Dean S. Rosenthal

Transient Poly(ADP-ribosyl)ation of Nuclear Proteins and Role of Poly(ADP-ribose) Polymerase in the Early Stages of Apoptosis

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP −/− fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP −/− fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.

A review of the local pathophysiologic bases of burn wound progression.

Burn wound progression refers to the phenomenon of continued tissue necrosis in the zone of stasis after abatement of the initial thermal insult. A multitude of chemical and mechanical factors contribute to the local pathophysiologic process of burn wound progression. Prolonged inflammation results in an accumulation of cytotoxic cytokines and free radicals, along with neutrophil plugging of dermal venules. Increased vascular permeability and augmentations of interstitial hydrostatic pressure lead to edema with vascular congestion. Hypercoagulability with thrombosis further impairs blood flow, while oxidative stress damages endothelial cells and compromises vascular patency. A number of studies have investigated the utility of various agents in modulating these mechanisms of burn wound progression. However, as many of studies have used animal models of burn injury, often with administration of therapy preburn, obscuring the clinical applicability of the results to burn patients is of questionable benefit. An understanding of the complex, interrelated mediators of burn wound progression and their ultimate point of convergence in effecting tissue necrosis—cell apoptosis or oncosis—will allow for the future development of therapeutic interventions.

Involvement of PARP and poly(ADP-ribosyl)ation in the early stages of apoptosis and DNA replication

We have focused on the roles of PARP and poly(ADP-ribosyl)ation early in apoptosis, as well as during the early stages of differentiation-linked DNA replication. In both nuclear processes, a transient burst of PAR synthesis and PARP expression occurs early, prior to internucleosomal DNA cleavage before commitment to apoptosis as well as at the round of DNA replication prior to the onset of terminal differentiation. In intact human osteosarcoma cells undergoing spontaneous apoptosis, both PARP and PAR decreased after this early peak, concomitant with the inactivation and cleavage of PARP by caspase-3 and the onset of substantial DNA and nuclear fragmentation. Whereas 3T3-L1, osteosarcoma cells, and immortalized PARP +/+ fibroblasts exhibited this early burst of PAR synthesis during Fas-mediated apoptosis, neither PARP-depleted 3T3-L1 PARP-antisense cells nor PARP -/- fibroblasts showed this response. Consequently, whereas control cells progressed into apoptosis, as indicated by induction of caspase-3-like PARP-cleavage activity, PARP-antisense cells and PARP -/- fibroblasts did not, indicating a requirement for PARP and poly(ADP-ribosyl)ation of nuclear proteins at an early reversible stage of apoptosis. In parallel experiments, a transient increase in PARP expression and activity were also noted in 3T3-L1 preadipocytes 24 h after induction of differentiation, a stage at which ~95% of the cells were in S-phase, but not in PARP-depleted antisense cells, which were consequently unable to complete the round of DNA replication required for differentiation. PARP, a component of the multiprotein DNA replication complex (MRC) that catalyzes viral DNA replication in vitro, poly(ADP-ribosyl)ates 15 of ∼40 MRC proteins, including DNA pol α, DNA topo I, and PCNA. Depletion of endogenous PARP by antisense RNA expression in 3T3-L1 cells results in MRCs devoid of any DNA pola and DNA pol δ activities. Surprisingly, there was no new expression of PCNA and DNA pol α, as well as the transcription factor E2F-1 in PARP-antisense cells during entry into S-phase, suggesting that PARP may play a role in the expression of these proteins, perhaps by interacting with a site in the promoters for these genes.

PARP-1 binds E2F-1 independently of its DNA binding and catalytic domains, and acts as a novel coactivator of E2F-1-mediated transcription during re-entry of quiescent cells into S phase.

The transcription factor E2F-1 is implicated in the activation of S-phase genes as well as induction of apoptosis, and is regulated by interactions with Rb and by cell cycle-dependent alterations in E2F-1 abundance. We earlier demonstrated a pivotal role for poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of E2F-1 expression and promoter activity during S-phase re-entry when quiescent cells re-enter the cell cycle. We now investigate the putative mechanism(s) by which PARP-1 may upregulate E2F-1 promoter activity during S-phase re-entry. DNase-1 footprint assays with purified PARP-1 showed that PARP-1 did not directly bind the E2F-1 promoter in a sequence-specific manner. In contrast to p53, a positive acceptor in poly(ADP-ribosyl)ation reactions, E2F-1 was not poly(ADP-ribosyl)ated by wild-type PARP-1 in vitro, indicating that PARP-1 does not exert a dual effect on E2F-1 transcriptional activation. Protein-binding reactions and coimmunoprecipitation experiments with purified PARP-1 and E2F-1, however, revealed that PARP-1 binds to E2F-1 in vitro. More significantly, physical association of PARP-1 and E2F-1 in vivo also occurred in wild-type fibroblasts 5 h after re-entry into S phase, coincident with the increase in E2F-1 promoter activity and expression of E2F-1-responsive S-phase genes cyclin A and c-Myc. Mapping of the interaction domains revealed that full-length PARP-1 as well as PARP-1 mutants lacking either the catalytic active site or the DNA-binding domain equally bind E2F-1, whereas a PARP-1 mutant lacking the automodification domain does not, suggesting that the protein interaction site is located in this central domain. Finally, gel shift analysis with end-blocked E2F-1 promoter sequence probes verified that the binding of PARP-1 to E2F-1 enhances binding to the E2F-1 promoter, indicating that PARP-1 acts as a positive cofactor of E2F-1-mediated transcription.

Sulfur Mustard Induces Apoptosis in Cultured Normal Human Airway Epithelial Cells: Evidence of a Dominant Caspase-8-Mediated Pathway and Differential Cellular Responses

We have shown that sulfur mustard (SM; bis-(2-chloroethyl) sulfide), an alkylating, vesicating chemical warfare agent, causes dermal toxicity, including skin microblisters, via the induction of both death receptor (DR) and mitochondrial pathways of apoptosis in human epidermal keratinocytes. While SM is known for its skin-vesicating properties, respiratory tract lesions are the main source of morbidity and mortality after inhalation exposure. We, therefore, investigated whether SM induces apoptotic cell death in normal human bronchial epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. Cells were exposed to various concentrations of SM (0, 50, 100, and 300 μM for 16 h) in the culture medium and then tested for the activation of apoptotic executioner caspase-3 and initiator caspases-8 and -9. Caspases-8 and -3 were activated by SM in both airway cell types, indicating the induction of a DR pathway of apoptosis in these cells; however, the levels of enzyme activation were different, depending on the cell type and the SM concentrations used. Consistent with enzyme activity results, immunoblot analyses revealed the proteolytic processing of the proenzymes to the active forms of caspases-8 and -3 in these cells after SM exposure. Interestingly, NHBE cells were found to be exquisitely sensitive to SM, compared to SAEC, with caspase-3 activities in SM-exposed NHBE cells ∼2-fold higher and caspase-8 activities ∼10-fold higher than in SAEC. Furthermore, SM activated caspase-9 in NHBE cells, but not in SAEC, indicating a possible role of the mitochondrial pathway only in the NHBE cells. The present study shows that both upper airway (NHBE cells) and deep lung (SAEC) epithelial cells undergo SM-induced apoptotic death in vitro, but distinct cell-type specific responses can be elicited, which may be attributed to intrinsic properties that characterize the response of these cells to SM. These findings need to be taken into consideration in the search for modulators of these pathways for the therapeutic intervention to reduce SM injury due to respiratory tract lesions.

Calmodulin, poly(ADP–ribose)polymerase and p53 are targets for modulating the effects of sulfur mustard

We describe two pathways by which the vesicating agent sulfur mustard (HD) may cause basal cell death and detachment: induction of terminal differentiation and apoptosis. Following treatment of normal human epidermal keratinocytes (NHEK) with 10 or 100 μmM HD, the differentiation‐specific keratin pair K1/K10 was induced and the cornified envelope precursor protein, involucrin, was cross‐linked by epidermal transglutaminase. Fibronectin levels were reduced in a time‐ and dose‐dependent manner. The rapid increase in p53 and decrease in Bcl‐2 levels was consistent not only with epidermal differentiation but with apoptosis as well. Further examination of biochemical markers of apoptosis following treatment of either NHEK or human papillomavirus (HPV)‐immortalized keratinocytes revealed a burst of poly(ADP–ribose) synthesis, specific cleavage of poly(ADP–ribose)polymerase (PARP) in vivo and in vitro into characteristic 89 and 24 kDa fragments, processing of caspase‐3 into its active form and the formation of DNA ladders. The intracellular calcium chelator BAPTA suppressed the differentiation markers, whereas antisense oligonucleotides and chemical inhibitors specific for calmodulin blocked both markers of differentiation and apoptosis. Modulation of p53 levels utilizing retroviral constructs expressing the E6, E7 or E6 + E7 genes of HPV‐16 revealed that HD‐induced apoptosis was partially p53‐dependent. Finally, immortalized fibroblasts derived from PARP −/− ‘knockout mice’ were exquisitely sensitive to HD‐induced apoptosis. These cells became HD resistant when wild‐type PARP was stably expressed in these cells. These results indicate that HD exerts its effects via calmodulin, p53 and PARP‐sensitive pathways. Copyright

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol α, is regulated by interactions with Rb and by cell-cycle dependent alterations in E2F-1 abundance. We have shown that depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA expression downregulates pol α and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol α and E2F-1 gene expression, we utilized immortalized mouse fibroblasts derived from wild-type and PARP knockout (PARP−/−) mice as well as PARP−/− cells stably transfected with PARP cDNA [PARP−/−(+PARP)]. After release from serum deprivation, wild-type and PARP−/−(+PARP) cells, but not PARP−/− cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h. Whereas [3H]thymidine incorporation remained negligible in PARP−/− cells, in vivo DNA replication maximized after 18 h in wild-type and PARP−/−(+PARP) cells. To investigate the effect of PARP on E2F-1 promoter activity, a construct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells. E2F-1 promoter activity in control and PARP−/−(+PARP) cells increased eightfold after 9 h, but not in PARP−/− cells. PARP−/− cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP−/−(+PARP) cells. RT – PCR analysis and pol α activity assays revealed the presence of pol α transcripts and a sixfold increase in activity in both wild-type and PARP−/−(+PARP) cells after 20 h, but not in PARP−/− cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity, which then positively regulates both E2F-1 and pol α expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum.

Proteomic Analysis of Pathways Involved in Estrogen-Induced Growth and Apoptosis of Breast Cancer Cells

Background Estrogen is a known growth promoter for estrogen receptor (ER)-positive breast cancer cells. Paradoxically, in breast cancer cells that have been chronically deprived of estrogen stimulation, re-introduction of the hormone can induce apoptosis. Methodology/Principal Findings Here, we sought to identify signaling networks that are triggered by estradiol (E2) in isogenic MCF-7 breast cancer cells that undergo apoptosis (MCF-7:5C) versus cells that proliferate upon exposure to E2 (MCF-7). The nuclear receptor co-activator AIB1 (Amplified in Breast Cancer-1) is known to be rate-limiting for E2-induced cell survival responses in MCF-7 cells and was found here to also be required for the induction of apoptosis by E2 in the MCF-7:5C cells. Proteins that interact with AIB1 as well as complexes that contain tyrosine phosphorylated proteins were isolated by immunoprecipitation and identified by mass spectrometry (MS) at baseline and after a brief exposure to E2 for two hours. Bioinformatic network analyses of the identified protein interactions were then used to analyze E2 signaling pathways that trigger apoptosis versus survival. Comparison of MS data with a computationally-predicted AIB1 interaction network showed that 26 proteins identified in this study are within this network, and are involved in signal transduction, transcription, cell cycle regulation and protein degradation. Conclusions G-protein-coupled receptors, PI3 kinase, Wnt and Notch signaling pathways were most strongly associated with E2-induced proliferation or apoptosis and are integrated here into a global AIB1 signaling network that controls qualitatively distinct responses to estrogen.

Role of poly(ADP–ribose) polymerase (PARP) in DNA repair in sulfur mustard-exposed normal human epidermal keratinocytes (NHEK)†‡

We previously reported that, in normal human epidermal keratinocytes (NHEK) cultures exposed to the alkylating compound sulfur mustard (bis‐(2‐chloroethyl) sulfide, HD, 0.3–1 mM), there is a rapid (≤1 h) activation (100% above unexposed control) of the DNA repair enzyme DNA ligase I (130 kD) followed by a first‐order decay (1–5 h). The DNA ligase activation is accompanied by a time‐dependent (0.5–4 h) and significant DNA repair. Inhibition of another putative DNA repair enzyme, poly(ADP–ribose) polymerase (PARP), by using 3‐amino benzamide does not affect DNA ligase activation following HD exposure, but increases the half‐life of the activated enzyme threefold. To examine the role of PARP in HD‐induced DNA ligase activation and subsequent DNA repair, we conducted studies using cultured keratinocytes in which the level of PARP had been selectively lowered (≥85%) by the use of induced expression of antisense RNA. In these cells, there was no stimulation of DNA ligase up to 3 h, and a small stimulation (ca. 30% above unexposed control at 5–6 h after HD exposure. A time‐course (0.5–6 h) study of DNA repair in HD‐exposed PARP‐deficient keratinocytes revealed a much slower rate of repair compared with HD‐exposed NHEK. The results suggest an active role of PARP in DNA ligase activation and DNA repair in mammalian cells, and also indicate that modulation of PARP‐mediated mechanisms may provide a useful approach in preventing HD toxicity. Published in 2000 by John Wiley & Sons, Ltd.

Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.