UNIPROT:P04637 - FACTA Search

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Query: UNIPROT:P04637 (p53)
77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophage P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70 degrees C. The resulting library, designated DMPC-HFF#1 series A, consists of approximately 130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date we have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human alpha-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, beta-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert.
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PMID:Preparation and screening of an arrayed human genomic library generated with the P1 cloning system. 814 66

p53 can be isolated from cells in a form that is inert for binding to DNA but that can be stimulated dramatically by phosphorylation, antibody binding, or short single strands of DNA. This suggests that upon genotoxic stress, cells can convert latent p53 to one that is active for DNA binding. Surprisingly, we observed that latent p53 is as effective in activating transcription in vitro as is active p53. We found that HeLa nuclear extracts can stimulate DNA binding by latent p53 and have purified from them a p53-stimulating protein that we have determined to be the product of the Ref-1 gene. Interestingly, Ref-1 is a dual function protein that can both regulate the redox state of a number of proteins and function as a DNA repair (A/P) endonuclease. We observed that oxidized forms of full-length and carboxy-terminally truncated p53 (p53 delta30), which are inactive for DNA binding, are both stimulated by the Ref-1 protein. However, in the presence of reducing agent, Ref-1 is an extremely potent stimulator of full-length p53 but not p53 delta30. These and additional data indicate that Ref-1 protein stimulates p53 by both redox-dependent and -independent means and imply a key role for it in p53 regulation. Importantly, we have also determined that Ref-1 can stimulate p53 transactivation in vivo. This is the first example of a noncovalent protein modifier of p53 function identified in cells.
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PMID:Identification of redox/repair protein Ref-1 as a potent activator of p53. 911 21

Transgenic systems, both cell lines and mice with gain or loss of function, are being used in order to modulate the expression of DNA repair proteins, thus allowing to assess their contribution to the defense against genotoxic mutagens and carcinogens. In this review, questions have been addressed concerning the use of transgenic systems in elucidating critical primary DNA lesions, their conversion into genotoxic endpoints, low-dose effects, and the relative contribution of individual cellular functions in defense. It has been shown that the repair protein alkyltransferase (MGMT) is decisive for protection against methylating and chloroethylating compounds. Protection pertains also to tumor formation, as revealed by the response of MGMT transgenic and knockout mice. Overexpression of genes involved in base excision repair (N-methylpurine-DNA glycosylase, apurinic endonuclease, DNA polymerase beta) is in most cases not beneficial in increasing the protection level, whereas their down-modulation or inactivation increases cellular sensitivity. This indicates that non-repaired base N-alkylation lesions and/or repair intermediates possess genotoxic potential. Modulation of mismatch repair and poly(ADP)ribosyl transferase has also been shown to affect the cellular response to alkylating agents. Furthermore, the role of Fos, Jun and p53 in cellular defense against alkylating mutagens is discussed.
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PMID:Transgenic systems in studies on genotoxicity of alkylating agents: critical lesions, thresholds and defense mechanisms. 974 64

Ref-1 is a multifunctional protein that stimulates DNA binding by a number of transcription factors and serves as the abasic (A/P) endonuclease in base excision repair. Ref-1 was discovered to be a potent activator of p53 DNA binding in vitro. To address the physiological significance of the effects of Ref-1 on p53, we have analyzed its role in regulating p53 function in vivo. We found that Ref-1 over-expression enhances the ability of p53 to transactivate a number of p53 target promoters and increases the ability of p53 to stimulate endogenous p21 and cyclin G expression. Additionally, it was observed that Ref-1 associates with p53 in vivo and in vitro. Importantly, downregulation of Ref-1 (by antisense) causes a marked reduction in p53 induction of p21 mRNA and protein, as well as diminished ability of p53 to transactivate the p21 and Bax promoters. Moreover, Ref-1 levels are correlated with the extent of apoptosis induced by p53. Finally, we observed that Ref-1 cooperates with a DNA-damaging compound, camptothecin, to stimulate the transcriptional activity of p53. Together these data indicate that Ref-1 is a key cellular regulator of p53.
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PMID:Ref-1 regulates the transactivation and pro-apoptotic functions of p53 in vivo. 1052 5

The present study examined the immunohistochemical expression of human AP endonuclease 1 (HAP1/Ref-1), the major endonuclease in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA, in normal lung and lung carcinomas. Cellular expression of HAP1 was determined using a standard avidin-biotin-peroxidase complex (ABC) technique and an anti-HAP1 rabbit polyclonal antibody on paraffin-embedded tissue sections from normal lung and in 103 primary non-small cell lung carcinomas (NSCLCs). In normal lung, the staining for HAP1 was found to be both nuclear and cytoplasmic in the pneumocytes of the alveoli. Superficial ciliated cells of the bronchial epithelium presented cytoplasmic staining, while staining for the basal cells was mostly nuclear. Bronchial glandular cells demonstrated mixed nuclear and cytoplasmic staining. Lung carcinomas showed all patterns of expression for HAP1. Loss of HAP1 expression was associated with low proliferation index (p=0.01) and with squamous histology (p=0.04). In squamous carcinomas, a significant correlation was observed between positive nuclear HAP1 and negative p53 expression (p=0.03). A survival benefit was seen in patients presenting nuclear HAP1 expression and those presenting the nuclear HAP1+/p53- phenotype (p=0.01 and 0.007, respectively). It is concluded that nuclear HAP1 localization may be relevant to its role as a DNA repair protein and/or to the recently proposed role as an activator of wild-type p53, and thus to the better outcome seen in this group of patients.
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PMID:Nuclear localization of human AP endonuclease 1 (HAP1/Ref-1) associates with prognosis in early operable non-small cell lung cancer (NSCLC). 1054 96

Thioredoxin (TRX) is a dithiol-reducing enzyme that is induced by various oxidative stresses. TRX regulates the activity of DNA-binding proteins, including Jun/Fos and nuclear factor-kappaB. TRX also interacts with an intranuclear reducing molecule redox factor 1 (Ref-1), which enhances the activity of Jun/Fos. Here, we have investigated the role of TRX in the regulation of p53 activity. Electrophoretic mobility shift assay showed that TRX augmented the DNA binding activity of p53 and also further potentiated Ref-1-enhanced p53 activity. Luciferase assay revealed that transfection of TRX enhanced p53-dependent expression of p21 and further intensified Ref-1-mediated p53 activation. Furthermore, Western blot analysis revealed that p53-dependent induction of p21 protein was also facilitated by transfection with TRX. Overexpression of transdominant negative mutant TRX (mTRX) suppressed the effects of TRX or Ref-1, showing a functional interaction between TRX and Ref-1. cis-Diamminedichloroplatinum (II) (CDDP) induced p53 activation and p21 transactivation. The p53-dependent p21 transactivation induced by CDDP was inhibited by mTRX overexpression, suggesting that TRX-dependent redox regulation is physiologically involved in p53 regulation. CDDP also stimulated translocation of TRX from the cytosol into the nucleus. Hence, TRX-dependent redox regulation of p53 activity indicates coupling of the oxidative stress response and p53-dependent repair mechanism.
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PMID:Thioredoxin-dependent redox regulation of p53-mediated p21 activation. 1058 64

Mice that are genetically engineered are becoming increasingly more powerful tools for understanding the molecular pathology of many human hereditary diseases, especially those that confer an increased predisposition to cancer. We have generated mouse strains defective in the Xpc gene, which is required for nucleotide excision repair (NER) of DNA. Homozygous mutant mice are highly prone to skin cancer following exposure to UVB radiation, and to liver and lung cancer following exposure to the chemical carcinogen acetylaminofluorene (AAF). Skin cancer predisposition is significantly augmented when mice are additionally defective in Trp53 (p53) gene function. We also present the results of studies with mice that are heterozygous mutant in the Apex (Hap1, Ref-1) gene required for base excision repair and with mice that are defective in the mismatch repair gene Msh2. Double and triple mutant mice mutated in multiple DNA repair genes have revealed several interesting overlapping roles of DNA repair pathways in the prevention of mutation and cancer.
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PMID:Defective nucleotide excision repair in xpc mutant mice and its association with cancer predisposition. 1072 60

The DNA base excision repair (BER) pathway is responsible for the repair of cellular alkylation and oxidative DNA damage. A crucial and the second step in the BER pathway involves the cleavage of baseless sites in DNA by an AP endonuclease. The major AP endonuclease in mammalian cells is Ape1/ref-1. Ape1/ref-1 is a multifunctional protein that is not only responsible for repair of AP sites, but also functions as a reduction-oxidation (redox) factor maintaining transcription factors in an active reduced state. Ape1/ref-1 has been shown to stimulate the DNA binding activity of numerous transcription factors that are involved in cancer promotion and progression such as Fos, Jun, NF(B, PAX, HIF-1(, HLF and p53. Ape1/ref-1 has also been implicated in the activation of bioreductive drugs which require reduction in order to be active and has been shown to interact with a subunit of the Ku antigen to act as a negative regulator of the parathyroid hormone promoter, as well as part of the HREBP transcription factor complex. Ape1/ref-1 levels have been found to be elevated in a number of cancers such as ovarian, cervical, prostate, rhabdomyosarcomas and germ cell tumors and correlated with the radiosensitivity of cervical cancers. In this review, we have attempted to try and assimilated as much data concerning Ape1/ref-1 and incorporate the rapidly growing information on Ape1/ref-1 in a wide variety of functions and systems.
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PMID:Going APE over ref-1. 1101 83

The temporospatial expression pattern of the nuclear DNA repair enzyme redox factor-1 (ref-1), the p53-activated gene (pag) 608 and the effector caspase-3 was examined by in situ hybridization histochemistry in gerbils subjected to two 10-min episodes of unilateral common carotid artery occlusion, separated by 5h. Gene responses were correlated with the metabolic state, as revealed by regional adenosine 5'-triphosphate bioluminescent imaging, and with the degree of histological damage, as assessed by haematoxylin-eosin staining and terminal deoxynucleotidyl transferase-mediated-dUTP nick end labeling (TUNEL), in order to evaluate the role of these genes in the maturation of injury. Focal infarcts developed in the dorsolateral cerebral cortex at the bregma level and the nucleus caudate-putamen within four days after repeated unilateral ischemia, as indicated by a secondary adenosine 5'-triphosphate loss after initial adenosine 5'-triphosphate recovery and by histomorphological signs of pannecrosis. The more caudal cortex at hippocampal levels and the hippocampus (CA1>CA3 area), however, exhibited selective neuronal injury without adenosine 5'-triphosphate depletion. TUNEL+ cells appeared starting 5h after repeated unilateral ischemia. TUNEL+ cells reached maximum levels in the caudate-putamen at 12-24h, but much later in the cortex and hippocampus at two days after ischemia. Remarkably few TUNEL+ cells were noticed in the thalamus, where adenosine 5'-triphosphate state did not recover after reperfusion. Following repeated unilateral ischemia, a transient elevation of ref-1 mRNA was detected after 5h in the cerebral cortex and hippocampal CA1 area. Ref-1 mRNA levels decreased within 12-24h, before the onset of tissue damage. Subsequently, pag608 and caspase-3 mRNA levels increased, closely in parallel with the appearance of DNA fragmented cells, but slightly prior to the deterioration of adenosine 5'-triphosphate state. In the caudate-putamen, pag608 and caspase-3 mRNAs reached maximum levels already 12-24h after repeated common carotid artery occlusion, when DNA fragmentation was most prominent, and declined thereafter. In the cortex and hippocampal CA1-3 areas, where DNA damage appeared more slowly, pag608 and caspase-3 mRNAs were induced starting 24h after ischemia, and remained elevated even after two to four days. The levels of pag608 and caspase-3 mRNAs were similar at rostral and caudal levels of the cortex, as well as in the hippocampal CA1 and CA3 area, although the degree of injury differed considerably between these structures. Notably, pag608 and caspase-3 mRNAs were not elevated in the thalamus after repeated unilateral ischemia. The present report shows a close temporal association between the induction of ref-1, pag608 and caspase-3 mRNAs, the manifestation of cell injury and the secondary adenosine 5'-triphosphate depletion in infarcting brain areas, suggesting (i) that de novo responses of these genes may be involved in the maturation of cell injury and (ii) that apoptotic programs and the secondary deterioration of cerebral energy state may interfere with each other after ischemia.
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PMID:Expression of redox factor-1, p53-activated gene 608 and caspase-3 messenger RNAs following repeated unilateral common carotid artery occlusion in gerbils--relationship to delayed cell injury and secondary failure of energy state. 1118 42

The human AP endonuclease (Ape1 or ref-1) DNA base excision repair (BER) enzyme is a multifunctional protein that has an impact on a wide variety of important cellular functions including oxidative signaling, transcription factor regulation, and cell cycle control. It acts on mutagenic AP (baseless) sites in DNA as a critical member of the DNA BER repair pathway. Moreover, Ape1/ref-1 stimulates the DNA-binding activity of transcription factors (Fos-Jun, nuclear factor-kappaB, Myb, ATF/cyclic AMP-responsive element binding protein family, HIF-1alpha, HLF, PAX, and p53) through a redox mechanism and thus represents a novel component of signal transduction processes that regulate eukaryotic gene expression. Ape1/ref-1 has also been shown to be closely linked to apoptosis associated with thioredoxin, and altered levels of Ape1/ref-1 have been found in some cancers. In a pilot study, we have examined Ape1/ref-1 expression by immunohistochemistry in sections of germ cell tumors (GCTs) from 10 patients with testicular cancer of various histologies including seminomas, yolk sac tumors, and malignant teratomas. Ape1/ref-1 was expressed at relatively high levels in the tumor cells of nearly all sections. We hypothesized that elevated expression of Ape1/ref-1 is responsible in part for the resistance to therapeutic agents. To answer this hypothesis, we overexpressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1 using retroviral gene transduction with the vector LAPESN. Using an oligonucleotide cleavage assay and immunohistochemistry to assess Ape1/ref-1 repair activity and expression, respectively, we found that the repair activity and relative Ape1/ref-1 expression in GCT cell lines are directly related. NT2/D1 cells transduced with Ape1/ref-1 exhibited 2-fold higher AP endonuclease activity in the oligonucleotide cleavage assay, and this was reflected in a 2-3-fold increase in protection against bleomycin. Lesser protection was observed with gamma-irradiation. We conclude that: (a) Ape1/ref-1 is expressed at relatively high levels in some GCTs; (b) elevated expression of Ape1/ref-1 in testicular cancer cell lines results in resistance to certain therapeutic agents; and (c) Ape1/ref-1 expression in GCT cell lines determined by immunohistochemistry and repair activity assays parallels the level of protection from bleomycin. We further hypothesize that elevated Ape1/ref-1 levels observed in human testicular cancer may be related to their relative resistance to therapy and may serve as a diagnostic marker for refractory disease. To our knowledge, this is the first example of overexpressing Ape1/ref-1 in a mammalian system resulting in enhanced protection to DNA-damaging agents.
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PMID:Altered expression of Ape1/ref-1 in germ cell tumors and overexpression in NT2 cells confers resistance to bleomycin and radiation. 1128 Jul 90

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