Prevention of human prostate tumor metastasis in athymic mice by antisense targeting of human angiogenin.

PURPOSE: Angiogenin is a potent positive mediator of neovascularization, a process required for both primary tumor growth and metastasis. In the present study, the effect of a fully phosphorothioated antisense oligodeoxynucleotide, designated JF2S, targeting the AUG translation initiation codon region of human angiogenin, on human prostate tumor development and metastasis in athymic mice was examined. EXPERIMENTAL DESIGN: JF2S was evaluated for its capacity to affect in vitro synthesis of angiogenin and subsequent tumorigenicity of transiently transfected prostate tumor cells in mice. In vivo treatment experiments were then conducted in which JF2S was used to prevent formation of tumors in an ectopic model and metastasis in an orthotopic model. RESULTS: Transient transfection of tumor cells with JF2S inhibited both angiogenin gene expression in vitro and tumorigenicity of these transfected cells in athymic mice. In therapy experiments, local treatment with JF2S completely protected mice from developing prostate tumors after s.c. injection of PC-3 human prostate tumor cells (P < 0.0001, survivor analysis). Most importantly, systemic prophylactic administration of JF2S prevented, in 47% of mice, formation of regional iliac lymph node micrometastases arising from primary tumors growing in the more natural orthotopic prostate setting (P = 0.0003, Fisher's exact test). Furthermore, total protection from regional metastasis occurred in those mice in which JF2S treatment successfully diminished human angiogenin expression in vivo. Tumor-associated angiogenesis was also impaired by JF2S treatment. When therapy was delayed until all of the mice harbored primary tumors in the prostate, the incidence of regional metastasis was still significantly decreased (P < 0.005, survivor analysis). CONCLUSIONS: These findings demonstrate that human prostate cancer establishment and spread in athymic mice is extremely susceptible to targeted disruption of tumor-derived human angiogenin gene expression. Therefore, angiogenin is a valid target against which to devise preventative strategies for prostate cancer metastasis.

Regulation of angiogenin expression in human HepG2 hepatoma cells by mediators of the acute-phase response.

Angiogenin is a potent inducer of neovascularization in vivo. However, like other angiogenic molecules, its specific physiologic roles and mechanisms regulating its expression remain to be elucidated. Angiogenin is a liver-derived component of normal serum whose concentration can increase in various disease states. This suggests that it might participate in the acute-phase response. In an initial study we showed that angiogenin protein and mRNA levels transiently increased in mice following an acute inflammatory stimulus. We now report that IL-6, a major inducer of acute-phase proteins, stimulates the synthesis and secretion of angiogenin protein in human HepG2 cells within 24 hr following treatment, an effect enhanced by dexamethasone. IL-6 also increases the amount of angiogenin mRNA without altering its half-life. This increase, suppressible by cycloheximide, peaks at 12 hr following stimulation and returns to basal levels by 48 hr. IL-1 alone slightly decreases the basal production of angiogenin protein and mRNA, but essentially abolishes the response to IL-6 in the absence or presence of dexamethasone. This antagonistic effect by IL-1 on IL-6 activity is not a result of changes in mRNA stability nor is it dependent on new protein synthesis. Thus, the combined effects of IL-6, IL-1, glucocorticoids, and perhaps other related factors may specifically control angiogenin expression. Since angiogenin is regulated in a manner similar to that of acute phase proteins both in vitro and in vivo, it may play a role in the host response to injury.

Chimeric anti-angiogenin antibody cAb 26-2F inhibits the formation of human breast cancer xenografts in athymic mice.

Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26-2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26-2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26-2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26-2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26-2F. Furthermore, the capacities of cAb 26-2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26-2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.

Angiogenin is regulated in vivo as an acute phase protein.

Angiogenin (Ang), a potent mediator of neovascularization, is secreted by and is critical for the growth of human tumor cells in experimental animals. However, control mechanisms that regulate its expression under normal physiological conditions have not been described. We have determined previously that Ang is present in normal human serum and that its concentration, normally falling within a narrow range, can vary widely in hospitalized patients. This observation, plus a report that Ang is synthesized in the adult liver, led us to investigate whether it can be regulated as an acute phase protein (APP). Ang concentration in the serum of mice placed into the acute phase by injection with 3% thioglycollate do indeed increase transiently as is typical for APPs. Moreover, a liver-specific rise and subsequent fall in Ang mRNA transcripts also follows entrance into acute inflammation. We conclude that Ang can be regulated in vivo in a manner that is characteristic of an APP and, therefore, may contribute to the angiogenic component of tissue repair that accompanies host response to inflammation and trauma. To our knowledge, this is the first demonstration that a well-characterized angiogenic mediator can be regulated as an APP.

Angiogenin single-chain immunofusions: influence of peptide linkers and spacers between fusion protein domains.

The gene for human angiogenin (Ang), a member of the ribonuclease superfamily, was fused to a gene encoding a single-chain antibody (sFv) against the human transferrin receptor. Three Ang single-chain immunofusion proteins (AngsFvs) were constructed with variations in the type of linker connecting the VL and VH chain [EGKSSGSGSESKEF, L1 or (GGGGS)3, L2] as well as with or without a spacer (FB) connecting the Ang and sFv (AngFBsFvL1 or L2; AngsFv(L2)]. Although the nature of the linker did not affect the enzymatic activity of the FB-containing fusion proteins, the fusion protein containing the L2 linker was 2.3-fold more effective than the L1 linker in competing with the labeled monoclonal IgG1 antibody for binding to the transferrin receptor. The fusion protein containing the L2 linker without the FB spacer exhibited a 13-fold decrease in binding to the transferrin receptor as well as a decrease in its capacity to degrade tRNA and to inhibit translation in the rabbit reticulocyte lysate compared to its counterpart containing the FB spacer. Binding of placental ribonuclease inhibitor (PRI) to Ang also was affected by the nature of the linker and by the presence or absence of a spacer. PRI bound to Ang and AngFBsFv(L2) and inhibited their ribonuclease activity. A 3-fold greater concentration of PRI, however, did not affect the activity of AngFBsFv(L1) or AngsFv(L2), suggesting that the conformation of these fusion proteins was altered. Binding of monoclonal and polyclonal anti-Ang antibodies to AngsFvs was also used to investigate conformational alterations of the fusion proteins. AngFBsFv(L2) was the least altered while AngFBsFv(L1) exhibited the greatest change in structure. Yet maximal concentrations of all AngsFvs elicited angiogenesis in the chick chorioallantoic membrane assay, demonstrating that Ang in all three fusion proteins remained functionally active. Consistent with all the activities, the fusion protein containing the FB spacer and L2 linker was the most cytotoxic to three different human tumor cell lines. The fusion protein lacking the FB spacer exhibited the least cytotoxicity. These data demonstrate that the linker connecting the VH-VL chains can affect the binding and cellular cytotoxicity of Ang immunofusions and that placement of a spacer between the antibody binding domains and Ang is necessary for optimal activity. Thus, a new class of targeted therapeutic agents containing Ang as the toxic moiety can be designed that potentially will be less immunogenic and less toxic than immunotoxins available currently.

Angiogenin antagonists prevent tumor growth in vivo.

A noncytotoxic neutralizing monoclonal antibody (mAb), 26-2F, to human angiogenin (Ang), a potent inducer of neovascularization, has been reported to prevent or delay the establishment of HT-29 human tumor xenografts in athymic mice. In the present study the tumor model was modified to increase sensitivity to Ang antagonists to facilitate further investigations and comparisons of their capacity to inhibit tumor growth. An increase in the percentage of tumor-free mice from 10-25% to 65% is observed in this modified model after treatment with mAb 26-2F. An additional neutralizing mAb, 36u, that interacts with a different epitope on Ang similarly prevents the appearance of tumors, both alone and in combination with mAb 26-2F. In those tumors that develop in mice treated with these agents, the number of vascular elements is reduced. Actin, an Ang antagonist that unlike the mAbs binds both human and mouse Ang, also prevents the establishment of tumors while exhibiting no toxic effects at daily doses > 50 times the molar amount of circulating mouse Ang. Ang antagonists also inhibit the appearance of tumors derived from two other Ang-secreting human tumor cell lines--i.e., A549 lung adenocarcinoma and HT-1080 fibrosarcoma. These results demonstrate that inhibition of the action of Ang is an effective therapeutic approach for the treatment of malignant disease.

A monoclonal antibody to human angiogenin suppresses tumor growth in athymic mice.

Human angiogenin, a potent inducer of neovascularization, is secreted by HT-29 colon adenocarcinoma cells. microgram doses of a monoclonal antibody that neutralizes the in vitro and in vivo activities of angiogenin prevent or delay the appearance of s.c. HT-29 tumors in athymic mice in a statistically significant, dose-dependent manner. The antibody is not cytotoxic to tumor cells in vitro, which indicates that inhibition of tumor growth most likely occurs by neutralization of the activity of angiogenin in vivo and further implies a critical role for angiogenin in the early development of HT-29 tumors. The results suggest a therapeutically useful approach to the treatment of angiogenin-dependent malignancy.

A monoclonal antibody to human angiogenin. Inhibition of ribonucleolytic and angiogenic activities and localization of the antigenic epitope.

A monoclonal antibody (mAb) to human angiogenin, a protein that induces formation of new blood vessels, was produced by somatic cell fusion techniques and designated as 26-2F. It is an IgGl kappa whose binding affinity, expressed as an IC50, is (1.6 +/- 0.1) x 10(-9) M as determined by a competition radioimmunoassay. mAb 26-2F neutralizes the ribonucleolytic activity of angiogenin as assessed by in vitro protein synthesis and tRNA degradation assays. It also effectively inhibits neovascularization induced by angiogenin on the chick chorioallantoic membrane. Epitope mapping indicates that the binding region of angiogenin recognized by mAb 26-2F is discontinuous and involves both Trp-89 and residues in the segment 38-41. This epitope is formed by two surface loops which are juxtaposed in the three-dimensional structure of human angiogenin recently determined by X-ray crystallography. Thus mAb 26-2F, along with similar antibodies under investigation, will facilitate structure/function studies of angiogenin, help define its physiological role, and lead to an understanding of the consequences of its inhibition in pathological situations in which angiogenin may be involved.

A cell-surface proteoglycan mediates human adenocarcinoma HT-29 cell adhesion to human angiogenin.

Human angiogenin is an excellent substrate for the adhesion of HT-29 human colon adenocarcinoma cells. These cells adhere more quickly to human angiogenin than to fibronectin, laminin, collagen I, and collagen IV. Anti-angiogenin antibodies and the angiogenesis inhibitors platelet factor-4 and placental ribonuclease inhibitor prevent adhesion of HT-29 cells to angiogenin. Calcium and magnesium ions are not required for adhesion and Arg-Gly-Asp-Ser has no effect, indicating that the interaction is integrin-independent. Instead, adhesion seems to involve a heparan/chondroitin sulfate proteoglycan. Treatment of the cells with heparinase or heparitinase decreases HT-29 cell adhesion onto angiogenin but not onto collagen I. Moreover, cell adhesion is decreased by the presence of heparin or chondroitin sulfates and by preincubation of the cells with inhibitors of proteoglycan synthesis or secretion. In addition, angiogenin binds tightly to heparin-Sepharose, requiring 0.78 M NaCl for elution. Angiogenin-affinity chromatography of a 35S-, 3H-labeled HT-29 cell fraction enriched in cell-surface proteoglycans yields a single, heparinase-sensitive component of apparent molecular mass > 200 kDa, as detected by autoradiography after SDS-polyacrylamide gel electrophoresis. These results suggest that angiogenin could be an effective substrate for tumor cell adhesion during metastasis and may provide a basis for the design of inhibitors of this process.

Actin is a surface component of calf pulmonary artery endothelial cells in culture.

An angiogenin binding protein isolated previously from endothelial cells has been shown to be a member of the actin family. Calf pulmonary artery endothelial (CPAE) cells were investigated for the presence of surface actin by immunoblotting of isolated surface proteins and by immunofluorescence. CPAE cell surface proteins were isolated by selective apical biotinylation and recovery of biotinylated proteins by avidin affinity chromatography. Immunoblotting with a specific smooth muscle alpha-actin antibody detected the presence of this type of actin among the isolated cell surface proteins. Immunofluorescence confirmed that smooth muscle alpha-actin is localized at the surface of nonpermeabilized CPAE cells. Exposure of CPAE cells to angiogenin prior to cell surface immunostaining diminished the signal. When CPAE and rat aortic smooth muscle cells were made permeable before staining, stress fibers could be recognized by the antibody in smooth muscle cells but not CPAE cells. The results indicate that a smooth muscle type of alpha-actin is localized specifically on the surface of cultured CPAE cells where it might interact with angiogenin and other actin binding proteins present in the extracellular environment.

Actin is a binding protein for angiogenin.

The 42-kDa angiogenin binding protein isolated previously has been purified to electrophoretic homogeneity. It has been identified as a member of the actin family by peptide mapping and partial amino acid sequencing. The interaction of bovine muscle actin with angiogenin is similar to that of the angiogenin binding protein. Angiogenin induces the polymerization of actin below the critical concentration for spontaneous polymerization. The interaction occurs both in solution and on a poly(vinylidene difluoride) membrane. It is inhibited by excess unlabeled angiogenin and also by platelet factor 4 and protamine, which are known inhibitors of angiogenesis. Two other angiogenic molecules, basic fibroblast growth factor and tumor necrosis factor alpha, bind to 125I-labeled actin and can be crosslinked by a water-soluble carbodiimide. Both actin and an anti-actin antibody inhibit the angiogenic activity of angiogenin in the chicken embryo chorioallantoic membrane assay. The results indicate that the angiogenin binding protein is a cell surface actin and suggest that the reaction between angiogenin and this actin is an essential step in the angiogenesis process induced by angiogenin.

C-terminal angiogenin peptides inhibit the biological and enzymatic activities of angiogenin.

Synthetic peptides corresponding to the C-terminal region of angiogenin (Ang) inhibit the enzymatic and biological activities of the molecule while peptides from the N-terminal region do not affect either activity. The peptide Ang(108-121) transiently abolishes the inhibition of cell-free protein synthesis caused by angiogenin coincidentally with its cleavage of reticulocyte RNA. Several C-terminal peptides also inhibit nuclease activity of angiogenin when tRNA is the substrate. Furthermore, peptide Ang(108-123) significantly decreases neovascularization elicited by angiogenin in the chick chorioallantoic membrane assay.

Expression of human angiogenin in cultured baby hamster kidney cells.

Baby hamster kidney cells were transformed with DNA sequences derived from the gene for human angiogenin. Expression was under the transcriptional control of the inducible mouse metallothionein 1 promoter. Recombinant angiogenin was purified and shown to be chemically, biologically, and enzymatically indistinguishable from the natural product. The large-scale production of recombinant angiogenin achieved should facilitate detailed studies into the structure-function relationships of this potent angiogenic molecule.

Comparison of the effects of class 1 and class 2 heparin-binding growth factors on protein synthesis and actin mRNA expression in BALB/c-3T3 cells.

Biological effects of class 1 or class 2 heparin-binding growth factors (HBGFs) were compared in BALB/c-3T3 cells. Changes in protein synthesis, as monitored by two-dimensional gel electrophoresis, reveal that while both HBGFs induce the same changes in the synthesis of intracellular proteins, class 2 HBGF selectively increases the synthesis of a 43-kD extracellular protein. Heparin, which potentiates the mitogenic activity of class 1 but not class 2 HBGF, does not potentiate the changes in protein synthesis elicited by HBGF-1. Since each HBGF increases actin synthesis, regulation of actin mRNA expression was examined. Actin mRNA levels increase rapidly and transiently in response to either HBGF, and similar superinduction responses are observed in the presence of HBGF and cycloheximide. Although the maximum increase in actin mRNA stimulated by either HBGF is similar, the levels of mRNA induced by class 2 HBGF remain elevated up to 48 hours compared to the level induced by class 1 HBGF. These results imply that in the same cell type class 1 and class 2 HBGFs may modulate some biological effects differently.

Induction of angiogenesis by mixtures of two angiogenic proteins, angiogenin and acidic fibroblast growth factor, in the chick chorioallantoic membrane.

The chick chorioallantoic membrane assay was employed to assess the angiogenic response induced by mixtures of human angiogenin with bovine heparin-binding acidic fibroblast growth factor. Statistical evaluation of data accumulated at several molar ratios of the two proteins indicate that the angiogenic activity observed is neither an additive nor a synergistic resultant of the activities of the proteins separately. The possibility exists, however, that at an approximately 1:1 mole ratio an apparent inhibitory effect can be observed. Mechanisms which could underlie such observed effects are discussed.

Angiogenin mRNA in human tumor and normal cells.

Angiogenin mRNA was characterized in HT-29 human colon adenocarcinoma cells and its distribution in other human cell types was studied. Several RNA species ranging from 800 to 6000 nucleotides hybridized to the angiogenin probes with the smallest being the major poly(A)-containing transcript. This transcript was detected in tumor cells of diverse cellular origin. Expression of angiogenin mRNA is not limited to neoplastic cells and is detected in normal epithelial cells, fibroblasts and peripheral blood cells. The latter, when induced by mitogens, expressed more mRNA than did unstimulated or HT-29 cells. Transformed fibroblasts did not contain higher levels of angiogenin mRNA than their normal counterparts, demonstrating that increased angiogenin mRNA expression does not necessarily occur upon neoplastic transformation. This study demonstrates that angiogenin mRNA is expressed in a wide spectrum of cells and is not correlated to a particular cell phenotype.

Isolation and characterization of a human colon carcinoma-secreted enzyme with pancreatic ribonuclease-like activity.

A ribonuclease was isolated from serum-free supernatants of the human colon adenocarcinoma cell line HT-29. It was purified by cation-exchange and C18 reversed-phase high-performance liquid chromatography. The protein is basic, has a molecular weight of approximately 16,000, and has an amino acid composition that is significantly different from that of human pancreatic ribonuclease. The amino terminus is blocked, and the carboxyl-terminal residue is glycine. The catalytic properties of this ribonuclease resemble those of the pancreatic ribonucleases in numerous respects. Thus, it exhibits a pH optimum of approximately 6 for dinucleotide cleavage and employs a two-step mechanism in which transphosphorylation to a cyclic 2',3'-phosphate is followed by slower hydrolysis to produce a 3'-phosphate. It does not cleave NpN' substrates in which adenosine or guanosine is at the N position and prefers purines at the N' position. Like bovine ribonuclease A, the HT-29-derived ribonuclease is inactivated by reductive methylation or by treatment with iodoacetate at pH 5.5 and is strongly inhibited by the human placental ribonuclease inhibitor. However, in contrast, the tumor enzyme does not cleave CpN bonds at an appreciable rate and prefers poly(uridylic acid) as substrate 1000-fold over poly(cytidylic acid). It also hydrolyzes cytidine cyclic 2',3'-phosphate at least 100 times more slowly than uridine cyclic 2',3'-phosphate and is inhibited much less strongly by cytidine 2'-monophosphate than by uridine 2'-monophosphate. Other ribonucleases known to prefer poly(uridylic acid) were isolated both from human serum and from liver and were compared with the tumor enzyme. The physical, functional, and chromatographic properties of the serum ribonuclease are essentially identical with those of the tumor enzyme. The liver enzymes, however, differ markedly from the HT-29 ribonuclease. The potential utility of the tumor ribonuclease in the diagnosis of cancer is considered.

Immunocytochemical and enzymatic detection of lysozyme in human colon carcinoma cell lines.

Six of a total of 14 human colon carcinoma cell lines produce and secrete lysozyme in vitro. Three also produce the enzyme when propagated in vivo in athymic mice. None of the lysozyme positive cells stained in a manner typical of Paneth cells. Additionally, lysozymes from all six colon lines possess identical molecular weights (approximately 14,000 daltons).

Lysates of two established human tumor lines contain heparin-binding growth factors related to bovine acidic brain fibroblast growth factor.

Cell lysates of two established human tumor lines, a medulloblastoma (TE671), and a rhabdomyosarcoma (RD), contain mitogenic activity which elutes from heparin-Sepharose under conditions typical of class 1 heparin-binding growth factors, such as acidic brain fibroblast growth factor. The presence of this class of mitogen in both cell lines was confirmed by their chromatographic behavior on reversed-phase C3 columns, and by the ability of heparin to enhance their mitogenic activity. Using a specific synthetic DNA probe, RNA's were isolated from both cell lines by hybridization-selection, translated in vitro, and translated proteins affinity fractionated on heparin-Sepharose. The results demonstrate that TE671 and RD cell lysates contain mRNA's for mitogens related to acidic brain fibroblast growth factor, and also suggest that high molecular weight proteins exist that are closely related to, or are precursor forms of, the class 1 mitogens.