Id3 induces a caspase-3- and -9-dependent apoptosis and mediates UVB sensitization of HPV16 E6/7 immortalized human keratinocytes.

Inhibitor of differentiation/DNA binding (Id) proteins comprise a class of helix-loop-helix transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. We have shown that while Id2 is induced by UVB in primary keratinocytes, Id3 is upregulated only in immortalized cells. We have now determined that the consequences of ectopic expression of Id3 protein are strikingly different between immortalized and primary keratinocytes. Overexpression of Id3 induces a significant increase in apoptotic cells as revealed by Annexin V positivity as well as proteolytic processing of caspase-3 in immortalized, but not in primary keratinocytes. Id3-green fluorescent protein (GFP)-positive cells exhibited a fivefold increase in apoptotic nuclear fragmentation compared to Id3-GFP-negative cells. These apoptotic responses were accompanied by activation of caspase-3, as shown by immunocytochemical staining with antibodies to active caspase-3. Immunostaining with antibodies to the active form of caspase-9 as well as to the active form of Bax further revealed that induction of apoptosis in Id3-overexpressing keratinocytes occurred via a mitochondrial-caspase-9-mediated pathway. Coexpression of dominant-negative caspase-9 with Id3 significantly suppressed apoptotic nuclear fragmentation, indicating that caspase-9 activation is essential for Id3-induced cell death. This response was also markedly attenuated by coexpression with the Bax antagonist antiapoptotic protein Bcl2, confirming a role for Bax activation in this apoptotic response. Id3-induced Bax activation may result from increased expression of Bax protein. Furthermore, reduction of Id3 expression by small interfering RNAs abrogated the UVB-induced proteolytic activation of caspase-3 in these cells. These data together suggest that UVB-induced apoptosis of immortalized keratinocytes is at least in part due to Id3 upregulation in these cells.

Poly(ADP-ribosyl)ation of p53 in vitro and in vivo modulates binding to its DNA consensus sequence.

The tumor-suppressor p53 undergoes extensive poly(ADP-ribosyl)ation early during apoptosis in human osteosarcoma cells, and degradation of poly(ADP-ribose) (PAR) attached to p53 coincides with poly(ADP-ribose)polymerase-1, (PARP-1) cleavage, and expression of p53 target genes. The mechanism by which poly(ADP-ribosyl)ation may regulate p53 function has now been investigated. Purified wild-type PARP-1 catalyzed the poly(ADP-ribosyl) of full-length p53 in vitro. In gel supershift assays, poly(ADP-ribosyl)ation suppressed p53 binding to its DNA consensus sequence; however, when p53 remained unmodified in the presence of inactive mutant PARP-1, it retained sequence-specific DNA binding activity. Poly(ADP-ribosyl)ation of p53 by PARP-1 during early apoptosis in osteosarcoma cells also inhibited p53 interaction with its DNA consensus sequence; thus, poly(ADP-ribosyl)ation may represent a novel means for regulating transcriptional activation by p53 in vivo.

Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy.

Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP(-/-) fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP(-/-) fibroblasts were exposed for 3 weeks to 20 microM GPI 6150 (1,11b-dihydro-[2H:]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (K(i) = 60 nM) and one of the most potent PARP inhibitors to date (IC(50) = 0.15 microM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by approximately 91%, to about the residual levels in PARP(-/-) cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.

Mechanisms of JP-8 jet fuel cell toxicity. II. Induction of necrosis in skin fibroblasts and keratinocytes and modulation of levels of Bcl-2 family members.

JP-8 induces apoptosis in rat lung epithelial cells, primary mouse T lymphocytes, Jurkat T lymphoma cells, and U937 monocytic cells (Stoica et al., 2001). Here, we have observed a different mechanism of cytotoxicity in human keratinocytes grown in culture as well as when grafted onto nude mice. At lower levels of JP-8 (80 microg/ml; 1 x 10(-4) dilution), sufficient to induce apoptosis in other cell types, including lung epithelial cells (Stoica et al., 2001), no apoptosis was observed. At higher levels (>200 microg/ml; 2.5 x 10(-4) dilution), JP-8 is cytotoxic to both primary and immortalized human keratinocytes, as evidenced by the metabolism of calcein, as well as by morphological changes such as cell rounding and cell detachment. There was no evidence of activation of caspases-3, -7, or -8 either by enzyme activity or immunoblot analysis, and the stable expression of a dominant-negative inhibitor of apoptosis (FADD-DN) did not increase the survival of keratinocytes to JP-8. The pattern of poly(ADP-ribose) polymerase (PARP) cleavage was also characteristic of necrosis. PARP has been also been implicated in necrosis via its ability to lower levels of ATP in damaged cells. However, fibroblasts derived from PARP-/- mice underwent necrotic cell death similar to those derived from PARP+/+ mice, indicating that the effects of JP-8 are independent of PARP. Immunoblot analysis further revealed that exposure of keratinocytes to the toxic higher levels of JP-8 markedly downregulates the expression of the prosurvival members of the Bcl-2 family, Bcl-2 and Bcl-x(L), and upregulates the expression of antisurvival members of this family, including Bad and Bak. Bcl-2 and Bcl-x(L) have been shown to preserve mitochondrial integrity and suppress cell death. In contrast, Bak and Bad both promote cell death by alteration of the mitochondrial membrane potential, in part by heterodimerization with and inactivation of Bcl-2 and Bcl-x(L), and either inducing necrosis or activating a downstream caspase program. High intrinsic levels of Bcl-2 and Bcl-x(L) may prevent apoptotic death of keratinocytes at lower levels of JP-8, while perturbation of the balance between pro- and antiapoptotic Bcl-2 family members at higher levels may ultimately play a role in necrotic cell death in human keratinocytes. Finally, when human keratinocytes were grafted to form a human epidermis on nude mice, treatment of these grafts with JP-8 revealed cytotoxicity and altered histology in vivo.

PARP determines the mode of cell death in skin fibroblasts, but not keratinocytes, exposed to sulfur mustard.

Sulfur mustard is cytotoxic to dermal fibroblasts as well as epidermal keratinocytes. We demonstrated that poly(ADP-ribose) polymerase (PARP) modulates Fas-mediated apoptosis, and other groups and we have shown that PARP plays a role in the modulation of other types of apoptotic and necrotic cell death. We have now utilized primary dermal fibroblasts, immortalized fibroblasts, and keratinocytes derived from PARP(-/-) mice and their wildtype littermates (PARP(+/+)) to determine the contribution of PARP to sulfur mustard toxicity. Following sulfur mustard exposure, primary skin fibroblasts from PARP-deficient mice demonstrated increased internucleosomal DNA cleavage, caspase-3 processing and activity, and annexin V positivity, compared to those derived from PARP(+/+) animals. Conversely, propidium iodide staining, PARP cleavage patterns, and random DNA fragmentation revealed a dose-dependent increase in necrosis in PARP(+/+) but not PARP(-/-) cells. Using immortalized PARP(-/-) fibroblasts stably transfected with the human PARP cDNA or with empty vector alone, we show that PARP inhibits markers of apoptosis in these cells as well. Finally, primary keratinocytes were derived from newborn PARP(+/+) and PARP(-/-) mice and immortalized with the E6 and E7 genes of human papilloma virus. In contrast to fibroblasts, keratinocytes from both PARP(-/-) and PARP(+/+) mice express markers of apoptosis in response to sulfur mustard exposure. The effects of PARP on the mode of cell death in different skin cell types may determine the severity of vesication in vivo, and thus have implications for the design of PARP inhibitors to reduce sulfur mustard pathology.

Misregulation of gene expression in primary fibroblasts lacking poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP) is implicated in the maintenance of genomic integrity, given that inhibition or depletion of this enzyme increases genomic instability in cells exposed to genotoxic agents. We previously showed that immortalized fibroblasts derived from PARP(-/-) mice exhibit an unstable tetraploid population, and partial chromosomal gains and losses in PARP(-/-) mice and immortalized fibroblasts are accompanied by changes in the expression of p53, Rb, and c-Jun, as well as other proteins. A tetraploid population has also now been detected in primary fibroblasts derived from PARP(-/-) mice. Oligonucleotide microarray analysis was applied to characterize more comprehensively the differences in gene expression between asynchronously dividing primary fibroblasts derived from PARP(-/-) mice and their wild-type littermates. Of the 11,000 genes monitored, 91 differentially expressed genes were identified. The loss of PARP results in down-regulation of the expression of several genes involved in regulation of cell cycle progression or mitosis, DNA replication, or chromosomal processing or assembly. PARP deficiency also up-regulates genes that encode extracellular matrix or cytoskeletal proteins that are implicated in cancer initiation or progression or in normal or premature aging. These results provide insight into the mechanism by which PARP deficiency impairs mitotic function, thereby resulting in the genomic alterations and chromosomal abnormalities as well as in altered expression of genes that may contribute to genomic instability, cancer, and aging.

Identification of poly(ADP-ribose) polymerase as a transcriptional coactivator of the human T-cell leukemia virus type 1 Tax protein.

Human T-cell leukemia virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose activity is believed to contribute significantly to cellular transformation. Tax stimulates transcription from the proviral promoter as well as from promoters for a variety of cellular genes. The mechanism through which Tax communicates to the general transcription factors and RNA polymerase II has not been completely determined. We investigated whether Tax could function directly through the general transcription factors and RNA polymerase II or if other intermediary factors or coactivators were required. Our results show that a system consisting of purified recombinant TFIIA, TFIIB, TFIIE, TFIIF, CREB, and Tax, along with highly purified RNA polymerase II, affinity-purified epitope-tagged TFIID, and semipurified TFIIH, supports basal transcription of the HTLV-1 promoter but is not responsive to Tax. Two additional activities were required for Tax to stimulate transcription. We demonstrate that one of these activities is poly(ADP-ribose) polymerase (PARP), a molecule that has been previously identified to be the transcriptional coactivator PC1. PARP functions as a coactivator in our assays at molar concentrations approximately equal to those of the DNA and equal to or less than those of the transcription factors in the assay. We further demonstrate that PARP stimulates Tax-activated transcription in vivo, demonstrating that this biochemical approach has functionally identified a novel target for the retroviral transcriptional activator Tax.

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 microM 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, 3 and PARP-sensitive pathways.

Chromosomal aberrations in PARP(-/-) mice: genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA.

Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP(-/-) mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP(-/-)mice and immortalized PARP(-/-)fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP(-/-) cells stably transfected with PARP cDNA [PARP(-/-)(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP(-/-) cells, p53 expression was partially restored in PARP(-/-) (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP(-/-) mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.

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

E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol alpha, 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 alpha and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol alpha 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 alpha activity assays revealed the presence of pol alpha 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 alpha expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum.

Poly(ADP-ribosyl)ation of p53 during apoptosis in human osteosarcoma cells.

Spontaneous apoptosis in human osteosarcoma cells was observed to be associated with a marked increase in the intracellular abundance of p53. Immunoprecipitation and immunoblot analysis revealed that, together with a variety of other nuclear proteins, p53 undergoes extensive poly(ADP-ribosyl)ation early during the apoptotic program in these cells. Subsequent degradation of poly(ADP-ribose) (PAR), attached to p53 presumably by PAR glycohydrolase, the only reported enzyme to degrade PAR, was apparent concomitant with the onset of proteolytic processing and activation of caspase-3, caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation during the later stages of cell death. The decrease in PAR covalently bound to p53 also coincided with the marked induction of expression of the p53-responsive genes bax and Fas. These results suggest that poly(ADP-ribosyl)ation may play a role in the regulation of p53 function and implies a regulatory role for PARP and/or PAR early in apoptosis.

Poly(ADP-ribose) polymerase activation mediates 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that causes parkinsonism in humans and nonhuman animals, and its use has led to greater understanding of the pathogenesis of Parkinson's disease. However, its molecular targets have not been defined. We show that mice lacking the gene for poly(ADP-ribose) polymerase (PARP), which catalyzes the attachment of ADP ribose units from NAD to nuclear proteins after DNA damage, are dramatically spared from MPTP neurotoxicity. MPTP potently activates PARP exclusively in vulnerable dopamine containing neurons of the substantia nigra. MPTP elicits a novel pattern of poly(ADP-ribosyl)ation of nuclear proteins that completely depends on neuronally derived nitric oxide. Thus, NO, DNA damage, and PARP activation play a critical role in MPTP-induced parkinsonism and suggest that inhibitors of PARP may have protective benefit in the treatment of Parkinson's disease.

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 approximately 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 approximately 40 MRC proteins, including DNA pol alpha, DNA topo I, and PCNA. Depletion of endogenous PARP by antisense RNA expression in 3T3-L1 cells results in MRCs devoid of any DNA pol alpha and DNA pol delta activities. Surprisingly, there was no new expression of PCNA and DNA pol alpha, 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.

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments.

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.

The E7 protein of human papillomavirus type 16 sensitizes primary human keratinocytes to apoptosis.

The 'high risk' human papillomaviruses are associated with the development of anogenital carcinomas and their E6 and E7 genes possess immortalizing and transforming functions in several in vitro culture systems. Recently the E6 gene has also been shown to enhance the apoptosis of human mammary epithelial cells. To determine the apoptotic activity of these oncogenes in the natural host cell, we infected genital keratinocytes with retroviruses expressing either HPV-16 E6, E7, or both the E6 and E7 (E6/7) genes. Apoptosis was quantitated under normal growth conditions or when induced by tumor necrosis factor alpha/cycloheximide or sulfur mustard. In contrast to previous findings with mammary epithelial cells, the E6 gene did not significantly augment either spontaneous or induced apoptosis. E6 also did not suppress apoptosis in normal keratinocytes (despite dramatically reducing their p53 levels), suggesting that p53-independent events mediated this effect. In contrast, E7 increased both spontaneous and induced apoptosis as well as the cellular levels of p53 and p21 protein. Interestingly, co-expression of E6 abrogated E7-facilitated apoptosis by tumor necrosis factor alpha nearly completely, but had only a minor protective effect on sulfur mustard induced apoptosis in these cells, demonstrating at least in part the p53-dependence and -independence of these two apoptotic pathways. Finally, our results indicate that the apoptosis of normal and E7-expressing keratinocytes is differentially affected by E6 expression and that E7, when unaccompanied by E6, sensitizes keratinocytes to apoptosis.

Regulation of the expression or recruitment of components of the DNA synthesome by poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.

Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a Ca2+-calmodulin and caspase-dependent pathway.

Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.

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.

Prolongation of the p53 response to DNA strand breaks in cells depleted of PARP by antisense RNA expression.

The observation that 3-aminobenzamide, which inhibits a variety of ADP-ribose transferases, prolongs the gamma-irradiation-induced increase in intracellular p53 concentration suggested that one or more of such enzymes may determine the duration of the p53 response during G1 arrest. The role of poly(ADP-ribose) polymerase (PARP), an abundant nuclear enzyme activated by DNA strand breaks, in the p53 response to y-irradiation was investigated in Burkitt's lymphoma AG876 cells stably transfected with an inducible PARP antisense construct. Immunoblot analysis revealed that the cellular content of PARP was reduced to virtually undetectable levels after incubation of transfected cells for 72 h with the inducer dexamethasone. In noninduced antisense cells, the p53 concentration reached a maximum 2 h after exposure to 6.3 Gy of gamma-radiation and returned to control values by 4 h. In contrast, the p53 response in PARP-depleted antisense cells peaked at 4 h, with the levels of p53 remaining elevated for up to 12 h after y-irradiation. The maximal increase in p53 concentration was similar in both induced and noninduced cells. These results thus indicate that PARP activity, in part, determines the duration, but not the magnitude, of the p53 response to DNA damage.