Survivin, a target to modulate the radiosensitivity of Ewing's sarcoma.

BACKGROUND AND PURPOSE: Radiotherapy constitutes an essential element in the multimodal therapy of Ewing's sarcoma. Compared to other sarcomas, Ewing tumors normally show a good response to radiotherapy. However, there are consistently tumors with a radioresistant phenotype, and the underlying mechanisms are not known in detail. Here we investigated the association between survivin protein expression and the radiosensitivity of Ewing's sarcoma in vitro. MATERIAL AND METHODS: An siRNA-based knockdown approach was used to investigate the influence of survivin expression on cell proliferation, double-strand break (DSB) induction and repair, apoptosis and colony-forming ability in four Ewing's sarcoma cell lines with and without irradiation. RESULTS: Survivin protein and mRNA were upregulated in all cell lines tested in a dose-dependent manner. As a result of survivin knockdown, STA-ET-1 cells showed reduced cell proliferation, an increased number of radiation-induced DSBs, and reduced repair. Apoptosis was increased by knockdown alone and increased further in combination with irradiation. Colony formation was significantly reduced by survivin knockdown in combination with irradiation. CONCLUSION: Survivin is a radiation-inducible protein in Ewing's sarcoma and its down-regulation sensitizes cells toward irradiation. Survivin knockdown in combination with radiation inhibits cell proliferation, repair, and colony formation significantly and increases apoptosis more than each single treatment alone. This might open new perspectives in the radiation treatment of Ewing's sarcoma.

NH2-terminal substitutions of basic amino acids induce translocation across the microsomal membrane and glycosylation of rabbit cytochrome P450IIC2.

Insertion of rabbit cytochrome P450IIC2 and its modified form, [2-lys,3-arg]P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. In contrast, when [2-lys,3-arg]-P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. After treatment with endoglycosidase H, the more slowly migrating species comigrated with [2-lys,3-arg]P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. Both the glycosylated and nonglycosylated forms of [2-lys,3-arg]P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. Similar results were obtained for a truncated species, [2-lys,3-arg]P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. In contrast to the proteolytic processing observed previously in a hybrid [2-lys,3-arg]P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of [2-lys,3-arg]P450IIC2 was observed in the presence of membranes. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from [2-lys,3-arg]P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.

Parathyroid secretion: discovery of a major calcium-dependent protein.

Bovine parathyroid tissue incubated in vitro secretes a protein that is distinct from both parathyroid hormone and proparathyroid hormone and comprises about 50 percent of the total secreted protein. This protein appears to be an aggregate consisting of two or more subunits of molecular weight 70,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Although the function of this protein is unknown, the secretion rates of both the protein and parathyroid hormone respond in parallel to changes in the concentration of calcium in the medium.

Proparathyroid hormone: biosynthesis by human parathyroid adenomas.

Biosynthesis of a precursor (proparathyroid hormone) to human parathyroid hormone was demonstrated during incubation of tissue from parathyroid adenomas. The proparathyroid hormone is labeled more rapidly than parathyroid hormone during incubation with amino acids labeled with carbon-14 and is progressively converted to the hormone. Apparent differences in the relative rate of conversion of precursor to hormone found in different tumors suggest that proparathyroid hormone may accumulate in some of the tumors and be secreted into the circulation.

Precession-band variance missing from East Asian monsoon runoff.

Speleothem CaCO3 δ18O is a commonly employed paleomonsoon proxy. However, inferring local rainfall amount from speleothem δ18O can be complicated due to changing source water δ18O, temperature effects, and rainout over the moisture transport path. These complications are addressed using δ18O of planktonic foraminiferal CaCO3, offshore from the Yangtze River Valley (YRV). The advantage is that the effects of global seawater δ18O and local temperature changes can be quantitatively removed, yielding a record of local seawater δ18O, a proxy that responds primarily to dilution by local precipitation and runoff. Whereas YRV speleothem δ18O is dominated by precession-band (23 ky) cyclicity, local seawater δ18O is dominated by eccentricity (100 ky) and obliquity (41 ky) cycles, with almost no precession-scale variance. These results, consistent with records outside the YRV, suggest that East Asian monsoon rainfall is more sensitive to greenhouse gas and high-latitude ice sheet forcing than to direct insolation forcing.

Pre - proparathyroid hormone identified by cell - free translation of messenger RNA from hyperplastic human parathyroid tissue.

An 8-15S fraction of RNA isolated from hyperplastic human parathyroid tissue (primary chief-cell hyperplasia) and translated in a cell-free extract of wheat germ directs the synthesis of a protein that shares antigenic determinants and tryptic peptides with parathyroid hormone and its previously recognized immediated precursor, proparathyroid hormone. In addition, the protein contains tryptic peptides not found in proparathyroid hormone and migrates more slowly than does proparathyroid hormone on both urea-acid and urea-sodium dodecyl sulfate polyacrylamide gels, indicating that it is more acidic and larger than proparathyroid hormone. Sequential Edman degradation of the cell-free protein, radiolabeled with [35S]methionine, for 25 cycles released [35S]methionine at cycles 1, 7, 11, and 14, indicating that the NH2-terminal peptide sequence of the protein differs from that of both proparathyroid hormone and parathyroid hormone. We propose that this protein is an early biosynthetic precursor of human parathyroid hormone, pre-proparathyroid hormone, analogous to that identified recently by in vitro translation of bovine parathyroid mRNA.

Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

Enzymatic mutation detection: enrichment of heteroduplexes from hybrid DNA mixtures by cleavage-deficient GST-tagged endonuclease VII.

A method for the enrichment of heteroduplex DNAs from hybrid DNA mixtures by endonuclease VII is reported. The procedure is based on the ability of a GST-fused cleavage-deficient mutant endonuclease VII (EVII-N62D(GST)) to bind to mismatching nucleotides in heteroduplex DNAs identical to the wild-type enzyme. The GST tag was used for stable immobilisation of the protein to Glutathione Sepharose 4B. This enables the material to withstand the repeated rounds of binding steps required for enrichment of heteroduplex molecules from appropriate samples.

Tissue-specific chromatin structure of the phenobarbital-responsive unit and proximal promoter of CYP2B1/2 and modulation by phenobarbital.

Phenobarbital induction of transcription of CYP2B genes is mediated by an enhancer, termed a phenobarbital responsive unit (PBRU), approximately 2000 bp 5' of the transcription start site. To further delineate the mechanism of phenobarbital induction, protein binding in native chromatin and the nucleosomal structure of the PBRU and proximal promoter were examined in liver and kidney, in which the CYP2B1/2 genes are expressed and not expressed, respectively. Protein binding to the PBRU in kidney chromatin was not detected even though in vitro DNase I footprints were not detectably different with nuclear extracts from liver and kidney. Likewise, protein binding to regulatory motifs was not detected in the proximal promoter region in kidney chromatin. In liver chromatin, however, DNase I hypersensitivity and partial protection of the regulatory motifs from DNase I digestion or reaction with dimethyl sulfate was observed and phenobarbital treatment increased the hypersensitivity but only modestly affected protection. Low resolution Southern analysis of micrococcal nuclease-digested chromatin from untreated rats revealed micrococcal nuclease hypersensitive regions in the proximal promoter and PBRU regions in liver, but not in kidney. Phenobarbital treatment increased hyper-sensitivity in liver in both regions. Micrococcal nuclease hypersensitivity in the PBRU was largely restricted to a linker region between phased nucleosomes while in the proximal promoter hypersensitivity extended over approximately 200 bp suggesting disruption of a nucleosome in this region. These data indicate that in liver phenobarbital treatment substantially alters protein binding to regulatory motifs in the PBRU, while not greatly affecting such binding in the proximal promoter, and substantially alters chromatin structure in both regions, presumably as a result of chromatin modifying factors recruited to the PBRU. In the kidney, chromatin is probably in a closed conformation that prevents binding of regulatory factors.

Regulation of cytochrome P450 gene transcription by phenobarbital.

The ability of phenobarbital to induce levels of drug metabolism in mammals has been known for over 40 years. However, the molecular mechanisms underlying increased expression of the genes of the key enzyme in drug metabolism, cytochrome P450, have not been elucidated, primarily because in vitro model systems in which the induction could be studied were not available. Transfected primary cultured hepatocytes, transfection of liver in situ, and transgenic mice now provide suitable models for phenobarbital induction. In this review, progress toward understanding the mechanism of phenobarbital induction of gene expression is discussed with an emphasis on the mammalian genes, CYP2B1, CYP2B2, and Cyp2b10, which are most highly inducible by phenobarbital. Barbiturate induction of P450s in Bacillus megaterium, which is the system best understood, and its relevance to mammalian mechanisms of induction are also discussed. In B. megaterium, the binding of a repressor to several motifs is reversed by direct effects of barbiturates and by induction of positively acting factors. One of the repressor binding sites, the barbie box, is present in many mammalian phenobarbital-inducible genes, including the promimal promoter regions of CYP2B1, CYP2B2, and Cyp2B10. In the mammalian P450 genes, evidence has been proposed for phenobarbital-regulated elements both in the proximal promoter region and in a distal enhancer region. The role of the proximal region is controversial. A positively acting element that overlaps the barbie box sequence and a negative element have been proposed to mediate induction of CYP2B1/2, based primarily on protein binding and cell-free transcription assays. In contrast, other investigators have not found differences in phenobarbital-dependent protein binding in the proximal promoter region nor mediation of phenobarbital induction by this region. A distal gene fragment, at about -2000 kb in CYP2B1, CYP2B2, and Cyp2b10, has been shown to be a phenobarbital-responsive enhancer independent of proximal promoter elements. This fragment contains several binding sites for proteins and several functional elements, including an NF-1 site, and, therefore, has been designated as a phenobarbital-responsive unit. Possible models are presented in which phenobarbital treatment induces altered chromatin structure, which allows the binding of positively acting factors, or activates factors already bound, to the distal enhancer and the proximal promoter.

Inverse relationship of cotranslational translocation with the hydrophobic moment of the bovine preproparathyroid hormone signal sequence.

To investigate the dependence of transmembrane translocation on the hydrophobic moment of the hydrophobic core of the preproparathyroid hormone signal sequence, amino acids were switched to maximize or minimize the hydrophobic moment without changing the length or overall hydrophobicity of the core. As assayed in an in vitro translation system with microsomal membranes, the efficiency of translocation of these mutants was inversely related to the hydrophobic moment, indicating the hydrophobic moment or a related property may contribute to translocational activity.

Bacteriophage T4 strand transfer protein UvsX tolerates symmetric and asymmetric heterologies in short double-stranded oligonucleotides.

The UvsX protein of bacteriophage T4 catalyzes strand transfer from double-stranded DNA to homologous single-stranded DNA to generate both paranemic and plectonemic joints. We demonstrate here that UvsX mediates strand transfer efficiently from synthetic double-stranded donor oligonucleotides of 30 to 117 bp in length to circular single-stranded recipient M13mp19 DNA. Recovery of a diagnostic BamHI-restriction site, activated in the recipient after strand transfer, demonstrates that recipient and donated strands are perfectly base-paired after the exchange reaction has taken place. The transfer reaction progresses with greatest efficiency using donor DNA with a 3' overhang. Use of donor DNA having recessed 3' ends or blunt ends reduces the transfer efficiency by half. Single-stranded heterologies, centrally located in either strand of the donor DNA and forming either heteroduplex loops or a bulge in the donor are transferred with 80 to 100% efficiency. Also, a centrally located C/C-mismatch in the donor does not affect the transfer efficiency. Double-stranded heterologies are tolerated by the UvsX-catalyzed reaction but have different effects on the transfer efficiencies, depending on length and location in the molecule. A heterology of 24 bp located at the proximal end (start of transfer), the distal end (termination of transfer) and at each end of the donor molecule results in transfer efficiencies of 100%, 50% and 50 to 60%, respectively. Strand transfer efficiency is markedly reduced to about 15% if the 24 bp heterology is at a central location. However, insertion of a 4 bp heterology at this position yields a transfer efficiency of about 30%. Also, large double-stranded heterologies of 187 bp at the proximal end or 590 bp at the distal end of control-donor DNAs derived from plasmid digests did not impair the transfer activity of UvsX. This result differs from published results obtained with strand transfer reactions with large distally located heterologies catalyzed by RecA of Escherichia coli in vitro.

T4 endonuclease VII resolves cruciform DNA with nick and counter-nick and its activity is directed by local nucleotide sequence.

Endonuclease VII of phage T4 resolves Holliday structures in vitro by nicking pairs of strands across the junction. We report here analyses of this reaction between endonuclease VII and a Holliday structure analogue, made in vitro from synthetic oligonucleotides. The enzyme cleaves the structure in a non-concerted way and nicks each strand independently. Combinations of nicks with counter-nicks in strands across the junction resolve the construct. The specificity of the enzyme for DNA secondary structures was tested with a series of branched molecules made from oligonucleotides with the same nucleotide sequence in one strand. Results show that the number, location and relative cleavage efficiencies depend largely on the local nucleotide sequence, rather than on the branch type. In particular, endonuclease VII cleaves a complete four-armed cruciform as efficiently as a three-armed Y-junction or its derivatives, a semi-Y, a fork with two single-strand overhangs, a single-strand overhang, and a nicked DNA. However, exchange or addition of one or more nucleotides within the cleavage area flanking the structural signal for endonuclease VII strongly affects the cleavage pattern as well as their relative efficiency of usage. Examples with a single-stranded overhang are presented and show in summary that the enzyme has a fivefold preference for pyrimidines rather than purines.

Association of holliday-structure resolving endonuclease VII with gp20 from the packaging machine of phage T4.

Endonuclease VII (endo VII) is the product of gene 49 (gp49) of bacteriophage T4. It is a Holliday-structure resolvase (X-solvase) responsible for clearing branched replicative DNA prior to packaging. Consequently, mutations in gene 49 are unable to fill heads to completion because unresolved branches stop translocation of DNA. A likely association of gp49 with heads or proheads, however, could not be shown in the past. We have investigated whether gp49 could be part of the transiently assembled packaging machine (the "packasome") located at the base of proheads. Using purified proteins gpl6, gpl7 and gp20, which are constituents of the packasome, we found that gp49 binds tightly to gp20 and does not bind to gpl6 or gpl7. Quantification revealed that one dimer of gp49 binds one monomer of gp20. Notably, dimerisation of gp49 was an essential prerequisite for complex formation with gp20, and the dimerisation-deficient point mutation His-EVII-W87R showed only residual affinity to gp20. Furthermore, truncated peptides of gp49 deficient in dimer formation to various degrees were found to be impaired in binding to gp20. In contrast, the cleavage-deficient mutation EVII-N62D bound normally to gp20. The cruciform DNA (cf-DNA) resolving activity typical of endo VII is maintained in gp20-gp49 complexes. Furthermore, the complexes bind cf-DNA in the absence of Mg2+ as demonstrated by electromobility shift assays. The binding of the complexes to cf-DNA occurs via gp49, since gp20 alone does not bind cf-DNA. In conclusion, these findings are consistent with a model in which gp49 is an integral part of the packaging machine of phage T4.

Localization and characterization of the dimerization domain of holliday structure resolving endonuclease VII of phage T4.

Endonuclease VII (Endo VII) is a Holliday structure resolving enzyme of bacteriophage T4. Its nucleolytic activity depends on subactivities, which in order of execution are: (i) dimerization, (ii) binding to DNA, (iii) and cleavage of DNA. In an effort to assign these subfunctions to the primary sequence of the protein, a series of spontaneous point mutations deficient in DNA cleavage was isolated. Some of these mutations affected the dimerization of Endo VII. Compared with wild-type protein, which dimerizes completely in solution, more than 95% of one of the mutant proteins (W87R) remained in the monomeric state. Only the dimeric fraction of this protein bound to DNA. The dimerization domain of Endo VII was mapped by truncating the gene from both ends and analysing the dimerization ability of the purified peptides by crosslinking with glutaraldehyde. The dimerization domain was thus determined to reside between amino acid residues 55 and 105. Computer analyses predicted two alpha-helices (H2 and H3) in this section of the protein. As demonstrated by heterodimer formation, two copies of helix H3, but only one copy of helix H2, are required for dimerization. Helical wheel analyses revealed that both helices expose a hydrophobic face along their axes, suggesting that hydrophobic interaction between helices H3 mediate formation of Endo VII dimers, while helices H2 stabilize them.

Inhibition of Holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY.

Proteins UvsX, UvsY and Endonuclease VII (Endo VII) of bacteriophage T4 are required for DNA recombination, replication and repair. Endo VII is the product of gene 49 (gp49) and essential for resolution of branches from newly made DNA, prior to packaging into preformed heads. The ability of Endo VII to resolve Holliday structures in vitro suggested an in vivo function for the resolution of recombination intermediates, generated by UvsX and UvsY during the early infection cycle. Here we report results which contrast with this hypothesis. It is shown that the potent endonucleolytic activity of Endo VII with branched DNAs is inhibited in strand transfer reactions by the strand transferase UvsX, and more strongly by the accessory protein UvsY in vitro. The inhibitory effect of UvsX or UvsY is also seen in reactions with Endo VII using two synthetic cruciform DNAs and a C/C-mismatch containing substrate. Low concentrations of UvsY protein (12 ng or 0,76 pmol) were sufficient to reduce the cleavage efficiency of 30 units of Endo VII (about 16 fmol) to 50%. The inhibition is due to a direct protein-protein interaction between Endo VII, UvsX and UvsY as suggested by electrophoretic mobility shift assays (EMSAs). These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immobilized on a NHS-activated Sepharose matrix. This is the first identification of phage-encoded proteins which modulate the potent endonucleolytic activity of gp49 in vitro.

Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding.

Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs.

Endonuclease VII of phage T4 triggers mismatch correction in vitro.

The reactivity of endonuclease VII (gp49 of phage T4) with DNA-loops of eight, four, or one nucleotide, or any of 12 possible base mismatches was tested in vitro. Endonuclease VII introduces double-strand breaks by nick and counter-nick within six nucleotides 3' from the mispairings. High relative cleavage efficiencies at mismatches in heteroduplexes correlate with their decreased thermal stability and vice versa. A delay between nick and counter-nick was sufficient to allow T4 DNA-polymerase and T4 DNA-ligase to correct a C/C-mismatch in vitro, thereby saving the DNA from double-strand breakage. Very short repair tracks of three to four nucleotides mapped between the mismatch and one of the formerly induced nicks, which were subsequently sealed by DNA ligase.

No braiding of Holliday junctions in positively supercoiled DNA molecules.

The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.