Pal I. Bauer

Destabilization of Zn2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function.

6‐Nitroso‐ 1,2‐benzopyrone, an oxidation product of 6‐amino‐ 1,2‐benzopyrone, binds to the DNA‐recognizing domain of the ADP‐ribose transferase protein and preferentially destabilizes Zn2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn2+ from the 65Zn2+‐isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc‐deficient enzyme still binds to a DNA template. consisting of a 17‐mer DNA primer annealed to M 13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I, Auto‐poly‐ADP‐ribosylated ADP‐ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer‐template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP‐ribose transferase in intact cells can induce significant alteration in DNA structure and replication.

Inhibition of HIV-1 IIIb replication in AA-2 and NT-2 cells in culture by two ligands of poly (ADP-ribose) polymerase: 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone

The effects of two adenosine diphosphoribose transferase (ADPRT) enzyme inhibitory ligands, 6-amino-1,2-benzopyrone and its 5-iodo-derivative, were determined in AA-2 and MT-2 cell cultures on the replication of HIV-1 IIIb, assayed by an immunochemical test for the HIV protein p24, and syncytium formation, characteristic of HIV-infected cells. Intracellular concentrations of both drugs were sufficient to inhibit poly(ADP-ribose) polymerase activity within the intact cell. Both drugs inhibited HIV replication parallel to their inhibitory potency on ADPRT, but distinct differences were ascertained between the two cell lines. In AA-2 cells both p24 and syncytium formation were depressed simultaneously, whereas in MT-2 cells only syncytium formation was inhibited by the drugs, and the p24 production, which remained unchanged during viral growth, was unaffected. Both drugs only moderately depressed the growth rate of the AA-2 and MT-2 cells and there was no detectable cellular toxicity. Results suggest the feasibility of the development of a new line of ADPRT ligand anti-HIV drugs that fundamentally differ in their mode of action from currently used chemotherapeutics.

Anti-cancer action of 4-iodo-3-nitrobenzamide in combination with buthionine sulfoximine: inactivation of poly(ADP-ribose) polymerase and tumor glycolysis and the appearance of a poly(ADP-ribose) polymerase protease.

E-ras 20 tumorigenic malignant cells and CV-1 non-tumorigenic cells were treated with a drug combination of 4-iodo-3-nitrobenzamide (INO(2)BA) and buthionine sulfoximine (BSO). Growth inhibition of E-ras 20 cells by INO(2)BA was augmented 4-fold when cellular GSH content was diminished by BSO, but the growth rate of CV-1 cells was not affected by the drug combination. Analyses of the intracellular fate of the prodrug INO(2)BA revealed that in E-ras 20 cells about 50% of the intracellular reduced drug was covalently protein-bound, and this binding was dependent upon BSO, whereas in CV-1 cells BSO did not influence protein binding. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the protein that covalently binds the reduction product of INO(2)BA, which is 4-iodo-3-nitrosobenzamide. Since only the enzymatically reduced drug INOBA bound covalently to GAPDH, the BSO-dependent covalent protein-drug association indicated an apparent nitro-reductase activity present in E-ras 20 cells, but not in CV-1 cells, explaining the selective toxicity. Covalent binding of INOBA to GAPDH inactivated this enzyme in vitro; INO(2)BA+BSO also inactivated cellular glycolysis in E-ras 20 cells because it provided the precursor to the inhibitory species: INOBA. Another event that occurred in INO(2)BA+BSO-treated E-ras 20 cells was the progressive appearance of a poly(ADP-ribose) polymerase protease. This enzyme was partially purified and characterized by the polypeptide degradation product generated from PARP I, which exhibited a 50kDa mass. This pattern of proteolysis of PARP I is consistent with a drug-induced necrotic cell killing pathway.

Evidence for the participation of histidine residues located in the 56 kDa C-terminal polypeptide domain of ADP-ribosyl transferase in its catalytic activity

Purified ADPRT protein was inactivated by the histidine specific reagent diethylpyrocarbonate, binding to two histidine residues, or by a relatively histidine selective photoinactivation method. Inactivation with up to 1.3 mM diethylpyrocarbonate was reversible by hydroxylamine. Enzymatic inactivation coincided with the loss of binding capacity of the enzyme protein to benzamide affinity matrix but not to DNA cellulose. Labelled diethylpyrocarbonate was identified exclusively in the 56 kDa carboxyl‐terminal polypeptide where 2 out of 13 histidine residues were modified by this reagent. It is proposed that histidine residues in the 56 kDa polypeptide may participate as initiator sites for polyADP‐ribosylation.

Isolation and identification of a proteinase from calf thymus that cleaves poly(ADP-ribose) polymerase and histone H1

A proteinase was isolated from calf thymus that degraded pADPRT, histone H1 and alpha-casein in a Ca(2+)-dependent manner. In a five-step procedure, a homogenous proteinase was obtained with a subunit structure of 80 and 30 kDa. The amino-acid homology of an internal sequence as well as kinetic and inhibitor assays identified the proteinase as calpain I. It is suggested that even though the general substrate alpha-casein is widely used for the assaying of calpains, more appropriately physiological cellular components (pADPRT and histone H1) specify the thymus proteinase.

Reversion of malignant phenotype by 5-iodo-6-amino-1,2-benzopyrone a non-covalently binding ligand of poly(ADP-ribose) polymerase

A non-covalently binding inhibitory ligand of poly(ADP-ribose) polymerase, 5-iodo-6-amino-1,2-benzopyrone, when incubated at 5-600 microM external concentration with an E-ras-transformed tumorigenic cell line or with human prostatic carcinoma cells for 40 to 60 days converts both cancer cells to a non-tumorigenic phenotype that is characterized by drastic changes in cell morphology, absence of tumorigenicity in nude mice, and a high rate of aerobic glycolysis.

Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-Amino-1,2-benzopyrone

The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethasone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. At a non-toxic concentrations (0.2 mM) 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. The drug had no direct effect on cellular DNA polymerases as tested in vitro, and the inhibition of DNA synthesis in permeabilized cells following drug treatment for 4 days was abolished by externally added DNA templates. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.

Inhibitory binding of adenosine diphosphoribosyl transferase to the DNA primer site of reverse transcriptase templates.

Purified adenosine diphosphoribose transferase protein binds to RNA-DNA hybrid templates of reverse transcriptase at the DNA primer site and inhibits RT activity of HIV and MMu RTs. This action is prevented by auto-poly-ADP-ribosylation of the transferase but is reinduced by inhibitory ligands of the enzyme.

Inactivation of the Polymerase but not the DNA Binding Function of ADPRT by Destabilization of one of its Zn2+ Coordination Centers by 6-Nitroso-1,2-Benzopyrone

In the course of studies on the metabolism of inhibitors of poly (ADP- ribose) transferase (ADPRT), we observed that the inhibitor 6-amino-1,2-benzopyrone (6-ABP) (1) when metabolized by rat liver microsomal preparations is oxidized to the corresponding 6-nitroso derivative (6-NOBP) as the main metabolite (2). Since ADPRT is located in the nuclear matrix (3,4) and most of the cytochrome P450 resides in the endoplasmic reticulum which is contiguous with the nuclear membrane (5), generation of 6-NOBP near ADPRT is likely. This suggested that the exact cellular significance of this oxidation path be investigated with respect to ADPRT.

Conversion of Poly(ADP-ribose) Polymerase Activity to NAD- Glycohydrolase During Retinoic Acid Induced Differentiation of HL60 Cells

Cells of the promyelocytic leukemia line HL60 can be induced to differentiate to mature granulocytes by polar-planar compounds (e.g.,DMSO)(1), or by retinoic acid (RA)(2). Involvement of poly ADP-ribose polymerase (ADPRT) in this process had been suggested by work from different Iaboratories; notably, it was observed (3) that during DMSO induced differentiation the cellular endogenous (ADPR)n polymer content increased sharply in the final stages of differentiation while simultaneously and in apparent contradiction ADPRT activity (with or without added DNAase I) declined and remained very low in the differentiated state. We now studied the changes of the ADPRT system that occur upon RA induced differentiation of HL60 cells (4).