Induction of endonuclease-mediated apoptosis in tumor cells by C-nitroso-substituted ligands of poly(ADP-ribose) polymerase.

Abstract

ABSTRACT6-Nitroso-1,2-benzopyrone and 3-nitrosobenzamide, two C-nitroso compounds that inactivate the eukaryotic nuclear protein poly(ADP-ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] at one zinc-ringer site, completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanism elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesiumdependent endonuclease. This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs derepresses the DNA-degrading activity. ADPRT is thus identified as a critical regulatory enzyme component of a DNA-binding multiprotein system that plays a central function in defining DNA structures in the intact cell.In pursuing the biological consequences of the inhibition of poly(ADP-ribose) polymerase [NAD+ :poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] within intact cells we have developed a group of C-nitroso-substituted molecules that are the oxidation products of previously described ligands containing amino groups (1). These C-nitroso ligands uniquely oxidize one of the zinc fingers of ADPRT, resulting in zinc ion ejection and concomitant inactivation of the poly(ADP-ribosyl)ation activity of ADPRT without abrogating its binding to DNA (1). In the present paper we demonstrate a cytostatic and apoptotic action of two of these ADPRT ligands, 6-nitroso-1,2-benzopyrone (NOBP) and 3-nitrosobenzamide (NOBA), ¶ toward malignant human cells. The apoptotic effect was traced to the derepression of a calcium/magnesiumdependent endonuclease that under resting conditions is known to be inhibited by poly(ADP-ribosyl)ation (2). (85 Ci/mmol; 1 Ci = 37 GBq) was added per well and its incorporation into DNA following an 18-hr incubation was determined radiochemically (6). Proliferation was also monitored by direct cell counting. Since both assays were in agreement, the more convenient radiochemical test was used routinely. The tetrazolium reduction assay for cell viability (7), which was originally developed as a test for mitochondrial succinate dehydrogenase (8), was compared with trypan blue uptake and plating efficiency, and on the basis of positive correlation among the three assays, the more quantitative dye reduction method was adopted. MATERIALS AND METHODSClonigenic Studies. Human 855-2 and HL-60 leukemic cells were plated at 105 per ml and human and rhesus bone marrow mononuclear cells and human peripheral stem cells at 4 x 105 per ml, and assays for colony-forming units were performed as reported (5). A semiquantitative assessment of malignant cells in bone marrow specimens was done by flow cytometry with labeled fluorescein-conjugated antibodies against ...

Temporal relationship of CDK1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells

“…Dr Ron Jemmerson of the University of Minnesota Medical School (Minneapolis, MN) kindly provided the monoclonal antibody to cyt c. 14 Anti-caspase-3 antibody was purchased from Transduction Laboratories (Lexington, KY, USA), whereas a polyclonal PARP antibody was kindly provided by Dr Ernest Kun of San Francisco State University (Tiburon, CA, USA). 22 Antibodies for detection of tyrosine phosphorylation of CDK1 were purchased from New England Biolabs (Beverly, MA, USA), whereas anti-cyclin B1 antibodies were purchased from DAKO (Carpinteria, CA, USA).…”

The interleukin 1β‐converting enzyme inhibitor CrmA prevents Apo1/Fas‐ but not glucocorticoid‐induced poly(ADP‐ribose) polymerase cleavage and apoptosis in lymphoblastic leukemia cells

“…In addition to its protective action on the genome (predominantly in response to environmental stress [26]), it influences polyADP-ribosylation of nucleases implicated in internucleosomal DNA degradation [27] and consumes NAD and its precursor ATP [28], an energy source required for apoptosis [29]. Due to these various effects, PARP and its degradation may both contribute to, or interfere with, the apoptotic process [30,31]. Apoptotic inactivation of PARP involves a single cleavage of the polypeptide after aspartate 216 [12], which separates the Nterminal Zn-fmger DNA-binding domain from the C-terminal automodification and polyADP-ribosylating domains.…”

Inhibition of the replication of native and 3′‐azido‐2′,3′‐dideoxy‐thymidine (AZT)‐resistant simian immunodeficiency virus (SIV) by 3‐nitrosobenzamide

“…It is important that the retroviral zinc finger destabilizing action of NOBA is not confined to the zinc fingers of gag proteins only, but includes one of the retroviral-type zinc fingers of eukaryotic poly(ADP-ribose) polymerase [16]. Inactivation of poly(ADP-ribose) polymerase by zinc ejection [16] derepresses an ADP-ribosylation inhibited CaZ+/Mg2+-dependent endonuclease in tumor cells and produces selective apoptosis [17]. The participation of poly(ADP-ribose) polymerase in the regulation of DNA, including retroviral DNA, is being analyzed.…”