Efficient inhibition of in vivo human malignant glioma growth and angiogenesis by interferon-beta treatment at early stage of tumor development.

Malignant gliomas are highly angiogenic and aggressive tumors. IFN-beta has been used for the treatment of patients with malignant glioma; however, its antitumor mechanism in vivo remains unclear. To understand the in vivo antitumor effect and mechanism of recombinant human IFN-beta (rhIFN-beta) depending on the stages of tumor development or progression, we used orthotopic xenograft brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days (group 1) and 21 days (group 2) postimplant were treated with 2 x 10(5) IU/day of rhIFN-beta or saline i.p. for 15 days, respectively. Tumor growth was suppressed by 69.6% in group 1 and 10.8% in group 2 compared with tumors of each control group treated with saline. rhIFN-beta-treated group 1 animals showed 38% reduction in vascularization along with a 2.5-fold increase of the apoptotic index and no change in the proliferative index as compared with untreated tumors. The expression level of vascular endothelial cell growth factor and basic fibroblast growth factor was not affected by rhIFN-beta treatment. rhIFN-beta showed inhibitory activity on proliferation of U-87 cells, human umbilical vein endothelial cells, and PAM 212 murine keratinocytes in vitro. Our results indicate that the in vivo antitumor effect of rhIFN-beta on malignant gliomas may be mediated, at least in part, via angiogenesis inhibition rather than antiproliferative activity and that rhIFN-beta may be more effective for the treatment of malignant glioma patients at an early stage with minimal or microscopic tumor burdens rather than at an advanced stage of tumor development.

Cloning and sequencing of a chick embryo cDNA encoding the 20-kDa hypusine-containing protein, eIF-5A.

Chick embryo contains 18- and 20-kDa isoforms of eukaryotic translation initiation factor 5A (eIF-5A). cDNA clones corresponding to the 20-kDa eIF-5A were isolated and sequenced. A full-length cDNA clone encodes a 153-amino-acid (aa) protein. The deduced aa sequence exactly matches with the partial aa sequence determined for this protein and shows high identity to that of human or rabbit eIF-5A. The results of Southern and Northern hybridization provide evidence for multiple transcripts for chick embryo eIF-5A or an eIF-5A-like protein that presumably derive from more than one gene.

Potential of adenoviral p53 gene therapy and irradiation for the treatment of malignant gliomas.

We investigated the combined effects of p53 gene transfer and irradiation and its still unclear interaction mechanism in human gliomas. Four human glioma cell lines expressing mutant type p53 (U373 and A172) and wild-type p53 (D54MG and EFC-2) were transfected by adenoviral vectors bearing p53 gene at 50 multiplicity of infection. Two days after transfection, cells were irradiated (3, 6, and 9 Gy). The cytotoxicity was evaluated by clonogenic assay. The quantitative analysis of apoptosis and cell cycle analysis were performed using flow cytometry. Irradiation combined with adenoviral p53 transfection significantly increased cytotoxicity, which was additive in cell lines with wild-type p53 and more than additive in cell lines with mutant p53. The combination of two modalities increased the apoptotic population by 14% in A172 cells and 20% in D54 MG cells, which were the sum of apoptosis from each modality. Adenoviral p53 transfection increased the G1 phase fraction and concomitant decrease of radioresistant S phase fraction in A172 and D54MG cells. Our study demonstrated that p53 gene transfer combined with irradiation increased absolute cytotoxicity in human glioma cells used in this experiment. The interaction mechanism for increased cytotoxicity involved, in part, increased apoptosis and change of cell cycle profile.

Purification and characterization of recombinant murine endostatin in E. coli.

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent.

Kanamycin acetyltransferase gene from kanamycin-producing Streptomyces kanamyceticus IFO 13414.

A kanamycin producer, Streptomyces kanamyceticus IFO 13414 is highly resistant to kanamycin. Cloning of the kanamycin resistance genes in S. lividans 1326 with pIJ702 gave several kanamycin resistant transformants. Two transformants, S. lividans SNUS 90041 and S. lividans SNUS 91051 showed similar resistance patterns to various aminoglycoside antibiotics. Gene mapping experiments revealed that plasmids pSJ5030 and pSJ2131 isolated from the transformants have common resistant gene fragments. Subcloning of pSJ5030 gave a 1.8 Kb gene fragment which showed resistance to kanamycin. Cell free extracts of S. lividans SNUS 90041, S. lividans SNUS 91051 and subclone a S. lividans SNUS 91064 showed kanamycin acetyltransferase activity. The detailed gene map is included.

Structural features of the eIF-5A precursor required for posttranslational synthesis of deoxyhypusine.

Eukaryotic translation initiation factor 5A (eIF-5A, older nomenclature, eIF-4D) is a highly conserved protein that contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). The biosynthesis of hypusine occurs posttranslationally in only this protein by modification of a single lysine residue (Lys50 in the human eIF-5A precursor). The basis for the specificity of this modification with respect to the substrate protein was investigated using fragments of eIF-5A precursor protein, each containing this lysine residue, as substrates for deoxyhypusine synthase, the first enzyme in hypusine synthesis. Proteolytic fragments (5-6 kDa) of ec-eIF-5A (the precursor form of eIF-5A produced in Escherichia coli by expression of the human eIF-5A cDNA) generated by specific cleavage by endoproteinases Arg-C, Asp-N, or Glu-C, did not act as substrates for deoxyhypusine synthesis. A series of truncated forms of the eIF-5A precursor protein generated by expression in E. coli of recombinant deletion constructs from the human eIF-5A cDNA were tested. Truncation of up to 9 amino acid residues (Met1-Thr9) from the NH2 terminus or 64 amino acid residues (Leu91-Lys154) from the COOH terminus did not significantly decrease the substrate reactivity, but removal of an additional 10 amino acids from either side did. Deletion of 34 amino acid residues (Met1-Lys34) from the NH2 terminus or of 84 amino acid residues (Asp71-Lys154) from the carboxyl terminus caused complete loss of substrate property. The results obtained thus far define the minimum domain of the eIF-5A precursor protein required for enzymatic deoxyhypusine synthesis as Phe30-Asp80, which corresponds to a region of high amino acid conservation in this protein throughout the eukaryotic kingdom.

Cloning and expression of human deoxyhypusine synthase cDNA. Structure-function studies with the recombinant enzyme and mutant proteins.

Deoxyhypusine synthase catalyzes the first step in the post-translational formation of hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). cDNA clones encoding deoxyhypusine synthase were isolated from a human HeLa cell library. Full-length cDNA clones encoding a 369-amino acid protein (calculated molecular mass of 40,970 Da) and a shorter cDNA clone that would potentially encode a protein with an internal deletion of 56 amino acids (Asp262-Ser317) were isolated. The deduced amino acid sequence of the human enzyme shows a high degree of identity to that of yeast deoxyhypusine synthase and to the known sequences of tryptic peptides from the rat and Neurospora crassa enzymes. The recombinant enzyme formed upon expression in Escherichia coli effectively catalyzed deoxyhypusine synthesis. Variant human recombinant proteins with (i) a truncation of 48 or 97 NH2-terminal amino acids, (ii) a truncation of 39 COOH-terminal amino acids, or (iii) an internal deletion (Asp262-Ser317) were inactive. A chimeric protein consisting of the complete human sequence and 16 amino acids of the yeast sequence (Gln197-Asn212, not present in the human enzyme) inserted between Glu193 and Gln194 exhibited moderate activity.

Hypusine is essential for eukaryotic cell proliferation.

Hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] occurs in all eukaryotes at one residue in a highly conserved protein, the putative eukaryotic translation initiation factor 5A (eIF-5A, old terminology eIF-4D). This unusual amino acid is produced in a unique posttranslational modification reaction that involves the conjugation of the 4-aminobutyl moiety of the polyamine spermidine to the epsilon-amino group of a specific lysine residue of the eIF-5A precursor protein to form the deoxyhypusine [N epsilon-(4-aminobutyl)lysine] residue and its subsequent hydroxylation. The strict specificity of hypusine synthesis, its derivation from spermidine and its requirement for the activity of eIF-5A and for eukaryotic cell proliferation have raised keen interest in the physiological function of the hypusine-containing protein, eIF-5A.

Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae.

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. The null mutation in the single copy gene, yDHS, encoding deoxyhypusine synthase results in the loss of viability in the yeast Saccharomyces cerevisiae. Upon depletion of deoxyhypusine synthase, and consequently of eIF-5A, cessation of growth was accompanied by a marked enlargement of cells, suggesting a defect in cell cycle progression or in cell division. Two residues of the yeast enzyme, Lys308 and Lys350, corresponding to Lys287 and Lys329, respectively, known to be critical for the activity of the human enzyme, were targeted for site-directed mutagenesis. The chromosomal ydhs null mutation was complemented by the plasmid-borne yDHS wild-type gene, but not by mutated genes encoding inactive proteins, including that with Lys350-->Arg substitution or with substitutions at both Lys308 and Lys350. The mutated gene ydhs (K308R) encoding a protein with diminished activities (< 1% of wild type) could support growth but only to a very limited extent. These findings provide strong evidence that the hypusine modification is indeed essential for the survival of S. cerevisiae and imply a vital function for eIF-5A in all eukaryotes.

Enzyme-substrate intermediate at a specific lysine residue is required for deoxyhypusine synthesis. The role of Lys329 in human deoxyhypusine synthase.

Deoxyhypusine synthase catalyzes the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine] in eukaryotic translation initiation factor 5A. We recently reported biochemical evidence for a covalent enzyme-substrate intermediate involving a specific lysine residue (Lys329) in human deoxyhypusine synthase (Wolff, E. C., Folk, J. E., and Park, M. H. (1997) J. Biol. Chem. 272, 15865-15871). In an effort to evaluate the role of this enzyme-substrate intermediate in catalysis, we carried out site-directed mutagenesis (Lys to Arg and/or Ala) of the conserved lysine residues in human deoxyhypusine synthase. A drastic reduction in enzyme intermediate formation and enzymatic activities was observed with mutant proteins with substitution at Lys287 but not with those with mutations at residues 141, 156, 205, 212, 226, 251, or 338. Lys to Ala or Lys to Arg substitution at Lys329 totally abolished covalent enzyme-substrate intermediate formation and deoxyhypusine synthesis activity, indicating that Lys329 is the unique site for the enzyme intermediate and that it is absolutely required for deoxyhypusine synthesis in the eukaryotic translation initiation factor 5A precursor. The K329A mutant showed spermidine cleavage activity ( approximately 6% of the wild type enzyme) suggesting that in contrast to deoxyhypusine synthesis, spermidine cleavage can occur without enzyme intermediate formation.

The polyamine-derived amino acid hypusine: its post-translational formation in eIF-5A and its role in cell proliferation.

The unusual amino acid hypusine [N (ε) -(4-amino-2-hydroxybutyl)lysine] is a unique component of one cellular protein, eukaryotic translation initiation factor 5A (eIF-5A, old terminology, eIF-4D). It is formed posttranslationally and exclusively in this protein in two consecutive enzymatic reactions, (i) modification of a single lysine residue of the eIF-5A precursor protein by the transfer of the 4-aminobutyl moiety of the polyamine spermidine to itsε-amino group to form the intermediate, deoxyhypusine [N (ε) -(4-aminobutyl)lysine] and (ii) subsequent hydroxylation of this intermediate to form hypusine. The amino acid sequences surrounding the hypusine residue are strictly conserved in all eukaryotic species examined, suggesting the fundamental importance of this amino acid throughout evolution. Hypusine is required for the activity of eIF-5Ain vitro. There is strong evidence that hypusine and eIF-5A are vital for eukaryotic cell proliferation. Inactivation of both of the eIF-5A genes is lethal in yeast and the hypusine modification appears to be a requirement for yeast survival (Schnier et al., 1991 [Mol Cell Biol 11: 3105-3114]; Wöhl et al., 1993 [Mol Gen Genet 241: 305-311]). Furthermore, inhibitors of either of the hypusine biosynthetic enzymes, deoxyhypusine synthase or deoxyhypusine hydroxylase, exert strong anti-proliferative effects in mammalian cells, including many human cancer cell lines. These inhibitors hold potential as a new class of anticancer agents, targeting one specific eukaryotic cellular reaction, hypusine biosynthesis.

Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.

Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd

Disruption of interkringle disulfide bond of plasminogen kringle 1-3 changes the lysine binding capability of kringle 2, but not its antiangiogenic activity.

Kringle 1-3 of human plasminogen is a potent inhibitor of endothelial cell proliferation. To understand a possible role for the unique cystine bridge between kringle 2 and kringle 3, we disrupted the interkringle disulfide bond by mutating Cys(169) and Cys(297) to serine residues. The yield of the mutant during the refolding process was decreased significantly. Anti-endothelial cell proliferative activity of the mutant was similar to that of the wild type. There was no significant difference in in vivo antiangiogenic activity between the wild type and the mutant in chorioallantoic membrane assay. However, in the mutant, the weak lysine binding capability of kringle 2 was not detected and its mobility in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is different from that of the wild type. These results support the notion that the overall antiangiogenic function of angiostatin is mediated by individual kringles, and suggest that the lysine binding capability of kringle 2 is likely not important for the antiangiogenic activity of kringle 1-3.

Inhibition of human malignant glioma growth in vivo by human recombinant plasminogen kringles 1-3.

Human malignant gliomas are highly vascularized and aggressive tumors. Angiogenesis inhibitors have been shown to induce regression of a variety of primary and metastatic tumors in vivo. However, their usefulness in treating brain tumors is not well understood. Angiostatin, a multiple kringle (1-4 of 5)-containing fragment of plasminogen, is one of the highly effective natural cryptic angiogenesis inhibitors. In our study, the therapeutic efficacy of non-glycosylated and small molecular size recombinant kringles 1-3 (rPK1-3) was examined in the treatment of brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days post-implant were treated daily with rPK1-3 (100 mg/kg) s.c. for 21 days. Treated animals showed suppressed brain tumor growth by greater than 71.2% along with a 3-fold increase of apoptotic index and suppressed vascularization by 78.9%, without any observable signs of toxicity. Analysis of bFGF and VEGF expression in the tumors of treated animals using immuno-histochemical methods showed near complete absence of growth factors. Our results indicate that the non-glycosylated, small molecular size rPK1-3 is an efficient tumoristatic agent for the treatment of intracranial human glioma xenografts in mice and might provide new strategies for the treatment of brain tumors.

Potentials and limitations of adenovirus-p53 gene therapy for brain tumors.

We investigated the antineoplastic potentials of recombinant adenovirus containing wild-type p53 cDNA (Ad5CMV-p53) for malignant gliomas. In four human glioma cell lines (U-251 and LG expressing endogenous mutant p53, and U-87 and EFC-2 expressing wild-type p53) and two rat glioma cell lines (9L and C6, each expressing mutant and wild-type p53), gene transfer efficiency determined by X-gal staining and Western blotting was varied (10-99% at 10-500 multiplicity of infection, MOI). Growth inhibitory effect was drastic (>90% at 100 MOI) in U-251 cells and only moderate or minimal in other cell lines harboring wild-type p53 or low gene transfer efficiency. Ex vivo transduction of U-251 cells with Ad5CMV-p53 suppressed the in vivo tumorigenicity of the cells. Histopathologic examination for Ad5CMV-p53 toxicity to rat brains showed inflammatory reactions in half of the tested brains at 10(8) MOI. U-251 cells were inoculated intracerebrally in nude mice and injected Ad5CMV-p53 into the tumor, in which neither the tumor suppression nor the survival benefit was observed. In conclusion, heterogeneity of the cellular subpopulations of malignant glioma in p53 status, variable and insufficient gene delivery to tumor, and adenoviral toxicity to brain at higher doses may be limiting factors to be solved in developing adenovirus-p53 gene therapy for malignant gliomas.