Involvement of PLAGL2 in activation of iron deficient- and hypoxia-induced gene expression in mouse cell lines.

We searched iron-deficient inducible cDNA, using subtraction cloning and mRNA from desferrioxamine-treated mouse macrophage Raw264.7 cells. We identified a pleomorphic adenoma gene like 2 (PLAGL2), one of PLAG superfamily proteins exhibiting antiproliferative properties on tumor cells. Mouse PLAGL2 consists of 496 amino acids with seven C2H2 zinc-fingers. PLAGL2 mRNA was induced in RAW264.7 cells, mouse erythroleukemia cells and Balb/c 3T3 cells when they were treated with desferrioxamine. Hypoxia also increased PLAGL2 mRNA. Expression of PLAGL2 in COS-7 cells led to nuclear localization. PLAGL2 had potential binding ability to GC-rich oligonucleotide and activated transcription of a gene with the binding sequence in transient reporter assay, a finding consistent with a case seen in a PLAGL2 homolog, ZAC-1. Transient co-transfection of PLAGL2 or ZAC1 cDNA and a reporter containing a lactate dehydrogenase A (LDHA) promoter carrying the hypoxia inducible factor-1 responsive element led to an increase in the basal transcription in Balb/c 3T3 and HepG2 cells. Activation in transcription from the LDHA promoter increased by desferrioxamine treatment or hypoxia was further enhanced when PLAGL2 was expressed. We propose that PLAGL2 is involved in the cell cycle arrest and apoptosis of tumor cells by regulating iron depletion- or hypoxia-inducible gene expression.

DNA interaction and nucleotide sequence cleavage of copper-streptonigrin.

The copper-accelerated DNA binding and cleavage of streptonigrin have been investigated by 1H-NMR, ESR spectrometry and nucleotide sequence analysis. In the DNA breakage by the streptonigrin-Cu(II)-NADPH system, the somewhat preferred cleavage sites were several cytosine bases adjacent to purine bases such as GCGG(5'----3'), ACGC(5'----3') and GGCG(5'----3') sequences. The proton chemical shifts for the streptonigrin-Cu(I)-poly(dA-dT) complex demonstrated the interaction between the pyridine ring of the drug and the purine bases of the nucleic acid. Indeed, the temperature profile of adenine H-2 proton clearly showed the Tm to shift from 70 degrees C in the binary streptonigrin-poly(dA-dT) system to 75 degrees C in the ternary streptonigrin-Cu(I)-poly(dA-dT) system. The interaction of the streptonigrin-Cu(II) complex with DNA also induced the apparent change of ESR parameters. The tricyclic phenanthidium ring system including the copper chelate ring appears to significantly contribute to the present DNA interaction and cleavage of copper-streptonigrin.

Nucleotide sequence cleavages of manganese-bleomycin induced by reductant, hydrogen peroxide and ultraviolet light. Comparison with iron- and cobalt-bleomycins.

The prominent DNA breakages of bleomycin-Mn complex system were induced by reductant, hydrogen peroxide and ultraviolet light, and these three induction systems gave remarkably similar nucleotide sequence cleavage modes. The preferred DNA cleavage sites at guanine-cytosine(5'----3') and guanine-thymine(5'----3') sequences were appreciably comparable to those of the corresponding bleomycin-Fe complex systems, but not identical. In contrast, the bleomycin-Co complex system significantly degraded isolated DNA only by irradiation of ultraviolet light. The present results provide valuable information on the role of transition metals on DNA cleavage reaction of bleomycin.

Gene of rat Ca2+/calmodulin-dependent protein kinase II alpha isoform -- its cloning and whole structure.

The gene encoding the alpha isoform of rat Ca2+/calmodulin-dependent protein kinase II was cloned, and its exon-intron organization was analyzed. The coding region of cDNA consists of 18 exons spanning more than 50 kilobase pairs. Each of the discrete functional units, such as the ATP-binding site, the autophosphorylation site responsible for Ca2+-independent activity, the calmodulin-binding site, and link structure is encoded by a single exon. The largest and smallest exons consist of 229 and 41 base pairs, respectively. All splice junction sequences flanking the introns conform to the consensus splice junction sequence and the GT-AG splice rule.

Canine and feline aural hematoma: clinical, experimental, and clinicopathologic observations.

The pathogenesis of canine and feline aural hematoma (AH) was investigated. Clinical observations, selected experimental procedures, and clinicopathologic examinations were done on 40 dogs and 20 cats affected with AH. Eighteen healthy dogs and 14 healthy cats were used as the controls. The results of this investigation provide a valid basis for questioning the conventionally held view that AH is caused by trauma and for postulating that the actual cause is immune mediated.

Molecular biological analysis of sequence-specific DNA recognition and cleavage by metallobleomycins.

The DNase I footprinting analysis shows binding sites of approximately two or three base pairs, in particular 5'-XGC sequences, for the green-colored Co(III) and fully oxidized Fe(III) complexes of bleomycin (BLM). In contrast to covalent attachment of guanine N-7 with aflatoxin B1 or dimethyl sulfate, the modification of guanine 2-amino group with anthramycin remarkably inhibits the DNA cleavages at 5'-GC and 5'-GT sites by the iron and cobalt complex systems of BLM. The present results strongly indicate that metallobleomycin binds in minor groove of B-DNA and that the 2-amino group of guanine adjacent to 5'-side of the cleaved pyrimidine base is one key element of specific 5'-GC or 5'-GT recognition by metallobleomycin. On the basis of these experimental data, possible binding mode of metallobleomycin in B-DNA helix has been proposed by computer-constructed model building.

Sequence-specific recognition and cleavage of DNA by metallobleomycin: minor groove binding and possible interaction mode.

The DNase I cleavage-inhibition analysis shows binding sites of approximately 2 or 3 base pairs--in particular, 5' N-G-C sequences--for the green-colored CoIII and fully oxidized FeIII complexes of bleomycin. The apparent binding constant of the bleomycin-CoIII complex is quite similar for glucosylated and nonglucosylated phage T4 DNAs, whereas poly[d(I-C)] clearly gives a smaller binding constant than does poly[d(G-C)]. In contrast to the covalent attachment of the guanine N-7 with aflatoxin B1, the modification of the guanine 2-amino group with anthramycin remarkable inhibits the DNA cleavages at 5' G-C and 5' G-T sites by the FeIII and CoIII complex systems of bleomycin. These results strongly indicate that metallobleomycin binds in the minor groove of B-DNA and that the 2-amino group of guanine adjacent to the 5' side of the cleaved pyrimidine base is one key element of the specific 5' G-C or G-T recognition by the bleomycin-metal complex. A possible binding mode of metallobleomycin in the DNA helix has been proposed by computer-constructed model building.

Nuclear localization of transcription factor Sp1.

Transcription factor Sp1 is importantly related to expression of many cellular genes. In order to identify the nuclear localization signal of Sp1, various truncated regions of Sp1 were fused to green fluorescent protein (GFP) and were expressed in HeLa cell. The C-terminal region are required to localize Sp1 in the nucleus of HeLa cell.

Studies on antitumor drugs targeting DNA: photosensitive DNA cleavage of copper-camptothecin.

In combination with copper(II) ion and 365 nm-light, anti-tumor alkaloid camptothecin produced remarkable DNA strand scission. The DNA sequencing analysis revealed considerably random nucleotide sequence cleavage. The present DNA breakage reaction was strongly inhibited by catalase and bathocuproine, but not by superoxide dismutase, mannitol, and 1,4-diazabicyclo-[2.2.2]octane. The camptothecin-Cu(II)-UV light system also clearly induced bacteriophage-inactivation which is associated with the DNA degradation. On the basis of the experimental results, the reaction mechanism for the present DNA cleavage has been discussed.

Alteration of DNA binding specificity by nickel (II) substitution in three zinc (II) fingers of transcription factor Sp1.

Transcription factor Sp1, which has a DNA binding domain composed of three zinc fingers, binds to GC box (consensus sequence, G/T-GGGCGG-G/A-G/A-C/T) and activates the transcription by RNA polymerase II. Metal substitution of nickel(II) for zinc(II) in Sp1 causes no differences in the mode of protein-DNA interaction. However, sequence preference of Ni(II)Sp1 changes from 5'-GGGGCGGGGC to 5'-GGGGCGTGGC, and is distinct from that of Zn(II)Sp1. The result indicates an important effect of metal-induced folding on sequence-specific recognition of DNA by zinc-finger proteins.

Copper-bleomycin has no significant DNA cleavage activity.

In contrast to a very recent report [Ehrenfeld, G. M., Rodriguez, L. O., Hecht, S. M., Chang, C., Basus, V. J., & Oppenheimer, N. J. (1985) Biochemistry 24, 81-92], the present careful reexamination demonstrated that copper-bleomycin systems have no significant DNA cleavage activity. In the presence of dithiothreitol, the bleomycin-Cu(II) complex showed little activity for DNA degradation. The DNA strand scission by the Cu(I)-bleomycin-dithiothreitol system was remarkably depressed by deferoxamine rather than by bathocuproine, suggesting the effect of trace amounts of contaminating iron in the experiments. It seems highly unlikely that the DNA breakage activity due specifically to the Cu(I)-bleomycin complex system is substantially strong. Our results indicate that the metal really relevant to the DNA cleavage by bleomycin is iron not copper.

ESR characteristics of sulfhydryl-containing peptide-nickel (III) complexes: implication for nickel (III) center of hydrogenases.

The Ni(III) complexes of N-mercaptoacetylglycyl-L-histidine and N-mercaptoacetylglycylglycylglycine clearly show the rhombic ESR pattern and g-values similar to the Ni(III) chromophore of hydrogenases. The present results strongly suggest that the Ni(III) center of hydrogenases contains one cysteine sulfur coordination as equatorial ligand in a tetragonal geometry. In addition, axial nitrogen ligand and a sulfur-rich Ni(III) site as in an S4 donor set may be ruled out. Indeed, the Ni(III) ESR features are a useful probe of the Ni(III) center in hydrogenases.

Nucleotide sequence cleavage of guanine-modified DNA with aflatoxin B1, dimethyl sulfate, and mitomycin C by bleomycin and deoxyribonuclease I.

In guanine-modified DNA with aflatoxin B1, dimethyl sulfate, and mitomycin C, the nucleotide sequence cleavage of bleomycin or DNase I has been investigated using 32P-labeled DNA fragments. In contrast with cis-dichlorodiammine platinum (II) which induces intrastrand crosslinking of guanine N7 atoms, these mono-covalent modifiers did not give remarkable alteration of the DNA nucleotide cleavage mode by bleomycin and DNase I. From the viewpoint of combination drug, it is of interest that mitomycin C promoted the DNA cleavage activity of bleomycin.

Light-induced DNA cleavage by esperamicin and neocarzinostatin.

Ultraviolet radiation of the enediyne drugs is effective in causing nicks in supercoiled DNA. Of special interest is the fact that the observed nucleotide cleaving specificity for the UV light- and thiol-activated antibiotics was the same with esperamicin A1, but was different with neocarzinostatin. In addition to the preferred cutting of T and A bases, the light-activated neocarzinostatin attacked certain G bases which were rarely cleaved by the thiol-activated neocarzinostatin. It should be noted that these enediyne antibiotics lose the DNA breakage activity after light-exposure for 30 min.

Role of the zinc(II) ions in the structure of the three-finger DNA binding domain of the Sp1 transcription factor.

The transcription factor Sp1 from Hela cells contains near the C-terminus of this protein of 778 amino acids three contiguous repeats of an amino acid sequence, -Cys-X4-Cys-X12-His-X3-His-, typical of the Cys2His2-type zinc-finger DNA binding domain first found in transcription factor TFIIIA. A DNA sequence corresponding to the condons from residue 614 to residue 778 of Spl (encompassing the three zinc-finger motifs) has been cloned and overproduced in Escherichia coli. The fragment of Sp1 containing the C-terminal 165 residues plus 2 from the cloning vector, designated Sp1(167*), can be extracted with 5 M urea and then refolded in the presence of Zn(II) to a protein of specific conformation containing 3.0 +/- 0.2 mol of tightly bound Zn(II)/mol of protein. Gel retardation assays using a labeled 14-bp DNA sequence containing a consensus Sp1 binding site show that the refolded Zn(II) protein specifically recognizes the "GC box" sequence in the presence of a large excess of calf thymus DNA. Treatment of Zn(II)Sp1(167*) with 10 mM EDTA results in removal of Zn(II) and the formation of an apoprotein which does not specifically recognize DNA. Cd(II) can be exchanged for Zn(II) in the refolded protein with full retention of specific DNA recognition. This is the first Cys2His2-type "finger" protein where this substitution has been accomplished. Titration of the Zn(II) protein with 6 mol of p-mercuribenzenesulfonate/mol of protein results in the complete release of the three Zn(II) ions. Release of Zn(II) is highly cooperative.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA cleavages of bleomycin-transition metal complexes induced by reductant, hydrogen peroxide, and ultraviolet light: characteristics and biological implication.

In DNA strand scission of Mn-, Co-, and Fe-complexes of bleomycin-A2 (BLM), the Mn-BLM and Fe-BLM complex systems showed prominent DNA cleavage activity in combination with reductant, hydrogen peroxide, and ultraviolet light. The sequence-specific cleavage modes of the two metallobleomycins were remarkably similar. The present results suggest that the DNA breakage mechanism of the Mn-BLM resembles that of the Fe-BLM, and that Mn as well as Fe is an effective metal cofactor in the BLM drug action. In contrast, the Co -BLM complex system significantly degraded DNA only by irradiation of UV light. The DNA breakage of the tallysomycin(TLM)- and peplomycin(PEM)-metal complexes has also been investigated by nucleotide sequence analysis.

Effective DNA cleavage by bleomycin-vanadium(IV) complex plus hydrogen peroxide.

It has been firstly found that the bleomycin-vanadyl(IV) complex is effectively capable of cleaving DNA in the presence of hydrogen peroxide. The 1:1 bleomycin-VO(IV) complex has been characterized by ESR and electronic absorption spectra, and its ESR parameters (go = 1.982 and Ao = 93.5 G) are indicative of VO(N5) coordination type for the metal-binding environment. The mode of nucleotide sequence cleavage induced by the present bleomycin-VO(IV)-H2O2 complex system was appreciably different from the corresponding Fe(III) complex system. Of special interest is the fact that the bleomycin-vanadium complex system more preferentially attacked G-A(5'----3') sequences than the bleomycin-iron complex system.

Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin.

Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin-Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy.