Kalman G. Buki

Potential chemotherapeutic activity of 4-iodo-3-nitrobenzamide. Metabolic reduction to the 3-nitroso derivative and induction of cell death in tumor cells in culture.

A C-nitroso prodrug, 4-iodo-3-nitrobenzamide, was synthesized, and its action on a variety of tumor cells of human and animal origin tested. This prodrug was reduced transiently by tumor cells to 4-iodo-3-nitrosobenzamide at a very low rate, which was, however, sufficient to kill tumor cells. The final reduction product was 4-iodo-3-aminobenzamide, and no intermediates accumulated. No toxicity could be observed in hamsters even at 200 mg/kg, given i.p. daily for 7 days. The chemical reactivity of both 4-iodo-3-nitrosobenzamide and its noniodinated homolog with reduced ascorbate yielded the hydroxylamines. With glutathione, 4-iodo-3-aminobenzamide was formed, suggesting glutathione sulfinic acid formation. Confirming earlier studies, 4-iodo-3-nitrosobenzamide inactivated poly(ADP-ribose) polymerase by zinc ejection from the first zinc finger of this nuclear protein. The iodinated nitroso compound was more effective than its iodine-free analog. Selective tumoricidal action appeared to correlate with the reduction of the nitro group to nitroso in tumor cells, and with the previously described subsequent induction of tumor apoptosis by the C-nitroso intermediate. These processes were accelerated by buthionine sulfoximine, which diminishes cellular GSH.

Inactivation of estrogen receptor α in bone-forming cells induces bone loss in female mice

The role of the estrogen receptor α (ERα) in bone‐forming cells is incompletely understood at present. To examine the in vivo effects of ERα in these cells, we generated a mouse strain in which the ERα gene is inactivated in osteoblasts via osteocalcin promoter‐regulated cyclic recombinase (Cre) activity (ERαΔOB/ΔOB). This enabled micro‐computed tomography‐ and histomorphometry‐based analysis of ERα‐mediated effects in bone‐forming cells in mice, in which the systemic ERα‐mediated effects are intact. In female ERαΔOB/ΔOB mice, trabecular and cortical bone volumes were significantly reduced (31.5 and 11.4%, respectively) at 3.5 mo of age compared with control ERαfl/fl animals, and their response to ovariectomy was small compared with that of controls. In contrast with females, no differences could be detected in the bone phenotype of young males, whereas in 6‐mo‐old ERαΔOB/ΔOB males, trabecular bone volume (Tb.BV) was decreased (27.5%). The ERα inactivation‐related effects were compared with those of controls having a similar genetic background. Parental osteocalcin‐Cre mice did not show Cre‐related changes. Our results suggest that in female mice, Tb.BV and cortical bone volume are critically dependent on the ERα regulation of osteoblasts, whereas in male mice, osteoblastic ERα is not required for the regulation of bone formation during rapid skeletal growth, but it is involved in the maintenance of Tb.BV.—Määttä, J. A., Büki, K. G., Gu, G., Alanne, M. H., Vääräniemi, J., Liljenbäck, H., Poutanen, M., Härkönen, P., Väänänen, K. Inactivation of estrogen receptor α in bone‐forming cells induces bone loss in female mice. FASEB J. 27, 478–488 (2013). www.fasebj.org

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.

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.

Rab13 Is Upregulated During Osteoclast Differentiation and Associates with Small Vesicles Revealing Polarized Distribution in Resorbing Cells

Osteoclasts are bone-resorbing multinucleated cells that undergo drastic changes in their polarization due to heavy vesicular trafficking during the resorption cycle. These events require the precise orchestration of membrane traffic in order to maintain the unique characteristics of the different membrane domains in osteoclasts. Rab proteins are small GTPases involved in regulation of most, if not all, steps of vesicle trafficking. The investigators studied RAB genes in human osteoclasts and found that at least 26 RABs were expressed in osteoclasts. Out of these, RAB13 gene expression was highly upregulated during differentiation of human peripheral blood monocytic cells into osteoclasts. To study its possible function in osteoclasts, the investigators performed immunolocalization studies for Rab13 and various known markers of osteoclast vesicular trafficking. Rab13 localized to small vesicular structures at the superior parts of the osteoclast between the trans-Golgi network and basolateral membrane domain. Rab13 localization suggests that it is not involved in endocytosis or transcytosis of bone degradation products. In addition, Rab13 did not associate with early endosomes or recycling endosomes labeled with EEA1 or TRITC-conjugated transferrin, respectively. Its involvement in glucose transporter traffic was excluded as well. It is suggested that Rab13 is associated with a putative secretory function in osteoclasts.

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.