Repression of the c-myb gene by WT1 protein in T and B cell lines

The c-myb gene is primarily expressed in immature hematopoietic cells, and it is overexpressed in many leukemias. We have investigated the role of negative regulatory sites in the c-myb promoter in the Molt-4 T cell line and in the DHL-9 B cell line. A potential binding site for either the EGR-1 or WT1 protein was identified by in vivo footprinting in the 5'-flanking region of c-myb in a region of negative regulatory activity in T cells. We showed by electrophoretic mobility shift assay and electrophoretic mobility shift assay Western that WT1, EGR-1, and Sp1 bound to this site. A mutation of this site which prevented protein binding increased the activity of the c-myb promoter by 2.5-fold. In the DHL-9 B cell line, this site was nonfunctional; however, we found a potential EGF-1/WT1 site located more 3' in a region of negative regulatory activity. We showed that WT1, EGR-1, and Sp1 bound to this site, and that mutation of this site increased the activity of the c-myb promoter by 3.2-fold. Cotransfection of a WT1 expression vector repressed the activity of the c-myb promoter in both cell lines, and this repression was relieved when the EGR-1/WT1 sites were removed. Cotransfection of either an EGR-1 or Sp1 expression vector had no significant effect on the activity of the c-myb promoter. We conclude that WT1 is a negative regulator of c-myb expression in both T and B cell lines.     

Estrogen induces adenosine deaminase gene expression in MCF-7 human breast cancer cells: role of estrogen receptor-Sp1 interactions

Adenosine deaminase (ADA) gene expression is induced by 17beta-estradiol (E2) in MCF-7 human breast cancer cells, whereas the antiestrogens 4'-hydroxytamoxifen and ICI 182,780 exhibit partial estrogen receptor (ER) agonist/antagonist and antagonist activities, respectively. Previous studies have shown that the -211 to +11 region of the ADA gene promoter contains six GC-rich sites (I-VI) that bind Sp1 protein, and these elements are required for high basal expression. In transient transfection studies with pADA211, which contains the -211 to +11 ADA gene promoter linked to a bacterial chloramphenicol acetyl transferase (CAT) reporter gene, E2 and tamoxifen (but not ICI 182,780) induced CAT activity. Ligand-induced transactivation was observed only in cells cotransfected with expression plasmids for wild-type ER or HE11, which does not contain the DNA-binding domain of the ER. Cotransfection with HE15 and HE19, which contain the DNA-binding domain and activation function-1 (AF-1) and AF-2 of the ER, respectively, did not result in E2-induced activity. Subsequent deletion analysis of the ADA gene promoter showed that Sp1 binding site IV (-79 to -73) was primarily responsible for hormone responsiveness. ER activation of ADA gene expression is another example of an E2-induced gene that is dependent on ER/Sp1 interactions with a site-specific GC-rich motif.     

Control elements of Dnmt1 gene are regulated in cell-cycle dependent manner

The Dnmt1 gene is constitutively expressed and is required for the maintenance of global methylation after DNA replication. We investigated here the effects of histone deacetylase (HDAC) inhibitor and DNA demetylation agent on promoter activity of mouse Dnmt1 gene in somatic cells. The promoter activity of Dnmt1 gene was increased approximately 2-fold in the treatment of cells by Tricostatin A (TSA) at 1 x 10(-8) M, as compared with that without treatment of TSA. By contrast, treatment with 5-azacytidne (5aza-C) did not affect the promoter activity of the Dnmt1 gene. This result indicates the Dnmt1 gene is possibly regulated by histone acetylation. We also examined the expression levels of Dnmt1 gene and of its control elements like Sp1, Sp3 and p300 by the chromatin immunoprecipitation and Western blot analysis. The expression of Dnmt1 gene is observed at early S phase. Sp1 is recruited mainly at the G1 phase and Sp3 is recruited at the early S phase. p300 is also obviously recruited at the second S phase. These data indicated that the regulators of Dnmt1 gene were controlled in cell-cycle dependent manner.     

Formation of topoisomerase II alpha complexes with nascent DNA is related to VM-26-induced cytotoxicity

Several clinically active anticancer drugs are known to interfere with DNA topoisomerase II activity. However, the importance of the individual alpha (170 kDa) and beta (180 kDa) isozymes as targets of topoisomerase II-active drugs is not clear. To address this question, human CCRF-CEM leukemia cells were incubated with bromodeoxyuridine, and either the nascent DNA or bulk DNA not undergoing replication was purified by immunoprecipitation with an anti-bromodeoxyuridine antibody. The topoisomerase II isozymes that coprecipitated with either the nascent DNA or bulk DNA were analyzed by Western blotting. The alpha isozyme formed complexes with nascent DNA in cells pretreated with either VM-26 or mitoxantrone, while the beta isozyme was only bound to bulk DNA. At moderately cytotoxic concentrations, VM-26 enhanced the binding of topoisomerase II alpha to nascent DNA at least 5.2-fold compared to bulk DNA. However, in VM-26 resistant CEM/VM-1 cells incubated with equitoxic concentrations of VM-26, topoisomerase II alpha complex formation with nascent DNA was decreased at least 5.5-fold compared to bulk DNA. Drug-induced binding of topoisomerase II beta with bulk DNA in CEM/VM-1 cells did not correlate with cytotoxicity. Collectively, these results indicate that the formation of VM-26 stabilized complexes of topoisomerase II alpha with nascent DNA are critical to the development of cytotoxicity, and that resistance of CEM/VM-1 cells to VM-26 is related to impaired formation of these complexes. The results also provide indirect evidence that topoisomerase II alpha is involved in DNA, replication.     

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

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.     

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.