Crystal structure of Onconase at 1.1 Å resolution--insights into substrate binding and collective motion.

Onconase(®) (ONC) is an amphibian member of the pancreatic ribonuclease superfamily that is selectively toxic to tumor cells. It is a much less efficient enzyme than the archetypal ribonuclease A and, in an attempt to gain further insight, we report the first atomic resolution crystal structure of ONC, determined in complex with sulfate ions at 100 K. The electron density map is of a quality sufficient to reveal significant nonplanarity in several peptide bonds. The majority of active site residues are very well defined, with the exceptions being Lys31 from the catalytic triad and Lys33 from the B(1) subsite, which are relatively mobile but rigidify upon nucleotide binding. Cryocooling causes a compaction of the unit cell and the protein contained within. This is principally the result of an inward movement of one of the lobes of the enzyme (lobe 2), which also narrows the active site cleft. Binding a nucleotide in place of sulfate is associated with an approximately perpendicular movement of lobe 2 and has little further effect on the cleft width. Aspects of this deformation are present in the principal axes of anisotropy extracted from C(α) atomic displacement parameters, indicating its intrinsic nature. The three lowest-frequency modes of ONC motion predicted by an anisotropic network model are compaction/expansion variations in which lobe 2 is the prime mover. Two of these have high similarity to the cryocooling response and imply that the essential 'breathing' motion of ribonuclease A is conserved in ONC. Instead, shifts in conformational equilibria may contribute to the reduced ribonucleolytic activity of ONC.

Onconase responsive genes in human mesothelioma cells: implications for an RNA damaging therapeutic agent.

BACKGROUND: Onconase represents a new class of RNA-damaging drugs. Mechanistically, Onconase is thought to internalize, where it degrades intracellular RNAs such as tRNA and double-stranded RNA, and thereby suppresses protein synthesis. However, there may be additional or alternative mechanism(s) of action. METHODS: In this study, microarray analysis was used to compare gene expression profiles in untreated human malignant mesothelioma (MM) cell lines and cells exposed to 5 microg/ml Onconase for 24 h. A total of 155 genes were found to be regulated by Onconase that were common to both epithelial and biphasic MM cell lines. Some of these genes are known to significantly affect apoptosis (IL-24, TNFAIP3), transcription (ATF3, DDIT3, MAFF, HDAC9, SNAPC1) or inflammation and the immune response (IL-6, COX-2). RT-PCR analysis of selected up- or down-regulated genes treated with varying doses and times of Onconase generally confirmed the expression array findings in four MM cell lines. RESULTS: Onconase treatment consistently resulted in up-regulation of IL-24, previously shown to have tumor suppressive activity, as well as ATF3 and IL-6. Induction of ATF3 and the pro-apoptotic factor IL-24 by Onconase was highest in the two most responsive MM cell lines, as defined by DNA fragmentation analysis. In addition to apoptosis, gene ontology analysis indicated that pathways impacted by Onconase include MAPK signaling, cytokine-cytokine-receptor interactions, and Jak-STAT signaling. CONCLUSIONS: These results provide a broad picture of gene activity after treatment with a drug that targets small non-coding RNAs and contribute to our overall understanding of MM cell response to Onconase as a therapeutic strategy. The findings provide insights regarding mechanisms that may contribute to the efficacy of this novel drug in clinical trials of MM patients who have failed first line chemotherapy or radiation treatment.

Effect of Onconase on double-stranded RNA in vitro.

Onconase is a cytotoxic ribonuclease which targets tumor cells in vivo and in vitro. To date, cellular tRNA appeared to be the major target for Onconase mediated cytotoxic activity. Most recently we demonstrated that Onconase can also cleave double-stranded RNA (dsRNA). Incubation of Onconase at 37 degrees C with GAPDH gene-dsRNA (approximately 440 bp long) and dsRNA ladder showed degradation of dsRNA into a spectrum of smaller dsRNA fragments. Moreover, incubation of dsRNA substrates at 40 degrees C under similar conditions markedly potentiated further cleavage of dsRNAs. The recently discovered double-stranded RNase activity of Onconase suggests another mechanism for inducing cell death/apoptosis in malignant phenotypes via the RNA interference mechanism involving siRNA and miRNA.

Local controlled delivery of anti-neoplastic RNAse to the brain.

PURPOSE: Antineoplastic RNAse proteins, also known as Amphibinases, have been shown effective against various solid tumors but were found selectively neurotoxic to Purkinje cells in the cerebellum. This work describes the use of a waxy biodegradable poly(ricinoleic-co-sebacic acid) for the local controlled delivery of cytotoxic amphibinases in the parietal lobe of the brain in an attempt to overcome cerebellar neuronal toxicity while affecting glioma cells. METHODS: Amphibinase analogues were encapsulated in poly(ricinoleic-co-sebacic acid) formulations using mix-melt technology and loaded onto surgical foam. In-vitro release was monitored by BCA colorimetry and by RNAse specific bioactivity. The implants were inserted into rat brains bearing 9L glioma to assess toxicity and efficacy. RESULTS: The various formulations showed extended linear release for several weeks with minimal burst effect. Best in-vivo efficacy was obtained with ACC7201 containing implants, resulting in the extension of the median survival from 13 to 18 days with 13% long-term survivors. CONCLUSION: Antineoplastic proteins were released from a p(SA-RA) polyanhydride implants in a controlled manner, providing efficacy against 9L glioma, while evading neurotoxicity in the cerebellum. The controlled release of Amphibinases forms the potential for a new therapy against brain tumors.

The cytotoxic ribonuclease onconase targets RNA interference (siRNA).

Onconase (Onc), a ribonuclease from oocytes of Northern Leopard frogs (Rana pipiens) is cytostatic and cytotoxic to a variety of tumor lines in vitro, inhibits growth of tumors in animal in vivo models and enhances sensitivity of tumor cells to a number of other cytotoxic agents with diverse mechanism of action. In Phase III clinical trials Onc demonstrated significant efficacy in patients with malignant mesothelioma that failed prior chemotherapy. We previously postulated that the antitumor activity of Onc and the observed synergisms with other antitumor modalities at least in part may be mediated by targeting RNA interference (RNAi). In the present study we observed that the silencing of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene in human lung adenocarcinoma A549 cells by siRNA was effectively prevented by Onc. While transfection of cells with GAPDH siRNA reduced expression of this protein by nearly 70%, the expression was restored in the cells exposed to 0.8 muM Onc for 48 or 72 h. The data thus provide evidence that one of the targets of Onc is siRNA, likely within the RNA-induced silencing complex (RISC). In light of the findings that microRNAs are involved in tumor pathogenesis as well as in enhancing cell resistance to anticancer therapy the present data may provide explanation for both, the antitumor Onc activity and its propensity to enhance effectiveness of cytotoxic drugs.

Remarkable enhancement of cytotoxicity of onconase and cepharanthine when used in combination on various tumor cell lines.

Onconase (Onc), a ribonuclease from oocytes or early embryos of Northern Leopard frog (Rana pipiens), is cytostatic and cytotoxic to a variety of tumor lines in vitro, inhibits growth of tumors in animal in vivo models and is currently in Phase IIIb clinical trials for malignant mesothelioma where it displays antitumor activity with minor overall toxicity to the patient. One of the characteristic features of Onc is a synergism with a variety of other antitumor modalities. Cepharanthine (Cep), a biscoclaurine alkaloid from Stephania cepharantha Hayata, is widely used in Japan to treat variety of ailments. It also shows low toxicity to patients. The aim of the present study was to assess the interaction of these two drugs on different tumor cell lines. When human promyelocytic leukemia HL-60, histiomonocytic lymphoma U937, multiple myeloma RPMI-8228, prostate carcinoma DU 145 and prostate adenocarcinoma LNCaP cells were exposed to relatively low concentrations of Onc or Cep their growth rates were somewhat suppressed but the cells were still able to proliferate. Cell growth, however, was totally abolished in each of these cell lines when treated with Onc and Cep combined. The frequency of apoptosis was also many-fold higher in cultures treated with a combination of Onc and Cep than in respective cultures treated with Onc or Cep alone. The mechanism of the observed synergism is unclear but it may be associated with the Onc activity in targeting microRNAs and/or NFkappaB and Cep activity also targeting NFkappaB. The data suggest that the combination of these two drugs, that individually express a low toxic profile, may have strong antitumor potential.

Mechanisms of enhanced tumoricidal efficacy of multiple small dosages of ranpirnase, the novel cytotoxic ribonuclease, on lung cancer.

PURPOSE: The effect of multiple small dosages of the cytotoxic RNase, ranpirnase (ONCONASE, ONC), on lung cancer was studied. The possible mechanisms for the enhanced tumoricidal efficacy of multiple small dosages of ONC were also investigated. METHODS: Hematoxylin and eosin staining, TUNEL labeling, and caspase-3-antibody labeling were used for in vivo analysis of apoptosis. A growth-delay assay was applied to detect the therapeutic potential of small and multiple dosages of ONC in vivo. ONC-induced changes in blood flow in A549 tumors and the kidney were measured non-invasively by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). RESULTS: In cell culture studies, ONC significantly inhibited tumor growth of A549 human NSCLC cells without damaging non-cancerous cells (HLF-1 human lung fibroblast). Multiple small dosages of ONC significantly prolonged tumor growth delay of A549 tumors, with increased apoptosis in vivo from 0.5 +/- 0.3 to 70.1 +/- 1.1% (by TUNEL labeling, N = 3, P < 0.05). Interestingly, multiple small doses of ONC were more effective than a single large dose for the inhibition of tumor growth with reduced side effect. Using non-invasive DCE-MRI methods, we found that the mean of the K (trans) median values increased to 49.3 +/- 7.5% from the pre-ONC values by ONC (N = 4 mice, P < 0.05). A subsequent T (1) map of the kidney showed that T (1) values were temporarily decreased for up to 2 days (however, fully recovered approximately 4 days post-treatment). CONCLUSIONS: Multiple small dosages of ONC significantly inhibited tumor growth of A549 NSCLC cells in vivo, with markedly increased apoptosis. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.

Cytostatic and cytotoxic properties of Amphinase: a novel cytotoxic ribonuclease from Rana pipiens oocytes.

Onconase (Onc), is a novel amphibian cytotoxic ribonuclease with antitumor activity, and is currently in a confirmatory phase III clinical trial for the treatment of malignant mesothelioma. It was recently reported that Rana pipiens oocytes contain still another ribonuclease, named Amphinase (Amph). Amph shows 38-40% amino acid sequence identity with onconase, presents as four variants varying between themselves from 87-99% in amino acid sequence identity and has a molecular mass approximately 13,000. In the present study we describe the effects of Amph on growth of several tumor cell lines. All four variants demonstrated cytostatic and cytotoxic activity against human promyelocytic HL-60-, Jurkat T-cell- and U-937 monocytic leukemia cells. The pattern of Amph activity to certain extent resembled that of Onc. Thus, cell proliferation was suppressed at 0.5-10.0 mug/ml (40-80 nM) Amph concentration with distinct accumulation of cells in G(1) phase of the cell cycle. In addition, the cells were undergoing apoptosis, which manifested by DNA fragmentation (presence of "sub-G1" cells, TUNEL-positivity), caspases and serine proteases activation as well as activation of transglutaminase. The cytostatic and cytotoxic effects of Amph required its ribonuclease activity: the enzymatically inactive Amph-2 having histidine at the active site alkylated was ineffective. The effectiveness and cell cycle specificity was generally similar for all four Amph variants and at the equimolar concentrations was somewhat more pronounced than that of Onc. The observed cytostatic and cytotoxic activity of Amph against tumor cell lines suggests that similar to Onc this cytotoxic ribonuclease may have antitumor activity and find an application in clinical oncology.

The therapeutic mechanisms of ranpirnase-induced enhancement of radiation response on A549 human lung cancer.

AIM: The goal of this study was to investigate the therapeutic potential of combining radiation therapy and cytotoxic RNase, ranpirnase (ONCONASE; ONC), in human lung tumor models in vitro and in vivo. As translational implications, the non-invasive monitoring response to individual therapy with ONC was also investigated to determine the underlying therapeutic mechanisms. MATERIALS AND METHODS: A clonogenic survival assay was used to measure the effect of ONC and radiation on A549 human non-small cell lung carcinoma (NSCLC) cells. H&E staining, TUNEL staining and caspase-3-antibody labeling were used for in vivo analysis of apoptosis. A growth-delay assay was applied to detect the therapeutic potential of ONC as a radiation sensitizer in vivo. ONC-induced changes in blood flow and biochemical metabolites were measured by various noninvasive dynamic contrast enhanced magnetic resonance imaging (DCE MRI), non-localized 1H magnetic resonance spectroscopy (MRS), and near-infrared spectroscopy (NIRS) methods. RESULTS: ONC at 5-10 microg/ml sensitized the radiation response of A549 tumor cells in vitro. Remarkable increases in ONC-induced apoptosis in vivo were observed in caspase-3 antibody labeling and TUNEL staining assays. ONC significantly increased the radiation-induced tumor growth delay of A549 tumors. It was observed, when using a DCE MRI method, that there were significant increases in K(trans) values at the rim of tumor regions at 1.5 h post-injection of ONC. When using non-localized 1H MRS, an approximately 20% decrease in lactate levels with ONC was found. CONCLUSION: ONC may be a new and promising drug in the treatment of NSCLC as a radiation therapy enhancer.

Possible mechanisms of improved radiation response by cytotoxic RNase, Onconase, on A549 human lung cancer xenografts of nude mice.

The cytotoxic RNase, Onconase (ONC), isolated from amphibian oocytes, was used to study its effect on the radiation response in A549 human NSCLC in vitro and in vivo. In cell culture studies, we found that ONC increased the radiation response by ONC-induced inhibition of O2 consumption (QO2). The occurrence of apoptosis was increased by ONC and was dependent on dosages and time exposure (measured by a Tunnel in situ cell death detection assay). Moreover, ONC inhibited sublethal damage repair (SLDR), confirmed by a split dose experiment. In animal studies, ONC significantly increased the radiation-induced tumor growth delay of A549 tumors in vivo. Using a non-invasive DCE-MRI technology, ONC-induced changes of perfusion were observed in A549 tumors. We concluded that the ONC-induced enhancement in tumor oxygenation was mainly due to the reduction in QO2 rather than an increase in tumor blood flow. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.

The interdependence between catalytic activity, conformational stability, and cytotoxicity of onconase.

Onconase (ONC) is a cytotoxic ribonuclease of the pancreatic ribonuclease A superfamily isolated from oocytes or early embryos of the Northern leopard frog (Rana pipiens). It shows anticancer activity and currently is in Phase IIIb clinical trial for unresectable malignant mesothelioma. We generated several variants of ONC possessing mutations in selected structural regions of the molecule that have altered ribonucleolytic activity and/or conformational stability. The relationship between the stability and ribonucleolytic activity of these variants and their cytostatic and cytotoxic properties was investigated on several tumor cell lines. Similar to ONC, all variants were inducing reproductive cell death detected by reduced clonogenicity. The surviving cells proliferated at reduced rates as reflected by diminished size of colonies and prolongation of G(0/1) phase of the cell cycle. Some cells were undergoing apoptosis. The cytotoxic and cytostatic effects of ONC and its variants were predominantly determined by their catalytic activity rather than by conformational stability.

Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes.

Besides Onconase (ONC) and its V11/N20/R103-variant, oocytes of the Northern Leopard frog (Rana pipiens) contain another homologue of ribonuclease A, which we named Amphinase (Amph). Four variants (Amph-1-4) were isolated and sequenced, each 114 amino acid residues in length and N-glycosylated at two positions. Sequence identities (a) among the variants and (b) versus ONC are 86.8-99.1% and 38.2-40.0%, respectively. When compared with other amphibian ribonucleases, a typical pattern of cysteine residues is evident but the N-terminal pyroglutamate residue is replaced by a six-residue extension. Amph variants have relatively weak ribonucleolytic activity that is insensitive to human ribonuclease inhibitor protein (RI). Values of k(cat)/K(M) with hypersensitive fluorogenic substrates are 10(4) and 10(2)-fold lower than the maximum values exhibited by ribonuclease A and ONC, respectively, and there is little cytosine/uracil or adenine/guanine discrimination at the B(1) or B(2) subsites, respectively. Amph variants have cytotoxic activity toward A-253 carcinoma cells that requires intact ribonucleolytic activity. The glycan component has little or no influence over single-stranded RNA cleavage, RI evasion or cytotoxicity. The crystal structures of natural and recombinant Amph-2 (determined at 1.8 and 1.9 A resolution, respectively) reveal that the N terminus is unlikely to play a catalytic role (but an unusual alpha2-beta1 loop may do so) and the B(2) subsite is rudimentary. At the active site, structural features that may contribute to the enzyme's low ribonucleolytic activity are the fixture of Lys14 in an obstructive position, the accompanying ejection of Lys42, and a lack of constraints on the conformations of Lys42 and His107.

Antitumor efficacy of the cytotoxic RNase, ranpirnase, on A549 human lung cancer xenografts of nude mice.

BACKGROUND: The cytotoxic RNase, ranpirnase (ONCONASE, ONC), may have promising therapeutic implication as an alternative for cisplatin for the treatment of lung cancer, due to inhibition of protein synthesis by t-RNA cleavage. MATERIALS AND METHODS: A549 and NCI-H1975 human NSCLC cell lines were cultured in the presence and absence of ONC. Cytotoxicity was monitored using a clonogenic assay. Using an inverted phase and fluorescence microscope, we studied whether apoptosis was induced by ONC in gefitinib-induced apoptosis-resistant A549 tumor cells. The therapeutic effectiveness of ONC was studied via single and multiple administrations on A549 human non-small cell lung cancer (NSCLC), including tumors previously untreatable by cisplatin. ONC-induced changes in ATP levels were also monitored by non-localized phosphorus MR spectroscopy. RESULTS: ONC significantly inhibited the cell growth of A549 tumors. Apoptosis was significantly induced by ONC in a dose-dependent manner. In animal studies, multiple small doses of ONC were more effective than one large single dose for the inhibition of tumor growth with reduced side-effects, probably due to the normalization of leaky tumor vessels. ONC in combination with cisplatin significantly reduced tumor growth of A549 tumors. In large tumors, including those unsuccessfully treated with cisplatin, ONC showed inhibition of tumor growth, while a second treatment of cisplatin did not. During monitoring by non-localized phosphorus MR spectroscopy, ATP levels decreased, likely due to ONC-induced inhibition of oxygen consumption (QO2). CONCLUSION: ONC significantly inhibited tumor growth of A549 NSCLC cells in both in vitro and in vivo studies. This investigation suggests important potential clinical uses of ONC for the treatment of NSCLC cancer patients.

Mild hyperthermia predisposes tumor cells to undergo apoptosis upon treatment with onconase.

Onconase (ONC), (ranpirnase) a cytotoxic ribonuclease isolated from amphibian oocytes and early embryos targeting tumor cells in vitro and in vivo, is currently in a confirmatory Phase IIIb clinical trial for unresectable malignant mesothelioma where it demonstrates antitumor activity with relatively minor overall toxicity to patients. Since hyperthermia has been shown to be synergistic with certain antitumor modalities, the aim of the present study was to explore whether the cytotoxic effects of ONC can be enhanced under conditions of mild hyperthermia. Treatment of human lymphoblastoid TK6 cells with 2 or 5 microg/ml of ONC at 40 degrees C for 24 or 48 h led to 64-200% enhancement in incidence of apoptosis assessed by frequency of cells showing the presence of activated (cleaved) caspase-3 or activated serine proteases, compared to treatment at 37.5 degrees C. The incidence of apoptosis at 40 degrees C in the absence of ONC was unchanged compared to 37.5 degrees C, for up to 48 h. Although at 41 degrees C in absence of ONC the incidence of apoptosis was elevated compared to 37 degrees C the cytotoxicity of ONC was further enhanced and the overall pro-apoptotic effect was above the level of additive effects of ONC plus that of 41 degrees C-hyperthermia. While the mechanism of the observed enhancement of ONC cytotoxicity is currently under investigation, the findings suggest that a combination of ONC and mild hyperthermia should be explored to increase effectiveness of ONC in cancer treatment.

Onconase induces caspase-independent cell death in chemoresistant neuroblastoma cells.

The efficacy of Onconase on the growth of a panel of chemosensitive and chemoresistant neuroblastoma cell lines was investigated. Onconase decreased cell viability of chemosensitive (IMR-32, UKF-NB-3) and chemoresistant neuroblastoma cell lines characterised by high expression of P-glycoprotein (P-gp) (UKF-NB-3(r)DOX(20)) or by high P-gp expression in combination with mutated p53 (UKF-NB-3(r)VCR(10), Be(2)-C), in a similar manner. Moreover, Onconase caused cell cycle block in G1 phase and induced caspase-independent cell death. Transmission electron microscope investigations suggested that Onconase-induced autophagy contributes to Onconase-induced cell death. Antitumour activity of Onconase against naïve and drug-resistant neuroblastoma xenografts was confirmed in animals.

Treatment of Jurkat acute T-lymphocytic leukemia cells by onconase (Ranpirnase) is accompanied by an altered nucleocytoplasmic distribution and reduced expression of transcription factor NF-kappaB.

Onconase (Ranpirnase), a novel ribonuclease isolated from Rana pipiens oocytes, was reported to suppress cancer cell growth in vitro, reduce tumor size in animals, and augment cytotoxicity of several chemotherapeutic agents. Since onconase is currently in phase III clinical trials tested in treatment of mesothelioma, much emphasis has been placed on the mechanism of its anti-tumor activity. Previous studies have shown that onconase-responsive cells become arrested at the G1/S checkpoint of the cell cycle and also undergo apoptosis. A proposed mechanism for these effects is that the enzymatic activity of onconase targets cellular RNAs, in particular tRNA, with an accompanying inhibition of protein synthesis. In the present study, we have investigated the time- and dose-dependent effects of onconase on growth of Jurkat SN acute T-lymphocytic leukemia cells. Significant suppression of cell proliferation became evident after 72 and 96 h of treatment, and was most pronounced at the highest concentration (10 microg/ml; 8.3x10(-7) M) of onconase. This reduction of cell proliferation, however, was not accompanied by measurable changes in distribution of cells at different phases of the cell cycle, but was paralleled by the induction of apoptosis, as assayed by flow cytometry, and with a modest decrease in the expression of a cell cycle regulatory retinoblastoma protein (Rb). Further biochemical analysis revealed that growth suppression was closely coordinated with a down-regulation in the steady state and subcellular distribution of NF-kappaB, a transcription factor known to be functionally associated with cell survival. The reduction in expression of NF-kappaB by onconase appeared to coincide or even precede growth suppression, suggesting a causal relationship. To further test the hypothesis that cellular localization and expression of NF-kappaB may be critical to cellular response to onconase, we also studied the growth effects of onconase in Jurkat-BalphaM cells, which, unlike the parent SN T cells, contain a stably transfected dominant-negative IkappaB gene. Growth suppression by onconase in BalphaM cells was more pronounced and occurred earlier compared to SN cells, although still did not affect changes in cell cycle phase distribution. Contrary to expectation, however, diminution in NF-kappaB expression by onconase was even more pronounced in BalphaM cells, suggesting that this transcription factor, while presumably prevented from dissociation from its inhibitory protein IkappaB in these cells, is even more efficiently targeted for degradation by onconase. These results implicate NF-kappaB and its turnover as important determinants in the anti-proliferative/apoptotic effects of onconase in acute T-lymphocytic leukemia cells.

Effect of ONCONASE +/- tamoxifen on ASPC-1 human pancreatic tumors in nude mice.

To evaluate changes in tumor physiological parameters after treatment with ONCONASE (ONC), we used laser Doppler flowmetery for tumor blood flow (TBF) in legs of nude mice bearing AsPC-1 human pancreatic carcinoma. Tumor interstitial fluid pressure (TIFP) was measured using the wick-in-needle technique, while intratumoral pO2 utilized O2 sensitive needle electrodes. TBF was significantly increased by an i.p. injection of ONC, then returned to the untreated levels at 150 min post-treatment. Single and multiple intraperitoneal injections of ONC significantly reduced TIFP (post-treatment at 1-7 days). ONC significantly improved tumor oxygenation evidenced by an increase in median pO2 from 4.2 mm Hg to 8.2 mm Hg. Since ONC had a synergistic interaction with tamoxifen (TAMX) on the cytotoxic clonogenicity of AsPC-1 tumor cells in vitro, we evaluated the antitumoral effects in vivo by ONC +/- TAMX, using nude mice bearing AsPC-1 pancreatic tumor cells of ascite (intraperitoneally implanted) or solid (subcutaneously implanted to legs) tumors. ONC alone effectively retarded the tumor growth, but TAMX alone did not. ONC + TAMX was synergistically effective in inhibiting tumor growth.

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family.

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.

Phase II trial of a single weekly intravenous dose of ranpirnase in patients with unresectable malignant mesothelioma.

PURPOSE: A multicenter phase II trial of ranpirnase (Onconase; Alfacell Corp, Bloomfield, NJ) as a single agent was conducted to further assess the safety and clinical efficacy of this novel antitumor ribonuclease. Patients with unresectable and histologically confirmed malignant mesothelioma (MM) were eligible. PATIENTS AND METHODS: One hundred five patients with Eastern Cooperative Oncology Group performance status 0 to 2 were enrolled onto the study. Thirty-seven percent of patients had not responded to prior chemotherapy. The primary end point of the study was survival. Tumor responses and time to progression were also assessed. The Cancer and Leukemia Group B (CALGB) prognostic group criteria were used to define a treatment target group (TTG). Both the intent-to-treat (ITT) and the TTG populations were analyzed for survival. RESULTS: Median survival times of 6 months for the ITT and 8.3 months for the TTG populations were observed. The 1- and 2-year survival rates were 34.3% and 21.6% for ITT, respectively, and 42% and 26.8% for TTG, respectively. Among the 81 patients assessable for tumor response, four had partial responses, two had minor regressions, and thirty-five experienced stabilization of previously progressive disease. Patients with responses and stable disease demonstrated markedly prolonged survival. Ranpirnase was well tolerated in the majority of patients, and there were no drug-related deaths. CONCLUSION: Ranpirnase demonstrated activity and a tolerable toxicity profile in patients with unresectable MM. The prognostic value of the CALGB groups was confirmed.