Genotoxic Injury Research Articles

Critical Function for SIP, a Ubiquitin E3 Ligase Component of the β-Catenin Degradation Pathway, for Thymocyte Development and G1 Checkpoint

Beta-catenin has been implicated in thymocyte development because of its function as a coactivator of Tcf/LEF-family transcription factors. Previously, we discovered a novel pathway for p53-induced beta-catenin degradation through a ubiquitin E3 ligase complex involving Siah1, SIP (CacyBP), Skp1, and Ebi. To gain insights into the physiological relevance of this new degradation pathway in vivo, we generated mutant mice lacking SIP. We demonstrate here that SIP-/- thymocytes have an impaired pre-TCR checkpoint with failure of TCRbeta gene rearrangement and increased apoptosis, resulting in reduced cellularity of the thymus. Moreover, the degradation of beta-catenin in response to DNA damage is significantly impaired in SIP-/- cells. SIP-/- embryonic fibroblasts show a growth-rate increase resulting from defects in G1 arrest. Thus, the beta-catenin degradation pathway mediated by SIP defines an essential checkpoint for thymocyte development and cell-cycle progression.

The Role of Poly(ADP-ribose) Polymerase-1 in Ventilator-Induced Lung Injury

Background : Reactive oxygen species (ROS) take center stage as executers in ventilator-induced lung injury (VILI). The protein with DNA-damage scanning activity, poly (ADP-ribose) polymerase-1 (PARP1), signals DNA rupture and participates in base-excision repair. Paradoxically,overactivation of PARP1 in response to massive genotoxic injury such as ROS can induce cell death through -nicotinamide adenine dinucleotide () depletion, resulting in inflammation. The purpose of this study is to investigate the role of PARP1 and the effect of its inhibitor in VILI. Methods : Forty-eight male C57BL/6 mice were divided into sham, lung protective ventilation(LPV), VILI, and PARP1 inhibitor (PJ34)+VILI (PJ34+VILI) groups. Mechanical ventilator setting for the LPV group was + + RR 90/min + 2 hours. The VILI and PJ34+VILI groups were ventilated on a setting of + + RR 90/min + 2 hours. As a PARP1 inhibitor for the PJ34+VILI group, 20 mg/Kg of PJ34 was treated intraperitoneally 2 hours before mechanical ventilation. Wet-to-dry weight ratio and acute lung injury (ALI) score were measured to determine the degree of VILI. PARP1 activity was evaluated by using an immunohistochemical method utilizing biotinylated NAD. Myeloperoxidase (MPO) activity and the concentration of inflammatory cytokines such as tumor necrosis factor , interleukin , and IL-6 were measured in bronchoalveolar lavage fluid (BALF). Results : In the PJ34+VILI group, PJ34 pretreatment significantly reduced the degree of lung injury, compared with the VILI group (p, , and IL-6 were also significantly lower in the PJ34+VILI group (all, p

Liver cancer: the role of stem cells.

Studies of aggregation chimaeras and X-linked polymorphisms strongly suggest that liver tumours are derived from single cells (monoclonal), but the important question is, which cell? Stem cell biology and cancer are inextricably linked. In continually renewing tissues such as the gut mucosa and epidermis, where a steady flux of cells occurs from the stem cell zone to the terminally differentiated cells that are imminently to be lost, it is widely accepted that cancer is a disease of stem cells, since these are the only cells that persist in the tissue for a sufficient length of time to acquire the requisite number of genetic changes for neoplastic development. In the liver the identity of the founder cells for the two major primary tumours, hepatocellular carcinoma and cholangiocarcinoma, is more problematic. The reason for this is that no such obvious unidirectional flux occurs in the liver, although it is held that the centrilobular hepatocytes may be more differentiated (polyploid) and closer to cell senescence than those cells closest to the portal areas. Moreover, the existence of bipotential hepatic progenitor cells, along with hepatocytes endowed with longevity and long-term repopulating potential suggests there may be more than one type of carcinogen target cell. Cell proliferation at the time of carcinogen exposure is pivotal for 'fixing' any genotoxic injury into a heritable form, thus any proliferative cell in the liver can be susceptible to neoplastic transformation. Hepatocytes are implicated in many instances of hepatocellular carcinoma, direct injury to the biliary epithelium implicates cholangiocytes in some cases of cholangiocarcinoma, while hepatic progenitor cell/oval cell activation accompanies many instances of liver damage irrespective of aetiology, making such cells very likely carcinogen targets. Of course, we must qualify this assertion by stating that many carcinogens are both cytotoxic and cytostatic, and that hepatic progenitor cell proliferation may be merely a bystander effect of this toxicity. An in-depth discussion of causes of cancer in the liver is beyond the scope of this review, but infectious agents (e.g. hepatitis B and C viruses) play a major role, not just in transactivating or otherwise disrupting cellular proto-oncogenes (hepatitis B virus), but also in causing chronic inflammation (hepatitis C and B viruses). Sustained epithelial proliferation in a milieu rich in inflammatory cells, growth factors and DNA-damaging agents (reactive oxygen and nitrogen species--produced to fight infection), will lead to permanent genetic changes in proliferating cells. Up-regulation of the transcription factor NF-kappaB in transformed hepatocytes, through the paracrine action of TNF-alpha from neighbouring endothelia and inflammatory cells, may be critical for tumour progression given the mitogenic and antiapoptotic properties of proteins encoded by many of NF-kappaB's target genes.

β1-integrin-mediated signaling essentially contributes to cell survival after radiation-induced genotoxic injury

Integrin-mediated adhesion to extracellular matrix proteins confers resistance to radiation- or drug-induced genotoxic injury. To analyse the underlying mechanisms specific for beta1-integrins, wild-type beta1A-integrin-expressing GD25beta1A cells were compared to GD25beta1B cells, which express signaling-incompetent beta1B variants. Cells grown on fibronectin, collagen-III, beta1-integrin-IgG or poly-l-lysine were exposed to 0-6 Gy X-rays in presence or depletion of growth factors and phosphatidylinositol-3 kinase (PI3K) inhibitors (LY294002, wortmannin). In order to test the relevance of these findings in tumor cells, human A-172 glioma cells were examined under the same conditions after siRNA-mediated silencing of beta1-integrins. We found that beta1A-integrin-mediated adhesion to fibronectin, collagen-III or beta1-IgG was essential for cell survival after radiation-induced genotoxic injury. Mediated by PI3K, pro-survival beta1A-integrin/Akt signaling was critically involved in this process. Additionally, the beta1-integrin downstream targets p130Cas and paxillin-impaired survival-regulating PI3K-dependent JNK. In A-172 glioma cells, beta1-integrin knockdown and PI3K inhibition confirmed the central role of beta1-integrins in Akt- and p130Cas/paxillin-mediated prosurvival signaling. These findings suggest beta1-integrins as critical regulators of cell survival after radiation-induced genotoxic injury. Elucidation of the molecular circuitry of prosurvival beta1-integrin-mediated signaling in tumor cells may promote the development of innovative molecular-targeted therapeutic antitumor strategies.

Liver Stem Cells: Implications for Hepatocarcinogenesis

Numerous studies point to the fact that liver tumors are derived from single cells (monoclonal), but the important question is, which cell? Stem cell biology and cancer are inextricably linked. In continually renewing tissues such as the intestinal mucosa and epidermis, in which a steady flux of cells occurs from the stem cell zone to the terminally differentiated cells that are imminently to be lost, it is widely accepted that cancer is a disease of stem cells, as these are the only cells that persist in the tissue for a sufficient length of time to acquire the requisite number of genetic changes for neoplastic development. In the liver the identity of the founder cells for the two major primary tumors, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), is more problematic. The reason for this is that no such obvious unidirectional flux occurs in the liver, though it is held that the centrilobular hepatocytes may be more differentiated (polyploid) and closer to cell senescence than those cells closest to the portal areas. Moreover the existence of bipotential hepatic progenitor cells (HPCs), along with hepatocytes endowed with longevity and long-term repopulating potential suggests there may be more than one type of carcinogen target cell. Irrespective of which target cell is involved, cell proliferation at the time of carcinogen exposure is pivotal for "fixation" of the genotoxic injury into a heritable form. Taking this view, any proliferative cell in the liver can be susceptible to neoplastic transformation. Thus, hepatocytes are implicated in many instances of HCC, direct injury to the biliary epithelium implicates cholangiocytes in some cases of CC, whereas HPC/oval cell activation accompanies very many instances of liver damage irrespective of etiology, making such cells very likely carcinogen targets. Of course, we must qualify this assertion by stating that many carcinogens are both cytotoxic and cytostatic, and that HPC proliferation may be merely a bystander effect of this toxicity. An indepth discussion of causes of cancer in the liver are beyond the scope of this review, but infectious agents (e.g., hepatitis B and C viruses) play a major role, not just in transactivating or otherwise disrupting cellular proto-oncogenes (hepatitis B virus [HBV]), but in also causing chronic inflammation (hepatitis C virus [HCV] and HBV). Sustained epithelial proliferation in a milieu rich in inflammatory cells, growth factors, and DNA-damaging agents (reactive oxygen and nitrogen species produced to fight infection), will lead to permanent genetic changes in proliferating cells. The upregulation of the transcription factor nuclear factor kappaB (NF-kappaB) in transformed hepatocytes, through the paracrine action of tumor necrosis factor-alpha from neighboring endothelia and inflammatory cells, may be critical for tumor progression given the mitogenic and anti-apoptotic properties of proteins encoded by many of NF-kappaB's target genes.

Growth differentiation Factor-15/Macrophage inhibitory cytokine-1 induction through P53- and Tnf-independent mechanisms in liver, kidney, and lung injury

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent TGF-β family member overexpressed in numerous malignancies. Expression of MIC-1 correlates with disease stage in pancreatic and colon cancer and has been proposed as a potential diagnostic and prognostic biomarker. We have previously shown that the murine ortholog of MIC-1, Growth/Differentiation Factor-15 (GDF-15), is highly expressed in response to various acute liver injuries including those associated with subsequent carcinogenesis. Here we show that MIC-1 is overexpressed in chronic human liver disease, and furthermore, that GDF-15 is expressed in liver, kidney, and lung in various models of acute organ injury, including toxic/genotoxic, ischemic, surgical, and hyperoxic. We also show that Gdf15 is an immediate-early gene. GDF-15 expression after injury may be induced by the inflammatory response, since administration of TNF and, to a lesser extent IL-6, induced expression of Gdf15. Injury-induced Gdf15 expression was not reduced in mice deficient for both TNF receptor subtypes, however, suggesting that cytokine signaling is not required for GDF-15 expression. As well, although the stress sensor p53 is widely recognized to induce MIC-1 expression, injury-induced Gdf15 expression was unchanged in p53 null mice. Our results demonstrate that GDF-15 expression following injury is regulated by both paracrine (cytokine) and autocrine (p53) mechanisms, and in more tissues than previously recognized. Taken together with its described activities, these data suggest that MIC-1/GDF-15 production may regulate inflammation, cell proliferation, apoptosis, and perhaps migration in injured tissues, and moreover, may influence carcinogenesis and cancer progression in settings of genotoxic injury.

CUGBP2 plays a critical role in apoptosis of breast cancer cells in response to genotoxic injury.

Posttranscriptional control of gene expression plays a key role in regulating gene expression in cells undergoing apoptosis. Cyclooxygenase-2 (COX-2) is a crucial enzyme in the conversion of arachidonic acid to prostaglandin E2 (PGE(2)) and is significantly upregulated in many types of adenocarcinomas. COX-2 overexpression leads to increased PGE(2) production, resulting in increased cellular proliferation. PGE(2) enhances the resistance of cells to ionizing radiation. Accordingly, understanding mechanisms regulating COX-2 expression may lead to important therapeutic advances. Besides transcriptional control, COX-2 expression is significantly regulated by mRNA stability and translation. We have previously demonstrated that RNA binding protein CUGBP2 binds AU-rich sequences to regulate COX-2 mRNA translation. In the current study, we have determined that expression of both COX-2 mRNA and CUGBP2 mRNA are induced in MCF-7 cells, a breast cancer cell line, following exposure to 12 Gy gamma-irradiation. However, only CUGBP2 protein is induced, but COX-2 protein levels were not altered. Silencer RNA (siRNA)-mediated inhibition of CUGBP2 reversed the block in COX-2 protein expression. Furthermore, MCF-7 cells underwent apoptosis in response to radiation injury, which was also reversed by CUGBP2 siRNAs. These data suggest that CUGBP2 is a critical regulator of the apoptotic response to genotoxic injury in breast cancer cells.

Double-stranded RNA activates a p38 MAPK-dependent cell survival program in biliary epithelia.

Double-stranded RNA (dsRNA) is produced during replicative viral infection or genotoxic stress. Thus knowledge of the cellular response to dsRNA is necessary to understand the effects of DNA damage or viral infection in biliary epithelia. We assessed the effect of dsRNA on biliary epithelial cell proliferation and apoptosis and the role of the stress-activated p38 MAPK signaling pathway in these responses. dsRNA did not induce apoptosis or proliferation in Mz-ChA-1 human malignant cholangiocytes, but decreased cytotoxicity induced by camptothecin or tumor necrosis factor-related apoptosis inducing ligand and decreased activity of caspases 3, 8, and 9. Furthermore, dsRNA increased p38 MAPK and JNK kinase active site phosphorylation but had no effect on either MAPK kinase (MEK)1/2 or protein kinase R phosphorylation. Inhibition of p38 MAPK with SB-203580 increased basal caspase activity. Thus dsRNA stimulates a p38 MAPK-dependent cell-survival pathway in biliary epithelial cells that may modulate the response of the biliary epithelia to dsRNA produced during genotoxic injury or virus infection.

Transcriptional Activators Stimulate DNA Repair

To counteract the deleterious effects of genotoxic injury, cells have set up a sophisticated network of DNA repair pathways. We show that Gal4-VP16 and RAR transcriptional activators stimulate nucleotide excision repair (NER). This DNA repair activation is not coupled to transcription since it occurs in Cockayne syndrome cells (which are transcription-coupled repair deficient) and is observed in vitro in the presence of α-amanitin and in the absence of the basal transcription factors. Using a reconstituted dual incision assay, we also show that binding of activators to their cognate sequences induces a local chromatin remodeling mediated by ATP-driven chromatin remodeling and acetyltransferase activities to facilitate DNA repair.

Modulating poly (ADP-ribose) polymerase activity: potential for the prevention and therapy of pathogenic situations involving DNA damage and oxidative stress.

Poly (ADP-ribose) polymerase is a zinc-finger DNA-binding enzyme which detects and signals DNA strand breaks generated either directly during base excision repair, or indirectly by genotoxic agents such as oxygen radicals. In response to genotoxic injury, PARP catalyses the synthesis of poly (ADP-ribose), from its substrate beta-NAD+ and this polymer is covalently attached to several nuclear proteins and PARP itself. As a result, PARP converts DNA breaks into intracellular signals which activate DNA repair programs or cell death options. Several studies have also shown that PARP is involved in either necrosis and subsequent inflammation or apoptosis. Although this enzyme is not indispensable during the latter cell death program, it has been demonstrated that PARP plays a facilitating role in this process. PARP is activated at an intermediate stage of apoptosis and is then cleaved and inactivated at a late stage by apoptotic proteases, namely caspase-3/CPP-32/Yama/apopain and caspase-7. This cleavage prevents necrosis during apoptosis, avoiding inflammation. All these functions, and the observation that PARP is an abundant and highly conserved enzyme, suggest that this enzyme plays a pivotal role, particularly in the maintenance of genomic DNA stability, apoptosis and in the response to oxidative stress. Since these situations are found in cancer, inflammation, autoimmunity (such as diabetes), myocardial dysfunction, certain infections, ageing and radiation/chemical exposure, attempts have been made to modulate PARP activity. With regard to the increasing interest towards PARP, the aim of this review is to explain the cellular role of PARP and the advantages of modulating its activity in diverse preventive or therapeutic strategies.

TFIIH: from transcription to clinic

Once a large proportion of the genes responsible for genetic disorders are identified in the post-genome era, the fundamental challenge is to establish a genotype/phenotype relationship. Our aim is to explain how mutations in a given gene affect its enzymatic function and, in consequence, disturb the life of the cell. Genome integrity is continuously threatened by the occurrence of DNA damage arising from cellular exposure to irradiation and genotoxic chemicals. This mutagenic or potentially lethal DNA damage induces various cellular responses including cell cycle arrest, transcription alteration and processing by DNA repair mechanisms, such as the nucleotide excision repair (NER) pathway. Disruption of NER in response to genotoxic injuries results in autosomal recessive hereditary diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). One of the most immediate consequences of the induction of strand-distorting lesions is the arrest of transcription in which TFIIH plays a role in addition to its role in DNA repair. The observations made by clinicians close to XP, TTD and CS patients, suggested that transcription defects responsible for brittle hair and nails for TTD, or developmental abnormalities for CS, resulted from TFIIH mutations. Here a story will be related which could be called ‘a multi-faceted factor named TFIIH’. As biochemists, we have characterized each component of TFIIH, three of which are XPB and XPD helicases and cdk7, a cyclin-dependent kinase. With the help of structural biologists, we have characterized most of the specific three-dimensional structures of TFIIH subunits and obtained its electron microscopy image. Together these approaches help us to propose a number of structure–function relationships for TFIIH. Through transfection and microinjection assays, cell biology allows us to determine the role of TFIIH in transcription and NER. We are thus in a position to explain, at least in part, transcription initiation mechanisms and their coupling to DNA repair. We now know how the XPB helicase opens the promoter region for RNA synthesis and that one of the roles of XPD helicase is to anchor the cdk7 kinase to the core-TFIIH. In XP and CS associated patients, we have demonstrated that some XPD mutations prevent an optimal phosphorylation of nuclear receptors by cdk7 with, as a consequence, a drop in the expression of genes sensitive to hormone action. We have thus shown that hormonal responses operate through TFIIH. Careful analysis of each TFIIH subunit also shows how the p44 Ring finger participates in certain promoter escape reactions. We are also able to localize the action of TFIIH in the sequence of events that lead to the elimination of DNA lesions. Thanks to the combination of these different approaches we are obtaining a much clearer picture of the TFIIH complex and its integration into the life of the cell.

Siah-1, SIP, and Ebi Collaborate in a Novel Pathway for β-Catenin Degradation Linked to p53 Responses

Destruction of β-catenin is regulated through phosphorylation-dependent interactions with the F box protein β-TrCP. A novel pathway for β-catenin degradation was discovered involving mammalian homologs of Drosophila Sina (Siah), which bind ubiquitin-conjugating enzymes, and Ebi, an F box protein that binds β-catenin independent of the phosphorylation sites recognized by β-TrCP. A series of protein interactions were identified in which Siah is physically linked to Ebi by association with a novel Sgt1 homolog SIP that binds Skp1, a central component of Skp1–Cullin–F box complexes. Expression of Siah is induced by p53, revealing a way of linking genotoxic injury to destruction of β-catenin, thus reducing activity of Tcf/LEF transcription factors and contributing to cell cycle arrest.

Disruption of cyclooxygenase-1 gene results in an impaired response to radiation injury.

Prostaglandins may play an important role in regulating normal renewal of gastrointestinal epithelium, epithelial injury repair, and initiation or progression of intestinal neoplasia. Synthesis of prostaglandins is catalyzed by either of two cyclooxygenase isoforms, Cox-1 and Cox-2. Cox-1 is the predominant cyclooxygenase isoform found in the normal intestine. In contrast, Cox-2 is present at low levels in normal intestine but is elevated at sites of inflammation and in adenomas and carcinomas. To determine directly whether prostaglandins synthesized by Cox-1 or Cox-2 regulate crypt epithelial cell fate after genotoxic or cytotoxic injury, we examined apoptosis, prostaglandin synthesis, and crypt stem cell survival after gamma-irradiation in Cox-1(-/-) and Cox-2(-/-) mice. Cox-1(-/-) mice had increased crypt epithelial cell apoptosis and decreased clonogenic stem cell survival compared with wild-type littermates. PGE(2) synthesis was also diminished in Cox-1(-/-) mice compared with wild-type controls in unstressed intestine and after radiation injury. In contrast, apoptosis, stem cell survival, and intestinal PGE(2) synthesis in Cox-2(-/-) mice after irradiation were the same as in wild-type littermates. Crypt stem cell survival after irradiation was inhibited by a highly specific neutralizing antibody to PGE(2), suggesting that this prostaglandin mediates stem cell fate in vivo. These data suggest that prostaglandins synthesized by Cox-1 regulate multiple steps that determine the fate of crypt epithelial cell after genotoxic or cytotoxic injury.

How does radiation kill cells?

Recent advances in the understanding of intracellular signaling after genotoxic injury have led to a better understanding of the pathways that influence radiation-induced cell death. Particular progress has been made in defining molecular controls of apoptosis and radiation-induced cell cycle arrest, as well as the possible role of telomerase activity in stabilizing DNA breaks.

P53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1.

The Drosophila seven in absentia (sina) gene is required for R7 photoreceptor cell formation during Drosophila eye development, where it functions within the Ras/Raf pathway and targets other proteins for degradation via associations with a ubiquitin-conjugating enzyme. Recently, a mammalian sina homologue was reported to be a p53-inducible gene in a myeloid leukemia cell line. To explore the function of human SINA-homologous (Siah) proteins, expression plasmids encoding Siah-1A were transiently transfected into 293 epithelial cells and GM701 fibroblast cells, resulting in growth arrest without induction of apoptosis. We discovered that BAG-1, a ubiquitin-like Hsp70/Hsc70-regulating protein, is a negative regulator of Siah-1A. Siah-1A was identified as a BAG-1-binding protein via yeast two-hybrid methods. Specific interaction of BAG-1 with Siah-1A was also demonstrated by in vitro binding experiments using glutathione S-transferase fusion proteins and co-immunoprecipitation studies. Siah-1A-induced growth arrest in 293 and GM701 cells was abolished by co-transfection of wild-type BAG-1 with Siah-1A but not by a C-terminal deletion mutant of BAG-1 that fails to bind Siah-1A. Over-expression of BAG-1 significantly inhibited p53-induced growth arrest in 293 cells without preventing p53 transactivation of reporter gene plasmids. BAG-1 also prevented growth arrest following UV-irradiation-induced genotoxic injury without interfering with accumulation of p53 protein or p21(waf-1) expression. BAG-1 functions downstream of p53-induced gene expression to inhibit p53-mediated suppression of cell growth, presumably by suppressing the actions of Siah-1A. We suggest that Siah-1A may be an important mediator of p53-dependent cell-cycle arrest and demonstrate that Siah-1A is directly inhibited by BAG-1.

Age-dependent change of metabolic capacity and genotoxic injury in rat intestine

The ontogeny of intestinal phase I and II xenobiotic metabolising enzymes and influence on susceptibility to genotoxic injury, are unclear. This study assessed expression of cytochrome P450 monooxygenases (CYP1A, CYP2B, CYP2C, CYP3A, CYP4A), glutathione- S-transferase (GSTA1/2, GSTA3, GSTA4 and GSTM1), and uridine diphosphate glucuronosyl transferase (UGT) in rat intestine, between fetal life and maturity. Enzyme induction and DNA adduct formation were assessed after 3-methylcholanthrene (MC) exposure. Untreated rat intestine expressed CYP2B, GSTA1/2, GSTA4 and UGT at all stages of maturation, although CYP2B and GSTA1/2 increased in postnatal life. MC induced new expression of CYP1A, GSTA3 and enhanced expression of GSTA1/2 and UGT. Age-dependent differences of enzyme induction and DNA adduct formation between pre- and postnatal intestine and during postnatal maturation, were observed. Rat intestinal epithelium shows variable competence for MC metabolism and sustains disparate levels of DNA adducts during pre- and postnatal development.

Bax Involvement in p53-Mediated Neuronal Cell Death

The tumor suppressor gene p53 has been implicated in the loss of neuronal viability, but the signaling events associated with p53-mediated cell death in cortical and hippocampal neurons are not understood. Previous work has shown that adenovirus-mediated delivery of the p53 gene causes cortical and hippocampal neuronal cell death with some features typical of apoptosis. In the present study we determined whether p53-initiated changes in neuronal viability were dependent on members of the Bcl-2 family of cell death regulators. Primary cultures of cortical neurons were derived from animals containing Bax (+/+ and +/-) or those deficient in Bax (-/-). Cell damage was assessed by direct cell counting and by measurements of MTT activity. Neurons containing at least one copy of the Bax gene were damaged severely by exposure to excitotoxins or by the induction of DNA damage. In contrast, Bax-deficient neurons (-/-) exhibited significant protection from both types of injury. Bax protein expression was elevated significantly by glutamate exposure, but not by camptothecin-induced DNA damage in wild-type neurons. The glutamate-induced increase in Bax protein was dependent on the presence of the p53 gene. However, increased p53 expression, using adenovirus-mediated transduction, was not sufficient by itself to elevate Bax protein levels. These results demonstrate that Bax is required for neuronal cell death in response to some forms of cytotoxic injury and further support the key role for p53 activation in response to excitotoxic and genotoxic injury.

Metabolic competence and susceptibility of intestinal epithelium to genotoxic injury during regeneration

The carcinogenic potency of many mutagens is increased in conditions of tissue regeneration. This involves fundamental changes of cellular division and differentiation, in intestinal epithelium. However, effects on epithelial capacity for carcinogen metabolism and susceptibility to genotoxic injury are unknown. Using a novel rat model, this study assessed expression of cytochrome P450 mono-oxygenases (Cyps), glutathione S-transferases (GSTs) and uridine diphosphoglucuronosyl transferase (UGT) in intestinal epithelium during sequential stages of regeneration. Enzyme induction and DNA adduct formation were also assessed after benzo[a]pyrene (BaP) exposure. Control assays were carried out in normal intestinal epithelium. Fewer phase I and II xenobiotic metabolizing enzymes were expressed in regenerating intestinal epithelium than in normal control intestinal epithelium (GSTA3, UGT in regeneration vs Cyp2B, GSTA1/2, GSTA4, GSTP1, UGT in control). Benzo[a]pyrene induced GSTA3 and UGT in regeneration vs Cyp1A, Cyp2B, GSTA1/2, GSTA3, GSTA4, GSTP1 and UGT in control normal intestinal epithelium. Benzo[a]pyrene induced low levels of GSTA3 in early regenerating intestinal epithelium but induction increased by >2-fold at late stage regeneration. Higher levels of benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE) DNA adducts were formed at early stages of regeneration, than at later stages. Intestinal epithelium displayed reduced metabolic competence and differential susceptibility to genotoxic injury from BaP, during regeneration.

UV but not gamma-irradiation induces specific transcriptional activity of p53 in primary hepatocytes.

The mechanisms are poorly understood by which p53 can stimulate different downstream events, including growth arrest, DNA repair, and apoptosis, after DNA damage. Changes in protein levels do not predict a particular p53 response, but it is possible that differences in functional activities such as transactivation are important. The present report describes the successful use of a specific p53 reporter plasmid transfected into primary murine hepatocytes to evaluate p53 transactivation activity over time after two different genotoxic injuries (gamma-irradiation, 15 Gy and UV-c irradiation, 10 J/m2) known to produce p53-dependent growth arrest in this cell type. The results show that UV injury to hepatocytes was followed by a transient increase in transcriptional activation of the reporter plasmid by p53 and that this response preceded changes in p53 protein levels, as assessed by immunocytochemistry. By contrast, gamma-irradiation injury failed to induce detectable changes in either transactivation activity or hepatocyte p53 protein levels. The data show that p53 responses to DNA damage are dependent on both cell and injury type and suggest that in hepatocytes they can be independent of protein concentration and specific transcriptional activity. The results have implications for how particular dysfunctional p53 mutations in carcinogenesis could alter hepatocyte responses to different DNA injuries.

Specific Triggering of the Fas Signal Transduction Pathway in Normal Human Keratinocytes

The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.