See help. --------------------------------------------------------------- 22: Mol Pharmacol. 2003 Nov;64(5):1259-69. Cloning and Characterization of the Murine and Rat mrp1 Promoter Regions. Muredda M, Nunoya K, Burtch-Wright RA, Kurz EU, Cole SP, Deeley RG. Queen's University Cancer Research Institute, Botterell Hall Room A315C, Queen's University, Kingston, Ontario, Canada K7L 3N6. The ATP-binding cassette transporter multidrug resistance protein 1 (MRP1) confers resistance to a number of clinically important chemotherapeutic agents. The proximal promoter region of MRP1 is GC-rich and contains binding sites for members of the Sp1 family of trans-acting factors that seem to be important for basal expression. As an approach to searching for other elements that may contribute to expression, we have sequenced and functionally compared the promoters of the murine and rat mrp1 genes with that of the human gene. All three promoters are GC-rich, TATA-less, and CAAT-less. Conservation of sequence between rodent and human promoters is limited to a proximal region of 100 nucleotides containing binding sites for members of the Sp1 family and a putative activator protein-1 element. The 5'-untranslated region (UTR) of human MRP1 contains an insertion of approximately 160 nucleotides comprising a GCC-triplet repeat and a GC-rich tandem repeat that is absent from the rodent sequences. Transient transfection analyses demonstrated that the conserved GC-boxes of all three genes are the major determinants of basal activity. Based on electrophoretic mobility shift assays, each GC-box can be bound by Sp1 or Sp3. Unlike the rodent genes, the human MRP1 5'UTR also binds Sp1 but not Sp3, and the human promoter retains substantial activity even in the absence of the conserved GC-boxes. Finally, we show that the tumor suppressor protein p53 can repress the human and rodent promoters by a mechanism that is independent of the Sp1 elements. PMID: 14573776 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Oncogene. 2003 Sep 1;22(37):5813-27. Transcription factors activated in mammalian cells after clinically relevant doses of ionizing radiation. Criswell T, Leskov K, Miyamoto S, Luo G, Boothman DA. Department of Radiation Oncology and Program in Molecular Basis of Disease, Laboratory of Molecular Stress Responses, Ireland Comprehensive Cancer Center, Case Western Reserve University and University Hospitals of Cleveland, OH 44106-4942, USA. Over the past 15 years, a wealth of information has been published on transcripts and proteins 'induced' (requiring new protein synthesis) in mammalian cells after ionizing radiation (IR) exposure. Many of these studies have also attempted to elucidate the transcription factors that are 'activated' (i.e., not requiring de novo synthesis) in specific cells by IR. Unfortunately, all too often this information has been obtained using supralethal doses of IR, with investigators assuming that induction of these proteins, or activation of corresponding transcription factors, can be 'extrapolated' to low-dose IR exposures. This review focuses on what is known at the molecular level about transcription factors induced at clinically relevant (< or =2 Gy) doses of IR. A review of the literature demonstrates that extrapolation from high doses of IR to low doses of IR is inaccurate for most transcription factors and most IR-inducible transcripts/proteins, and that induction of transactivating proteins at low doses must be empirically derived. The signal transduction pathways stimulated after high versus low doses of IR, which act to transactivate certain transcription factors in the cell, will be discussed. To date, only three transcription factors appear to be responsive (i.e. activated) after physiological doses (doses wherein cells survive or recover) of IR. These are p53, nuclear factor kappa B(NF-kappaB), and the SP1-related retinoblastoma control proteins (RCPs). Clearly, more information on transcription factors and proteins induced in mammalian cells at clinically or environmentally relevant doses of IR is needed to understand the role of these stress responses in cancer susceptibility/resistance and radio-sensitivity/resistance mechanisms. Publication Types: Review PMID: 12947388 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Radiat Res. 2003 Sep;160(3):273-90. ATM-dependent and -independent gene expression changes in response to oxidative stress, gamma irradiation, and UV irradiation. Heinloth AN, Shackelford RE, Innes CL, Bennett L, Li L, Amin RP, Sieber SO, Flores KG, Bushel PR, Paules RS. Growth Control and Cancer Group, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA. Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiencies, telangiectasias, sensitivity to ionizing radiation, and high predisposition for malignancies. The ataxia telangiectasia mutated (ATM) gene encodes a protein (ATM) with serine/threonine kinase activity. DNA-double strand breaks are known to increase its kinase activity. While cells from individuals with AT are attenuated in their G(1)-, S- and G(2)-phase cell cycle checkpoint functions in response to gamma irradiation and oxidative stress, their response to UV irradiation appears to be equivalent to that of wild-type cells. In this study, we investigated changes in gene expression in response to gamma irradiation, oxidative stress, and UV irradiation, focusing on the dependence on ATM. Doses for all three treatments were selected that resulted in roughly an equivalent induction of a G(1) checkpoint response and inhibition of progression through S phase. To investigate gene expression changes, logarithmically growing wild-type and AT dermal diploid fibroblasts were exposed to either gamma radiation (5 Gy), oxidative stress (75 micro M t-butyl-hydroperoxide), or UV radiation (7.5 J/m(2)), and RNA was harvested 6 h after treatment. Gene expression analysis was performed using the NIEHS Human ToxChip 2.0 with approximately 1900 cDNA clones representing known genes and ESTs. All three treatments resulted in distinct patterns of gene expression changes, as shown previously. ATM-dependent and ATM-independent components were detected within these patterns, as were novel indications of involvement of ATM in regulation of transcription factors such as SP1, AP1 and MTF1. PMID: 12926986 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Oncogene. 2003 Aug 14;22(34):5315-24. Role of protein kinase C and the Sp1-p53 complex in activation of p21(WAF-1) expression by 12-O-tetradecanoylphorbol-13-acetate in human T cells. Schavinsky-Khrapunsky Y, Huleihel M, Aboud M, Torgeman A. Department of Microbiology and Immunology, Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel. Previous reports have shown that, in certain cell types, p21(WAF-1), which plays a central role in cell proliferation, can be activated by HTLV-I Tax protein and by TPA. Tax and TPA are also known to stimulate HTLV-I gene expression. Since cell proliferation has a major impact on HTLV-I replication, it was of interest to investigate their effect on p21(WAF-1) in human T cells, which are the main target of HTLV-I in human infection. This study demonstrates that p21(WAF-1) is activated in such cells by both factors, each acting through a different mechanism that does not influence the other. The effect of TPA is shown to require PKC activity. Notably, however, examination of different PKC isoforms revealed that PKC-alpha and PKC-epsilon stimulated p21(WAF-1) expression, whereas PKC-eta was rather inhibitory and PKC-beta1 and beta2 were ineffective. All these isoforms were found to be activated by TPA in the employed T cells, but this apparent paradox was resolved by the observation that when coexpressed together in these cells, the stimulatory PKCs override the inhibitory isoform. Further experiments demonstrated that the PKC-induced p21(WAF-1) activation was mediated by binding of Sp1-p53 complex to the second most upstream of the six Sp1 recognition sites present in its promoter and that this effect did not require the cooperation of an p53-binding site. PMID: 12917633 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Biol Chem. 2003 Sep 19;278(38):36496-504. Epub 2003 Jul 7. Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter. Di Padova M, Bruno T, De Nicola F, Iezzi S, D'Angelo C, Gallo R, Nicosia D, Corbi N, Biroccio A, Floridi A, Passananti C, Fanciulli M. Laboratory B. Che-1 is a recently identified human RNA polymerase II binding protein involved in the regulation of gene transcription and cell proliferation. We previously demonstrated that Che-1 inhibits the Rb growth-suppressing function by interfering with Rb-mediated HDAC1 recruitment on E2F target gene promoters. By hybridization of cancer profile arrays, we found that Che-1 expression is strongly down-regulated in several tumors, including colon and kidney carcinomas, compared with the relative normal tissues. Consistent with these data, Che-1 overexpression inhibits proliferation of HCT116 and LoVo human colon carcinoma cell lines by activation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 in a p53-independent manner and by promoting growth arrest at the G1 phase of the cell cycle. Che-1 activates p21WAF1/Cip1 by displacing histone deacetylase (HDAC)1 from the Sp1 binding sites of the p21WAF1/Cip1 gene promoter and accumulating acetylated histone H3 on these sites. Accordingly, Che-1-specific RNA interference negatively affects p21WAF1/Cip1 transactivation and increases cell proliferation in HCT116 cells. Taken together, our results indicate that Che-1 can be considered a general HDAC1 competitor and its down-regulation is involved in colon carcinoma cell proliferation. PMID: 12847090 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: J Nutr. 2003 Jul;133(7 Suppl):2456S-2460S. ZBP-89 mediates butyrate regulation of gene expression. Merchant JL, Bai L, Okada M. Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA. merchanj@umich.edu Inducible p53-independent regulation of the cyclin-dependent kinase inhibitor p21(Waf1) transcription is mediated through its proximal GC-rich sites. Prior studies have shown that Sp1, Sp3 and the histone acetyltransferase coactivator p300 are components of the complexes that bind to these sites. Although Sp1 and Sp3 collaborate with p300, a direct interaction between Sp1 and p300 does not occur. Zinc-finger binding protein-89 (ZBP-89, also known as BFCOL1, BERF-1 and ZNF-148) is a Kruppel-type zinc-finger transcription factor that binds to the same GC-rich sequences as Sp1. We sought to determine whether ZBP-89 is a target of p300 during butyrate induction of p21(Waf1). This review summarizes the evidence that supports a crucial role for ZBP-89 in butyrate regulation of p21(Waf1). Adenovirus-mediated expression of ZBP-89 in HT-29 cells reveals that ZBP-89 potentiates butyrate induction of endogenous p21(Waf1) gene expression. DNA-protein interaction assays demonstrate that Sp1, Sp3 and ZBP-89 bind the p21(Waf1) promoter at -245 to -215. Coprecipitation assays reveal that p300 preferentially binds to the N-terminus of ZBP-89. ZBP-89 also induces p21(Waf1) through stabilization of p53. Although ZBP-89 binds mutant and wild-type p53, only wild-type p53 is stabilized. Moreover, mutant p53 shifts the subnuclear location of ZBP-89 to the nuclear periphery, which is a domain rich in heterochromatin. This finding led to the conclusion that mutant p53 exerts a dominant negative effect on ZBP-89. We propose that gene silencing by mutant p53 might be mediated by sequestering ZBP-89 within heterochromatin regions at the nuclear periphery. Overall, ZBP-89 is a butyrate-regulated coactivator of p53 and is able to induce p21(Waf1) gene expression through both p53-dependent and -independent mechanisms to inhibit cell growth. Publication Types: Review Review, Tutorial PMID: 12840224 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: FEBS Lett. 2003 Jun 5;544(1-3):38-44. Transcriptional repression of cyclin-dependent kinase inhibitor p21 gene by phospholipase D1 and D2. Kwun HJ, Lee JH, Min do S, Jang KL. Department of Microbiology, College of Natural Sciences, Pusan National University, South Korea. Phospholipase D (PLD) is known to stimulate cell cycle progression and to transform murine fibroblast cells into tumorigenic forms, although the precise mechanisms are not elucidated. In this report, we demonstrated that both PLD1 and PLD2 repressed expression of cyclin-dependent kinase inhibitor p21 gene in an additive manner. The phospholipase activity of PLDs was important for the effect. PLD1 repressed the p21 promoter by decreasing the level of p53, whereas PLD2 via a p53-independent pathway through modulating Sp1 activity. Taken together, we suggest that PLD isozymes stimulate cell growth by repressing expression of p21 gene, which may ultimately lead to carcinogenesis. PMID: 12782287 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Cell Biol. 2003 Jun;23(12):4056-65. Methylation of adjacent CpG sites affects Sp1/Sp3 binding and activity in the p21(Cip1) promoter. Zhu WG, Srinivasan K, Dai Z, Duan W, Druhan LJ, Ding H, Yee L, Villalona-Calero MA, Plass C, Otterson GA. Department of Internal Medicine. Department of Pathology, The Ohio State University-Comprehensive Cancer Center, Columbus, Ohio 43210, USA. DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21(Cip1) expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C-->W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21(Cip1) expression because the promoter of p21(Cip1) in these cells is hypermethylated. By analyzing luciferase activity of transfected p21(Cip1) promoter vectors, we demonstrate that depsipeptide functions on Sp1-binding sites to induce p21(Cip1) expression. We hypothesize that hypermethylation may interfere with Sp1/Sp3 binding. By using an electrophoretic mobility shift assay, we show that, although methylation within the consensus Sp1-binding site did not reduce Sp1/Sp3 binding, methylation outside of the consensus Sp1 element induced a significant decrease in Sp1/Sp3 binding. Depsipeptide induced p21(Cip1) expression was reconstituted when cells were pretreated with 5-aza-2'-deoxycytidine. Our data suggest, for the first time, that hypermethylation around the consensus Sp1-binding sites may directly reduce Sp1/Sp3 binding, therefore leading to a reduced p21(Cip1) expression in response to depsipeptide treatment. PMID: 12773551 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Biol Chem. 2003 Jul 25;278(30):27593-604. Epub 2003 May 14. The human caspase-8 promoter sustains basal activity through SP1 and ETS-like transcription factors and can be up-regulated by a p53-dependent mechanism. Liedtke C, Groger N, Manns MP, Trautwein C. Department of Gastroenterology, Hepatology and Endocrinology, Medical School of Hannover, D-30625 Hannover, Germany. Caspase-8, also known as MACH/FLICE/Mch5, is the most upstream-located cysteine-aspartyl-protease (caspase) in a caspase cascade involved in apoptosis triggered by members of the tumor necrosis factor receptor superfamily or other stimuli such as chemotherapeutic agents. Regulation of caspase-8 expression on a post-translational level has been studied in detail, whereas only little information is available on its control by gene transcription. We identified and cloned the human caspase-8 promoter, determined the transcriptional start site of the caspase-8 gene, and examined the regulatory mechanisms of the promoter with respect to its basal activity as well as to its inducibility upon apoptotic stimuli in human hepatoma cells. We identified two minimal sequences essential for basal transcription of caspase-8 and demonstrate that a single SP1 and an ETS-like binding motif mediate this effect. We further show that the caspase-8 promoter is inducible and demonstrate that adenoviral infection increases caspase-8 mRNA levels. However, the increase in caspase-8 gene transcription after adenoviral infection absolutely depends on the p53 status of the hepatoma cell line, implying that caspase-8 is a target gene of p53. We show that delivery of exogenous p53 alone is sufficient to induce the caspase-8 promoter even in p53-deficient Hep3B hepatoma cells. Subsequent promoter deletion analysis in combination with luciferase reporter assays identified a p53-responsive element downstream of the transcriptional start site. We demonstrate that this p53-responsive sequence overlaps with the ETS-like binding site and suggest that an additional p53-inducible, yet unknown factor interacts with this region of the caspase-8 promoter. In summary, our study contributes to the understanding of the transcriptional regulation of the caspase-8 gene by basal (SP1- and ETS-dependent) and inducible (p53-dependent) mechanisms. PMID: 12748179 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: J Biol Chem. 2003 Jul 18;278(29):26589-96. Epub 2003 May 5. The tumor suppressor interferon regulatory factor 1 interferes with SP1 activation to repress the human CDK2 promoter. Xie RL, Gupta S, Miele A, Shiffman D, Stein JL, Stein GS, van Wijnen AJ. Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. Cell growth control by interferons (IFNs) involves up-regulation of the tumor suppressor interferon regulatory factor 1 (IRF1). To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here we show that the G1/S phase-related cyclin-dependent kinase 2 (CDK2) gene is a novel proliferation-related downstream target of IRF1. We find that IRF1, but not IRF2, IRF3, or IRF7, selectively represses CDK2 gene transcription in a dose- and time-dependent manner. We delineate the IRF1-responsive repressor element between nt -68 to -31 of the CDK2 promoter. For comparison, the tumor suppressor p53 represses CDK2 promoter activity independently of IRF1 through sequences upstream of nt -68, and the CDP/cut/Cux1 homeodomain protein represses transcription down-stream of -31. Thus, IRF1 repression represents one of three distinct mechanisms to attenuate CDK2 levels. The -68/-31 segment lacks a canonical IRF responsive element but contains a single SP1 binding site. Mutation of this element abrogates SP1-dependent enhancement of CDK2 promoter activity as expected but also abolishes IRF1-mediated repression. Forced elevation of SP1 levels increases endogenous CDK2 levels, whereas IRF1 reduces both endogenous SP1 and CDK2 protein levels. Hence, IRF1 represses CDK2 gene expression by interfering with SP1-dependent transcriptional activation. Our findings establish a causal series of events that functionally connect the anti-proliferative effects of interferons with the IRF1-dependent suppression of the CDK2 gene, which encodes a key regulator of the G1/S phase transition. PMID: 12732645 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Mol Cell Biol. 2003 Apr;23(8):2669-79. The tumor suppressor p53 and histone deacetylase 1 are antagonistic regulators of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 gene. Lagger G, Doetzlhofer A, Schuettengruber B, Haidweger E, Simboeck E, Tischler J, Chiocca S, Suske G, Rotheneder H, Wintersberger E, Seiser C. Institute of Medical Biochemistry, Division of Molecular Biology, Vienna Biocenter, University of Vienna, A-1030 Vienna, Austria. The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs. PMID: 12665570 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Exp Cell Res. 2003 Apr 1;284(2):264-73. Prolonged progestin treatment induces the promoter of CDK inhibitor p21 Cip1,Waf1 through activation of p53 in human breast and endometrial tumor cells. Kester HA, Sonneveld E, van der Saag PT, van der Burg B. Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands. Progestins are frequently used in the treatment of advanced breast and endometrial cancer. The human breast carcinoma cell line T47D shows a biphasic response to progestins. Short-term progestin treatment leads to enhanced DNA synthesis, while this line is growth inhibited upon prolonged exposure. An important protein involved in growth regulation by progestins in this cell is the CDK inhibitor p21(Cip1,Waf1). We show that after 1 day of progestin treatment in T47D cells, the p21 promoter-proximal region containing Sp1 binding sites is crucial in the induction by progestins. However, after 3 days the activity of the promoter-distal region becomes predominant in T47D cells or the endometrial carcinoma cell line ECC1. This is dependent upon two domains within this region that contain p53 response elements. In ECC1 and T47D cells 3-day progestin treatment induces a reporter containing a p53 response element, but not a mutated version. This induction is due to activation of p53 by progestin, which may be caused by nuclear translocation of p53. These data indicate that upon prolonged exposure, progestins activate p53, in human breast and endometrial tumor cells, which up-regulates the p21(Cip1,Waf1) promoter. This may be an important mechanism involved in progestin-inhibited cellular proliferation in these cells. PMID: 12651158 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Carcinogenesis. 2003 Jan;24(1):121-32. Perturbations of the Ink4a/Arf gene locus in aflatoxin B1-induced mouse lung tumors. Tam AS, Devereux TR, Patel AC, Foley JF, Maronpot RR, Massey TE. Queen's University, Department of Pharmacology and Toxicology, Kingston, Ontario K7L 3N6, Canada. Lung tumors from AC3F1 mice treated with aflatoxin B(1) (AFB(1)), were examined for loss of alleles, point mutations and hypermethylation of CpG sites within the promoters of the two genes in the Ink4a/Arf gene locus. Loss of microsatellite alleles in the Ink4a/Arf region occurred in 22 of 74 (30%) AFB(1)-induced lung tumors. Fifty-one of 61 (83%) tumors had at least partial methylation of CpG sites within the p16Ink4a promoter-exon 1alpha region. At least partial methylation of CpG sites was observed in 43 of 49 (88%) tumors analyzed for p19Arf promoter hypermethylation, with methylation of identified transcription factor binding sites or consensus sequences occurring in 21 tumors (DMP1/Ets in two tumors, CTCF in four tumors, E2F in three tumors, Sp1 in 16 tumors). Two tumors contained point mutations in the p19Arf promoter. Nuclear staining for p19(Arf) was decreased by 80-100% in 41 of 71 (58%) tumors. The concordance between p19Arf molecular perturbations and altered protein expression was 63%. However, upon comparing p19Arf promoter perturbations (i.e. methylation of functional transcription factor binding sites and point mutations) and altered p19(Arf) expression, the concordance was 86%, suggesting a mechanism for changes in protein expression in some tumors. There was an absence of a mutually exclusive relationship between disruption of p53 and p19(Arf), since the concordance was 62%. Similarly, no evidence was found of inverse relationships between perturbation of p16Ink4a and p19(Arf) (43% concordance) or p16Ink4(a) and p53 (37% concordance), suggesting that inactivation of these genes occurs independently and provides evidence that, although these genes may participate in cooperative cellular pathways, they also have functions in independent pathways that are important in mouse lung tumorigenesis. PMID: 12538357 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Apoptosis. 2003 Jan;8(1):11-8. Roles of the stress-induced gene IEX-1 in regulation of cell death and oncogenesis. Wu MX. Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA. mxwu@bu.edu In response to changes in the external environment cells must initiate a coordinated program of gene expression for them to adapt. IEX-1 (immediate early response gene X-1) is precisely regulated by multiple transcription factors among which p53, NF-kappaB/rel, Sp1 and c-Myc play central roles, to ensure rapid and transient expression of IEX-1 in cells under a variety of stress conditions. Overexpression of IEX-1 renders some cells sensitive to apoptosis and accelerates cell cycle progression, but reduces proliferation of other cells, whereas disruption of IEX-1 expression is associated with decreases in both apoptosis and cell cycle progression. In sharp contrast to in vitro studies, in vivo constitutive expression of IEX-1 prevents activated T cells but not B cells from apoptosis, as shown using IEX-1-transgenic mice that target IEX-1 expression specifically to lymphocytes driven by the Emu enhancer. The animals developed a lupus-like disease and subsequently a high incidence of T cell lymphomas when they aged, due to insufficient apoptosis of T cells. These varied effects of IEX-1 on cell death and cell cycle progression in a cell-context dependent fashion implicate that IEX-1 is involved in more than one signaling pathway, understanding of which will certainly improve our knowledge with respect to cancer biology, cell death and cell cycle regulation. Publication Types: Review Review, Tutorial PMID: 12510147 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Mol Biol (Mosk). 2002 Nov-Dec;36(6):1035-43. [Identification of two new p53 target genes through implementation of the modified chromatin immunoprecipitation method and inverse PCR] [Article in Russian] Burgess R, Luniak V, Noskin L, Giliano N. TransGenetics Incorporated, University of California at San Diego, La Jolla, California, USA. Direct target loci for the transcription factor p53 were identified through the employment of a combination of a modified version of chromosomal immunoprecipitation and inverse PCR. Irradiation of Hela cells to drive DNA damage response was followed by sequential chromosomal immunoprecipitation utilizing antibodies which recognize the large subunit of RNA polymerase II and p53. Inverse PCR with degenerate oligonucleotides specific for the p53 binding site was subsequently performed on immunoprecipitated DNA and fragments containing putative p53 target genes were subcloned and sequenced. Two sequences were identified which contain near-consensus p53 binding sites as well as recognition sites for the core transcriptional machinery including RNA polymerase II and Sp1. Cotransfections of vectors containing these sequences linked to a reporter with p53 expression vectors resulted in stimulation of transcription. Application of the technology described herein may result in the identification of target loci for a wide variety of transcription factors. PMID: 12500542 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: J Biol Chem. 2003 Jan 3;278(1):462-70. Epub 2002 Oct 25. DNA damage-induced inhibition of securin expression is mediated by p53. Zhou Y, Mehta KR, Choi AP, Scolavino S, Zhang X. Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA. Tumor suppressor p53 induces the cellular response to DNA damage mainly by regulating expression of its downstream target genes. The human securin is an anaphase inhibitor, preventing premature chromosome separation through inhibition of separase activity. It is also known as the product of the human pituitary tumor-transforming gene, pttg, a proto-oncogene. Here we report that the expression of human securin is suppressed in cells treated with the DNA-damaging drugs doxorubicin and bleomycin. This suppression requires functional p53. Analysis of the human securin promoter reveals that DNA-binding sites for Sp1 and NF-Y are both required for activation of securin expression; however, only the NF-Y site is essential for the suppression by p53. Our study indicates that securin is a p53 target gene and may play a role in p53-mediated cellular response to DNA damage. PMID: 12403781 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Nucleic Acids Res. 2002 May 1;30(9):1967-76. A single cell cycle genes homology region (CHR) controls cell cycle-dependent transcription of the cdc25C phosphatase gene and is able to cooperate with E2F or Sp1/3 sites. Haugwitz U, Wasner M, Wiedmann M, Spiesbach K, Rother K, Mossner J, Engeland K. Department of Internal Medicine II, University of Leipzig, Max Burger Research Center, Johannisallee 30, D-04103 Leipzig, Germany. The cdc25C phosphatase participates in regulating transition from the G2 phase of the cell cycle to mitosis by dephosphorylating cyclin-dependent kinase 1. The tumor suppressor p53 down-regulates expression of cdc25C as part of G2/M checkpoint control. Transcription of cdc25C oscillates during the cell cycle with no expression in resting cells and maximum transcription in G2. We had identified earlier a new mechanism of cell cycle-dependent transcription that is regulated by a cell cycle-dependent element (CDE) in conjunction with a cell cycle genes homology region (CHR). The human cdc25C gene was the first example. CDE/CHR tandem elements have since been found in promoters of many cell cycle genes. Here we show that the mouse cdc25C gene is regulated by a CHR but does not hold a CDE. Therefore, it is the first identified gene with CHR-dependent transcriptional regulation during the cell cycle not relying on a CDE located upstream of it. The CHR leads to repression of cdc25C transcription early in the cell cycle and directs a release of this repression in G2. Furthermore, we find that this CHR can cooperate in cell cycle-dependent transcription with elements placed directly upstream of it binding E2F, Sp1 or Sp3 transcription factors. PMID: 11972334 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Blood. 2002 May 1;99(9):3367-75. MDM2 induces NF-kappaB/p65 expression transcriptionally through Sp1-binding sites: a novel, p53-independent role of MDM2 in doxorubicin resistance in acute lymphoblastic leukemia. Gu L, Findley HW, Zhou M. Division of Pediatric Hematology/Oncology/BMT, Emory University School of Medicine, Atlanta, GA 30322, USA. MDM2 protein is thought to exhibit tumorigenic activity by binding to the p53 tumor-suppressor protein and inhibiting its function. Alternatively, MDM2 may have oncogenic roles other than those resulting from p53 interactions. Here we report that MDM2 can induce expression of the p65 subunit of NF-kappaB, which is an anti-apoptotic factor expressed in certain neoplastic cells in response to chemotherapy. Initially, we noted that the overexpression of MDM2 protein in leukemic bone marrow cells of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and an ALL cell line (EU-4) transfected with the MDM2 gene was associated with elevated expression of p65 and in vitro resistance to doxorubicin (Adriamycin). By cotransfection of the MDM2 gene and p65-promoter-reporter constructs into EU-4 cells, we found that transient and high-level MDM2 expression induced p65 promoter activity. In the presence of wild-type (wt) p53, MDM2 increased p65 promoter activity by reversing p53-mediated suppression of p65. In the absence of p53, MDM2 directly increased p65 promoter activity. Deletion and mutation analysis of the p65 promoter indicated that the region between nt -575 and -178, which contains the first and second Sp1-binding sites, was required for activation by MDM2. Further studies using chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) showed that MDM2 was able to directly bind to the Sp1 site of the p65 promoter. Our findings suggest that by inducing p65 expression, MDM2 has a p53-independent role in tumorigenesis, which may further elucidate the association between MDM2 overexpression and resistant disease in childhood ALL. PMID: 11964305 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Endocrinology. 2002 May;143(5):1778-88. Differential activation of the IGF binding protein-3 promoter by butyrate in prostate cancer cells. Tsubaki J, Hwa V, Twigg SM, Rosenfeld RG. Department of Pediatrics, School of Medicine, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201-3402, USA. Sodium butyrate (NaB), a dietary micronutrient, is a potent growth inhibitor that initiates cell differentiation in many cell types, including prostate cancer cells. The molecular mechanisms by which these effects occur remain largely unknown. In this study, we investigated the effects of NaB on the expression of IGF binding protein (IGFBP)-3, a known growth regulator, in two human prostate cancer cell lines (PC-3 and LNCaP). Treatment with NaB (0-10 mM) caused a dose-dependent stimulation of IGFBP-3 mRNA expression and parallel increases in protein levels. A specific histone deacetylase inhibitor, trichostatin A (TSA) similarly induced IGFBP-3 expression, indicating that histone hyperacetylation may be critical in the regulation of IGFBP-3 expression. To investigate the molecular mechanism of NaB-regulated IGFBP-3 expression, 1.87 kb of the human IGFBP-3 gene promoter was cloned into the pGL2-basic luciferase reporter vector. In both PC-3 and LNCaP cells, NaB (10 mM) significantly increased luciferase activity 20- to 30-fold, compared with the untreated control. However, using 5' sequential deletion constructs of the IGFBP-3 promoter, the NaB response sequences in the IGFBP-3 promoter were different in PC-3 and LNCaP cells. Our studies identified a region, -75 to +69 from the start of transcription (+1), that is fully inducible by NaB treatment in LNCaP cells, but not in PC-3 cells. Unlike other well characterized NaB-regulated genes, Sp1 DNA sequences are not involved in NaB up-regulation of IGFBP-3 gene in LNCaP cells. Further deletion studies identified two independent regions critical for NaB-induced transactivation in LNCaP cells. These regions contain consensus binding sites for p53 and GATA, respectively, but mutational analyses and gel shift assays suggested that, while the p53 response element is required for NaB responsiveness, neither p53 nor GATA are involved. In summary, we have demonstrated that 1) NaB significantly up-regulates IGFBP-3 mRNA and protein levels in PC-3 and LNCaP prostate cancer cells; and 2) novel butyrate- responsive elements lacking consensus Sp1 sites are used in LNCaP cells. PMID: 11956160 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: FEBS Lett. 2002 Feb 13;512(1-3):223-6. The characterization of the human Siah-1 promoter(1). Maeda A, Yoshida T, Kusuzaki K, Sakai T. Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, 602-8566, Kyoto, Japan. Siah-1, the human homologue of Drosophila seven in absentia, is related to apoptosis and tumor suppression. Although it was reported that the expression of Siah-1 is induced by p53 and p21/WAF1, little is known about the transcriptional regulation of the Siah-1 gene. To investigate the transcriptional regulation, we isolated and sequenced the genomic fragment of the Siah-1 promoter region. The Siah-1 promoter has no typical TATA box or CCAAT box. Transient transfection assays using reporter plasmids in which the promoter region of the Siah-1 gene was deleted or mutated showed that one Sp1 site was responsible for the basal promoter activity. In Northern blotting analysis, the expression of the Siah-1 gene was upregulated by p53, but activation of the reporter plasmid by the p53 co-transfection assay was not shown, suggesting that a p53 responsive element does not exist in the promoter region we examined in this study but might be present in another region. PMID: 11852084 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: J Biol Chem. 2002 Apr 26;277(17):14612-21. Epub 2002 Feb 13. Divergent regulation of the growth-promoting gene IEX-1 by the p53 tumor suppressor and Sp1. Im HJ, Pittelkow MR, Kumar R. Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA. IEX-1, a recently discovered early response gene, regulates cell growth and apoptosis. IEX-1 gene expression is regulated by a variety of factors such as x-irradiation, ultraviolet radiation, steroids, growth factors, and inflammatory stimuli. By systematic examination of the IEX-1 promoter, we show that IEX-1 gene expression is controlled by multiple conserved gene regulatory elements and that IEX-1 is a downstream target of the p53 tumor suppressor and Sp1. In addition, p300, Sox, nuclear factor-kappaB, and AP4 appear to be modulators of IEX-1 gene expression to a lesser degree. We found that there is at least one Sp1 element that functions as an activator and contributes to high basal transcriptional levels of the IEX-1 gene. We demonstrate the presence of a p53 response element that represses IEX-1 promoter activity in HaCaT keratinocytes, indicating that Sp1 and p53 have opposite effects on IEX-1 gene expression. We conclude that IEX-1 expression in cells is regulated by the p53 tumor suppressor and Sp1, thus providing a direct mechanism for control of cell proliferation. PMID: 11844788 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: J Biol Chem. 2002 Mar 1;277(9):7157-64. Epub 2001 Dec 17. HMGB1 and HMGB2 cell-specifically down-regulate the p53- and p73-dependent sequence-specific transactivation from the human Bax gene promoter. Stros M, Ozaki T, Bacikova A, Kageyama H, Nakagawara A. Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic. stros@ibp.cs The recently cloned gene p73 is a close homologue of p53, which is a crucial tumor suppressor gene for preventing the malignant transformation of cells by inducing cell cycle arrest and apoptosis. Previous reports have shown that architectural DNA-bending/looping chromosomal proteins HMGB1 and HMGB2 (formerly known as HMG1 and HMG2), which function in a number of biological processes including transcription and DNA repair, interact in vitro with p53 and stimulate p53 binding to DNA containing p53 consensus sites. Here, we report that HMGB1 physically interacts with two splicing variants of p73, alpha and beta (pull-down assay), and enhances binding of p73 to specific cognate DNA sites (gel-shift assay). Both HMG box domains of HMGB1, A and B, interact with p73alpha. Association of HMGB1 with p73, like the demonstrated ability of HMGB1 to stimulate p73 binding to different p53-responsive elements, requires the oligomerization region and/or region between DNA-binding domain and oligomerization domain of p73 (residues 312-381). Transient transfections revealed that ectopically expressed or endogenous HMGB1 and HMGB2 (antisense strategy) significantly inhibit in vivo both p73alpha/beta- and p53-dependent transactivation from the Bax gene promoter (and much less from Mdm2 and p21(waf1) promoters) in p53-deficient SAOS-2 cells. In contrast, HMGB1 and HGMB2 stimulate p73- or p53-dependent transactivation in p53-deficient H1299 cells, irrespective of the promoter used. Our results suggest that ubiquitously expressed HMGB1 and HMGB2 have potential to cell- and promoter-specifically down- or up-regulate in vivo transcriptional activity of different members of the p53 family. A possible mechanism of HMGB1-mediated modulation of p73- and p53-dependent transactivation is discussed. PMID: 11748232 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Cancer Res. 2001 Dec 1;61(23):8416-21. The constitutive photomorphogenesis 9 signalosome directs vascular endothelial growth factor production in tumor cells. Pollmann C, Huang X, Mall J, Bech-Otschir D, Naumann M, Dubiel W. Division of Molecular Biology, Department of Surgery, Medical Faculty Charite, Humboldt University, Berlin, Germany. christianpollman@hotmail.com Angiogenesis is a prerequisite for solid tumor growth and metastasis. Elucidation of the signaling pathways that control tumor angiogenesis constitutes the basis for a rational antiangiogenic tumor therapy. Here we show that the production of vascular endothelial growth factor (VEGF) in HeLa and HL-60 cells is directed by the constitutive photomorphogenesis 9 signalosome (CSN). The CSN is a kinase complex that cooperates with the ubiquitin/26S proteasome system in regulating the stability of proteins involved in signal transduction. VEGF expression is controlled by the transcription factors activator protein (AP)-1, AP-2, SP-1, and hypoxia-inducible factor 1. Inhibition of CSN kinase activity by 50 microM curcumin for 2 h decreases the cellular c-Jun concentration, resulting in a reduction of the VEGF production by approximately 75%. The removal of the inhibitor from the cells led to a time-dependent recovery of endogenous c-Jun that is paralleled by increasing VEGF production. Elevated cellular CSN activity induced by CSN subunit 2 overexpression causes increased VEGF production in HeLa cells. A competitor of CSN-dependent c-Jun phosphorylation, the NH(2)-terminal c-Jun fragment Deltac-Jun(1-226), inhibits VEGF production in HeLa cells. The transcription factors AP-2 and SP-1 act independently of the CSN. They contribute less than a quarter to basal VEGF production. Under our experimental conditions, hypoxia-inducible factor 1alpha protein was not detected. Overexpression of the tumor suppressor p53 reduces VEGF production in HeLa cells. p53 competes with c-Jun for CSN-specific phosphorylation with the consequence of c-Jun destabilization. We conclude that CSN-directed c-Jun signaling mediates high VEGF production in HeLa and HL-60 cells. The data provide an explanation for the known antiangiogenic and antitumorigenic activities of curcumin. Because the CSN regulates the major part of VEGF production in the tested tumor cells, it constitutes a potentially important target for tumor therapy. PMID: 11731421 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Cancer Gene Ther. 2001 Oct;8(10):803-14. Hammerhead ribozyme against gamma-glutamylcysteine synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both the glutathione synthesis and the expression of multidrug resistance proteins. Iida T, Kijima H, Urata Y, Goto S, Ihara Y, Oka M, Kohno S, Scanlon KJ, Kondo T. Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Nagasaki 852-8523, Japan. Multidrug resistance in cancer cells is often associated with an elevation in the concentration of glutathione (GSH) and the expression of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme for GSH. We constructed a hammerhead ribozyme against a gamma-GCS heavy subunit (gamma-GCSh) mRNA transcript and transfected it to human colonic cancer cells (HCT8DDP) resistant to cisplatin (CDDP). The effect of the ribozyme transfection on the drug resistance of cancer cells was studied. (a) Transfection of the ribozyme decreased the GSH level and the efflux of CDDP-GSH adduct, resulting in higher sensitivity of the cells to CDDP. (b) The transfection suppressed the expression of ATP-binding cassette (ABC) family of transporters such as MRP1, MRP2, and MDR1, and stimulated the expression of mutant p53. (c) An electrophoretic mobility shift assay showed that mutant p53 suppresses the SP1-DNA binding activity, suggesting that this mutant p53 is functional and it, in turn, suppresses the expression of ABC transporters. Collectively, transfection of anti-gamma-GCSh ribozyme reduced the synthesis of GSH and the expression of ABC transporters, which causes an increase in the sensitivity of cancer cells to anticancer drugs. Suppression of the SP1-DNA binding activity by p53 may be a factor of down-regulation of ABC transporters. PMID: 11687904 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Gene. 2001 Sep 5;275(1):163-8. Transcriptional repression of p21(waf1) promoter by hepatitis B virus X protein via a p53-independent pathway. Ahn JY, Chung EY, Kwun HJ, Jang KL. Department of Microbiology, College of Natural Sciences, Pusan National University, Pusan 609-735, South Korea. The X-gene product of hepatitis B virus (HBx) has been implicated in hepatitis B virus (HBV)-mediated hepatocellular carcinoma through its ability to induce liver cancer in some transgenic mice and to transactivate a variety of viral and cellular promoters. In this study, we demonstrated that the level of p21(waf1) RNA was decreased in the HBx-expressing cells and this effect was due to the transcriptional repression of the p21(waf1) gene by HBx via a p53-independent pathway. As the Sp1 binding sites of the p21(waf1) promoter were sufficient to confer HBx responsiveness to a previously non-responsive promoter, we suggested that HBx represses the transcription of p21(waf1) by downregulating the activity of Sp1. Because the tumor repressor p21(waf1) protein is a universal inhibitor of cyclin-CDK complexes and DNA replication that induces cell cycle arrest at the G1-S checkpoint, the repression of p21(waf1) by HBx might play an important role in a HBV-mediated pathogenesis. PMID: 11574165 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Cancer Res. 2001 Sep 15;61(18):6952-7. Central role of p53 on regulation of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) expression in mammary carcinoma. Pal S, Datta K, Mukhopadhyay D. Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA. The process of angiogenic switching is one of the most important factors in the growth and development of breast tumors. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is considered to be the most important directly acting angiogenic protein that has been shown to be up-regulated in breast cancer cells. Hypoxia seems to be an important stimulus for inducing VPF/VEGF mRNA expression in human mammary tumors. Here, we have studied the roles of the tumor suppressor gene p53 and the proto-oncogene c-Src in regulating the transcription of VPF/VEGF in breast cancer cell lines MCF-7 and MDA-MB 435 under both normoxic and hypoxic conditions. p53 significantly inhibited the transcription of VPF/VEGF involving the transcription factor Sp1. Increased binding of Sp1 to the VPF/VEGF promoter has been observed when the cells were exposed to hypoxia. It has been shown that p53 makes a complex with Sp1 and inhibits its binding to the VPF/VEGF promoter to prevent the transcriptional activation. Furthermore, c-Src kinase activity was found to be increased in the hypoxic condition, and in the presence of antisense of Src, there was down-regulation of the total mRNA level and also the promoter activity of VPF/VEGF. The present study indicates that p53 can also inhibit the hypoxic induction of Src kinase activity and thereby may prevent VPF/VEGF transcription. Taken together, our data suggest a central role of p53, through which it can inhibit VPF/VEGF expression by regulating the transcriptional activity of Sp1 and also by down-regulating the Src kinase activity, under both normoxic and hypoxic conditions. PMID: 11559575 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: J Biol Chem. 2001 Aug 3;276(31):29116-25. Epub 2001 May 30. Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene by the p53 tumor suppressor protein. Koutsodontis G, Tentes I, Papakosta P, Moustakas A, Kardassis D. Department of Basic Sciences, University of Crete Medical School, Heraklion GR-71110, Greece. In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Sp1 but have a mutated p53 form. In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Sp1 or related factors. Cotransfection of SL2 cells with p53 and Sp1 resulted in a synergistic transactivation of the p21 promoter. Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Sp1-binding site indicating functional cooperation between Sp1 and p53 in the transactivation of the p21 promoter. Synergistic transactivation was also decreased by mutations in the transactivation domain of p53. Physical interactions between Sp1 and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Sp1 is required for its physical interaction with p53. In conclusion, Sp1 must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis. PMID: 11384995 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: J Biol Chem. 2001 Aug 10;276(32):29729-39. Epub 2001 May 25. Transcriptional regulation of the human DNA polymerase delta catalytic subunit gene POLD1 by p53 tumor suppressor and Sp1. Li B, Lee MY. Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA. The DNA polymerase delta catalytic subunit gene (POLD1) was studied as a transcriptional target of p53. Northern blotting showed that a significantly decreased steady-state level of POLD1 mRNA was associated with increased wild-type p53 expression in cells treated with methyl methanesulfonate. When ectopic wild-type p53 expression was induced to a physiologically relevant level in "tet-off" cultured cells in which p53 expression was tightly regulated by tetracycline, it was found that POLD1 steady-state mRNA was repressed by about 65%. Transient cotransfection experiments using a POLD1 promoter luciferase reporter construct showed that: (i) POLD1 promoter activity was inhibited by transfected wild-type p53 plasmid to a maximum of about 86%; (ii) p53 mediated a large part of the transcriptional repression through a sequence-specific interaction with a site identified as the P4 site of the POLD1 promoter; (iii) tumor-derived p53 mutations in the p53 DNA-binding domain completely abolished the p53 transrepression activity. Moreover, transfection assays demonstrated that p53 was able to repress Sp1-stimulated POLD1 promoter activity and that this repression was largely due to the loss of the sequence-specific interaction between Sp1 protein and the P4 Sp1-binding site, which overlaps the P4 p53-binding site. Finally, gel shift assays suggested that p53 competes with Sp1 protein for binding to the P4 sequence of the POLD1 promoter. PMID: 11375983 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: J Biol Chem. 2001 May 25;276(21):18193-9. Epub 2001 Mar 14. p53-dependent transcriptional regulation of the APC promoter in colon cancer cells treated with DNA alkylating agents. Jaiswal AS, Narayan S. Department of Anatomy and Cell Biology and the Shands Cancer Center, College of Medicine, The University of Florida, Gainesville, Florida 32610, USA. The APC (adenomatous polyposis coli) gene product is involved in cell cycle arrest and in apoptosis. The loss of APC function is associated with the development of colorectal carcinogenesis. In previous studies, we have shown that the APC gene is inducible and that the DNA damage-induced level of APC mRNA requires p53. In the present study, we examined the role of p53 in the transcriptional regulation of APC promoter and characterized two p53-binding sites on the cloned APC promoter (pAPCP). Results of electrophoretic mobility shift assay showed specific interactions of p53 protein with p53-binding site oligonucleotides. The DNA-protein complex formed in electrophoretic mobility shift assay was competed with unlabeled excess of p53-binding site oligonucleotide, unaffected with p53-binding site mutant or Sp1-binding site oligonucleotides, and supershifted with anti-p53 antibodies. In a transient transfection assay, the pAPCP promoter activity was lower in HCT-116(p53(+/+)) cells versus HCT-116(p53(-/-)) cells. p53-dependent down-regulation was further confirmed after co-transfection of pAPCP plasmid with pCMV-p53 into HCT-116(p53(-/-)) and SAOS-2 (p53-negative) cells. However, the treatment of cells with DNA alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine, which cause phosphorylation of p53 at Ser(15) and Ser(392), induced pAPCP promoter activity in HCT-116(p53(+/+)) cells. Other than p53-binding sites, using deletion mutation constructs, we have shown that N-methyl-N'-nitro-N-nitrosoguanidine-induced transcriptional activation of the pAPCP promoter in HCT-116(p53(+/+)) cells depended upon the Sp1-binding site and the E-box B site. From these results, we conclude that unphosphorylated p53 can down-regulate and phosphorylated p53 can up-regulate the pAPCP promoter activity involving the p53, Sp1, or E-box B elements. These studies are important to understanding the role of p53 and APC in DNA damage-induced cell cycle arrest and/or apoptosis of cancer cells. PMID: 11279192 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: J Biol Chem. 2001 May 11;276(19):15598-608. Epub 2001 Feb 13. The tumor suppressor protein p53 requires a cofactor to activate transcriptionally the human BAX promoter. Thornborrow EC, Manfredi JJ. Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, USA. An important regulator of the proapoptotic BAX is the tumor suppressor protein p53. Unlike the p21 gene, in which p53-dependent transcriptional activation is mediated by a response element containing two consensus p53 half-sites, it previously was reported that activation of the BAX element by p53 requires additional sequences. Here, it is demonstrated that the minimal BAX response element capable of mediating p53-dependent transcriptional activation consists of two p53 half-sites plus an adjacent 6 base pairs (5'-GGGCGT-3'). This GC-rich region constitutes a "GC box" capable both of binding members of the Sp family of transcription factors, including Sp1 in vitro, and of conferring Sp1-dependent transcriptional activation on a minimal promoter in cells. Mutations within this GC box abrogated the ability of p53 to activate transcription without affecting the affinity of p53 for its binding site, demonstrating that these 6 bases are required for p53-dependent activation. In addition, a positive correlation was observed between the ability of p53 to activate transcription in cells and the ability of Sp1 to bind this response element in vitro. Mutations that inhibited Sp1 binding also blocked the ability of p53 to activate transcription through this element. Together, these results suggest a model in which p53 requires the cooperation of Sp1 or a Sp1-like factor to mediate transcriptional activation of the human BAX promoter. PMID: 11278953 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: Virology. 2001 Mar 1;281(1):10-20. Sp1-p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C. Torgeman A, Mor-Vaknin N, Zelin E, Ben-Aroya Z, Lochelt M, Flugel RM, Aboud M. Department of Microbiology and Immunology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel. We have previously demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) activates human T-cell leukemia virus type-I long terminal repeat (LTR) in Jurkat cells by a protein kinase C (PKC)-independent mechanism involving a posttranslational activation of Sp1 binding to an Sp1 site located within the Ets responsive region-1 (ERR-1). By employing the PKC inhibitor, bisindolylmaleimide I and cotransfecting the reporter LTR construct with a vector expressing PKC-alpha, we demonstrated, in the present study, that this effect of TPA was not only independent of, but actually antagonized by, PKC. Electrophoretic mobility shift assays together with antibody-mediated supershift and immuno-coprecipitation analyses, revealed that the posttranslational activation of Sp1 was exerted by inducing the formation of Sp1-p53 heterocomplex capable of binding to the Sp1 site in ERR-1. Furthermore, we demonstrated that Jurkat cells contain both wild-type (w.t.) and mutant forms of p53 and we detected both of them in this complex at variable combinations; some molecules of the complex contained either the w.t. or the mutant p53 separately, whereas others contained the two of them together. Finally, we showed that the Sp1-p53 complexes could bind also to an Sp1 site present in the promoter of another gene such as the cyclin-dependent kinase inhibitor p21(WAF-1), but not to consensus recognition sequences of the w.t. p53. Therefore, we speculate that there might be several other PKC-independent biological effects of TPA which result from interaction of such Sp1-p53 complexes with Sp1 recognition sites residing in the promoters of a wide variety of cellular and viral genes. Copyright 2001 Academic Press. PMID: 11222091 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: Biochem Biophys Res Commun. 2000 Dec 20;279(2):383-90. Mutant p53 forms a complex with Sp1 on HIV-LTR DNA. Chicas A, Molina P, Bargonetti J. Department of Biological Sciences, Institute for Biomolecular Structure and Function, Hunter College, 695 Park Avenue, New York, New York, 10021, USA. Many mutants of p53 activate HIV-LTR driven transcription and promote HIV replication. The region of the HIV-LTR containing Sp1-binding sites is important for this effect. In this study we test the hypothesis that mutant p53 interacts with DNA-bound Sp1 and in this way can increase transcription from Sp1-dependent promoters. We have used the breast cancer cell line MDA-MB-468 that expresses endogenous mutant p53(His273) as our source of p53 protein. First, we demonstrated that this mutant p53 participates in activating transcription from the HIV-LTR by showing that HIV-LTR-directed transcription in MDA-MB-468 cells is inhibited in a dominant-negative manner by p53(Val135). Using HIV-LTR DNA affinity chromatography, we detected coelution of p53(His273) and Sp1. We also demonstrated that this mutant p53 binds sequence specifically to the super consensus sequence (SCS) and that Sp1 coeluted with p53(His273) from a column containing this site. These data indicate that p53(His273) can associate with DNA-bound Sp1 suggesting that activated HIV-LTR transcription associated with mutant p53 occurs through a DNA driven multi-protein complex. Copyright 2000 Academic Press. PMID: 11118296 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Biochem Biophys Res Commun. 2000 Nov 11;278(1):125-33. Cloning and initial analysis of the human multidrug resistance-related MVP/LRP gene promoter. Lange C, Walther W, Schwabe H, Stein U. Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13092 Berlin, Germany. The lung resistance-related protein (LRP) was identified as the human major vault protein (MVP), and is overexpressed in various multidrug-resistant cancer cell lines and clinical samples. We characterized DNA sequences upstream to the transcription initiation site of the MVP gene in the human non-small cell lung cancer cell line SW-1573. A 1.9-kb and a shortened 0.7-kb fragment of the 5'-upstream genomic region show strong promoter activity in chloramphenicol acetyltransferase (CAT) reporter assays. The promoter is TATA-less and contains an inverted CCAAT-box and a Sp1 site located near to a p53 binding motif. An alternative 3'-splice site of intron 1 results in a splicing variant within the 5'-untranslated region of MVP mRNA. Copyright 2000 Academic Press. PMID: 11071864 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: Oncogene. 2000 Oct 26;19(45):5123-33. Downregulation of telomerase reverse transcriptase mRNA expression by wild type p53 in human tumor cells. Xu D, Wang Q, Gruber A, Bjorkholm M, Chen Z, Zaid A, Selivanova G, Peterson C, Wiman KG, Pisa P. Department of Medicine, Division of Hematology, Radiumhemmet, Karolinska Hospital, SE-171 76 Stockholm, Sweden. The p53 tumor suppressor protein inhibits the formation of tumors through induction of cell cycle arrest and/or apoptosis. In the present study we demonstrated that p53 is also a powerful inhibitor of human telomerase reverse transcriptase (hTERT), a key component for telomerase. Activation of either exogenous temperature-sensitive (ts) p53 in BL41 Burkitt lymphoma cells or endogenous wild type (wt) p53 at a physiological level in MCF-7 breast carcinoma cells triggered a rapid downregulation of hTERT mRNA expression, independently of the induction of the p53 target gene p21. Co-transfection of an hTERT promoter construct with wt p53 but not mutant p53 in HeLa cells inhibited the hTERT promoter activity. Furthermore, the activation of the hTERT promoter in Drosophila Schneider SL2 cells was completely dependent on the ectopic expression of Sp1 and was abrogated by wt p53. Finally, wt p53 inhibited Sp1 binding to the hTERT proximal promoter by forming a p53-Sp1 complex. Since activation of telomerase, widely observed in human tumor cell lines and primary tumors, is a critical step in tumorigenesis, wt p53-triggered inhibition of hTERT/telomerase expression may reflect yet another mechanism of p53-mediated tumor suppression. Our findings provide new insights into both the biological function of p53 and the regulation of hTERT/telomerase expression. PMID: 11064449 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Biochem Biophys Res Commun. 2000 Oct 5;276(3):1203-9. Molecular cloning and functional analysis of the promoter region of rat nonmuscle myosin heavy chain-B gene. Yam JW, Chan KW, Li N, Hsiao WL. Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, Kowloon, China. Rat nonmuscle myosin heavy chain-B (r-nmMHC-B) mRNA was previously found downregulated in Rat 6 fibroblasts transformed by mutant p53(val135) [J. W. P. Yam, J. Y. Zheng, and W. L. W. Hsiao (1987) Biochem. Biophys. Res. Commun. 266, 472-480]. Overexpression of exogenous r-nmMHC-B could partially reverse the transforming phenotypes both in vitro and in vivo. The downregulation of r-nmMHC-B was also observed in Rat 6 transformed by c-H-ras and v-myc oncogenes. We cloned a 5.2-kb r-nmMHC-B promoter region. Sequence analysis of -1248 to +1 revealed no TATA box, but did show that it contained CAAT boxes, E12/E47, MyoD, MEF, E2F, CREB, and SP1 binding sites. Based on transient reporter assays, the promoter/enhancer activities were unusually extended to the entire 5.2 kb region in normal Rat 6 cultures, but markedly suppressed in p53(val135)-, and c-H-ras-transformed cells. The activity detected by the reporter assay corresponded to levels of mRNA as analyzed previously by Northern blots in each respective cell line. Thus, the switch-off of the r-nmMHC-B in the transformed cells is very likely controlled by upstream transcriptional factors, which might have been altered in the course of neoplastic transformation. Copyright 2000 Academic Press. PMID: 11027611 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Oncogene. 2000 Aug 3;19(33):3717-26. Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma. Lee YI, Lee S, Das GC, Park US, Park SM, Lee YI. Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon. Aflatoxin B1 (AFB1) induced mutation of the p53 gene at codon 249 (p53mt249) is critical during the formation of hepatocellular carcinoma (HCC) following hepatitis B virus (HBV) infection. p53mt249 markedly increases insulin-like growth factor II (IGF-II) transcription largely from promoter 4, accumulating the fetal form of IGF-II. Modulation of the transcription factor binding to IGF-II P4 by wild-type p53 and p53mt249 was identified. Wild-type p53 inhibited binding of transcription factors Sp1 and TBP on the P4 promoter, while p53mt249 enhanced the formation of transcriptional complexes through enhanced DNA-protein (Sp1 or TBP) and protein-protein (Sp1 and TBP) interactions. p53mt249 stimulates transcription factor Sp1 phosphorylation which might be a cause of increased transcription factor binding on the P4 promoter while wild-type p53 does not. Transfection of hepatocytes with p53mt249 impaired induction of apoptosis by the HBV-X protein and TNF-alpha. Therefore, the blocking of apoptosis through enhanced production of IGF-II should provide a favorable opportunity for the selection of transformed hepatocytes. These results explain the molecular basis for the genesis of HCC by p53mt249 which was found to be induced by a potent mutagen, AFB1. PMID: 10949925 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Int J Radiat Biol. 2000 Aug;76(8):1045-53. Radiation-induced transcription factor activation in the rat cerebral cortex. Raju U, Gumin GJ, Tofilon PJ. Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA. PURPOSE: To determine the effects of ionising radiation on the DNA-binding activity of the injury-related transcription factors AP-1, Sp-1, p53 and NFkappaB in the rat brain. MATERIALS AND METHODS: Male Sprague-Dawley rats were irradiated with 137Cs gamma-rays at 3.8Gy/min and the cerebral cortex was isolated at intervals up to 24h. Nuclear protein extract of the cerebral cortex was analysed by electrophoretic mobility shift assay for DNA-binding activity of AP-1, Sp-1, p53 and NFkappaB. In addition, total RNA was extracted from the cerebral cortex and subjected to northern analysis. RESULTS: The DNA-binding activity of each of the transcription factors increased in a time- and dose-dependent manner after irradiation. Maximum increase in the activity of AP-1, Sp-1, and p53 DNA binding was seen after exposure to 10Gy and then decreased after higher doses. In contrast, NFkappaB DNA-binding activity continued to increase out to at least 30Gy. The levels of bFGF and p21WAF-1 mRNA increased after irradiation, suggesting an increase in the transactivating activity of AP-1, Sp-1, and p53. CONCLUSIONS: These data indicate that the response of the CNS to irradiation includes the activation of a similar set of transcription factors as previously observed after other types of insults. PMID: 10947117 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: J Biol Chem. 2000 Oct 27;275(43):33395-403. Modulation of glutathione S-transferase alpha by hepatitis B virus and the chemopreventive drug oltipraz. Jaitovitch-Groisman I, Fotouhi-Ardakani N, Schecter RL, Woo A, Alaoui-Jamali MA, Batist G. Lady Davis Institute of the Sir Mortimer B. Davis Jewish General Hospital, The Center for Translational Research in Cancer, Department of Medicine, McGill University, Montreal, Quebec H3T 1E2, Canada. Persistent infection by hepatitis B virus (HBV) and exposure to chemical carcinogens correlates with the prevalence of hepatocellular carcinoma in endemic areas. The precise nature of the interaction between these factors is not known. Glutathione S-transferases (GST) are responsible for the cellular metabolism and detoxification of a variety of cytotoxic and carcinogenic compounds by catalysis of their conjugation with glutathione. Diminished GST activity could enhance cellular sensitivity to chemical carcinogens. We have investigated GST isozyme expression in hepatocellular HepG2 cells and in an HBV-transfected subline. Total GST activity and selenium-independent glutathione peroxidase activity are significantly decreased in HBV transfected cells. On immunoblotting, HBV transfected cells demonstrate a significant decrease in the level of GST Alpha class. Cytotoxicity assays reveal that the HBV transfected cells are more sensitive to a wide range of compounds known to be detoxified by GST Alpha conjugation. Although no significant difference in protein half-life between the two cell lines was found, semi-quantitative reverse transcription-polymerase chain reaction shows a reduced amount of GST Alpha mRNA in the transfected cells. Because the HBV x protein (HBx) seems to play a role in HBV transfection, we also demonstrated that expression of the HBx gene into HepG2 cells decreased the amount of GST Alpha protein. Transient transfection experiments using both rat and human GST Alpha (rGSTA5 and hGSTA1) promoters in HepG2 cells show a decreased CAT activity upon HBx expression, supporting a transcriptional regulation of both genes by HBx. This effect is independent of HBx interaction with Sp1. Treatment with oltipraz, an inducer of GST Alpha, partially overcomes the effect of HBx on both promoters. Promoter deletion studies indicate that oltipraz works through responsive elements distinct from AP1 or NF-kappaB transcription factors. Thus, HBV infection alters phase II metabolizing enzymes via different mechanisms than those modulated by treatment with oltipraz. PMID: 10934196 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Cancer Res. 2000 Jul 1;60(13):3655-61. Wild-type p53 suppresses angiogenesis in human leiomyosarcoma and synovial sarcoma by transcriptional suppression of vascular endothelial growth factor expression. Zhang L, Yu D, Hu M, Xiong S, Lang A, Ellis LM, Pollock RE. Department of Surgical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA. Our recent studies (R. Pollock et al., Clin. Cancer Res., 4: 1985-1994, 1998; M. Milas et al., Cancer Gene Ther., in press, 2000) have shown that the restoration of wild-type (wt) p53 enhances cell cycle control in vitro and inhibits the growth of human soft-tissue sarcoma in severe combined immunodeficient mice. We hypothesized that the antitumor effect of wt p53 overexpression in sarcoma cells is attributable not only to enhanced cell cycle control but also to inhibition of angiogenesis. We evaluated the effect of restoring wt p53 function on angiogenesis in human soft-tissue sarcoma harboring mutant p53. Restoration of wt p53 expression in human leiomyosarcoma SKLMS-1 cells that contain mutant p53 markedly inhibited angiogenesis induced by tumor cells in vivo. Angiogenesis assays using an in vivo Matrigel plug assay demonstrated that less neovascularization in severe combined immunodeficient mice was observed with conditioned medium (CM) from human synovial sarcoma cells expressing wt p53 compared with CM from human synovial sarcoma cells expressing mutant p53. Microvessel density and microvessel counts were lower in tumor xenografts from cells containing wt p53 than in tumor xenografts from cells containing mutant p53. The growth and migration of murine lung endothelial cells were decreased when cells were treated with CM from sarcoma cells expressing wt p53 compared with CM from sarcoma cells expressing mutant p53. The introduction of wt p53 into sarcoma cells containing mutant p53 significantly reduced the expression of vascular endothelial growth factor (VEGF), which is a key mediator of tumor angiogenesis. Stimulation of endothelial cell migration by CM from cells expressing mutant p53 was significantly reduced after anti-VEGF neutralizing antibody was added to the CM. Using luciferase as the reporter of VEGF promoter activity, we found that wt p53 inhibited VEGF promoter activity in SKLMS-1 cells. Deletion analysis defined an 87-bp region (bp -135 to -48) in the VEGF promoter that is necessary for inhibiting VEGF promoter activity by wt p53. The transcription factor Sp1 may be involved in the repression of VEGF promoter activity by wt p53 in SKLMS-1 cells. These data indicated that wt p53 can suppress angiogenesis in human soft-tissue sarcomas by transcriptional repression of VEGF expression. PMID: 10910082 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: Clin Cancer Res. 2000 Apr;6(4):1239-47. Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. Kanaya T, Kyo S, Hamada K, Takakura M, Kitagawa Y, Harada H, Inoue M. Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, Ishikawa, Japan. Telomerase activation is a critical step in cellular immortality and oncogenesis. The activity of telomerase is known to be correlated with cell proliferation, but its regulation by cell cycle regulators is not well understood. In the present study, we examined the effects of p53 on telomerase activity. Wild-type p53 was introduced into SiHa cells via a recombinant adenoviral vector, Ad5CMV-p53, and change in telomerase activity was examined by quantitative telomerase assay. Telomerase activity in the Ad5CMV-p53-infected cells was significantly repressed 36 h after infection following down-regulation of human telomerase catalytic subunit [human telomerase reverse transcriptase (hTERT)] mRNA expression, whereas no change in telomerase activity was observed in the cells infected with control vector AdSCMV-beta-gal. Interestingly, repression of telomerase activity was an early event that preceded cell growth inhibition or apoptosis induced by p53 overexpression, suggesting that p53 directly regulates telomerase activity. Transient expression assays using hTERT-promoter reporter constructs revealed that overexpression of p53 significantly repressed promoter activity of hTERT. 5'-Truncation of the promoter sequences revealed that the proximal core promoter region containing multiple binding sites for transcription factor Spl was responsible for p53-mediated transcriptional repression. Mutations in these binding sites for Spl led to failure of p53 to repress transcription. These findings suggest that p53 repressed telomerase activity through down-regulation of hTERT transcription and that interaction of p53 with Sp1 or other transcription factors may be involved in this regulation. PMID: 10778946 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: J Biol Chem. 2000 Jun 16;275(24):18391-8. The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter. Zhang W, Geiman DE, Shields JM, Dang DT, Mahatan CS, Kaestner KH, Biggs JR, Kraft AS, Yang VW. Departments of Medicine and Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1) gene. We show that the gene encoding the gut-enriched Kruppel-like factor (GKLF, KLF4) is concurrently induced with p21(WAF1/Cip1) during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21(WAF1/Cip1) due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21(WAF1/Cip1), suggesting that GKLF may be involved in the induction of p21(WAF1/Cip1). Indeed, GKLF activates p21(WAF1/Cip1) through a specific Sp1-like cis-element in the p21(WAF1/Cip1) proximal promoter. The same element is also required by p53 to activate the p21(WAF1/Cip1) promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21(WAF1/Cip1) promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21(WAF1/Cip1) promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21(WAF1/Cip1) is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21(WAF1/Cip1) in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21(WAF1/Cip1) and may be part of a novel pathway by which cellular responses to stress are modulated. PMID: 10749849 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------
See help. --------------------------------------------------------------- 64: FASEB J. 2000 Feb;14(2):379-90. Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation. Yang CR, Wilson-Van Patten C, Planchon SM, Wuerzberger-Davis SM, Davis TW, Cuthill S, Miyamoto S, Boothman DA. Departments of Radiation Oncology and Pharmacology and the Ireland Comprehensive Cancer Center, Laboratory of Molecular Stress Responses, Case Western Reserve University, Cleveland, Ohio 44106-4942, USA. Regulation of transcriptional responses in growth-arrested human cells under conditions that promote potentially lethal damage repair after ionizing radiation (IR) is poorly understood. Sp1/retinoblastoma control protein (RCP) DNA binding increased within 30 min and peaked at 2-4 h after IR (450-600 cGy) in confluent radioresistant human malignant melanoma (U1-Mel) cells. Increased phosphorylation of Sp1 directly corresponded to Sp1/RCP binding and immediate-early gene induction, whereas pRb remained hypophosphorylated. Transfection of U1-Mel cells with the human papillomavirus E7 gene abrogated Sp1/RCP induction and G(0)/G(1) cell cycle checkpoint arrest responses, increased apoptosis and radiosensitivity, and augmented genetic instability (i.e., increased polyploidy cells) after IR. Increased NF-kappaB DNA binding in U1-Mel cells after IR treatment lasted much longer (i.e., >20 h). U1-Mel cells overexpressing dominant-negative IkappaBalpha S32/36A mutant protein were significantly more resistant to IR exposure and retained both G(2)/M and G(0)/G(1) cell cycle checkpoint responses without significant genetic instability (i.e., polyploid cell populations were not observed). Nuclear p53 protein levels and DNA binding activity increased only after high doses of IR (>1200 cGy). Disruption of p53 responses in U1-Mel cells by E6 transfection also abrogated G(0)/G(1) cell cycle checkpoint arrest responses and increased polyploidy after IR, but did not alter radiosensitivity. These data suggest that abrogation of individual components of this coordinate IR-activated transcription factor response may lead to divergent alterations in cell cycle checkpoints, genomic instability, apoptosis, and survival. Such coordinate transcription factor activation in human cancer cells is reminiscent of prokaryotic SOS responses, and further elucidation of these events should shed light on the initial molecular events in the chromosome instability phenotype.-Yang, C.-R., Wilson-Van Patten, C., Planchon, S. M., Wuerzberger-Davis, S. M., Davis, T. W., Cuthill, C., Miyamoto, S., Boothman, D. A. Coordinate modulation of Sp1, NF-kappa B, and p53 in confluent human malignant melanoma cells after ionizing radiation. PMID: 10657994 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Cell Death Differ. 1999 Sep;6(9):873-82. The activity of the murine Bax promoter is regulated by Sp1/3 and E-box binding proteins but not by p53. Schmidt T, Korner K, Karsunky H, Korsmeyer S, Muller R, Moroy T. Institut fur Zellbiologie (Tumorforschung), I F Z, Universitatsklinikum Essen, Virchowstrasse 173, Germany. In human cells the expression of the pro-apoptotic protein Bax appears to be regulated through p53-dependent transcriptional activation. However, in the mouse, p53-deficiency does not affect Bax expression. To shed more light on the transcriptional regulation of the bax gene we have analyzed the murine bax promoter. We find several E-box and Sp1/Sp3 binding sites as well as three putative p53 binding sites that are conserved in the human promoter sequence. We can show that both the Sp1 and the E-box binding sites are necessary for proper regulation of bax transcription and show by genomic DMS footprinting that all these sites are occupied in vivo. In contrast, the putative p53 binding sites were not occupied by protein in vivo in primary murine thymocytes either before or after induction of p53 by DNA damage. Moreover, p53 was unable to regulate the transcription of bax promoter fragments up to 6.5 kb in length. Further, steady state levels of bax mRNA did not correlate with Bax protein expression levels in DNA damage-induced cell death. Our findings exclude a direct transcriptional transactivation of the bax gene by p53 in murine cells suggesting a dominance of p53 independent mechanisms for the regulation of Bax protein expression. PMID: 10510469 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Nucleic Acids Res. 1999 Jul 15;27(14):2889-97. The Wilms' tumor suppressor gene (wt1) product represses different functional classes of transcriptional activation domains. Lee TH, Moffett P, Pelletier J. Department of Biochemistry, McGill Cancer Center, McGill University, Montreal, Quebec H3 1 Y6, Canada. We have studied the ability of the wt1 tumor suppressor gene product to repress different classes of activation domains previously shown to stimulate the initiation and elongation steps of RNA polymerase II transcription in vivo. Repression assays revealed that WT1 represses all three classes of activation domains: Sp1 and CTF, which stimulate initiation (type I), human immunodeficiency virus type I Tat fused to a DNA-binding domain, which stimulates predominantly elongation (type IIA), and VP16, p53 and E2F1, which stimulate both initiation and elongation (type IIB). WT1 is capable of exerting its repression effect over a significant distance when positioned approximately 1700 bp from the core promoter. Deletion analysis of WT1 indicates that the responsible domain resides within the first 180 N-terminal amino acids of the protein. Nuclear run-ons analyzing the effects of WT1 on initiation of transcription demonstrate inhibition of this process. Our observations imply that WT1 can repress activators that stimulate initiation and/or elongation. PMID: 10390530 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: Exp Cell Res. 1999 Apr 10;248(1):272-9. UV radiation is a transcriptional inducer of p21(Cip1/Waf1) cyclin-kinase inhibitor in a p53-independent manner. Haapajarvi T, Kivinen L, Heiskanen A, des Bordes C, Datto MB, Wang XF, Laiho M. Department of Virology, University of Helsinki, Haartmaninkatu 3, Helsinki, FIN-00014, Finland. p53 target genes p21(Cip1/Waf1) cyclin-kinase inhibitor (p21 CKI), GADD45, bax, and cyclin G and genes affecting the redox state of the cells are implicated in p53 damage control responses. In order to attribute their functions and dependency of p53 in UV-damaged cells we undertook an analysis of UVC responses of fibroblasts derived from p53 knock-out mice. UVC radiation efficiently and rapidly inhibited DNA replication in both p53 -/- and +/+ cells. The arrest was persistent in p53 -/- fibroblasts and cells underwent apoptosis, whereas p53 +/+ cells recovered and reentered the cycle. Protein and mRNA analyses of p21 expression showed that it was induced up to sixfold with similar kinetics both in the presence and in the absence of p53. However, high doses of UV abrogated the p21 response in p53 -/- cells, whereas it was maintained in cells with normal p53. UVC radiation transcriptionally activated p21 expression as demonstrated by luciferase reporter assays using deletion constructs of the p21 promoter. The promoter assays further confirmed the independency of p53-binding sites in the activation and linked UV-responsive transcriptional regulation of p21 to two Sp1 consensus binding sites within -61 bp of the transcription initiation site. A weaker regulation was mediated by elements between -1300 to -500 bp relative to the transcription initiation site. The results suggest that in fibroblasts UVC radiation is a rapid and efficient inducer of p21 expression also in a p53-independent manner. Copyright 1999 Academic Press. PMID: 10094833 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Cancer Res. 1998 Dec 15;58(24):5762-9. Transcriptional suppression of multidrug resistance-associated protein (MRP) gene expression by wild-type p53. Wang Q, Beck WT. Division of Developmental Therapeutics, Cancer Center, College of Medicine, University of Illinois at Chicago, 60607, USA. Multidrug resistance is a major obstacle to the success of cancer chemotherapy. The multidrug resistance-associated protein (MRP) has been shown to confer multidrug resistance. To study MRP gene expression at the transcriptional level, we have fused the MRP gene promoter with the luciferase reporter gene and studied its regulation. Cotransfection of MRP promoter constructs with p53 expression plasmids in p53-null human H1299 and mouse (10)1 cells demonstrated that the wild-type (wt) p53 markedly suppressed MRP promoter activity, whereas mutant p53 had little inhibitory effect. Transfections using 5' deletion mutant constructs of the MRP promoter showed that inhibition of the promoter activity by wt p53 mainly resided in the region from -91 to +103 bp, where several Sp1 transcription factor binding sites are localized. Cotransfection of the MRP promoter into Drosophila SL2 cells with an Sp1 expression vector increased the promoter activity in a dose-related manner up to approximately 200-fold. The stimulation of MRP promoter activity by Sp1 was attenuated by the cotransfection of a wt p53-expression plasmid. Furthermore, we have determined that endogenous MRP mRNA levels were down-regulated by restoration of wt p53-expression in a human lung cancer cell line. The relevance of MRP regulation in drug resistance was studied in a drug-resistant cell line, CEM/VM-1-5, that is approximately 140-fold more resistant to the epipodophyllotoxin, teniposide (VM-26), than the parental CEM cells. CEM/VM-1-5 cells express a much higher amount of MRP mRNA and protein than CEM cells, indicating that the resistant phenotype is at least partly due to increased MRP production. Transient transfection of the promoter constructs revealed that CEM/VM-1-5 cells had higher (7-fold) MRP promoter activity than CEM cells. Cotransfection of a wt p53-expression plasmid caused a reduction of MRP promoter activity in both CEM and CEM/VM-1-5 cells, but the inhibition was more than double in CEM/VM-1-5 cells compared with CEM cells. Our results demonstrated that wt p53 acts as a negative regulator of MRP gene transcription, at least in part by diminishing the effect of a powerful transcription activator Sp1. Therefore, a loss of wt p53 function and/or an increase in Sp1 activity in tumor cells could contribute to an up-regulation of the MRP gene. PMID: 9865734 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: J Biol Chem. 1998 Nov 27;273(48):31644-7. Regulation of RNA polymerase II-dependent transcription by poly(ADP-ribosyl)ation of transcription factors. Oei SL, Griesenbeck J, Schweiger M, Ziegler M. Institut fur Biochemie, Freie Universitat Berlin-Dahlem, Thielallee 63, D-14195 Berlin, Germany. Poly(ADP-ribosyl) transferase (ADPRT) is a nuclear protein that modifies proteins by forming and attaching to them poly(ADP-ribose) chains. Poly(ADP-ribosyl)ation represents an event of major importance in perturbed cell nuclei and participates in the regulation of fundamental processes including DNA repair and transcription. Although ADPRT serves as a positive cofactor of transcription, initiation of its catalytic activity may cause repression of RNA polymerase II-dependent transcription. It is demonstrated here that ADPRT-dependent silencing of transcription involves ADP-ribosylation of the TATA-binding protein. This modification occurs only if poly(ADP-ribosyl)ation is initiated before TATA-binding protein has bound to DNA and thereby prevents formation of active transcription complexes. Specific DNA binding of other transcription factors including Yin Yang 1, p53, NFkappaB, Sp1, and CREB but not c-Jun or AP-2 is similarly affected. After assembly of transcription complexes initiation of poly(ADP-ribosyl)ation does not influence DNA binding of transcription factors. Accordingly, if bound to DNA, transcription factors are inaccessible to poly(ADP-ribosyl)ation. Thus, poly(ADP-ribosyl)ation prevents binding of transcription factors to DNA, whereas binding to DNA prevents their modification. Considering its ability to detect DNA strand breaks and stimulate DNA repair, it is proposed that ADPRT serves as a molecular switch between transcription and repair of DNA to avoid expression of damaged genes. PMID: 9822623 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: Biol Pharm Bull. 1998 Sep;21(9):899-904. Identification of active substances from Streptomyces culture fluids using p53-independent expression of p21/WAF1/Cip1 gene and their mode of action. Egawa K, Nishigori H, Kunimoto S, Takeuchi T, Nose K. Department of Microbiology, Showa University School of Pharmaceutical Sciences, Tokyo, Japan. An assay system was constructed to identify chemicals that have a potential to induce p21/WAF1 gene, a target of the tumor suppressor p53 critical for negative growth regulation. Screening of about 1300 culture fluids of Streptomyces resulted in identification of active substances which induced the p21 gene in a p53-independent manner; one was a mixture of four members of the actinomycin group, and the other was trichostatin A. Transcriptional regulatory regions of p21 gene for induction by actinomycin D and trichostatin A were determined by transient expression of luciferase constructs in cells which are p53-deficient (Saos-2) or express a mutated form of p53 (TMK-1). The essential transcriptional elements for the response to these drugs localize within 210 bp of the 5'-upstream region of human p21 gene, and Sp1 elements were determined to be critical for the induction. DNA-binding activity of Sp1 was not increased in cells treated with these drugs, but kinase inhibitors such as staurosporin and wortmannin inhibited the induction. PMID: 9781835 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Mol Cell Biol. 1998 Nov;18(11):6457-73. The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53. Robertson KD, Jones PA. Norris Comprehensive Cancer Center, The University of Southern California, Los Angeles, California 90033, USA. The INK4a/ARF locus encodes two proteins involved in tumor suppression in a manner virtually unique in mammalian cells. Distinct first exons, driven from separate promoters, splice onto a common exon 2 and 3 but utilize different reading frames to produce two completely distinct proteins, both of which play roles in cell cycle control. INK4a, a critical element of the retinoblastoma gene pathway, binds to and inhibits the activities of CDK4 and CDK6, while ARF, a critical element of the p53 pathway, increases the level of functional p53 via interaction with MDM2. Here we clone and characterize the promoter of the human ARF gene and show that it is a CpG island characteristic of a housekeeping gene which contains numerous Sp1 sites. Both ARF and INK4a are coordinately expressed in cells except when their promoter regions become de novo methylated. In one of these situations, ARF transcription could be reactivated by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, and the reactivation kinetics of ARF and INK4a were found to differ slightly in a cell line in which both genes were silenced by methylation. The ARF promoter was also found to be highly responsive to E2F1 expression, in keeping with previous results at the RNA level. Lastly, transcription from the ARF promoter was down-regulated by wild-type p53 expression, and the magnitude of the effect correlated with the status of the endogenous p53 gene. This finding points to the existence of an autoregulatory feedback loop between p53, MDM2, and ARF, aimed at keeping p53 levels in check. PMID: 9774662 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: Mol Cell Biol. 1998 Nov;18(11):6191-200. Sp1-mediated transcription of the Werner helicase gene is modulated by Rb and p53. Yamabe Y, Shimamoto A, Goto M, Yokota J, Sugawara M, Furuichi Y. AGENE Research Institute, Kamakura, Kanagawa 247, Japan. The regulation of Werner's syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5' upstream region (2.8 kb) of WRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs. A region consisting of nucleotides -67 to +160 was identified as the principal promoter of WRN by reporter gene assays in HeLa cells, using a series of WRN promoter-luciferase reporter (WRN-Luc) plasmids that contained the 5'-truncated or mutated WRN upstream regions. In particular, two Sp1 elements proximal to the transcription initiation site are indispensable for WRN promoter activity and bind specifically to Sp1 proteins. The RCE enhances WRN promoter activity. Coexpression of the WRN-Luc plasmids with various dosages of plasmids expressing Rb or p53 in Saos2 cells lacking active Rb and p53 proteins showed that the introduced Rb upregulates WRN promoter activity a maximum of 2. 5-fold, while p53 downregulates it a maximum of 7-fold, both dose dependently. Consistently, the overexpressed Rb and p53 proteins also affected the endogenous WRN mRNA levels in Saos2 cells, resulting in an increase with Rb and a decrease with p53. These findings suggest that WRN expression, like that of other housekeeping genes, is directed mainly by the Sp1 transcriptional control system but is also further modulated by transcription factors, including Rb and p53, that are implicated in the cell cycle, cell senescence, and genomic instability. PMID: 9774636 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Mol Biol Rep. 1998 Jul;25(3):189-91. The 3-flanking region of the mouse c-fos gene contains a cluster of GGTCA hormone response-like elements. Hyder SM, Chiappetta C, Stancel GM. Department of Integrative Biology, Pharmacology and Physiology, University of Texas Medical School, Houston 77225, USA. We previously identified an estrogen response element in the 3'-flanking region of the c-fos protooncogene [1, 2]. This element, GGTCAnnnCAGCC, has one half-site identical to that of the consensus ERE (GGTCAnnnTGACC) but only limited homology to the second half-site. Because of this non-canonical sequence and atypical location in the 3'-untranslated region of an estrogen target gene, we decided to analyze sequences adjacent to this element for the possible presence of other regulatory elements. We now report that the 635 base pairs downstream of the c-fos ERE contain: (i) an unusual cluster of 7 GGTCA hormone response-like elements; (ii) potential binding sites for other known DNA binding proteins; and (iii) a sequence specific binding site for a non-estrogen receptor protein present in hormone target tissues. PMID: 9700055 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Nippon Geka Gakkai Zasshi. 1998 Jun;99(6):373-8. [A novel approach for cancer chemoprevention referred to as "gene-regulating chemoprevention"] [Article in Japanese] Nakano K, Yamagishi H, Oka T, Sakai T. Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Japan. We examined the effect of butyrate on the expression of WAF1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1 mRNA was rapidly induced by treatment with 5.0 mM butyrate, and significant WAF1 protein induction was detected. Using several mutant WAF1 promoter fragments, we found that the butyrate responsive elements are specific Sp1 sites. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1 promoter through specific Sp1 sites in a p53-independent fashion. Based on our results, we propose a novel approach for cancer chemoprevention, which we term "gene-regulating chemoprevention". Our strategy is to activate the potent function of growth-inhibitory genes, which are preserved in cancer cells. WAF1 is a good candidate, because it rarely appears to be mutated in common human tumors, while the p53 gene is frequently mutated. Therefore the p53-independent activation of WAF1 by butyrate could be applied to the prevention of cancer when p53 is mutated. Publication Types: Review Review, Tutorial PMID: 9695075 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: J Biol Chem. 1998 Aug 7;273(32):20052-7. Cooperative actions of HIV-1 Vpr and p53 modulate viral gene transcription. Sawaya BE, Khalili K, Mercer WE, Denisova L, Amini S. Center for NeuroVirology and NeuroOncology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA. Transcription of the human immunodeficiency virus type-1 (HIV-1) genome is controlled by cooperative interaction of viral encoded proteins and host regulatory proteins. In this study, we have examined the capacity of the viral auxiliary protein, Vpr, to modulate transcriptional activity of the HIV-1 promoter sequence located within the long terminal repeat (LTR). We demonstrate that ectopic expression of Vpr in human astrocytic cells, U-87MG, enhances the basal activity of the viral promoter in transfected cells and that the GC-rich sequences, spanning nucleotides -80 to -43, are important for this activity. Since this region serves as the target for p53-induced suppression of LTR activity and interacts with the ubiquitous transcription factor, Sp1, we examined the cooperative activity of Vpr, p53, and Sp1 upon LTR transcription. Results from co-transfection studies indicated that overexpression of wild type p53, but not mutant p53, decreases the level of activation of the LTR by Vpr. Transcriptional activation of the LTR by Vpr required the presence of Sp1 since overexpression of Vpr in cells with no endogenous Sp1 failed to augment LTR activity. Results from protein-protein interaction studies indicated that Vpr is associated with both p53 and Sp1 in cells with ectopic expression of these proteins. Moreover, it was evident that p53 and Sp1 interact with each other in these cells. These functional and structural studies provided a working model on the cooperative interaction of Vpr with cellular proteins Sp1 and p53 and control of viral gene transcription at immediate early stage of infection prior to the participation of other viral regulatory proteins. PMID: 9685344 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: Endocrinology. 1998 Mar;139(3):1101-7. p53 regulates insulin-like growth factor-I (IGF-I) receptor expression and IGF-I-induced tyrosine phosphorylation in an osteosarcoma cell line: interaction between p53 and Sp1. Ohlsson C, Kley N, Werner H, LeRoith D. Diabetes Branch, National Institute of Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1770, USA. claes@ss.gu.se The insulin-like growth factor-I receptor (IGF-IR) is involved in tumorigenesis. The aim of the present study was to investigate whether the IGF-IR is a physiological target for p53 in osteosarcoma cells. The p53-induced regulation of IGF-IR levels was studied in a tetracycline-regulated expression system. When expressed in Saos-2, osteosarcoma cells that lack p53, wild-type p53 decreased, whereas mutated p53 increased IGF-IR expression, and IGF-I-induced tyrosine phosphorylation of the IGF-IR. Similarly, wild-type p53 decreased IGF-I-induced tyrosine phosphorylation of IRS-1. A functional and physical interaction between p53 and Sp1, in the regulation of the IGF-R, was studied in osteosarcoma cells. Expression of p53 decreased IGF-IR promoter activity, whereas no effect on promoter activity was seen by Sp1 expressed alone. However, Sp1 counteracted the inhibitory effect of p53 on IGF-IR promoter activity in a dose-dependent manner. Furthermore, wild-type and mutated p53 were coimmunoprecipitated with Sp1, indicating a physical interaction between p53 and Sp1. In conclusion, p53 regulates IGF-IR expression, as reflected by a reduction in IGF-IR protein and a parallel reduction in IGF-I-induced tyrosine phosphorylation of the IGF-IR and IRS-1 in an osteosarcoma cell line. These data indicate that the IGF-I receptor is a physiological target for p53 in osteosarcoma cells. Furthermore, data supporting an interaction between p53 and Sp1 in the regulation of the promoter activity of IGF-IR are presented. PMID: 9492043 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Cell Mol Biol (Noisy-le-grand). 1997 Nov;43(7):935-49. p53 represses Sp1 DNA binding and HIV-LTR directed transcription. Bargonetti J, Chicas A, White D, Prives C. Department of Biological Sciences, Hunter College and Graduate School, CUNY 10021, USA. The HIV-LTR region contains binding sites for, and is regulated by, a number of transcription factors including Sp1 and NF-kB. The wild-type p53 tumor suppressor protein represses transcription from the HIV-LTR promoter while oncogenic mutant forms of p53 stimulate expression from the HIV-LTR. We have shown previously that wild-type p53 is a site specific DNA binding protein that binds to a region of the SV40 virus which contains GC-box DNA binding sites for the ubiquitously expressed transcription factor Sp1. In this study using DNase I footprinting, we have shown that purified p53 is able to protect the Sp1 binding sites and the adjacent NF-kB site of the HIV-LTR. Furthermore we have demonstrated that when p53 and Sp1 are mixed together both proteins change each other's interaction with DNA. Interestingly, we noted that oncogenic mutant p53 is also able to change the interaction of Sp1 with DNA. We confirmed p53 dependent repression of HIV-LTR driven transcription by comparing the expression from an HIV-LTR reporter construct in the presence and absence of p53. EMSA of an oligonucleotide sequence derived from the HIV-LTR sequence demonstrated a slight decrease in Sp1 DNA binding activity with nuclear extract derived from the cell line expressing a high level of wild-type p53. These data suggest that the influence of p53 on the transcription of promoters with Sp1 binding sites may be partially due to a change in the DNA binding ability of Sp1. PMID: 9449526 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: J Biol Chem. 1997 Aug 29;272(35):22199-206. Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line. Nakano K, Mizuno T, Sowa Y, Orita T, Yoshino T, Okuyama Y, Fujita T, Ohtani-Fujita N, Matsukawa Y, Tokino T, Yamagishi H, Oka T, Nomura H, Sakai T. Department of Preventive Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan. Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mM butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at -82 and -69 relative to the transcription start site. We also found that a TATA element at -46 and two overlapping consensus Sp1 sites at -60 and -55 are essential for the basal promoter activity of WAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion. PMID: 9268365 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: Eur J Biochem. 1997 May 1;245(3):730-7. Redox-mediated regulation of p21(waf1/cip1) expression involves a post-transcriptional mechanism and activation of the mitogen-activated protein kinase pathway. Esposito F, Cuccovillo F, Vanoni M, Cimino F, Anderson CW, Appella E, Russo T. Dipartimento di Biochimica e Biotecnologie Mediche, Universita degli Studi di Napoli Federico II, Naples, Italy. p21(waf1/cip1) gene expression is induced by DNA damage in cells with wild-type p53 and contributes to the arrest of cell growth. It was demonstrated that under many experimental conditions, including oxidative stress, p21(waf1/cip1) expression can be induced through p53-independent pathways. Since most of these experimental conditions induce the phosphorylation of mitogen-activated protein kinase (MAPK) and thus its activation, we evaluated p21(waf1/cip1) mRNA levels in cells exposed to an oxidative stress, induced by diethylmaleate (Et2Mal), and in which the MAPK pathway was blocked. The expression of a dominant-negative mutant of MEK, the MAPK kinase that phosphorylates and activates MAPK, and of a dominant-negative [Asn17]Ras mutant prevented the Et2Mal-induced accumulation of p21(waf1/cip1) mRNA. Similarly, the expression of MEK- and of [Asn17]Ras mutants decreased the 12-O-tetradecanoyl-phorbol 13-acetate (TPA)-mediated p21(waf1/cip1) induction. Furthermore, TPA-induced and serum-induced p21(waf1/cip1) mRNA accumulation was blocked by pretreating the cells with the antioxidant compound N-acetylcysteine, suggesting that oxidative stress is involved in these responses. p21(waf1/cip1) mRNA levels reached a maximum within 2 h of adding Et2Mal or TPA; however, the rate of transcription from a p21(waf1/cip1)-promoter construct did not increase during this period. In contrast, cells treated with actinomycin D show an increase of p21(waf1/cip1) mRNA stability after Et2Mal treatment. This result suggests that the increase in p21(waf1/cip1) mRNA at early times results from post-transcriptional regulatory events. Longer exposure to TPA may activate p21(waf1/cip1) gene transcription through an Sp1-dependent mechanism, while Et2Mal treatment gradually inhibits p21(waf1/cip1) gene transcription through oxidative changes that affect Sp1 binding to DNA. PMID: 9183012 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: Carcinogenesis. 1997 Feb;18(2):239-44. Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences. Butler AP, Johnson DG, Kumar AP, Narayan S, Wilson SH, MacLeod MC. Department of Carcinogenesis, University of Texas, M.D. Anderson Cancer Center, Smithville 78957, USA. Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression. PMID: 9054613 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Nucleic Acids Res. 1996 Nov 1;24(21):4281-8. Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein. Farmer G, Friedlander P, Colgan J, Manley JL, Prives C. Department of Biological Sciences, Columbia University, New York, NY 10027, USA. In addition to serving a role as a DNA binding-dependent transcriptional activator, p53 has been reported to repress a variety of promoters that lack p53 binding sites. Data from recent studies have suggested that this activity is mediated via an interaction between p53 and the TATA box binding protein (TBP). To investigate the functional relevance of this interaction in vivo, we have performed transient transfection assays in Drosophila Schneider cells. Wild-type p53 was found to repress expression from TATA box- but not initiator (Inr)-containing promoters activated by GAL4-VP16, GAL4-ftzQ or Sp1. A mutant p53(His175), defective in DNA binding and transcriptional activation, also inhibited TATA-dependent transcription activated by Sp1. However, p53 was unable to repress a basal TATA promoter stimulated by overexpression of TBP. Furthermore, overexpression of TBP failed to rescue the p53-mediated repression of activated transcription and a p53 mutant with its N-terminal TBP interaction domain intact, but defective in transcriptional activation and binding to TBP-associated factors (TAFs), was similarly defective in transcriptional repression. These data suggest that a p53-TBP interaction is not sufficient for transcriptional repression by p53 and that repression involves an interaction between p53 and other factors, such as TAFs, that are required for activated but not basal transcription. We suggest that p53-mediated repression results from squelching of a factor limiting for activated transcription from TATA- but not Inr-containing promoters. PMID: 8932384 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: Gene. 1996 Oct 17;176(1-2):259-62. Analysis of intron 4 of the p53 gene in human cutaneous melanoma. Shamsher M, Montano X. Skin Tumour Laboratory, London Hospital Medical College, UK. DNA sequencing of intron 4 of the p53 gene from seven cutaneous melanoma cell lines showed an absence of mutations. However, both control and melanoma cell lines sequences were different from the reference source obtained from GenBank databank (accession No. X54156). Base pairs 101 and 689 were determined to be T (instead of A) and C (instead of G). Also, an additional C was not detected at position 371. Comparative analysis with p53 DNA-binding sequences, a sequence recognized by a p53 intron 4-binding protein and consensus sequences recognized by transcription factors demonstrated that intron 4 contains putative sequences for NF-kappa B, SP1, AP1 and TFIID binding. Binding of transcription factors could be one of the mechanisms by which intron 4 modulates human p53 expression. PMID: 8918263 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Mol Cell Biol. 1996 Aug;16(8):4295-304. Functional interaction between p53, the TATA-binding protein (TBP), andTBP-associated factors in vivo. Farmer G, Colgan J, Nakatani Y, Manley JL, Prives C. Department of Biological Sciences, Columbia University, New York, New York 10027, USA. The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAFII230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAFII40 and TAFII60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAFII110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53. PMID: 8754830 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Oncogene. 1996 Jul 18;13(2):323-32. Interaction of human polyomavirus BK with the tumor-suppressor protein p53. Shivakumar CV, Das GC. Department of Molecular Biology, University of Texas Health Center At Tyler 75710, USA. We have been studying the interaction of the oncogenic human polyomavirus BK (BKV) with the tumor-suppressor protein p53 to understand the biology of this virus as well as to understand the basic mechanisms of p53 transactivation. We here demonstrate that p53 binds specifically to the viral promoter at two different sites, S-I (np 361-383) and S-II (np 314-336) in the late region. Site S-I is a 23 bp domain comprising an unique combination of a 10 bp consensus monomer binding site (Pu Pu Pu C (A/T) (T/A) G Py Py Py) which is contiguous with a GC-rich Sp-1 motif that binds p53 in the SV40 promoter. Site S-II also spans a 23 bp sequence containing two tandem consensus binding sites with three base pair mismatches in each and a one base pair deletion. A dimer of a 100 bp region spanning both the binding sites or the site S-I alone induced p53 responsiveness to a basal promoter when cloned upstream from the TATA box, but a similar construct using S-II did not. One tumor-derived mutant protein, p53-175 H, which is defective in DNA binding, also failed to transactivate the reporter gene. We further show that p53 binding-dependent transactivation is abrogated by BKV large T antigen, thereby suggesting an interaction between these two proteins in vivo. In contrast to the isolated p53 binding site, viral early promoter is repressed by p53 in H 1299 cells and the mutants are defective in this function to varying extent. This is suggestive of an involvement of cellular factors in modulating p53's function in the context of the whole promoter. p53 binding sites in BKV are flanked by the binding sites for transcription factors Sp-1 and NF-1 and we show that these transcription factors are present in the immunocomplex with purified p53, implicating modification of p53's transactivation function by protein-protein interaction. Thus, oncoprotein synthesis in this virus might be modulated by p53 in vivo by a complex mechanism other than simple DNA binding and sequestration of the TATA binding protein. Together with SV40 and polyomavirus, which also harbor p53 binding sites, this viral system will serve as a model to understand the role of p53 in viral infection. PMID: 8710371 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: Cancer Res. 1996 Jun 15;56(12):2781-8. Repression of the insulin receptor promoter by the tumor suppressor gene product p53: a possible mechanism for receptor overexpression in breast cancer. Webster NJ, Resnik JL, Reichart DB, Strauss B, Haas M, Seely BL. Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, California 92093, USA. There is strong evidence to suggest that insulin and insulin-like growth factor (IGF)-I may be important for tumor growth. Both the insulin and IGF-I receptors (IGF-IR) are overexpressed in breast cancer, and antibody blockade of the IGF-IR inhibits the growth of some breast cancer cell lines. Furthermore, expression of an insulin receptor (IR) in a normal mammary epithelia] cell line causes insulin-dependent transformation. Functional inactivation of p53 is also very frequent in many tumors. In this paper, we investigated whether inactivation of p53 might be involved in the overexpression of the IR in malignancy, specifically breast cancer. We demonstrate a positive correlation between IR and IGF-IR levels and p53 overexpression in primary human breast malignancies. To examine possible mechanisms by which p53 may regulate IR gene expression, we show that p53 can repress the IR promoter and that a dominant-negative p53 (248Q) can de-repress the promoter in cells containing normal p53. The p53 effect was shown to be mediated by C/EBP and Sp1 transcription factors. We also documented that p53-null mice had elevated levels of Sp1, but not C/EBPalpha, and that insulin binding to liver extracts was increased compared to wild-type controls. These results suggest that p53 inactivation may lead to an up-regulation of genes, such as the IR, that are dependent on these transcription factors. PMID: 8665514 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):470-4. Selective repression of transcriptional activators by Pbx1 does not require the homeodomain. Lu Q, Kamps MP. Department of Pathology, University of California, San Diego, School of Medicine, La Jolla 92093, USA. PBX1 is a homeobox-containing gene identified as the chromosome 1 participant of the t(1;19) chromosomal translocation of childhood pre-B-cell acute lymphoblastic leukemia. This translocation produces a fusion gene encoding the chimeric oncoprotein E2A-Pbx1, which can induce both acute myeloid and T-lymphoid leukemia in mice. The binding of Pbx1 to DNA is weak; however, both Pbx1 and E2A-Pbx1 exhibit tight binding to specific DNA motifs in conjunction with certain other homeodomain proteins, and E2A-Pbx1 activates transcription through these motifs, whereas Pbx1 does not. In this report, we investigate potential transcriptional functions of Pbx1, using transient expression assays. While no segments of Pbx1 activated transcription, an internal domain of Pbx1 repressed transcription induced by the activation domain of Sp1, but not by the activation domains of VP16 or p53. This Pbx1 domain, which lies upstream of the homeodomain and is highly conserved among Pbx proteins, is thus predicted to bind a specific transcription factor. Surprisingly, the repression activity of Pbx1 did not require homeodomain-dependent DNA binding. Thus, Pbx1 may be able to alter gene transcription by both DNA-binding-dependent and DNA-binding-independent mechanisms. PMID: 8552663 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Oncogene. 1995 Oct 5;11(7):1299-307. p53 represses SV40 transcription by preventing formation of transcription complexes. Perrem K, Rayner J, Voss T, Sturzbecher H, Jackson P, Braithwaite A. Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia. There is now much evidence to suggest that the p53 tumour suppressor protein functions to monitor the integrity of the genome. When DNA damage is detected, p53 suppresses cell growth to allow repair or directs the cell into apoptosis. The mechanism of action of p53 is as yet unclear but recent evidence has accumulated to suggest that p53 might act by regulating gene expression. Consistent with this model, p53 can both activate and repress a number of viral and cellular promoters. p53 has also been shown to bind to the CCAAT-binding Factor and TATA-binding protein (TBP), and there is direct evidence that p53 represses in vitro transcription by preventing TBP from binding DNA. We now provide evidence that p53 can repress transcription from the SV40 promoter by disrupting DNA/protein complexes involving transcription factor Sp1. PMID: 7478550 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: J Biol Chem. 1995 Aug 25;270(34):19680-3. p53 and Sp1 interact and cooperate in the tumor necrosis factor-induced transcriptional activation of the HIV-1 long terminal repeat. Gualberto A, Baldwin AS Jr. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599, USA. Tumor necrosis factor alpha (TNF) is a potent activator of transcription directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). We have recently reported that the p53 tumor suppressor gene product binds to a site within the Sp1 binding region of the HIV-1 LTR and contributes to the TNF induction of this promoter. In this study we show that the transcription factor Sp1 cooperates with p53 in the transcriptional activation directed by the HIV-1 LTR. The presence of Sp1 increased p53 binding to its recognition sequence in the HIV-1 LTR, and experiments in Drosophila cells show that Sp1 is necessary for full transactivation by mutant p53. Importantly, TNF induced the association between p53 and Sp1 in Jurkat T cells. These data demonstrate a synergistic role for these proteins in the mechanism of TNF induction of HIV-1 LTR-mediated transcription and suggest that Sp1 may play an important role in modulating certain functions of p53. PMID: 7649977 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: J Biol Chem. 1995 Apr 14;270(15):8837-43. The retinoblastoma susceptibility gene product represses transcription when directly bound to the promoter. Adnane J, Shao Z, Robbins PD. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA. Rb represses E2F-mediated transcription in part by blocking the trans-activation domain of E2F. In addition, Rb can convert an E2F binding site from a positive to a negative element. To examine the effect of a Rb-DNA-bound complex on transcription, full-length Rb was fused to the DNA binding domain of GAL4. Here, we report that GAL4-Rb can repress transcription mediated by either Sp1, AP-1, or p53, dependent upon the presence of both the GAL4 DNA binding domain and GAL4 binding sites. Moreover, GAL4-Rb inhibited the activity of the herpes simplex virus tk promoter from GAL4 binding sites located at a distance from the promoter. In contrast, GAL4-Rb was unable to repress basal transcription. Cotransfection of specific cyclins and cyclin-dependent kinases or SV40 T-antigen abolished the repressive activity of GAL4-Rb. The domains of Rb involved in mediating the repression of transcription were mapped to regions that are overlapping, but not identical, to those required for the interaction with E2F. We propose that Rb can function as a general repressor of transcription when bound to the promoter region. PMID: 7721791 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: J Virol. 1994 Jul;68(7):4302-13. The tumor suppressor protein p53 strongly alters human immunodeficiency virus type 1 replication. Duan L, Ozaki I, Oakes JW, Taylor JP, Khalili K, Pomerantz RJ. Dorrance H. Hamilton Laboratories, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107. The p53 tumor suppressor gene product, a sequence-specific DNA-binding protein, has been shown to act as a transcriptional activator and repressor both in vitro and in vivo. Consistent with its role in regulating transcription are recent observations that the N-terminal acidic domain of p53 binds directly to the TATA box-binding protein subunit of the general transcription factor, TF IID. It is now demonstrated that wild-type p53 (wt-p53) inhibits human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed chloramphenicol acetyltransferase activity in a cotransfection assay system. Importantly, this effect of wt-p53 on the HIV-1 LTR was also demonstrated by in vitro transcription assays. In addition, the Sp1 sites and the TATA box of the HIV-1 LTR are demonstrated to be the primary sites involved with p53-induced effects on this viral promoter. The upstream elements of the HIV-1 LTR, including the nuclear factor kappa B (NF-kappa B) binding sites, decrease the p53-induced inhibitory effects on viral transcription. In the presence of the HIV-1 TAR sequence and Tat protein, the HIV-1 LTR also becomes less sensitive to wt-p53-induced inhibition. By using a retroviral vector delivery system, mutant forms of p53 genes were expressed in two HIV-1 latently infected cell lines, ACH-2 and U1. In the ACH-2 cell line, which is now demonstrated to contain an endogenous mutant form of p53 (amino acid 248, Arg to Gln), additional mutant p53 proteins did not alter HIV-1 replication. In U1 cells, which completely lack endogenous p53, overexpression of mutant p53 led to an increase in HIV-1 replication. Thus, these data indicate a possible functional role for wt-p53 and mutant p53 proteins in the control of HIV-1 replication patterns and proviral latency. PMID: 8207805 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5957-61. Sequence-independent induction of Sp1 transcription factor activity by phosphorothioate oligodeoxynucleotides. Perez JR, Li Y, Stein CA, Majumder S, van Oorschot A, Narayanan R. Division of Oncology, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ 07110. Modified analogues of antisense oligodeoxynucleotides (ODNs), particularly phosphorothioates ([S]ODNs), have been extensively used to inhibit gene expression. The potential sequence specificity of antisense oligomers makes them attractive as molecular drugs for human diseases. The use of antisense [S]ODNs to inhibit gene expression has been complicated by frequent nonspecific effects. In this study we show in diverse cell types that [S]ODNs, independent of their base sequence, mediated the induction of an Sp1 nuclear transcription factor. The [S]ODN-mediated Sp1 induction was rapid and was associated with elevated levels of Sp1 protein. This induction was dependent on NF-kappa B activity, since inhibition of NF-kappa B activity abolished the [S]ODN-induced Sp1 activity. [S]ODN-induced Sp1 activity was seen in mouse spleen cells following in vivo administration. Sp1 activity induced by [S]ODNs required the tyrosine kinase pathway and did not have transactivating potential. These results may help to explain some of the non-specific effects often seen with [S]ODNs. PMID: 8016096 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: G Batteriol Virol Immunol. 1994 Jan-Dec;86(1-12):43-54. [Protein p53 inhibits the activity of the enhancer of the immediate-early genes of murine cytomegalovirus] [Article in Italian] Lembo D, Angeretti A, Cavallo R, Gariglio M, Gribaudo G, Landolfo S. Istituto di Microbiologia, Universita degli Studi di Torino. The protein encoded by the tumor suppressor gene p53 can complex and functionally interact with cytomegalovirus proteins produced during the immediate-early phase of infection. The functions of these complex are unclear but there is some evidence to suggest that binding of p53 to these viral proteins may inactivate p53 functions. Recent reports have shown that p53 is involved in regulation of transcription. In this study we have considered the possibility that p53 may regulate transcription of cytomegalovirus immediate early genes which play a crucial role for virus replication. Here we report that experiments in which NIH 3T3 cells were cotransfected with a p53 expression plasmid together with a reporter gene linked to the mouse cytomegalovirus immediate-early enhancer/promoter revealed that wild type p53 could efficiently reduce the transcriptional activity of this viral regulatory sequence. By contrast expression of a mutated p53 correlated with a much smaller reduction of transcription. Deletion mutants analysis of the enhancer revealed that repression of transcription by p53 requires a minimal promoter containing an SP1 consensus sequence and a TATA box. PMID: 8706975 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: Exp Cell Res. 1994 Jan;210(1):94-101. Effect of tumor suppressors on cell cycle-regulatory genes: RB suppresses p34cdc2 expression and normal p53 suppresses cyclin A expression. Yamamoto M, Yoshida M, Ono K, Fujita T, Ohtani-Fujita N, Sakai T, Nikaido T. Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104. We show that expression of the p34cdc2 and cyclin A genes is induced by interleukin-2 in normal human T cells and present evidence to support the idea that these genes are deregulated in leukemic T cells. Our DNA sequencing data indicate that the promoter region of the p34cdc2 gene contains putative E2F-like binding sites which are recognized by Rb and binding sites for c-myb, Sp1, and ATF, and that the promoter region of the cyclin A gene contains binding sites for p53, Sp1, and ATF. In this study we focus on the effect of p53 and Rb on these cell cycle-regulatory genes. Cotransfection of Y79 human retinoblastoma cells with a p34cdc2 promoter-luciferase expression vector and a plasmid expressing the retinoblastoma gene (RB) indicated that RB suppresses p34cdc2 expression. Cotransfection of B104 rat neuroblastoma cells with a cyclin A promoter-luciferase expression vector and a plasmid expressing the normal or mutant p53 indicated that only the normal p53 suppresses cyclin A expression. In normal T cells, PHA stimulation reduces the amount of complexes in the p34cdc2 promoter between the E2F-like binding site and the RB gene product. These complexes were not detected in leukemic T cells. Our data support the idea that tumor suppressors modulate the expression of cell cycle-regulatory genes: RB regulates p34cdc2 expression and p53 regulates cyclin A expression. PMID: 8270002 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: J Virol. 1994 Jan;68(1):103-10. Activation of the human immunodeficiency virus type 1 long terminal repeat by transforming mutants of human p53. Subler MA, Martin DW, Deb S. Department of Microbiology, University of Texas Health Science Center, San Antonio 78284. We have studied the effects of human wild-type and mutant p53s on the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV). HeLa cells were cotransfected with a wild-type or mutant p53 expression plasmid and a plasmid containing a chloramphenicol acetyltransferase reporter gene under HIV LTR promoter control. As expected, expression of wild-type p53 inhibited promoter function. Expression of a p53 mutated at any one of the four amino acid positions 175, 248, 273, and 281 correlated with a significant increase of the HIV promoter activity. The HIV LTR was also significantly activated in Saos-2 cells that do not express endogenous p53. This finding suggests a gain-of-transactivation function by mutation of the p53 gene. Cotransfection of wild-type and mutant p53-281G expression plasmids indicated that either the wild type or the mutant was dominant in inhibiting or enhancing promoter activity, respectively, when transfected in excess of the other. Transfection experiments showed transactivation even when the Sp1, NF-kappa B, and TATA sites in the LTR were individually mutated. Synthetic minimal promoter constructs containing two Sp1 sites or two NF-kappa B sites or an ATF site are also significantly activated by the mutant p53-281G. Thus, the mutant protein may activate transcription through interaction with either a general transcription factor or a common factor that bridges the basal transcription machinery and the transcription factors Sp1, NF-kappa B, and ATF. PMID: 8254719 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Cold Spring Harb Symp Quant Biol. 1994;59:207-13. DNA-binding properties of the p53 tumor suppressor protein. Prives C, Bargonetti J, Farmer G, Ferrari E, Friedlander P, Wang Y, Jayaraman L, Pavletich N, Hubscher U. Department of Biological Sciences, Columbia University, New York, New York 10027, USA. PMID: 7587071 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: Trends Biochem Sci. 1993 Nov;18(11):433-7. DNA damage and the DNA-activated protein kinase. Anderson CW. Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000. DNA-activated protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is activated in vitro by DNA fragments. The cellular targets of DNA-PK are nuclear, DNA-binding, regulatory proteins including Sp1, Fos, Jun, Myc, the tumor suppressor protein p53, and RNA polymerase II. These characteristics suggest a role for DNA-PK in coordinating nuclear processes and as a modulator of checkpoint mechanisms activated by DNA damage. Publication Types: Review Review, Tutorial PMID: 8291090 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: J Biol Chem. 1993 Apr 15;268(11):7923-8. Induction of Sp1-p53 DNA-binding heterocomplexes during granulocyte/macrophage colony-stimulating factor-dependent proliferation in human erythroleukemia cell line TF-1. Borellini F, Glazer RI. Department of Pharmacology, Georgetown University Medical Center, Washington, D.C. 20007. The involvement of Sp1 in regulating cell proliferation in myeloid leukemia cells was determined by measuring the levels and DNA binding activity of Sp1 in TF-1 cells, a human erythroleukemia cell line dependent on granulocyte/macrophage colony-stimulating factor (GM-CSF) for viability and cell growth. DNA binding of Sp1 to a specific double-stranded oligodeoxynucleotide was increased markedly in a dose-dependent manner in proliferating cells in response to GM-CSF compared with growth-arrested or apoptotic cells. Competition experiments and mobility shift interference assays with antibodies against Sp1 as well as wild-type or mutant p53 indicated that GM-CSF-inducible DNA-binding complexes contained both Sp1 and p53 and that these heterocomplexes bound to both p53- and Sp1-binding sequences with high affinity. Immunoprecipitation of nuclear extracts with a p53 antibody indicated that Sp1 was associated as a heterocomplex with p53. Formation of this complex was dependent on the level of p53 since p53 was more abundant in proliferating cells and decreased upon induction of growth arrest and apoptosis by withdrawal of GM-CSF while Sp1 levels remained unchanged. These results suggest that the association of Sp1 with p53 may represent a novel mechanism of growth regulation in cytokine-dependent leukemia cells. PMID: 8463313 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: Nucleic Acids Res. 1993 Mar 25;21(6):1439-47. Identification of a DNA structural motif that includes the binding sites for Sp1, p53 and GA-binding protein. MacLeod MC. Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957. We have analyzed predicted helical twist angles in the 21-bp repeat region of the SV40 genome, using a semi-empirical model previously shown to accurately predict backbone conformations. Unexpectedly, the pattern of twist angles characteristic of the six GC-boxes is repeated an additional five times at positions that are regularly interspersed with the six GC-box sequences. These patterns of helical twist angles are associated with a second, imperfectly-repeated sequence motif, the TR-box 5'-RRNTRGG. Unrelated DNA sequences that interact with trans-acting factors (p53 and GABP) exhibit similar twist angle patterns, due to elements of the general form 5'-RRRYRRR that occur as interspersed arrays with a spacing of 10-11 bp and an offset of 4-6 bp. Arrays of these elements, which we call pyrimidine sandwich elements (PSEs), may play an important role in the interaction of trans-acting factors with DNA control regions. In 13 human proto-oncogenes analyzed, we identified 31 PSE arrays, 11 of which were in the 5'-flanking regions of the genes. The most extensive array was found in the promoter region of the K-ras gene. Extending over 80 bp of DNA, it contained 16 PSEs that showed an average deviation from the SV40 criterion pattern of angles of only 1.2 degrees. PMID: 8385318 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------