Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain.

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

Nonhistone proteins HMG1 and HMG2 change the DNA helical structure.

Two chromatin nonhistone proteins (from calf thymus) of the high mobility group, HMG1 and HMG2, reduce the linking number (topological winding number) of a circular DNA if the covalent closure of the DNA is carried out in their presence. This indicates that these proteins can either unwind the double helix, or induce a supercoiling of the DNA.

Effects of angiogenesis inhibitors on multistage carcinogenesis in mice.

Solid tumors depend on angiogenesis for their growth. In a transgenic mouse model of pancreatic islet cell carcinogenesis (RIP1-Tag2), an angiogenic switch occurs in premalignant lesions, and angiogenesis persists during progression to expansive solid tumors and invasive carcinomas. RIP1-Tag2 mice were treated so as to compare the effects of four angiogenesis inhibitors at three distinct stages of disease progression. AGM-1470, angiostatin, BB-94, and endostatin each produced distinct efficacy profiles in trials aimed at preventing the angiogenic switch in premalignant lesions, intervening in the rapid expansion of small tumors, or inducing the regression of large end-stage cancers. Thus, anti-angiogenic drugs may prove most efficacious when they are targeted to specific stages of cancer.

Broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of HIV-1.

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV)-1 resides within the V3 loop of the envelope protein. Antibodies elicited by peptides of this region were able to neutralize diverse isolates. Serum from one of three animals immunized with the human T cell lymphoma virus (HTLV)-IIIMN PND peptide, RP142, neutralized MN and the sequence-divergent HTLV-IIIB isolate. Serum from one of three animals immunized with a 13-amino acid IIIB PND peptide (RP337) also neutralized both of these isolates. Characterization of these sera revealed that the cross-neutralizing antibodies bound the amino acid sequence GlyProGlyArgAlaPhe (GPGRAF) that is present in both isolates. This sequence is frequently found in the PNDs analyzed in randomly selected HIV-1 isolates. Sera from two rabbits immunized with a peptide containing only the GPGRAF residues neutralized divergent isolates, including IIIB and MN.

Endostatin therapy reveals a U-shaped curve for antitumor activity.

Developing continuous systemic delivery of endostatin has been a goal of many laboratories. We have employed a method of gene therapy utilizing different viral constructs. Here, we report that a new serotype of adeno-associated viruses, which incorporates canine endostatin, provides dose-dependent transgene expression in the circulation after intramuscular injection in mice. Elevated levels of endostatin remained stable in the circulation for at least 4 months. In vitro assays determined that the protein expressed was biologically active. Antitumor activities of the above construct demonstrated a U-shape curve, where the maximum activity was observed within a certain critical concentration range. These data suggest that an optimum dose range may be required to achieve therapeutic efficacy in large animal models.

Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice.

BACKGROUND: Angiogenesis is essential for tumor growth and progression. Therefore, inhibition of angiogenesis is being studied as a new anticancer therapy. Because cytotoxic chemotherapy is more effective on rapidly growing tumors than on slowly growing tumors, it has been assumed that antiangiogenic therapy will also be effective only on rapidly growing, highly vascularized tumors. We compared the effects of two angiogenesis inhibitors, TNP-470 and angiostatin, on slowly growing, poorly vascularized and rapidly growing, highly vascularized human tumors in mice. METHODS: Slowly growing (RT-4) and rapidly growing (MGH-U1) human bladder carcinoma cell lines were grown in severe combined immunodeficiency mice. Established tumors were treated with one of the two angiogenesis inhibitors. Tumor volumes, vascularity, and proliferation indices were determined. The in vitro effects of TNP-470 and of angiostatin on the proliferation of RT-4 and MGH-U1 cells were also investigated. All statistical tests were two-sided. RESULTS: RT-4 and MGH-U1 tumor growth was statistically significantly inhibited by both angiogenesis inhibitors (P<.001). Both inhibitors decreased the blood vessel density in both tumor types but did not alter the in vivo proliferation indices of the tumors. TNP-470, but not angiostatin, marginally decreased the in vitro proliferation of MGH-U1 cells. CONCLUSION: Slowly growing, poorly vascularized tumors in animal models respond as well as rapidly growing, highly vascularized tumors to therapy with the angiogenesis inhibitors TNP-470 and angiostatin.

Physicochemical studies of non-histone protein HMG17 with DNA.

Non-histone protein high mobility group-17 (HMG17) isolated from calf thymus consists of 89 amino acids and the complete sequence is known (Walker, J.M., Hastings, J.R.B. and Johns, E.W. (1977) Eur. J. Biochem. 176, 461-468). We have studied its conformation and interaction with DNA by a variety of technics. The results show that the protein has a random structure. It binds non-cooperatively, non-specifically and reversibly to DNA. It is estimated that each molecule of protein binds to 57 nucleotides of calf thymus DNA. The equilibrium constant for binding is approx. 1 - 10(6) M-1. HMG17 induces conformational changes in DNA similar to some of the histones in particular H1.

Random copolymers containing specific ratios of negatively charged and aromatic amino acids bind V3 disulfide loop and neutralize diverse HIV type 1 isolates.

Random copolymers of polyamino acids containing negatively charged and aromatic residues at specific ratios appear to bind HIV type 1 V3 loop and neutralize diverse laboratory isolates. At least the putative heparin binding domain and isoleucine residues in the amino half of V3 are involved in the interactions with these polymers. There are a number of interesting features common between these polymer's modes of binding to the V3 and the protease inhibition drug ABT-538.

Alanine substitution of two arginines in amino terminus of V3 of SIV disrupts CD4 binding whereas a similar replacement of two amino acids, lysine and arginine, in the carboxyl half of V3 prevents binding of a neutralizing monoclonal antibody.

A series of amino acid substitutions were carried out in the V3 loop of SIV gp120 to investigate their effects on binding of the envelope to CD4 and neutralizing monoclonal antibodies. Alanine replacement of two adjacent arginines at the amino terminus of V3 resulted in a molecule that bound neither sCD4 nor conformation-dependent neutralizing monoclonal KK5 and KK9. A similar substitution of two amino acids, lysine and arginine, in the carboxyl half of V3 disrupted binding to KK9 without affecting CD4 binding. Removal of V3 from the envelope gave rise to a molecule that was not secreted. These data suggest a close linkage between V3 and CD4 binding domains of gp120, although neutralizing antibodies directed to V3 do not block binding of gp120 to CD4. We propose that differences in the modes of interactions of the V3 disulfide loops with CD4 in SIV and HIV may be responsible for the observed different neutralizing properties of the two V3 loops.

Neutralizing monoclonal antibody against a external envelope glycoprotein (gp110) of SIVmac251.

Three monoclonal antibodies (M318T, M56S and M815) against an external envelope glycoprotein (gp110) of simian immunodeficiency virus (SIV) mac251 were obtained by immunizing BALB/c mice with recombinant gp110 (rgp110). All three monoclonal antibodies reacted with the surface of cells infected with SIVmac251 but not with that of uninfected counterparts. The binding activity of these monoclonal antibodies against native gp110 was confirmed by means of Western blotting. One of them, M318T neutralized SIVmac251 infection both by cell-free and cell-associated viruses. M318T cross-reacted with human immunodeficiency virus type 2 strains (HIV-2 GH1 and ROD isolates) and SIVmac239 isolates. However, the antibody did not cross-neutralize these viral strains. Epitope mapping revealed that the neutralizing epitope recognized by M318T was localized at 8 residues between amino acids 178 and 185 (KRDKTKEY) in gp110, corresponding to the V2 region of human immunodeficiency virus type 1 (HIV-1). Because some antibodies against the V2 region of HIV-1 reportedly neutralize virus infection by interfering with CD4-gp120 interaction, we tested the activity of M318T against the binding of CD4-gp110. However, M318T did not inhibit CD4-gp110 interaction, suggesting the involvement of another unknown mechanism of M318T-mediated neutralization. In analogy with the V2 region of HIV-1, the V2 region of SIV contains a type specific neutralizing epitope recognized by M318T. Although some amino acid sequence in the epitope was conserved for the isolates of SIV and HIV-2 and there was cross-reactivity of the antibody against the strains, neutralization by M318T was associated with a single amino acid (182 T) in the epitope.

Immune responses induced by prototype vaccines for AIDS in rhesus monkeys.

A battery of assay systems was used to profile both humoral and cell-mediated immune responses induced by immunization with candidate vaccines consisting of recombinant simian immunodeficiency virus (SIV) glycoproteins rgp110 (nondenatured) with SAF-M adjuvant (gp110 + SAF-M) or rgp140 (denatured) with Freund's adjuvant (gp140 + FA). All of the monkeys became infected after intravenous challenge. However, 16 days following infection, viral antigenemia was reduced in both groups of vaccinates compared to controls. After 23 days antigenemia in the gp110 + SAF-M group remained at the same level as on day 16, whereas antigenemia in the gp140 + FA group was significantly reduced further than the level observed on day 16. Both vaccines induced blastogenic responses in PBMC cultures stimulated with rgp140, which decreased after repeated immunizations. Both vaccines induced high ELISA titers of IgG antibody against rgp140 that were equivalent to the titers in asymptomatic long-term survivors (LTSs). gp110 +/- SAF-M induced high titers of neutralizing antibody. In contrast, gp140 + FA failed to induce neutralizing antibody, suggesting that the natural conformation of the antigen may be essential for the induction of neutralizing antibody. High titers of antibodies capable of complement-mediated cytolysis (ACC) were induced by gp110 + SAF-M, whereas minimal ACC antibodies were induced by gp140 + FA. In spite of high titers of antibodies by ELISA, neither gp110 + SAF-M nor gp140 + FA vaccines induced detectable levels of antibody capable of antibody dependent cell-mediated cytolysis (ADCC). Detectable amounts of MHC class I-restricted, CD8+ cytotoxic T lymphocytes (CTLs) were not induced in immunized monkeys before challenge. After challenge and infection, antibody responses to glycoprotein (detected by ELISA and ACC) as well as glycoprotein-specific CTLs were induced in gp140 + FA vaccinates at levels higher than in nonimmunized control animals, indicating a priming effect by gp140 + FA immunization. No priming effect for ADCC antibody induction was observed in monkeys vaccinated with either gp110 + SAF-M or gp140 + FA. Rhesus monkey groups immunized with two different SIV envelope vaccines differed regarding potentially protective humoral and cell-mediated immune responses. The physical state of the immunogens, the type of adjuvant used, and/or the immunization protocol apparently affected these responses in both a qualitative and quantitative manner.

C-terminal fragments of gp120 and synthetic peptides from five HTLV-III strains: prevalence of antibodies to the HTLV-III-MN isolate in infected individuals.

The immunoreactivity of HTLV-III-infected individuals and virus-inoculated chimpanzees with gp120 synthetic peptides of the HTLV-III gp120 envelope principle neutralizing domain (amino acid 301-324 sequences), derived from the HTLV-III isolates 3B, RF, MN, WMJ2, and SC were determined. Sequential bleeds from an infected lab worker and chimpanzees, both infected with the HTLV-IIIB, were immunoreactive only with the 3B peptide. In contrast, 33 HTLV-III-infected individuals were immunoreactive with the HTLV-III(MN) peptide. Of these 33 individuals, 23 were also immunoreactive with the HTLV-III(SC) peptide, and 18 with the HTLV-III(WMJ2) peptide. The data suggest that HTLV-III strains related to MN are most prevalent among HTLV-III-infected individuals. The binding specificities of goat sera generated against either of these synthetic peptides or the C-terminal fragment of gp120 (PB-1, amino acid 287-467, derived from the HTLV-III isolates 3B, RF, MN, WMJ2, and SC) were also determined. Four different ELISA formats (peptide sera/peptide antigens, peptide sera/PB-1 antigens, PB-1 sera/PB-1 antigens, and PB-1 sera/peptide antigens) were utilized to determine the cross-reactivity patterns of goat sera with the antigens. Goat sera generated against MN and SC sequences (PB-1 proteins, as well as synthetic peptides) were highly cross reactive. Thus, patient sera cross reactivity to multiple strains of the principal neutralizing domain may reflect the antigenic relatedness of the virus isolates rather than multiple infection events or strains generated during disease progression.

Characteristics of a neutralizing monoclonal antibody to the HIV envelope glycoprotein.

We have studied the biologic and physical properties of a monoclonal antibody that binds to gp120, the exterior envelope glycoprotein of the human immunodeficiency virus (HIV) strain HTLV-IIIB. Designated 9284, the antibody possesses viral neutralizing activity and inhibits syncytium formation by infected cells. The antibody recognized a region of the polypeptide backbone previously described as an important neutralizing epitope. This region lies 307-330 residues from amino terminus of the glycoprotein. We have compared the biologic and physical properties of this antibody to those of the recently described 0.5 beta monoclonal antibody to gp120. The 0.5 beta antibody was biologically more potent and bound an epitope slightly downstream to that of the 9284 antibody. The antibodies did not differ significantly in their affinity for gp120. In competition studies, the 0.5 beta antibody was displaced by the 9284 antibody, but the binding of the latter was unaffected by 0.5 beta.

Nick translation of HeLa cell nuclei as a probe for locating DNase I-sensitive nucleosomes.

The technique of nick translation of nuclei (Levitt, A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) has been used in HeLa cells to label DNase I-sensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion.

Association of eukaryotic DNA topoisomerase I with nucleosomes and chromosomal proteins.

A DNA topoisomerase activity is found to be associated with the nucleosomes released by the Staphylococcal nuclease digestion of HeLa nuclei. Such an association is found to be salt dependent. A number of criteria have established that this DNA topoisomerase activity is due to HeLa topo I (Liu, L. F. and Miller, K. G. (1980) Proc. Natl. Acad. Sci. USA 78, 3489-3491). A similar association has been demonstrated from the in vitro studies using purified mononucleosomes and eukaryotic DNA topoisomerase I. Nonhistone HMG proteins and histone H1 are found to stimulate topoisomerase activity in vitro and form tight complexes with eukaryotic DNA topoisomerase I. The intimate interactions of topoisomerase I with chromosomal proteins and nucleosomes may be an essential feature of the topoisomerase function in vivo.

HMG17 protein facilitates the DNA catenation reaction catalyzed by DNA topoisomerases.

HMG17 protein is shown to greatly facilitate the catention of double-stranded DNA rings catalyzed by DNA topoisomerases. Even at low DNA concentrations such that catenanes are not observable in the absence of HMG17, the addition of the protein promotes the catenation of greater than 95% of the input DNA into networks that do not enter the gel upon electrophoresis. Electron microscopy and restriction enzyme cleavage experiments indicate that these networks are large structures containing many catenated DNA rings. The HMG17-promoted DNA network formation has been observed with calf thymus type II DNA topoisomerase and the type I topoisomerases of Escherichia coli, Micrococcus luteus, and calf thymus.