Selective Loss of Poly(ADP-ribose) and the 85-kDa Fragment of Poly(ADP-ribose) Polymerase in Nucleoli during Alkylation-induced Apoptosis of HeLa Cells

Abstract

Here, we performed a time-course analysis of (i) poly(ADP-ribose) synthesis and degradation as well as (ii) the subnuclear localization of PARP and its fragments by using confocal laser scanning immunofluorescence microscopy. PARP was activated within 15 min post-treatment, as revealed by nuclear immunostaining with antibody 10H (recognizing poly-(ADP-ribose)). This was followed by a late, time-dependent, progressive decline of 10H signals that coincide with the time of PARP cleavage. Strikingly, nucleolar immunostaining with antibodies 10H and C-II-10 (recognizing the 85-kDa PARP fragment) was lost by 15 min post-treatment, whereas F-I-23 signals (recognizing the 29-kDa fragment) persisted. We hypothesize that the 85-kDa PARP fragment is translocated, along with covalently bound poly(ADP-ribose), from nucleoli to the nucleoplasm, whereas the 29-kDa fragment is retained, because it binds to DNA strand breaks. Our data (i) provide a link between the known time-dependent bifunctional role of PARP in apoptosis and the subcellular localization of PARP fragments and also (ii) add to the evidence for early proteolytic changes in nucleoli during apoptosis.Higher eucaryotic organisms have developed a sophisticated signaling system to eliminate nonfunctional cells in a highly coordinated sequence of "suicidal" events. This tightly regulated process is known as "programmed cell death" or "apoptosis" (1-3). Superfluous cell populations that undergo apoptosis include genetically damaged and/or aging cells. The death program is executed in three chronologically distinct phases (4). First to occur is "the condemned phase" where a fully reversible sequence of metabolic and cell cycle adaptation(s) take place with mitochondrial components, such as cytochrome c and the bcl-2 gene produced, playing a decisive role. The second phase of "commitment" is irreversible and derives from unleashing a cascade of proteolytic signals emanating from the mitochondrion to the nucleus. Finally, the "execution phase" of apoptosis is manifested by the macromolecular degradation of chromosomal DNA catalyzed by caspase (cysteine-aspartase)-activated death-factor (5, 6). In this phase, key nuclear proteins are degraded by caspase-3, the main "executioner" of nuclear disassembly (7), and this leads to the disintegration of the nucleus. One of the primary targets for caspase-3 is poly(ADPribose)polymerase (PARP) 1 (E.C. 2.4.2.30) (8 -10). PARP is a nuclear DNA-binding protein of 1,014 amino acid residues (113 kDa) (11, 12) that is constitutively expressed in eucaryotes and comprises up to 1% of the total nuclear protein. This enzyme displays a multimodular domain structure that can be dissected into three functionally distinct domains from the amino terminus to the carboxyl terminus (13). The first module is the "DNA-binding domain" (DBD), a fragment of 46-kDa that contains two zinc fingers, which mediate binding to DNA strands breaks as well as a bipartite nuclear localization signal (14). The two karyophilic regions of the nuclear localization signa...

Apoptosis in Leukemia Cells Is Accompanied by Alterations in the Levels and Localization of Nucleolin

“…In accord with our observations, previous reports have also described redistribution of 89-kDa PARP-1 in response to apoptosis (53)(54)(55). Indeed, PARP-1 can be localized in the nucleolus under some circumstances (53)(54)(55)(56), and there is some evidence to suggest that the nucleolus is central to the process of apoptosis and the earliest site of caspase-mediated proteolysis (57,58).…”

Differential regulation of CXC ligand 1 transcription in melanoma cell lines by poly(ADP-ribose) polymerase-1

“…A 54 kDa domain of PARP-1 located in the carboxyl terminus represents the b-nicotinamide adenine dinucleotide (NAD þ )-binding domain. Between the DNA-binding domain and the NAD þ -binding domain is a 22 kDa 'automodification domain', which facilitates the homoand/or heterodimerization of PARP-1 with other proteins (Alvarez-Gonzalez et al, 1999).…”

Fine Tuning the Transcriptional Regulation of the CXCL1 Chemokine

“…A 54-kDa domain of PARP located in the carboxyl terminus represents the β-nicotinamide adenine dinucleotide (NAD + )-binding domain containing a highly conserved "PARP signature" sequence comprising the catalytically crucial amino acid residue Glu-988. Between the DNA-binding domain and the NAD + -binding domain lies a 22-kDa "automodification domain," which facilitates the homodimerization and/or heterodimerization of PARP with other chromatin proteins (156).…”

“…It has a 46-kDa DNA-binding domain at the N-terminus that contains two Zn fingers, facilitating the binding of PARP to DNA strand breaks, as well as bipartite nuclear localization signal. The two regions of nuclear localization signals are separated by the DEVD sequence, which is the target of caspase-3 during apoptosis and subsequently gets cleaved, and thus inactivated, resulting in the formation of two proteolytic fragments of PARP, a 29-kDa amino terminus and an 85-kDa carboxyl terminus (155,156). A 54-kDa domain of PARP located in the carboxyl terminus represents the β-nicotinamide adenine dinucleotide (NAD + )-binding domain containing a highly conserved "PARP signature" sequence comprising the catalytically crucial amino acid residue Glu-988.…”