PubMed · 10775501
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M Sohail, E M Southern. 2000. Antisense arrays.. https://doi.org/10.1006/mcbr.2000.0178
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Ewing sarcoma (ES) and peripheral primitive neuroectodermal tumors (PNETs) are associated with a chromosomal translocation resulting in a fusion of the amino-terminus of EWS with the DNA-binding domain of an ETS transcription factor (most commonly FLI1 or ERG). Although previous reports suggested that these chimera proteins would act as aberrant transcription factors, their downstream targets have not been fully elucidated. To identify downstream targets of these EWS-ETS fusion proteins, we introduced EWS-ETS fusion constructs into a human fibrosarcoma cell line, HT-1080, by retroviral transduction. Here we report that Tenascin-C (TNC) is induced to a significantly higher level in cells expressing EWS-ETSs than in cells expressing normal ETSs. Furthermore, through use of an antisense cDNA expression vector we show that expression of endogenous TNC mRNA and protein were reduced coordinately with attenuation of EWS-FLI1 fusion protein expression. A chromatin immunoprecipitation assay showed direct interaction between the TNC promoter and the EWS-FLI1 fusion protein in vivo. In addition, a luciferase reporter assay revealed that EWS-ETSs upregulated the TNC gene through four ETS binding sites in the TNC promoter. High levels of TNC expression were observed in a subset of ES cell lines (3 of 6) and primary tumors (4 of 6). Together with previous studies showing that TNC expression is involved in the invasive and malignant phenotype of several tumor types, our data suggest that the oncogenic effect of EWS-ETS may be mediated in part by upregulating of TNC expression.
AIM: To study the effects of transcriptional factor Sp1 antisense oligodeoxynucleotide (ODN) on telomerase activity and human telomerase reverse transcriptase (hTERT) expression. METHODS: Antisense oligodeoxynucleotide (ODN) was designed to inhibit Sp1 expression and transferred to Jurkat T cells by lipofectamin. Telomerase PCR-ELISA was used to detect telomerase activity. RT-PCR analysis was used to assess the mRNA expression of Sp1 and hTERT, and Western blot was used to analyze the levels of Sp1 protein. RESULTS: Treatment of Jurkat T cells with Sp1 antisense ODN (1 micromol/L) dramatically reduced Sp1 mRNA and protein levels. The inhibition rate was 44.8 % (P <0.05) and 57 % (P <0.01), respectively. Following the transcriptional factor Sp1 functionally altering, hTERT mRNA expression were suppressed with a 43.7 % inhibition rate (P <0.01). A dose-dependent inhibition of telomerase activity by antisense Sp1 ODN was also discovered. From 0.25 to 2.0 micromol/L, telomerase activity was reduced from 27.1 % to 64.6 %. CONCLUSION: Antisense Sp1 ODN decreases telomerase activity by inhibiting hTERT mRNA expression in Jurkat T cells.
In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.