Inhibition of effector cell function in human antibody-dependent cellular cytotoxicity by sera from cancer patients.

The effect of sera from 256 human cancer patients on the human antibody-dependent cellular cytotoxicity (ADCC) assay was studied. The cancer sera were compared to normal sera for their ability to alter the effector function of lymphocytes following 30 minutes' treatment at 37 degrees C. Even with this brief treatment, 74% of the 256 cancer sera inhibited effector activity. In most instances this inhibition was greater in patients with metastatic disease than in those without. Patients with colon and prostate cancer showed a statistically significant increase in inhibition among patients with disseminated disease (P less than 0.02 and P less than 0.01, respectively). An opposite effect was noted only in melanomas. It is suggested that the inhibition of the effector function in ADCC is a potential in vitro measure of the immunologic status of cancer patients.

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.

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.

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.

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.

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.

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.

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.

Aspergillus vegetative endocarditis and complete heart block in a patient with acute leukemia.

Aspergillus flavus vegetative endocarditis together with myocardial abscesses and pneumonitis developed in a patient with acute lymphocytic leukemia. The initial diagnosis was not suspected until 67gallium imaging revealed a radiographically undetectable thoracic abnormality. Despite apparently "early" diagnosis, antifungal therapy was inadequate to prevent disruption of the bundle of His, complete heart block and death.

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.

Synergism between a novel amphibian oocyte ribonuclease and lovastatin in inducing cytostatic and cytotoxic effects in human lung and pancreatic carcinoma cell lines.

A novel anti-tumour amphibian oocyte RNase, ONCONASER (ONC), previously known as P-30 Protein, is in the clinical trials. The effect of ONC alone and in combination with lovastatin (LVT), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme of mevalonate (MVA) and cholesterol synthesis pathway, in three human tumour cell lines ASPC-1 pancreatic, A-549 lung, and HT-520 lung carcinomas, has been presently studied. A synergism between ONC and LVT in inducing the cytostatic and cytotoxic effects was observed. The cytostatic effect, seen during the early phase of the treatment with this combination of drugs was manifested as prolongation of the cell cycle duration, especially of the G1 phase; cell death was apparent after 72 h of treatment. The synergistic effect of ONC and LVT was also evident in the clonogenicity assays. Both LVT lactone and its in vitro activated beta-hydroxy acid form, alone and in respective combinations with ONC, exerted similar degree of growth suppression. The effects of both forms of LVT (used alone or in combination with ONC) were reversed by MVA, which suggests that HMG-CoA reductase inhibition is a primary mechanism of LVT action. The data indicate that the LVT lactone can be activated intracellularly by tumour cells studied, and that the combination of ONC with LVT can produce significantly enhanced anti-tumour activities.

Amino acid sequence of an anti-tumor protein from Rana pipiens oocytes and early embryos. Homology to pancreatic ribonucleases.

Rana pipiens oocytes and early embryos contain large amounts of a basic protein with antiproliferative/cytotoxic activity against several tumor cell lines in vitro (Darzynkiewicz, Z., Carter, S. P., Mikulski, S. M., Ardelt, W., and Shogen, K. (1988) Cell Tissue Kinet. 21, 169-182; Mikulski, S.M., Viera, A., Ardelt, W., Menduke, H., and Shogen, K. (1990) Cell Tissue Kinet. 23, 237-246), as well as antitumor activity in vivo (Mikulski, S. M., Ardelt, W., Shogen, K., Bernstein, E. H., and Menduke, H. (1990) J. Natl. Cancer Inst. 82, 151-153). The protein, provisionally named P-30 Protein, was purified to homogeneity from early embryos and characterized. It is a single-chain protein consisting of 104 amino acid residues in the following sequence: less than Glu1-Asp-Trp-Leu-Thr-Phe-Gln-Lys-Lys-His-Ile-Thr-Asn-Thr- Arg15-Asp-Val-Asp-Cys-Asp-Ans-Ile-Met-Ser-Thr-Asn-Leu-Phe-His-C ys30-Lys-Asp-Lys - Asn-Thr-Phe-Ile-Tyr-Ser-Arg-Pro-Glu-Pro-Val-Lys45-Ala-Ile-Cys-Lys- Gly-Ile-Ile- Ala-Ser-Lys-Asn-Val-Leu-Thr-Thr60-Ser-Glu-Phe-Tyr-Leu-Ser-Asp -Cys-Asn-Val-Thr-Ser-Arg-Por-Cys75-Lys-Tyr-Lys-Leu-Lys-Lys-Ser-Thr -Asn-Lys-Phe- Cys-Val-Thr-Cys90-Glu-Asn-Gln-Ala-Pro-Val-His-Phe-Val-Gly-Val-Gly- Ser-Cys104-OH . Its molecular weight calculated from the sequence is 11,819. The sequence homology clearly indicates that the protein belongs to the superfamily of pancreatic ribonuclease. It is also demonstrated that it indeed exhibits a ribonucleolytic activity against highly polymerized RNA and that this activity seems to be essential for its antiproliferative/cytotoxic effects.

Cytostatic and cytotoxic effects of Pannon (P-30 Protein), a novel anticancer agent.

P-30 Protein is a novel protein, of molecular weight approximately 15 KD, obtained from the extract of a vertebrate tissue showing in vivo antitumour activity. Cytostatic and cytotoxic effects of this product in its purified form (P-30 Protein) or in partially purified extracts (Pannon) were studied in vitro on human leukaemic HL-60, human submaxillary carcinoma A-253, human colon adenocarcinoma Colo 320 CM and murine erythroleukaemia (Friend leukaemia) cell lines. Of these cells, HL-60, A-253 and Colo 320 CM were sensitive and Friend leukaemia resistant to this agent. The effects were time- and concentration-dependent. During the initial 24-48 h of treatment, a slowdown in cell proliferation was apparent but cell death was not extensive. After 24-48 h, there was a reduction in the proportion of cells in S phase of the cell cycle and the cells became preferentially arrested in G1 phase. The G1 cells showed high heterogeneity with respect to RNA content and some cells were characterized by very low RNA content. Progressive cell death occurred in cultures maintained with Pannon for up to 7 d in proportion to its concentration. Reductions of 50 and 90% in clonogenicity of A-253 cells were observed during their growth in the presence of 0.13 and 1.5 micrograms/ml of this protein, respectively. Exponentially growing cells were more sensitive to Pannon compared with cells from confluent cultures. Colonies of A-253 cells growing in the presence of Pannon were much smaller in size compared with control colonies, indicating that the rate of proliferation of clonogens is reduced by this agent. It appears that P-30 Protein induces cytostatic effects via modulation of cell transition to quiescence or differentiation. The mechanism of its cytotoxic activity is unclear.

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.