LOCALIZATION OF DNA REPAIR FUNCTIONS TO THE NUCLEAR MATRIX

The nucleus of a eukaryotic cell contains both structural and functional elements that contribute to the controlled operation of the cell. In this context, functional components refers to those nuclear constituents that perform metabolic activities such as DNA replication and RNA transcription. Structural nuclear components, designated nuclear matrix, organize the DNA into loops or domains and appear to provide a framework for nuclear DNA organization. However, the boundary between structural and functional components is not clear cut as evinced by reports of associations between metabolic functions and the nuclear matrix. The studies reported here attempt to determine the relationship of another nuclear function, DNA repair, to the nuclear matrix.^ One objective of these studies was to study the initiation of DNA repair by directly measuring the UV-incision activities in human cells and determine the influence of various extractable nuclear components on these activities. The assay for incision activities required the development of a nuclear isolation protocol that produced nuclei with intact DNA; the conformation of the nuclear DNA and its physical characteristics in response to denaturing conditions were determined.^ The nuclei produced with this protocol were then used as substrates for endogenous UV-specific nuclease activities. The isolated nuclei were shown to contain activities that cause breaks in nuclear DNA in response to UV-irradiation. These UV-responsive activities were tightly associated with nuclear components, being unextractable with salt concentration of up to 0.6 M.^ The tight association of the incision activities with salt-extracted nuclei suggested that other repair function might also be associated with salt-stable components of the nucleus. The site of unscheduled DNA synthesis (UDS) was determined in salt-extracted nuclei (nucleoids) using autoradiography and fluorescent microscopy. UDS was found to occur in association with the nuclear matrix following low-doses (2.55 J/M('2)) of ultraviolet light, but the association became looser after higher doses of ultraviolet light (10-30 J/m('2)). ^

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

BACKGROUND: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. PRINCIPAL FINDINGS: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2. SIGNIFICANCE: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved 'O-helix' of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.

{\it In vivo\/} induction of DNA changes in cervicovaginal epithelium by perinatal estrogen exposure

Epidemiological studies have associated estrogens with human neoplasm such as the endometrium, cervix, vagina, breast, and liver. Perinatal exposure to natural (17$\beta$-estradiol (17$\beta$-E$\sb2)\rbrack$ and synthetic (diethylstilbestrol (DES)) estrogens induces neoplastic changes in humans and rodents. Previous studies demonstrated that neonatal 17$\beta$-E$\sb2$ treatment increased the nuclear DNA content of mouse cervicovaginal epithelium that preceded histologically evident neoplasia. In order to determine whether this effect was specific to 17$\beta$-E$\sb2,$ associated with chromosomal changes, and relevant to the human, female BALB/c mice were treated neonatally with either 17$\alpha$-estradiol (17$\alpha$-E$\sb2)$ and 5$\beta$-dihydrotestosterone ($5\beta$-DHT), both inactive steroids in adult reproductive tissue, or 17$\beta$-E$\sb2.$ Ten-day-old mice received pellet implants of 17$\beta$-E$\sb2,$ 17$\alpha$-E$\sb2,$ $5\beta$-DHT, or cholesterol. Seventy-day-old cervicovaginal tracts were examined histologically and flow cytometrically. 17$\beta$-E$\sb2$-treated animals were evaluated by fluorescent in situ hybridization (FISH) using a probe specific for chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was evaluated by FISH in cervicovaginal material from 19 DES-exposed and 19 control patients.^ $17\beta$-E$\sb2, 17\alpha$-E$\sb2$, and $5\beta$-DHT-induced dramatic developmental and histological changes in the cervicovaginal tract, including hypospadia, hyperplasia, and persistent cornification. The changes induced by 17$\alpha$-E$\sb2$ were equivalent to 17$\beta$-E$\sb2.$ Neonatal 17$\alpha$-E$\sb2$-induced adenosquamous cervicovaginal tumors at 24 months. 17$\alpha$-E$\sb2$ and $5\beta$-DHT significantly increased the nuclear DNA content over control animals, but at significantly lower levels than 17$\beta$-E$\sb2.$ DNA ploidy changes were highest (80%) in animals treated neonatally and secondarily with 17$\beta$-E$\sb2.$ Secondary 17$\alpha$-E$\sb2$ and $5\beta$-DHT administration, unlike 17$\beta$-E$\sb2,$ didn't significantly increase DNA content. Chromosome 1 trisomy incidence was 66% in neonatal 17$\beta$-E$\sb2$-treated animals. Trisomy was evident in 4 DES-exposed patients: one patient with trisomy of chromosomes 1, 7, and 11; one patient with chromosome 7 trisomy; and two patients with chromosome 1 trisomy. These data demonstrated the biological effects of 17$\alpha$-E$\sb2$ and $5\beta$-DHT were age-dependent, 17$\alpha$-E$\sb2$ was equivalent to 17$\beta$-E$\sb2$ and tumorigenic when administered neonatally, and histological changes were not steroid specific. Chromosomal changes were associated with increased nuclear DNA content and chromosomal changes may be an early event in the development of tumors in human DES-exposed tissues. ^

Statistical characterization and development of summary measures of non-normal distributions of nuclear morphometry data from prostate cancer cells for use as prognostic indicators

Nuclear morphometry (NM) uses image analysis to measure features of the cell nucleus which are classified as: bulk properties, shape or form, and DNA distribution. Studies have used these measurements as diagnostic and prognostic indicators of disease with inconclusive results. The distributional properties of these variables have not been systematically investigated although much of the medical data exhibit nonnormal distributions. Measurements are done on several hundred cells per patient so summary measurements reflecting the underlying distribution are needed.^ Distributional characteristics of 34 NM variables from prostate cancer cells were investigated using graphical and analytical techniques. Cells per sample ranged from 52 to 458. A small sample of patients with benign prostatic hyperplasia (BPH), representing non-cancer cells, was used for general comparison with the cancer cells.^ Data transformations such as log, square root and 1/x did not yield normality as measured by the Shapiro-Wilks test for normality. A modulus transformation, used for distributions having abnormal kurtosis values, also did not produce normality.^ Kernel density histograms of the 34 variables exhibited non-normality and 18 variables also exhibited bimodality. A bimodality coefficient was calculated and 3 variables: DNA concentration, shape and elongation, showed the strongest evidence of bimodality and were studied further.^ Two analytical approaches were used to obtain a summary measure for each variable for each patient: cluster analysis to determine significant clusters and a mixture model analysis using a two component model having a Gaussian distribution with equal variances. The mixture component parameters were used to bootstrap the log likelihood ratio to determine the significant number of components, 1 or 2. These summary measures were used as predictors of disease severity in several proportional odds logistic regression models. The disease severity scale had 5 levels and was constructed of 3 components: extracapsulary penetration (ECP), lymph node involvement (LN+) and seminal vesicle involvement (SV+) which represent surrogate measures of prognosis. The summary measures were not strong predictors of disease severity. There was some indication from the mixture model results that there were changes in mean levels and proportions of the components in the lower severity levels. ^

AN ANALYSIS OF THE DNA DAMAGE INDUCED BY NICKEL COMPOUNDS IN CHINESE HAMSTER OVARY CELLS (CARCINOGENESIS, HETEROCHROMATIN, CYTOTOXICITY, REPLICATION, PROTEIN CROSSLINKING)

The carcinogenic activity of water-insoluble crystalline nickel sulfide requires phagocytosis and lysosome-mediated intracellular dissolution of the particles to yield Ni('2+). This study investigated the extent and nature of the DNA damage in Chinese hamster ovary cells treated with various nickel compounds using the technique of alkaline elution. Crystalline NiS and water-soluble NiCl(,2) induced single strand breaks that were repaired quickly and DNA-protein crosslinks that persisted up to 24 hr after exposure to nickel. The induction of single strand breaks was concentration dependent at both noncytotoxic and lethal amounts of nickel. The induction of DNA-protein crosslinks was concentration dependent but was absent at lethal amounts of nickel. The cytoplasmic and nuclear uptake of nickel was concentration dependent even at the toxic level of nickel. However, the induction of DNA-protein crosslinks by nickel required active cell cycling and occurred predominantly in mid-late S phase of the cell cycle, suggesting that the lethal amounts of nickel inhibited DNA-protein crosslinking by inhibiting active cell cycling. Since the DNA-protein crosslinking induced by nickel was resistant to DNA repair, the nature of this lesion was investigated using various methods of DNA isolation and chromatin fractionation in combination with SDS-polyacrylamide gel electrophoresis. High molecular weight, non-histone chromosomal proteins and possibly histone 1 were preferentially crosslinked to DNA by nickel. The crosslinked proteins were concentrated in a magnesium-insoluble fraction of sonicated chromatin (5% of the total) that was similar to heterochromatin in solubility and protein composition. Alterations in DNA structure and function, brought about by the effect of nickel on protein-DNA interactions, may be related to the carcinogenicity of nickel compounds. ^

Elucidation of the role of 53BP1 in DNA double strand break (DSB) repair and tumor suppression

The protein p53 binding protein one (53BP1) was discovered in a yeast two-hybrid screen that used the DNA binding domain of p53 as bait. Cloning of full-length 53BP1 showed that this protein contains several protein domains which help make up the protein, which include two tandem BRCT domains and a amino-terminal serine/glutamine cluster domain (SCD). These are two protein domains are often seen in factors that are involved in the cellular response to DNA damage and control of cell cycle checkpoints and we hypothesize that 53BP1 is involved in the cellular response to DNA damage. In support of this hypothesis we observe that 53BP1 is phosphorylated and undergoes a dramatic nuclear re-localization in response to DNA damaging agents. 53BP1 also interacts with several factors that are important in the cellular response to DNA damage, such as the BRCA1 tumor suppressor, ATM and Rad3 related (ATR), and the phosphorylated version of the histone variant H2AX. Mice deficient in 53BP1 display increased sensitivity ionizing radiation (IR), a DNA damaging agent that introduces DNA double strand breaks (DSBs). In addition, 53BP1-deficient mice do not properly undergo the process of class switch recombination (CSR). We also observe that when a defect in 53BP1 is combined with a defect in p53; the resulting mice have an increased rate of formation of spontaneous tumors, notably the formation of B and T lineage lymphomas. The T lineage tumors arise by two distinct mechanisms: one driven by defects in cell cycle regulation and a second driven by defects in the ability to repair DNA DSBs. The B lineage tumors arise by the inability to repair DNA damage and over-expression of the oncogene c-myc. ^ With these observations, we conclude that not only does 53BP1 function in the cellular response to DNA damage, but it also works in concert with p53 to suppress tumor formation. ^

The molecular mechanisms of sensing nucleoside analogue-induced DNA damage

Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematological malignancies. Upon being metabolized to their active triphosphate form, these agents are incorporated into DNA during replication or excision repair synthesis. Because DNA polymerases have a greatly decreased affinity for primers terminated by most nucleoside analogues, their incorporation causes stalling of replication forks. The molecular mechanisms that recognize blocked replication may contribute to drug resistance but have not yet been elucidated. Here, several molecules involved in sensing nucleoside analogue-induced stalled replication forks have been identified and examined for their contribution to drug resistance. ^ The phosphorylation of the DNA damage sensor, H2AX, was characterized in response to nucleoside analogues and found to be dependent on both time and drug concentration. This response was most evident in the S-phase fraction and was associated with an inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1-Cdc25A-Cdk2). Exposure of the Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), to cultures previously treated with nucleoside analogues caused increased apoptosis, clonogenic death, and a further log-order increase in H2AX phosphorylation, suggesting enhanced DNA damage. Ataxia-telangiectasia mutated (ATM) has been identified as a key DNA damage signaling kinase for initiating cell cycle arrest, DNA repair, and apoptosis while the Mre11-Rad50-Nbs1 (MRN) complex is known for its functions in double-strand break repair. Activated ATM and the MRN complex formed distinct nuclear foci that colocalized with phosphorylated H2AX after inhibition of DNA synthesis by the nucleoside analogues, gemcitabine, ara-C, and troxacitabine. Since double-strand breaks were undetectable, this response was likely due to stalling of replication forks. A similar DNA damage response was observed in human lymphocytes after exposure to ionizing radiation and in acute myelogenous leukemia blasts during therapy with the ara-C prodrug, CP-4055. Deficiencies in ATM, Mre11, and Rad50 led to a two- to five-fold increase in gemcitabine sensitivity, suggesting that these molecules contribute to drug resistance. Based on these results, a model is proposed for the sensing of nucleoside analogue-induced stalled replication forks that includes H2AX, ATM, and the Mre11-Rad50-Nbs1 complex. ^

The effects of DNA cytosine methylation on H -ras promoter structure and function

The effect of DNA cytosine methylation on H-ras promoter activity was assessed using a transient expression system employing the plasmid H-rasCAT (NaeI H-ras promoter linked to the chloramphenicol acetyltransferase (CAT) gene). This 551 bp promoter is 80% GC rich, enriched with 168 CpG dinucleotides, and contains six functional GC box elements which represent major DNA methylation target sites. Prokaryotic methyltransferases HhaI (CGm$\sp5$CG) and HpaII (Cm$\sp5$CGG) alone or in combination with a human placental methyltransferase (HP MTase) were used to introduce methyl groups at different CpG sites within the promoter. To test for functional promoter activity, the methylated plasmids were introduced into CV-1 cells and CAT activity assessed 48 h post-transfection. Methylation at specific HhaI and HpaII sites reduced CAT expression by 70%, whereas more extensive methylation at generalized CpG sites with HP MTase inactivated the promoter $>$95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in nonpromoter regions. We also observed that DNA cytosine methylation of a 360 bp promoter fragment by HP MTase induced a local change in DNA conformation. Using three independent methodologies (nitrocellulose filter binding assays, gel mobility shifts, and Southwestern blots), we determined that this change in promoter conformation affected the interaction of nuclear proteins with cis-regulatory sequences residing in the promoter region. The results provide evidence to suggest that DNA methylation may regulate gene expression by inducing changes in local promoter conformation which in turn alters the interactions between DNA and protein factors required for transcription. The results provide supportive evidence for the hypothesis of Cedar and Riggs, who postulated that DNA methylation may regulate gene expression by altering the binding affinities of proteins for DNA. ^

Characterization of the MLC box: A conserved {\it cis\/}-acting DNA element present in the chicken MLC1s/1c gene

The slow/cardiac alkali myosin light chain (MLC1s/1c) is a member of a multigene family whose protein products are essential for activation of the myosin ATPase. In the adult, the MLC1s/1c isoform is expressed in both cardiac and slow-twitch skeletal muscles, while it is expressed by all skeletal muscles during development.^ To elucidate the molecular mechanisms that underlie the transcriptional regulation of MLC1s/1c gene expression, the immediate 5$\sp\prime$ flanking region of the gene was isolated and shown to be capable of directing reporter gene expression. Analysis of this region revealed a 110 bp muscle-specific enhancer that includes a myocyte-specific enhancer-binding factor 2 (MEF-2) site, E-boxes, which are potential binding sites for the basic-helix-loop-helix proteins such as MyoD, and a MLC box. The focus of the thesis was to identify the role of the MLC box in expression of the MLC1s/1c gene.^ The MLC box is a member of the family of CArG box containing cis-acting DNA elements. Mutagenesis showed that the MLC box is necessary, but not sufficient, for the expression of a reporter gene linked to the 5$\sp\prime$ flanking region of the MLC1s/1c gene. Linker scanner and site-directed mutagenesis identified a number of potential sites within the 110 bp muscle-specific enhancer that may cooperate with the MLC box. These are the MEF-2 site, the E-box site, and a 10 bp element located upstream of the MEF-2 site that does not have sequence similarity with any known cis-acting element. The MLC box is capable of binding to factors present in muscle nuclear extracts, as well as to human recombinant serum response factor (SRF). Binding of SRF to the MLC box was correlated with the ability of the 5$\sp\prime$ flanking region of the MLC1s/1c gene to drive reporter gene expression. Results suggest a model in which binding of SRF to the MLC box activates expression of the MLC1s/1c gene while binding of the factors present in the nuclear extracts suppresses the expression of the gene. (Abstract shortened with permission of author.) ^

The yeast STM1 protein binds G*G multiplex nucleic acids in vitro and associates with ribosomes in vivo

The formation of triple helical, or triplex DNA has been suggested to occur in several cellular processes such as transcription, replication, and recombination. Our laboratory previously found proteins in HeLa nuclear extracts and in S. cerevisiae whole cell extracts that avidly bound a Purine-motif (Pu) triplex probe in gel shift assays, or EMSA. In order to identify a triplex DNA-binding protein, we used conventional and affinity chromatography to purify the major Pu triplex-binding protein in yeast. Peptide microsequencing and data base searches identified this protein as the product of the STM1 gene. Confirmation that Stm1p is a Pu triplex-binding protein was obtained by EMSA using both recombinant Stm1p and whole cell extracts from stm1Δ yeast. Stm1p had previously been identified as G4p2, a G-quartet DNA- and RNA-binding protein. To study the cellular role and identify the nucleic acid ligand of Stm1p in vivo, we introduced an HA epitope at either the N- or C-terminus of Stm1p and performed immunoprecipitations with the HA.11 mAb. Using peptide microsequencing and Northern analysis, we positively identified a subset of both large and small subunit ribosomal proteins and all four rRNAs as associating with Stm1p. DNase I treatment did not affect the association of Stm1p with ribosomal components, but RNase A treatment abolished the association with all ribosomal proteins and RNA, suggesting this association is RNA-dependent. Sucrose gradient fractionation followed by Western and EMSA analysis confirmed that Stm1p associates with intact 80S monosomes, but not polysomes. The presence of additional, unidentified RNA in the Stm1p-immunoprecipitate, and the absence of tRNAs and elongation factors suggests that Stm1p binds RNA and could be involved in the regulation of translation. Immunofluorescence microscopy data showed Stm1p to be located throughout the cytoplasm, with a specific movement to the bud during the G2 phase of the cell cycle. A dramatically flocculent, large cell phenotype is observed when Stm1p has a C-terminal HA tag in a protease-deficient strain background. When STM1 is deleted in this background, the same phenotype is not observed and the deletion yeast grow very slowly compared to the wild-type. These data suggest that STM1 is not essential, but plays a role in cell growth by interacting with an RNP complex that may contain G*G multiplex RNA. ^

Impact of BCR -ABL on DNA repair

The BCR-ABL fusion gene is the molecular hallmark of Philadelphia-positive leukemias. Normal Bcr is a multifunctional protein, originally localized to the cytoplasm. It has serine kinase activity and has been implicated in cellular signal transduction. Recently, it has been reported that Bcr can interact with xeroderma pigmentosum group B (XPB/ERCC3)—a nuclear protein active in UV-induced DNA repair. Two major Bcr proteins (p160 Bcr and p130Bcr) have been characterized, and our preliminary results using metabolic labeling and immunoblotting demonstrated that, while both the p160 and p130 forms of Bcr localized to the cytoplasm, the p130 form (and to a lesser extent p160) could also be found in the nucleus. Furthermore, electron microscopy confirmed the presence of Bcr in the nucleus and demonstrated that this protein associates with metaphase chromatin as well as condensed interphase heterochromatin. Since serine kinases that associate with condensed DNA are often cell cycle regulatory, these observations suggested a novel role for nuclear Bcr in cell cycle regulation and/or DNA repair. However, cell cycle synchronization analysis did not demonstrate changes in levels of Bcr throughout the cell cycle. Therefore we hypothesized that BCR serves as a DNA repair gene, and its function is altered by formation of BCR-ABL. This hypothesis was investigated using cell lines stably transfected with the BCR-ABL gene, and their parental counterparts (MBA-1 vs. M07E and Bcr-AblT1 vs. 4A2+pZAP), and several DNA repair assays: the Comet assay, a radioinimunoassay for UV-induced cyclobutane pyrimidine dimers (CPDs), and clonogenic assays. Comet assays demonstrated that, after exposure to either ultraviolet (UV)-C (0.5 to 10.0 joules m −2) or to gamma radiation (200–1000 rads) there was greater efficiency of DNA repair in the BCR-ABL-transfected cells compared to their parental controls. Furthermore, after UVC-irradiation, there was less production of CPDs, and a more rapid disappearance of these adducts in BCR-ABL-bearing cells. UV survival, as reflected by clonogenic assays, was also greater in the BCR-ABL-transfected cells. Taken together, these results indicate that, in our systems, BCR-ABL confers resistance to UVC-induced damage in cells, and increases DNA repair efficiency in response to both UVC- and gamma-irradiation. ^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

A role for p53 in sensing DNA damage and triggering apoptotic responses to anticancer agents

The p53 tumor suppressor protein plays a major role in cellular responses to anticancer agents that target DNA. DNA damage triggers the accumulation of p53, resulting in the transactivation of genes, which induce cell cycle arrest to allow for repair of the damaged DNA, or signal apoptosis. The exact role that p53 plays in sensing DNA damage and the functional consequences remain to be investigated. The main goal of this project was to determine if p53 is directly involved in sensing DNA damage induced by anticancer agents and in mediating down-stream cellular responses. This was tested in two experimental models of DNA damage: (1) DNA strand termination caused by anticancer nucleoside analogs and (2) oxidative DNA damage induced by reactive oxygen species (ROS). Mobility shift assays demonstrated that p53 and DNA-PK/Ku form a complex that binds DNA containing the anticancer nucleoside analog gemcitabine monophosphate in vitro. Binding of the p53-DNA-PK/Ku complex to the analog-containing DNA inhibited DNA strand elongation. Furthermore, treatment of cells with gemcitabine resulted in the induction of apoptosis, which was associated with the accumulation of p53 protein, its phosphorylation, and nuclear localization, suggesting the activation of p53 to trigger apoptosis following gemcitabine induced DNA strand termination. The role of p53 as a DNA damage sensor was further demonstrated in response to oxidative DNA damage. Protein pull-down assays demonstrated that p53 complexes with OGG1 and APE, and binds DNA containing the oxidized DNA base 8-oxoG. Importantly, p53 enhances the activities of APE and OGG1 in excising the 8-oxoG residue as shown by functional assays in vitro. This correlated with the more rapid removal of 8-oxoG from DNA in intact cells with wild-type p53 exposed to exogenous ROS stress. Interestingly, persistent exposure to ROS resulted in the accelerated onset of apoptosis in cells with wild-type p53 when compared to isogenic cells lacking p53. Apoptosis in p53+/+ cells was associated with accumulation and phosphorylation of p53 and its nuclear localization. Taken together, these results indicate that p53 plays a key role in sensing DNA damage induced by anticancer nucleoside analogs and ROS, and in triggering down-stream apoptotic responses. This study provides new mechanistic insights into the functions of p53 in cellular responses to anticancer agents. ^