the K41R/G88R variant has a low affinity for RI, which apparently compensates for its low ribonucleolytic activity. In contrast, K41A/G88R RNase A, which has the same affinity for RI as does the K41R/G88R variant, is not cytotoxic. The nontoxic K41A/G88R variant is a much less active catalyst than is the toxic K41R/G88R variant. These data indicate that maintaining sufficient ribonucleolytic activity in the presence of RI is a requirement for a homolog or variant of RNase A to be cytotoxic. This principle can guide the design of new chemotherapeutics based on homologs and variants of RNase A.
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PMID:A ribonuclease A variant with low catalytic activity but high cytotoxicity. 1074 60

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.
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PMID:Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis. 1076 89

Onconase (Onc) is a ribonuclease from amphibian oocytes that is cytostatic and cytotoxic to many tumour lines. It shows in vivo antitumour activity in mouse tumour models and is currently in Phase III clinical trials. The present study was designed to test whether cytotoxic effects of ONC can be modulated by differentiating agents. Human leukaemic HL-60 and prostate cancer LNCaP and JCA-1 cells were treated with Onc in the absence and presence of several inducers of differentiation and frequency of apoptosis was assessed using three different cytometric methods and confirmed by analysis of cell morphology. A moderate degree of apoptosis observed after 48-72 h incubation of HL-60 cells in the presence of 0.42 microM Onc alone was markedly potentiated by administration of retinoic acid (all trans), sodium butyrate or dimethylsulfoxide at concentrations known to induce differentiation but be minimally cytotoxic. Likewise, the frequency of apoptosis of LNCaP and JCA-1 cells treated with Onc was increased in the cultures to which phenylbutyrate was added. Although cell treatment with Onc alone, with each of the differentiating agents alone or with Onc in combination with the differentiating agents led to an increase in the proportion of G1 cells, no specific cell cycle phase preference in induction of apoptosis was observed. The data suggest that cells undergoing differentiation are particularly vulnerable to Onc; a combination of Onc and differentiating agents should be considered for further in vivo tests to assess its possible usefulness in the clinic.
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PMID:Induction of differentiation of leukaemic (HL-60) or prostate cancer (LNCaP, JCA-1) cells potentiates apoptosis triggered by onconase. 1110 Oct 12

Onconase, an anticancer ribonuclease, damages cellular tRNA and causes caspase-dependent apoptosis in targeted cells (M. S. Iordanov, O. P. Ryabinina, J. Wong, T. H. Dinh, D. L. Newton, S. M. Rybak, and B. E. Magun. Cancer Res. 60, 1983-1994, 2000). The proapoptotic action of onconase depends on its RNase activity, but the molecular mechanisms leading to RNA damage-induced caspase activation are completely unknown. In this study, we have investigated whether onconase activates two signal-transduction pathways commonly stimulated by conventional chemo- and radiotherapy, namely the stress-activated protein kinase (SAPK) cascade and the pathway leading to the activation of nuclear factor-kappa B (NF-kappaB). We found that, in all cell types tested, onconase is a potent activator of SAPK1 (JNK1 and JNK2) and SAPK2 (p38 MAP kinase), but that it is incapable of activating NF-kappaB. Inhibition of p38 MAP kinase activity with a pharmacological inhibitor, SB203580, demonstrated that p38 MAP kinase is not required for onconase cytotoxicity. Using explanted fibroblasts from mice that contain targeted disruption of both jnk1 and jnk2 alleles, we found that JNKs are important mediators of onconase-induced cytotoxicity. Surprisingly, following the immortalization of these same cells with human papilloma virus (HPV16) gene products E6 and E7, additional proapoptotic pathways (exclusive of JNK) were provoked by onconase. Our results demonstrate that onconase may activate proapoptotic pathways in tumor cells that are not able to be accessed in normal cells. These results present the possibility that the cytotoxic activity of onconase in normal cells may be reduced by blocking the activity of JNKs.
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PMID:Differential requirement for the stress-activated protein kinase/c-Jun NH(2)-terminal kinase in RNAdamage-induced apoptosis in primary and in immortalized fibroblasts. 1117 Aug 43

Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.
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PMID:Endowing human pancreatic ribonuclease with toxicity for cancer cells. 1155 55

Ranpirnase (Onconase) is the first ribonuclease to enter cancer clinical trials. In prior phase II trials, responses were seen in mesothelioma and other solid tumors. This phase II trial tested ranpirnase (480 microg/m2/w) in 14 patients with refractory advanced renal cell cancer. The median performance status was zero and the median age was 55. All patients had prior immunotherapy and three had prior chemotherapy. No responses were seen in 14 patients. The median survival from on study was 16 months (range two to 28 months). At this dose and schedule ranpirnase has minimal activity in metastatic renal cell cancer.
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PMID:A phase II trial of weekly intravenous ranpirnase (Onconase), a novel ribonuclease in patients with metastatic kidney cancer. 1156 84

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have constructed bootstrap-supported phylogenetic trees that reorganize these ribonucleases into five distinct lineages--the pancreatic ribonucleases (RNases 1), the eosinophil-associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases--with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with positive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contributing to the rapid diversification of ribonucleases within each cluster. This pattern of evolution-rapid diversification via positive selection among sequences of a multigene cluster-bears striking resemblance to what we have described for the eosinophil-associated ribonuclease genes of the rodent Mus musculus, a finding that may have implications with respect the physiologic function of this unique family of proteins.
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PMID:Rapid diversification of RNase A superfamily ribonucleases from the bullfrog, Rana catesbeiana. 1168 20

Onconase(ONC) is an amphibian ribonuclease that is in clinical trials as a cancer chemotherapeutic agent. ONC is a homolog of ribonuclease A (RNase A). RNase A can be made toxic to cancer cells by replacing Gly(88) with an arginine residue, thereby enabling the enzyme to evade the endogenous cytosolic ribonuclease inhibitor protein (RI). Unlike ONC, RNase A contains a KFERQ sequence (residues 7-11), which signals for lysosomal degradation. Here, substitution of Arg(10) of the KFERQ sequence has no effect on either the cytotoxicity of G88R RNase A or its affinity for RI. In contrast, K7A/G88R RNase A is nearly 10-fold more cytotoxic than G88R RNase A and has more than 10-fold less affinity for RI. Up-regulation of the KFERQ-mediated lysosomal degradation pathway has no effect on the cytotoxicity of these ribonucleases. Thus, KFERQ-mediated degradation does not limit the cytotoxicity of RNase A variants. Moreover, only two amino acid substitutions (K7A and G88R) are shown to endow RNase A with cytotoxic activity that is nearly equal to that of ONC.
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PMID:KFERQ sequence in ribonuclease A-mediated cytotoxicity. 1180 5

Onconase (ONC) is a ribonuclease isolated from amphibian oocytes that is cytostatic and cytotoxic to numerous tumor lines. ONC shows in vivo anti-tumor activity in mouse tumor models and is currently in Phase III clinical trials. Previous studies indicated that ONC induces apoptosis of the target cells most likely along the mitochondrial pathway involving caspase-9 as the initiator caspase. We have recently developed an approach to detect the activation of serine (Ser) proteases during apoptosis. The method is based on affinity labeling of Ser protease active centers with fluorochrome-tagged inhibitors. The aim of the present study was to reveal whether Ser proteases are activated during apoptosis induced by ONC. Human leukemic HL-60 cells were treated with ONC for up to 72 h and then exposed to 5(6)-carboxyfluoresceinyl-L-phenylalanylchloromethyl ketone (FFCK) or 5(6)-carboxyfluoresceinyl-L-leucylchloromethyl ketone (FLCK), the fluorescing green reagents reactive with active centers of the chymotrypsin-like enzymes that cleave proteins at the Phe (FFCK) or Leu (FLCK) site. Activation of caspases was assayed in the same cells using sulforhodamine-labeled (fluorescing red) pan-caspases inhibitor (SR-VAD-FMK). Administration of 1.67 microM ONC into cultures of HL-60 cells led to the appearance of cells that bound SR-VAD-FMK as well as FFCK and FLCK. Most labeled cells had features characteristic of apoptosis. We interpret the binding of these ligands, which was irreversible and withstood cell fixation, as revealing activation of caspases and chymotrypsin-like Ser proteases. Because the induction of binding of each of the three ligands occurred at approximately the same time, the data suggest that during apoptosis caspases and Ser proteases may transactivate each other. The intercellular and subcellular pattern of binding SR-VAD-FMK vs FFCK or vs FLCK was different indicating a variability in abundance and localization of these enzymes within individual apoptotic cells. The FFCK- and FLCK-reactive proteins were of similar molecular mass, approximately 59 and approximately 57 kDa, respectively.
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PMID:Activation of caspases and serine proteases during apoptosis induced by onconase (Ranpirnase). 1212 58


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