1: Cancer Res. 2005 Jun 15;65(12):5133-43. Early growth response 1 acts as a tumor suppressor in vivo and in vitro via regulation of p53. Krones-Herzig A, Mittal S, Yule K, Liang H, English C, Urcis R, Soni T, Adamson ED, Mercola D. Sidney Kimmel Cancer Center, San Diego, California 92121, USA. The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFbeta1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor. PMID: 15958557 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Cancer Genet Cytogenet. 2005 May;159(1):1-9. A combination of molecular cytogenetic analyses reveals complex genetic alterations in conventional renal cell carcinoma. Strefford JC, Stasevich I, Lane TM, Lu YJ, Oliver T, Young BD. Cancer Research UK Medical Oncology Unit, Queen Mary University of London, Charterhouse Square, London, UK. jcs@soton.ac.uk Here we report the complex pattern of genomic imbalances and rearrangements in a panel of 19 renal cell carcinoma cell lines detected with molecular cytogenetic analysis. Consistent heterogeneity in chromosome number was found, and most cell lines showed a near-triploid chromosome complement. Several cell lines showed deletions of the TP53 (alias p53), CDKN2A (alias p16), and VHL genes. Multiplex fluorescence in situ hybridization (M-FISH) analysis revealed chromosome 3 translocated to several other partners chromosomes, as well as breakage events commonly affecting chromosomes 1, 5, 8, 10, and 17. The most common abnormality detected with comparative genomic hybridization (CGH) was deletions of chromosome 3p, with loss of the RASSF1, FHIT, and p44S10 loci frequently involved. CGH gain of 5q showed overrepresentation of the EGR1 and CSF1R genes. Recurrent alterations to chromosome 7 included rearrangement of 7q11 and gains of the EGFR, TIF1, and RFC2 genes. Several lines exhibited rearrangement of 12q11 approximately q14 and overrepresentation of CDK4 and SAS loci. M-FISH revealed several other recurrent translocations, and CGH findings included loss of 9p, 14q, and 18q and gain of 8q, 12, and 20. Further genomic microarray changes included loss of MTAP, IGH@, HTR1B, and SMAD4 (previously MADH4) and gains of MYC and TOP1. An excellent correlation was observed between the genomic array and FISH data, demonstrating that this technique is effective and accurate. The aberrations detected here may reflect important pathways in renal cancer pathogenesis. PMID: 15860350 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Cancer Res. 2004 Nov 15;64(22):8318-27. Transactivation of the EGR1 gene contributes to mutant p53 gain of function. Weisz L, Zalcenstein A, Stambolsky P, Cohen Y, Goldfinger N, Oren M, Rotter V. Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel. Tumor-associated mutants of the p53 tumor suppressor protein exert biological activities compatible with an oncogenic gain of function. To explore the underlying molecular mechanism, we performed microarray analysis, comparing p53-null cells to mutant p53-expressing cells. One of the genes up-regulated in the presence of mutant p53 was EGR1, a transcription factor implicated in growth control, apoptosis, and cancer. EGR1 induction by various types of stress is markedly augmented in cells expressing mutant p53. Moreover, chromatin immunoprecipitation analysis indicates that mutant p53 is physically associated with the EGR1 promoter. Functional assays indicate that induction of EGR1 by mutant p53 contributes to enhanced transformed properties and resistance to apoptosis. We propose that EGR1 is a significant contributor to mutant p53 gain of function. PMID: 15548700 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Biol Chem. 2004 Jun 25;279(26):27494-501. Epub 2004 Apr 12. Early single cell bifurcation of pro- and antiapoptotic states during oxidative stress. Nair VD, Yuen T, Olanow CW, Sealfon SC. Department of Neurology, Mount Sinai School of Medicine, New York, New York 10029, USA. In a population of cells undergoing oxidative stress, an individual cell either succumbs to apoptotic cell death or maintains homeostasis and survives. Exposure of PC-12-D(2)R cells to 200 microm hydrogen peroxide (H(2)O(2)) induces apoptosis in about half of cells after 24 h. After 1-h exposure to 200 microm H(2)O(2), both antiapoptotic extracellular regulated kinase (ERK) phosphorylation and pro-apoptotic Ser-15-p53 phosphorylation are observed. Microarray and real-time PCR assays of gene expression after H(2)O(2) exposure identified several transcripts, including egr1, that are rapidly induced downstream of ERK. Single cell analysis of egr1 induction and of phospho-ERK and phospho-p53 formation revealed the presence of two distinct cellular programs. Whereas the proportion of cells activating ERK versus p53 at 1 h depended on H(2)O(2) concentration, individual cells showed exclusively either phospho-p53 formation or activation of ERK and egr1 induction. Exposure to H(2)O(2) for 1 h also elicited these two non-overlapping cellular responses in both dopaminergic SN4741 cells and differentiated postmitotic PC-12-D(2)R cells. Repressing p53 with pifithrin-alpha or small interfering RNA increased ERK phosphorylation by H(2)O(2), indicating that p53-dependent suppression of ERK activity may contribute to the bi-stable single cell responses observed. By 24 h, the subset of cells in which ERK activity was suppressed exhibit caspase 3 activation and the nuclear condensation characteristic of apoptosis. These studies suggest that the individual cell rapidly and stochastically processes the oxidative stress stimulus, leading to an all-or-none cytoprotective or pro-apoptotic signaling response. PMID: 15078887 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Mol Cell Biol. 2004 Feb;24(3):1132-42. The tumor suppressor p53 inhibits Net, an effector of Ras/extracellular signal-regulated kinase signaling. Nakade K, Zheng H, Ganguli G, Buchwalter G, Gross C, Wasylyk B. Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, 67404 Illkirch cedex, France. The tumor suppressor function of p53 is linked to its ability to repress gene expression, but the mechanisms of specific gene repression are poorly understood. We report that wild-type p53 inhibits an effector of the Ras oncogene/mitogen-activated protein (MAP) kinase pathway, the transcription factor Net. Tumor-associated mutant p53s are less efficient inhibitors. p53 inhibits by preventing phosphorylation of Net by MAP kinases. Loss of p53 in vivo leads to increased Net phosphorylation in response to wound healing and UV irradiation of skin. Our results show that p53 can repress specific gene expression by inhibiting Net, a factor implicated in cell cycle entry. PMID: 14729959 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Biochem J. 2003 Aug 1;373(Pt 3):739-46. The transcription factor early growth response factor-1 (EGR-1) promotes apoptosis of neuroblastoma cells. Pignatelli M, Luna-Medina R, Perez-Rendon A, Santos A, Perez-Castillo A. Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientificas-Universidad Autonoma, 28029 Madrid, Spain. Early growth response factor-1 (EGR-1) is an immediate early gene, which is rapidly activated in quiescent cells by mitogens or in postmitotic neurons after depolarization. EGR-1 has been involved in diverse biological functions such as cell growth, differentiation and apoptosis. Here we report that enforced expression of the EGR-1 gene induces apoptosis, as determined by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) analysis, in murine Neuro2A cells. In accordance with this role of EGR-1 in cell death, antisense oligonucleotides increase cell viability in cells cultured in the absence of serum. This apoptotic activity of the EGR-1 appears to be mediated by p73, a member of the p53 family of proteins, since an increase in the amount of p73 is observed in clones stably expressing the EGR-1 protein. We also observed an increase in the transcriptional activity of the mdm2 promoter in cells overexpressing EGR-1, which is paralleled by a marked decrease in the levels of p53 protein, therefore excluding a role of this protein in mediating EGR-1-induced apoptosis. Our results suggest that EGR-1 is an important factor involved in neuronal apoptosis. PMID: 12755686 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Life Sci. 2003 May 16;72(26):2975-92. The egr-1 gene is induced by DNA-damaging agents and non-genotoxic drugs in both normal and neoplastic human cells. Quinones A, Dobberstein KU, Rainov NG. Department of General Surgery, ECHO-Laboratory, Martin-Luther-University, D-06097, Halle, Germany. The human egr-1 gene encodes a zinc finger transcription factor induced by endogenous and exogenous stimuli such as growth factors, cytokines, and mitogens. Egr-1 regulates other genes involved in growth and differentiation. The present study investigated the influence of genotoxic agents, such as chemotherapy drugs and other DNA damaging agents, on egr-1 expression in normal and neoplastic cells. A transcriptional fusion between the human egr-1 promoter and the enhanced green fluorescent protein (EGFP) gene was used for direct visualization of intracellular Egr-1 regulation. The transcriptional activity of the egr-1 promoter in this reporter system faithfully reflects intrinsic egr-1 expression and induction, as demonstrated by FACS analysis of fluorescence and by RT-PCR for egr-1. EGFP was expressed under the control of the egr-1 promoter in stably transfected immortalized cell lines, such as HEK293, T98G, LNZ308, and 9L, which were then treated with genotoxic agents.A multitude of DNA damaging agents and therapeutic drugs caused significant upregulation of egr-1 transcription. Furthermore, cytotoxic compounds without a direct DNA damaging effect, such as resveratrol and vincristine, which interfere with DNA replication and cell division, were also able to activate egr-1 transcription. This suggests that cell cycle arrest rather than DNA damage seems to be the condition triggering egr-1 transcription. Moreover, treatment with the MAP kinase (MAPK) inhibitor SB203580, which specifically blocks the stress inducible p38/SAPK2 pathway, did not alter egr-1 induction. On the other hand, treatment with the inhibitor PD98059, which specifically blocks the MAPK/ERK pathway, partially suppressed the induction effect. In addition, the egr-1 induction effect caused by genotoxic stress was found to be at least in part independent from the cellular p53 status, as it was observed in p53-deficient as well as in wild type p53 cell lines.These results suggest that induction of egr-1, a gene to which until now no relation to DNA repair has been assigned, may belong to the fundamental cellular responses elicited by genotoxic and mitotic stress in normal as well as in neoplastic cells, and that enhanced levels of Egr-1 protein may be needed to regulate genes involved in DNA repair, cell survival, and apoptosis. PMID: 12706485 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3233-8. Epub 2003 Mar 10. Early growth response 1 protein, an upstream gatekeeper of the p53 tumor suppressor, controls replicative senescence. Krones-Herzig A, Adamson E, Mercola D. Sidney Kimmel Cancer Center, San Diego, CA 92121, USA. The proliferation of most primary cells in culture is limited by replicative senescence and crisis, p53-dependent events. However, the regulation of p53 itself has not been defined. We find that deletion of the early growth response 1 (EGR1) transcription factor leads to a striking phenotype, including complete bypass of senescence and apparent immortal growth consistent with loss of a suppressor gene. EGR1-null mouse embryo fibroblasts (MEFs) exhibit decreased expression of p53, p21(Cip1/Waf1), and other p53 "marker" proteins. Precrisis WT but not EGR1-null cells exhibit irradiation-induced arrest. WT MEFs that emerge from crisis exhibit a mutated p53 (sequence confirmed), colony formation, and tumorigenicity. In contrast, high-passage EGR1-null MEFs retain the WT p53 sequence but with much reduced expression, remain untransformed, and grow continuously. An EGR1-expressing retrovirus restores p53 expression and sencescence to EGR1-null but not p53-null MEFs or postcrisis WT cells. Taken together, the results establish EGR1 as a major regulator of cell senescence and previously undescribed upstream "gatekeeper" of the p53 tumor suppressor pathway. PMID: 12629205 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Genes Chromosomes Cancer. 2002 Sep;35(1):20-9. Loss of genetic material is more common than gain in acute myeloid leukemia with complex aberrant karyotype: a detailed analysis of 125 cases using conventional chromosome analysis and fluorescence in situ hybridization including 24-color FISH. Schoch C, Haferlach T, Bursch S, Gerstner D, Schnittger S, Dugas M, Kern W, Loffler H, Hiddemann W. Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University, Munich, Germany. Patients with acute myeloid leukemia (AML) and a complex aberrant karyotype have a poor outcome despite intensive antileukemic treatment. The aim of this study was to analyze in detail the genetic abnormalities in this subgroup of AML. Therefore, 125 AML cases with complex aberrant karyotype detected by G-banding were examined in addition with 24-color FISH and FISH with locus-specific probes for EGR1 (5q31), D7S522 (7q31), and TP53 (17p13), given that these regions are known to be commonly deleted in AML with a complex aberrant karyotype. The number of chromosome abnormalities per case varied from 3 to 30 (median 10). A gain of a whole chromosome was observed 131 times, with +8 (n = 30), +10 (n = 11), and +22 (n = 10) being the most frequent trisomies. A loss of a whole chromosome occurred 128 times. The chromosomes most often lost were 7 (n = 25), 18 (n = 24), and 17 (n = 17). Structural aberrations, leading to a gain or loss of chromosomal material, were detected 104 times and 433 times, respectively. Aberrations including only two chromosomes that seemed to be balanced were found only 19 times. Losses resulting from structural abnormalities most frequently involved 5q (n = 100), 17p (n = 47), and 12p (n = 29), whereas gains of 11q (n = 21), 21q (n = 19), and 8q (n = 11) were observed. Using locus-specific probes, deletions of the EGR1 locus (5q31), of 7q31, and the TP53 gene were observed in 103 (82%), 57 (46%), and 66 (53%) cases, respectively. In conclusion, in AML with a complex aberrant karyotype, loss of chromosomal material was observed much more often than gain. Unbalanced rearrangements leading to loss of chromosomal material are much more frequent than loss of whole chromosomes. These data suggest that in AML with a complex aberrant karyotype, loss of tumor-suppressor genes is a more important mechanism of leukemogenesis than activation of oncogenes, and that gene-dosage effects may play a significant role in the pathogenesis of this AML subtype. Copyright 2002 Wiley-Liss, Inc. PMID: 12203786 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Exp Cell Res. 2001 May 15;266(1):21-30. EGR-1, a UV-inducible gene in p53(-/-) mouse cells. Zhang W, Chen S. Laboratory for Cancer Research, College of Pharmacy, Rutgers, the State University of New Jersey, 164 Frelinghuysen Road, Piscataway, New Jersey 08854, USA. Changes in gene expression were examined in p53(-/-) and p53(+/+) mouse cells after ultraviolet (UV) irradiation. Differential display was used to identify differentially expressed gene(s) in UV-treated p53(-/-) and p53(+/+) cells. One of the differentially expressed genes was EGR-1 (early growth response gene-1), which was shown to be induced only in p53(-/-) cells. The induction of this gene by UV was detected as early as 0.5 h, peaked at 2 h, and returned to normal levels by 4 h. De novo protein synthesis was not required for UV-induced EGR-1 expression in p53(-/-) cells. Pretreatment of p53(-/-) cells with suramin, an inhibitor of growth factor receptors, completely suppressed UV-induced EGR-1 expression, suggesting that the induction may be mediated via the growth factor receptors. The presence of wild-type p53 suppressed the induction of EGR-1 after UV treatment. Overexpression of EGR-1 promoted the UV-induced transformation in p53(+/+) cells, but not in p53(-/-) cells. These data suggested that EGR-1 may be an important player in the UV responses of mammalian cells and may influence UV-induced transformation. Copyright 2001 Academic Press. PMID: 11339821 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Biol Chem. 2001 Feb 2;276(5):3279-86. Epub 2000 Oct 16. Ionizing radiation down-regulates p53 protein in primary Egr-1-/- mouse embryonic fibroblast cells causing enhanced resistance to apoptosis. Das A, Chendil D, Dey S, Mohiuddin M, Mohiuddin M, Milbrandt J, Rangnekar VM, Ahmed MM. Department of Radiation Medicine and Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536, USA. In this study, we sought to investigate the mechanism of the proapoptotic function of Egr-1 in relation to p53 status in normal isogenic cell backgrounds by using primary MEF cells established from homozygous (Egr-1(-/-)) and heterozygous (Egr-1(+/-)) Egr-1 knock-out mice. Ionizing radiation caused significantly enhanced apoptosis in Egr-1(+/-) cells (22.8%; p < 0.0001) when compared with Egr-1(-/-) cells (3.5%). Radiation elevated p53 protein in Egr-1(+/-) cells in 3-6 h. However, in Egr-1(-/-) cells, the p53 protein was down-regulated 1 h after radiation and was completely degraded at the later time points. Radiation elevated the p53-CAT activity in Egr-1(+/-) cells but not in Egr-1(-/-) cells. Interestingly, transient overexpression of EGR-1 in p53(-/-) MEF cells caused marginal induction of radiation-induced apoptosis when compared with p53(+/+) MEF cells. Together, these results indicate that Egr-1 may transregulate p53, and both EGR-1 and p53 functions are essential to mediate radiation-induced apoptosis. Rb, an Egr-1 target gene, forms a trimeric complex with p53 and MDM2 to prevent MDM2-mediated p53 degradation. Low levels of Rb including hypophosphorylated forms were observed in Egr-1(-/-) MEF cells before and after radiation when compared with the levels observed in Egr-1(+/-) cells. Elevated amounts of the p53-MDM2 complex and low amounts of Rb-MDM-2 complex were observed in Egr-1(-/-) cells after radiation. Because of a reduction in Rb binding to MDM2 and an increase in MDM2 binding with p53, p53 is directly degraded by MDM2, and this leads to inactivation of the p53-mediated apoptotic pathway in Egr-1(-/-) MEF cells. Thus, the proapoptotic function of Egr-1 may involve the mediation of Rb protein that is essential to overcome the antiapoptotic function of MDM2 on p53. PMID: 11035041 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Clin Immunol. 1999 Nov;93(2):152-61. Curcumin causes the growth arrest and apoptosis of B cell lymphoma by downregulation of egr-1, c-myc, bcl-XL, NF-kappa B, and p53. Han SS, Chung ST, Robertson DA, Ranjan D, Bondada S. Department of Microbiology and Immunology, Department of General Surgery, Lexington, Kentucky 40536, USA. It has been well known that curcumin is a powerful inhibitor of proliferation of several tumor cells. However, the molecular basis of the anti-proliferative effect of curcumin has not been investigated in detail. In this paper, we present evidence to show that curcumin inhibited proliferation of a variety of B lymphoma cells. At low concentrations curcumin inhibited the proliferation of BKS-2, an immature B cell lymphoma, more effectively than that of normal B lymphocytes and caused the apoptosis of BKS-2 cells in a dose- and time-dependent manner. Furthermore, curcumin downregulated the expression of survival genes egr-1, c-myc, and bcl-X(L) as well as the tumor suppressor gene p53 in B cells. In addition, NF-kappaB binding activity was also downregulated almost completely by curcumin. Stimulation with CpG oligonucleotides or anti-CD40 overcame growth inhibition induced by low concentrations of curcumin. Our results suggest that curcumin caused the growth arrest and apoptosis of BKS-2 immature B cell lymphoma by downregulation of growth and survival promoting genes. Copyright 1999 Academic Press. PMID: 10527691 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Mol Cell Biol. 1998 May;18(5):2768-78. Erratum in: Mol Cell Biol 1998 Sep;18(9):5620. Tumor suppressor p53 can participate in transcriptional induction of the GADD45 promoter in the absence of direct DNA binding. Zhan Q, Chen IT, Antinore MJ, Fornace AJ Jr. Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892-4255, USA. The GADD45 gene is a growth arrest-associated gene that is induced by certain DNA-damaging agents and other stresses, such as starvation, in all mammalian cells. In addition to a strong p53-binding element in an intronic sequence, we have recently found that p53, while not required or sufficient alone, may contribute to the stress responsiveness of the promoter. Much of the responsiveness was localized to a GC-rich motif in the proximal promoter which contains multiple Egr1 sites and a larger WT1 site; this 20-bp WT1 motif is identical to the WT1-binding site in the PDGF-A gene. In extracts from a human breast carcinoma cell line expressing p53 and WT1, which is known to associate with p53 in vivo, evidence was obtained that these proteins are in a complex that binds this 20-bp element. A combination of p53 and WT1 expression vectors strongly induced a GADD45-reporter construct, while mutation of the WT1-Egr1 site in the promoter prevented this induction. Abrogation of p53 function by a dominant-negative vector or abrogation of WT1 function by an antisense vector markedly reduced the induction of this promoter. Since p53 does not bind directly to the promoter, these results indicate that p53 can contribute to the positive regulation of a promoter by protein-protein interactions. PMID: 9566896 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Br J Cancer. 1998;77(2):253-9. Wilms' tumour-suppressor protein isoforms have opposite effects on Igf2 expression in primary embryonic cells, independently of p53 genotype. Duarte A, Caricasole A, Graham CF, Ward A. Cancer Research Campaign Growth Factors, Zoology Department, University of Oxford, UK. The p53 protein has been proposed as a modulator of the Wilms' tumour-suppressor protein (WT1) transcriptional regulation activity. To investigate this putative p53 role, the promoter P3 of the mouse insulin-like growth factor II gene (Igf2) was used as a target for WT1 regulation in primary cell cultures derived from p53 wild-type (p53+/+) and knock-out (p53-/-) mouse embryos. In these cells, the WT1 transcriptional activity was observed to be independent of p53 genotype. Furthermore, the two WT1 zinc finger (ZF) isoforms were for the first time found to have opposite effects on gene expression from a single promoter in the same cell type, WT1[-KTS] activating Igf2 P3, whereas WT1[+KTS] repressed its activity. In addition, we have mapped the WT1 binding sites and investigated the effect on WT1 binding activity of individual ZF deletions and Denys-Drash syndrome point mutations to this target. PMID: 9460996 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Anticancer Res. 1996 Nov-Dec;16(6B):3483-9. Deoxyadenosine-resistant mouse leukemia L1210 cell lines with alterations in early response genes and p53. Cory JG, He AW, Cory AH. Department of Biochemistry, East Carolina University School of Medicine, Greenville, NC 27858, USA. L1210 cell lines selected for resistance to deoxyadenosine exhibit altered steady-state levels of the mRNA for the early response genes and p53. In the deoxyadenosine-resistant cell lines (Y8 and ED2), the levels of the mRNAs for p53 and c-jun were markedly decreased while the steady-state levels for mRNAs for c-myc, c-fos and jun B were elevated in the Y-8 and ED2 cell lines. The levels of the mRNAs for PCNA and c-myb were the same in the wild type and mutant cell lines. The levels of the mRNAs for krox-24 were extremely low in the wild type and mutant cell lines. Cycloheximide (CHX) treatment of the cells resulted in the increase in the mRNA levels for c-jun, jun B, krox 24 and p53 in the Y-8 and ED2 cell lines. The time courses and the extents of the increases in the mRNA levels following CHX treatment were not the same for all of these mRNAs. The level of p53 RNA increased with no lag following CHX treatment while the levels of the mRNAs for c-myc, c-jun and krox-24 increased after a one-hour lag period. The level of the mRNA for p53 and c-myc increased 20- and 7-fold, respectively while the mRNA level for knox-24 increased 80-fold following CHX treatment. The Y8 and ED2 cell lines that lack steady-state levels of p53 show decreased sensitivity to cisplatin and increased frequency of gene amplification as measured by PALA resistance in a manner similar to other cell lines lacking p53. On the other hand, the ED2 and Y8 cell lines do not show a G1-block in response to PALA treatment. The cell lines appear to offer an experimental system in which to study the interactions between/among these early response genes and the p53-dependent and independent pathways. PMID: 9042210 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: EMBO J. 1995 Oct 2;14(19):4662-75. WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis. Englert C, Hou X, Maheswaran S, Bennett P, Ngwu C, Re GG, Garvin AJ, Rosner MR, Haber DA. Laboratory of Molecular Genetics, Massachusetts General Hospital Cancer Center, Charlestown 02129, USA. The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles. PMID: 7588596 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Genes Dev. 1995 Sep 1;9(17):2143-56. The WT1 gene product stabilizes p53 and inhibits p53-mediated apoptosis. Maheswaran S, Englert C, Bennett P, Heinrich G, Haber DA. Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts, USA. The Wilms' tumor-suppressor gene product WT1 coimmunoprecipitates with p53 from baby rat kidney (BRK) cells and Wilms' tumor specimens, and expression of WT1 in BRK cells is associated with increased levels of endogenous wild-type p53 protein. To study the effect of WT1 on p53 function, we cotransfected expression constructs into Saos-2 cells, an osteosarcoma cell line without endogenous expression of either gene. Expression of WT1 resulted in increased steady-state levels of p53, attributable to a prolongation in protein half-life, and associated with protection against papillomavirus E6-mediated degradation of p53. This effect mapped to zinc fingers 1 and 2 of WT1 and was not observed with the closely related EGR1 protein. The stabilized p53 demonstrated enhanced binding to its target DNA sequence and increased trans-activation of a promoter containing this RGC site, but reduced transcriptional repression of a TATA-containing promoter lacking this site. Expression of WT1 inhibited p53-mediated apoptosis triggered by UV irradiation or by expression of temperature-sensitive p53 in the wild-type conformation, but did not affect p53-mediated cell cycle arrest. We conclude that WT1 protein can stabilize p53, modulate its trans-activational properties, and inhibit its ability to induce apoptosis. This effect may contribute to the elevated levels of wild-type p53 protein that are observed in Wilms' tumors. PMID: 7657166 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Klin Padiatr. 1994 Jul-Aug;206(4):196-200. [The EWS gene rearrangement in Ewing tumors: key to the disease] [Article in German] Kovar H, Zoubek A, Pfleiderer C, Jug G, Auinger A, Aryee D, Ambros PF, Salzer-Kuntschik M, Amann G, Windhager R, et al. Childrens Cancer Research Institute (CCRI), St. Anna Kinderspital, Wien. The family of Ewing tumors (ET) is characterised by a unique gene rearrangement which is represented by a translocation t(11;22) (q24;q12) or a deletion del 22q12 in most cytogenetically analysable cases. The recent cloning of the underlying gene fusion provides the basis for the diagnostic detection of minimal amounts of residual tumor cells at resection margins, in blood and bone marrow. In addition, the very first steps in ET tumorigenesis can be studied on a functional basis. In this study, a variety of fusion products were identified with a sensitivity of 10(-6) by means of RT-PCR. In 20 of 22 ET, a gene rearrangement was identified which resulted in the substitution of the effector domain of one of the closely related DNA-binding oncogenes, FLI-1 or ERG, by the transactivating domain of a new gene, EWS. Presumably, the oncogene and consequently its target genes are activated by this type of translocation. If the EWS domain was replaced with a transcriptionally irrelevant domain by transfection of a recombinant gene into ET cells, competition with the endogenous chimeric oncogene-product for DNA-binding was observed resulting in a partial growth inhibition. Activation of FLI-1 has been previously shown to occur as a primary event in Friend virus induced mouse erythroleukemia. During progression of this disease, inactivating p53 mutations have been observed frequently. In contrast, such aberrations were found to be extremely rare in ET. PMID: 7967416 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5100-4. Physical and functional interaction between WT1 and p53 proteins. Maheswaran S, Park S, Bernard A, Morris JF, Rauscher FJ 3rd, Hill DE, Haber DA. Massachusetts General Hospital Cancer Center, Charlestown. WT1 is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to the development of Wilms tumor, a pediatric kidney cancer. WT1 encodes a transcription factor which binds to the EGR1 consensus sequence, mediating transcriptional repression. We now demonstrate that p53, the product of a tumor-suppressor gene with ubiquitous expression, physically associates with WT1 in transfected cells. The interaction between WT1 and p53 modulates their ability to transactivate their respective targets. In the absence of p53, WT1 acts as a potent transcriptional activator of the early growth response gene 1 (EGR1) site, rather than a transcriptional repressor. In contrast, WT1 exerts a cooperative effect on p53, enhancing its ability to transactivate the muscle creatine kinase promoter. PMID: 8389468 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Immunology. 1992 Aug;76(4):642-7. Inhibition of the serine/threonine protein phosphatases PP1 and PP2A in lymphocytes: effect on mRNA levels for interleukin-2, IL-2R alpha, krox-24, p53, hsc70 and cyclophilin. Richards FM, Milner J, Metcalfe S. Department of Pathology, University of Cambridge, Addenbrookes Hospital, U.K. Lymphocyte activation requires signal transduction mediated by reversible phosphorylation. Changing profiles of phosphorylated intermediates relate to the progressive series of transduction pathways in cells moving from G0 to G1, and thereafter through the cell cycle. We have previously shown that transient inhibition of the serine/threonine protein phosphatases PP1 and PP2A by okadaic acid enhances early mitogenic stimulation. Thus target proteins of PP1/PP2A may be involved in regulation of early mitogenic signalling, with the phosphorylated form(s) being associated with signal enhancement. Later, pathways require dephosphorylation of these proteins, since continuous treatment with okadaic acid blocks lymphocyte progression through the cell cycle. Delayed addition of okadaic acid showed that this blockade occurs between 8 and 24 hr. Here we have furthered these observations to the level of gene induction by measuring messenger RNA (mRNA) levels for the following proteins: interleukin-2 (IL-2) and IL-2R alpha; p53, a tumour suppressor protein; the transcription factor krox-24; and two mediators of protein folding, namely cyclophilin and the heat-shock protein hsc70. An external standard was used to quantitate the mRNA levels per cell. We found that 24 hr exposure to okadaic acid has a general suppressive effect on concanavalin A (Con A)-stimulated gene induction. However, at 4 hr okadaic acid enhanced IL-2 mRNA levels induced by Con A. Moreover, in unstimulated lymphocytes, okadaic acid caused the induction of krox-24, indicating a role for PP1 and PP2A in the regulation of this gene in resting cells. PMID: 1328040 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Oncogene Res. 1991;6(1):1-12. Interleukin-3 and phorbol esters induce different patterns of immediate-early gene expression in an interleukin-3 dependent cell line. McCubrey JA, Steelman LS, McKearn JP. Department of Microbiology & Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858. The myeloid interleukin-3 (IL-3) dependent cell line, FDC-P1, enters the G0 stage of the cell cycle after IL-3 deprivation for 24 hr. The expression of 13 protooncogenes and immediate-early genes was compared with 4 "control" genes after the addition of either IL-3 or phorbol myristate acetate (PMA) to IL-3-deprived cells. mRNA transcripts encoding c-myc and the T-cell receptor c-gamma gene were induced to high levels only after IL-3 addition, whereas c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 were induced transiently only after PMA addition. The remaining genes (fra-1, p53, jun-D, c-Ha-ras, c-Ki-ras, c-raf, beta-actin, ornithine decarboxylase, and histone 2B) were detected after culture with either IL-3 or PMA. When cells were serum- and IL-3-deprived, c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 were detected after exposure to either serum or PMA. Moreover, culture with cycloheximide and PMA resulted in superinduction of these genes, whereas cycloheximide and IL-3 addition did not. mRNAs encoding these genes had half-lives of 10-20 min after PMA treatment, whereas that of beta-actin was longer (greater than 2 hr), suggesting that short mRNA half-lives contributed to the transient nature of these genes. Although c-fos, fos-B, c-jun, jun-B, Krox-20, and Krox-24 expression can be detected in IL-3-dependent cells after exposure to either PMA or serum, these genes were not detected after IL-3 addition, which allows cell-cycle progression. These results document the existence of IL-3 and PMA-responsive genes and demonstrate that IL-3 and protein kinase C agonists can induce distinct patterns of gene expression. PMID: 1705318 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------