Differential Requirement for the Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase in RNA Damage-Induced Apoptosis in Primary and in Immortalized Fibroblasts

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-κB). 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-κB. 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.     

Binase and other microbial RNases as potential anticancer agents

Some RNases possess preferential cytotoxicity against malignant cells. The best known of these RNases, onconase, was isolated from frog oocytes and is in clinical trials as anticancer therapy. Here we propose an alternative platform for anticancer therapy based on T1 RNases of microbial origin, in particular binase from Bacillus intermedius and RNase Sa from Streptomyces aureofaciens. We discuss their advantages and the most promising directions of research for their potential clinical applications.     

Effective expression and purification of recombinant onconase, an antitumor protein

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. One of these is onconase, the smallest member of the RNase A superfamily, which is at present undergoing phase III clinical trials. We report here the expression of recombinant onconase in Escherichia coli inclusion bodies, the correct processing of the protein, followed by its purification in high yields. The recombinant protein has biological and catalytic properties identical to those of the natural enzyme.     

Cytotoxic mechanism of the ribotoxin alpha-sarcin. Induction of cell death via apoptosis.

Alpha-sarcin is a ribosome-inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of alpha-sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for alpha-sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. alpha-Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with alpha-sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase-3-like activity and cleavage of poly(ADP-ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of alpha-sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive alpha-sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z-VAD-fmk does not suppress protein synthesis inhibition by alpha-sarcin. Together, these data suggest that alpha-sarcin-induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.     

Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups.

Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives, cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.     

Onconase: an unusually stable protein

Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity.     

In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.     

Arginine 121 is a crucial residue for the specific cytotoxic activity of the ribotoxin alpha-sarcin.

Alpha-sarcin, a cyclizing ribonuclease secreted by the mould Aspergillus giganteus, is one of the best characterized members of a family of fungal ribotoxins. This protein induces apoptosis in tumour cells due to its highly specific activity on ribosomes. Fungal ribotoxins display a three-dimensional protein fold similar to those of a larger group of microbial noncytotoxic RNases, represented by RNases T1 and U2. This similarity involves the three catalytic residues and also the Arg121 residue, whose counterpart in RNase T1, Arg77, is located in the vicinity of the substrate phosphate moiety although its potential functional role is not known. In this work, Arg121 of alpha-sarcin has been replaced by Gln or Lys. These two mutations do not modify the conformation of the protein but abolish the ribosome-inactivating activity of alpha-sarcin. In addition, the loss of the positive charge at that position produces dramatic changes on the interaction of alpha-sarcin with phospholipid membranes. It is concluded that Arg121 is a crucial residue for the characteristic cytotoxicity of alpha-sarcin and presumably of the other fungal ribotoxins.     

Development of a transferrin receptor-targeting HVJ-E vector

The development of more effective cancer treatments is anticipated. Tumor-targeted drug delivery is an important strategy in cancer therapy. We have developed an HVJ (hemagglutinating virus of Japan; Sendai virus) envelope (HVJ-E) vector using inactivated Sendai virus. The HVJ-E vector has been observed to target a number of cell lines since its hemagglutinin-neuraminidase (HN) protein recognizes the sialic acids of host cells. Thus, to reduce non-specific binding of the HVJ-E vector, we eliminated HN protein using HN-specific short interfering RNA (siRNA). Then, to further increase its tumor-targeting ability, we constructed HN-depleted HVJ containing the F-transferrin chimeric protein. The modified vectors containing Q-dots demonstrated 32-fold greater tumor-targeting efficiency than wild-type HVJ-E.     

Synthesis, structure-activity relationship and biological evaluation of anticancer activity for novel N-substituted sophoridinic acid derivatives

Sophoridine (1), a natural anticancer drug, has been used in China for decades. A series of novel N-substituted sophoridinic acid derivatives were synthesized and evaluated for their cytotoxicity with 1 as the lead. The structure-activity relationship indicated that introduction of an aliphatic acyl on the nitrogen atom might significantly enhance the anticancer activity. Among the compounds, 6b bearing bromoacetyl side-chain afforded a potential effect against four human tumor cell lines (liver, colon, breast, and lung). The mechanism of action of 6b is to inhibit the activity of DNA topoisomerase I, followed by the S-phase arrest and then cause apoptotic cell death, similar to that of its parent 1. We consider 6b promising for further anticancer investigation.     

Deletion of the NH2-terminal beta-hairpin of the ribotoxin alpha-sarcin produces a nontoxic but active ribonuclease

Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. alpha-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal beta-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Delta(7-22) of alpha-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that alpha-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the beta-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.     

Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo.

Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.     

Cytotoxicity of RNases is increased by cationization and counteracted by K(Ca) channels

K(Ca) channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of K(Ca) channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing K(Ca) channels was found. These results suggest that cells without the K(Ca) channel activity cannot counteract toxic effect of RNases.     

Ribonucleases and their antitumor activity

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of ‘humanized antitumor ribonuclease’ that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.     

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.     

Morphological changes in Ehrlich ascites tumor cells during the cell fusion reaction with HVJ (Sendai virus). III. Morphological characterization of HVJ glycoproteins integrated into the plasma membrane and their internalization by coated vesicles

On cell-cell fusion of Ehrlich ascites tumor (EAT) cells with HVJ, HVJ envelopes also fuse with the cell membrane, resulting in integration of the viral envelope glycoproteins into the fused cell membranes. Morphological characterization of the glycoproteins in the plasma membrane and the mode of their internalization were investigated in detail. In the fusion reaction, the glycoproteins were rapidly integrated into the cell membrane within 2 or 3 min on incubation at 37 °C and they remained at the fusion sites, not dispersing widely, during further incubation. Thus they were still present in clusters in the plasma membrane at the end of the fusion reaction. On culture of fused cells in culture medium, internalization of the viral glycoproteins was initiated by formation of coated vesicles and most of the integrated glycoproteins were endocytosed into the cytoplasm within 30 min. Soon after internalization, the coated vesicles fused with each other, losing their coat materials. The intact virions that remained unfused on the cell surface were also internalized, but coat materials did not appear on the inside surface of the cell membrane, unlike in the case of integrated glycoproteins.     

Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.     

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.