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.

A phase II trial of weekly intravenous ranpirnase (Onconase), a novel ribonuclease in patients with metastatic kidney cancer.

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.

Potent and specific antitumor effects of an anti-CD22-targeted cytotoxic ribonuclease: potential for the treatment of non-Hodgkin lymphoma.

LL2, an anti-CD22 monoclonal antibody against B-cell lymphoma, was covalently linked to the amphibian ribonuclease, onconase, a member of the pancreatic RNase A superfamily. LL2 increased in vitro potency (10 000-fold) and specificity against human Daudi Burkitt lymphoma cells while decreasing systemic toxicity of onconase. Monensin further increased potency of LL2-onconase on Daudi cells (IC(50), 20 and 1.5 pM, absence and presence of monensin, respectively). A 1-hour exposure to LL2-onconase was sufficient to kill Daudi cells in culture. These favorable in vitro properties translated to significant antitumor activity against disseminated Daudi lymphoma in mice with severe combined immunodeficiency disease. In mice inoculated with tumor cells intraperitoneally (ip), LL2-onconase (100 microg 5 times ip every day) increased the life span of animals with minimal disease 200%. The life span of mice with advanced disseminated Daudi lymphoma (tumor cells inoculated intravenously) was increased 135%. Mice injected with LL2-onconase tolerated a dose as high as 300 mg/kg. Because both onconase and LL2 are in clinical trials as cancer therapeutics, the covalently linked agents should be considered for treatment of non-Hodgkin lymphoma.

Induction of differentiation of leukaemic (HL-60) or prostate cancer (LNCaP, JCA-1) cells potentiates apoptosis triggered by onconase.

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.

Enhanced cellular radiation sensitivity of androgen-independent human prostate tumor cells by onconase.

The RNase-like onconase, isolated from amphibian oocytes, showed increases in median tumor pO2 in solid tumors (1). This led us to consider if onconase could decrease cellular O2 consumption (QO2) on 9L rat glioma as well as DU145 human prostate adenocarcinoma cells. Using a Clark-type electrode chamber, we observed that onconase significantly inhibited QO2 in both tumors we tested. Since onconase-induced reduction in QO2 could lead to increases in radiation sensitivity, due to the diffusion of O2 to previously hypoxic tumor cells, we used androgen-insensitive DU145 cells to study onconase-induced changes in radiation sensitivity in vitro. Radiation sensitization was achieved with > 5 micrograms/ml of onconase, regardless of the p53 status of tumor cells. Data presented here suggested that onconase-induced enhancement in radiation sensitization in vitro of androgen-insensitive prostate cancer cells warranted further studies of radiation responses in vivo, prior to clinical settings for the advanced-stages of prostate cancer.

Tumoricidal effects of onconase on various tumors.

BACKGROUND AND OBJECTIVES: The effects of Onconase (Onc) on the tumor growth in vitro and in vivo were examined. Because elevated tumor interstitial fluid pressure (TIFP) is one of the major causes of inadequate drug delivery into solid tumors, we tested if Onc could lower TIFP in solid tumors. METHODS: We used several assays including a clonogenic assay and a growth delay assay for the determination of anti-tumoricidal effects of Onc. We also measured Onc-induced changes in several tumor physiological parameters. RESULTS: Onc demonstrated cytotoxic effects in all eight exponentially growing cell lines in vitro. It effectively inhibited the growth of all four transplanted tumors in vivo, and significantly reduced TIFP in all four tumors. Onc also induced increases in tumor blood flow (TBF) as well as increases in median tumor oxygen partial pressure (pO(2)) in solid tumors. CONCLUSIONS: Onc showed anti-tumoral effects on various tumor cells in vitro as well as in vivo. We also gained some insight regarding the potential physiological benefit of Onc as a new therapeutic agent in cancer treatment. Due to increases in both TBF and tumor pO(2), Onc could be a potential candidate as a novel radiation enhancer; therefore, the study of the radiation response in vivo is warranted.

Interferon enhances the activity of the anticancer ribonuclease, onconase.

Interferons (IFN) are biologic agents involved in the antiviral response and the inhibition of tumor growth. Biochemical pathways of IFN action include the double-stranded RNA-activated oligoadenylate synthetase, RNase L, and double-stranded RNA-dependent protein kinase (PKR). Extracellular ribonucleases, especially onconase, also display antiviral and antitumor properties and involve degradation of RNA. We find that IFN increases the anticancer activity of onconase. These two agents work synergistically, and the effect is seen at the level of translation probably because of the degradation of tRNA.

Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases.

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.

Enhanced in vitro cytotoxicity and cytostasis of the combination of onconase with a proteasome inhibitor.

In proliferating cells the turnover rate of proteins responsible for regulation of the cell cycle progression, namely cyclins and inhibitors of the cyclin-dependent kinases (CDKs) and phosphatases, is rapid and their cellular level is modulated at the transcriptional, translational and/or degradation (via proteasome pathway) stages. Inhibition of proteasome function results in accumulation of rapidly turning over proteins and, thus, causes an imbalance of the cell cycle regulatory components, and loss of their regulatory function. Indeed, it has been shown that proteasome inhibitors perturb the cell cycle progression. Onconase, a novel RNase which has anti-tumor activity and is in clinical trials, has previously been shown to suppress protein synthesis, presumably by degradation of intracellular RNA, preferentially tRNA. By interfering with regulation of expression of cyclins and/or CDK-inhibitors, onconase also may induce the imbalance of these proteins and potentiate the effect of proteasome inhibitors. In the present study, we observed that the combinations of onconase with peptide-aldehyde inhibitors of calpain and proteasome such as the N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) and the N-acetyl-leucinyl-valinyl-phenylalaninal (LVP), but not N-acetyl-leucinyl-leucinyl-methioninal (LLM), were synergistic in suppressing cell proliferation and inducing apoptosis in three human tumor cell lines: A-549 lung adenocarcinoma, DU-145 prostatic carcinoma, and MDA-MB-231 breast carcinoma. The observed cytotoxicity may also be a result of prevention of the induction of the 'survival' genes by the nuclear factor kappaB (NFkappaB) by onconase and proteasome inhibitors. The data indicate that such combinations should be further tested as potential anti-cancer regimens.

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.

Molecular determinants in the plasma clearance and tissue distribution of ribonucleases of the ribonuclease A superfamily.

The similarities and differences among members of the RNase A superfamily provide an ideal opportunity to examine the molecular basis for differences in their pharmacokinetics and biodistribution. Plasma clearances in BALB/c mice are similar among the five RNases studied: human pancreatic RNase, angiogenin, eosinophil-derived neurotoxin, onconase, and bovine seminal RNase. The average clearance is 0.13 ml/min or 60% of the glomerular filtration rate (measured by [14C]inulin clearance during continuous infusion from an i.p. implanted osmotic pump). Angiogenin has a higher volume of distribution and plasma-to-muscle transport rate than the other RNases, suggestive of binding to endothelial cells. Organ distribution differs dramatically among these RNases. The RNase most toxic to tumor cells, onconase, exhibits the longest retention in the kidneys: at 180 min, 50% of the injected dose is found in the kidneys, whereas only 1% or less of the other RNases is retained in the kidneys. Slower elimination of onconase from the kidneys may be due to a higher degree of binding in the kidney or a resistance to proteolytic degradation. To elucidate the molecular determinants involved in tissue uptake, we examined the biodistribution of recombinant onconase and two onconasepancreatic RNase chimeric proteins. The tissue retention property of onconase appears to be located in at least two regions, one of which is in the NH2-terminal 9-amino acid alpha-helix. The NH2-terminal pyroglutamate of onconase, a residue essential for ribonucleolytic activity and cytotoxicity, does not play a role in kidney retention.

Inhibition of HIV-1 production and selective degradation of viral RNA by an amphibian ribonuclease.

Ribonucleases appear to have physiologic roles in host defense against cancer, viruses, and other parasites. Previously it was shown that select ribonucleases added to cells concurrently with virions blocked human immunodeficiency virus, type I (HIV-1) infection of H9 cells. We now report that a ribonuclease homologous to RNase A, named onconase, inhibits virus replication in chronically HIV-1-infected human cells without killing the virally infected cell. Examining the mechanism of this inhibition shows that onconase enters the infected cells and degrades HIV-1 RNA without degrading ribosomal RNA or the three different cellular messenger RNAs analyzed. The homologous human pancreatic RNase lacks anti-viral activity. Comparing recombinant forms of onconase and a onconase-human RNase chimera shows that the N-terminal 9 amino acids and the pyroglutamyl residue of onconase are required for full anti-viral activity. Thus extracellular ribonucleases can enter cells, metabolize select RNAs, and inhibit HIV virion production within viable replicating cells.

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.

Anti-tumor ribonuclease, combined with or conjugated to monoclonal antibody MRK16, overcomes multidrug resistance to vincristine in vitro and in vivo.

Onconase, a ribonuclease isolated from Rana pipiens oocytes and early embryos, is a member of the RNase A superfamily. Onconase has anti-neoplastic properties both in vitro and in vivo, and is undergoing clinical evaluation. In the present study, Onconase was combined with or conjugated to MRK16, an anti-P-glycoprotein (Pgp) monoclonal antibody. The interaction of these combinations with vincristine (VCR) against parental and multidrug resistant (MDR), Pgp expressing, human colon carcinoma cells caused increased VCR cytotoxicity in vitro and enhanced survival of athymic nude mice given transplants of drug resistant HT-29(mdr1) cells in vivo. The results suggest that combination treatment with Onconase and other agents that modulate the chemosensitivity of Pgp-expressing human tumor cells has the potential to overcome MDR.

Tamoxifen in patients with advanced pancreatic adenocarcinoma.

Estrogen and somatostatin receptors have been found in pancreatic carcinoma cells, The growth of pancreatic carcinomas has been shown to be inhibited by various endocrine treatments. In view of these findings, a well known anti-estrogen tamoxifen has been used, both as a single agent and in combination with other agents, in clinical trials in patients with unresectable carcinoma of the exocrine pancreas. The impact on survival of tamoxifen in patients with advanced pancreatic adenocarcinoma has been assessed among patients from the previously reported trials of tamoxifen as a single agent, and compared with the survival of patients treated with the synergistic combination of intravenous Onconase plus oral tamoxifen. While a few patients treated with tamoxifen as a single agent may survive for prolonged period of time, the significance of such results remains uncertain in view of the lack of significant survival difference between patients treated with tamoxifen vs. untreated controls in prospectively randomized trials, and vis a vis clearly multifactorial determination of survival. On the other hand, the synergistic combinations of agents that demonstrate favorable toxicity profiles may offer significant survival advantage, at least in some patients with advanced pancreatic cancer.

A study of the intracellular routing of cytotoxic ribonucleases.

Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and indicating that degradation of RNA is the intracellular lesion causing toxicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility.

Treatment of malignant mesothelioma (review).

Malignant mesothelioma (MM) has a very variable natural history. Its known relationship to asbestos exposure is often difficult to establish in a particular patient in view of a long preceding latency period of up to 40+ years. Survival depends on several prognostic factors including histological type, stage, performance status, resectability, and response to radiation and chemotherapy (CT). The assessment of the therapeutic impact on survival is usually difficult due to the different treatment philosophies and selections of patients in various clinical trials. Combined modality treatment approaches seem to offer the best outcomes, with occasional long-term disease-free survivors. Unresectable MM presents a difficult therapeutic challenge. In patients treated with systemic CT the median survival time (MST) in the range of 6 to 12 months and the 2 year survival of <20% are typical. The MST in excess of 20 months and the 2 year survival in the range of 40% have been reported in a few multimodality therapy trials in patients with at least partially resectable MM. Despite some encouraging therapeutic results in selected patients, the overall response to treatment(s) remains unsatisfactory, and new treatments must be sought.

Relationship between response rate and median survival in patients with advanced nonsmall cell lung-cancer - comparison of onconase(r) with other anticancer agents.

The role of systemic cytotoxic therapy for the treatment of advanced non-small cell lung cancer (NSCLC) remains controversial. The response rate (RR) and the median survival time (MST) are the two most frequently used parameters for the assessment of efficacy of the anti-cancer therapies. The relationship between the previously reported RRs and MSTs from published chemotherapy trials in patients with advanced NSCLC was examined using linear regression analysis. The MST of the thirty patients with advanced NSCLC treated with ONCONASE (ONC) as a single agent was 7.7 months which compared favorably with the MSTs of patients treated with a variety of chemotherapeutic regimens either as single agents or combinations, as well as placebo and supportive care only. Moreover, the toxicity profile of ONC compared favorably to the profiles of other chemotherapy regimens. ONC had a favorable impact on the overall MST, including patients with stage IV disease, patients with poor performance status, and patients previously treated with radiotherapy and chemotherapy. The MST of 5 patients who had a stabilization of previously progressive disease was 9.3 months. Based on its positive impact on the MST, ONC appears to have a single agent activity in patients with advanced NSCLC, and it should be further investigated, particularly in combination with synergistic drugs, in concurrently controlled and prospectively randomized clinical trials. The duration and the quality of survival should be considered as the most meaningful parameters in assessing clinical efficacy of anti-cancer agents.

A novel re-regulator of cancer growth - a commentary.

Tumor cells should not necessarily be viewed as 'alien invaders' that must be killed in order to cure the host harboring them. Rather, the successful re-regulation of their growth, even without total tumor eradication, could provide much greater overall benefit to the host. Therefore, the criteria for a meaningful evaluation of clinical benefit of cancer treatment must be re-assessed, and the length of survival and its quality should be considered the two most important parameters. Tumor cells differ from the normal 'self' counterparts only in certain respects, often very narrowly, and primarily with regard to the de-regulation of cell growth and proliferation. The potentially reversible overall malignant phenotype is manifested by the growth largely independent of external factors, increased invasiveness, loss of contact inhibition, and development of the metastatic capabilities. This phenotype evolves in time and represents an inherently heterogeneous process. ONCONASE(R), a novel RNase from the eggs of the leopard bb frog (Rana pipiens) has demonstrated, both pre-clinically in vitro and in vivo as well as in clinical trials, a broad spectrum of anti-cancer activity. The favorable safety profile (e.g., lack of myelosuppression), broad spectrum of anti-cancer activity, synergistic interactions with multiple drugs, and the ability to induce apoptosis and to overcome multiple drug resistance, make ONCONASE(R) especially attractive as a re-regulator of the cancerous growth.