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Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), in human glioma cells

Oncogene volume 22, pages 4509–4516 (2003)Cite this article

Abstract

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2′-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

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Abbreviations

5-azacytidine:

5aC

trichostatin A:

TSA

DNA methyltransferase:

DNMT

methyl-CpG binding protein:

MBD

chromatin immunoprecipitation:

ChIP

reverse transcriptase polymerase chain reaction:

RT-PCR

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Authors and Affiliations

  1. Program of Cancer Biology, University of Illinois, Peoria, IL, USA

    Santhi D Konduri, Niranjan Yanamandra, Shakuntala Kondraganti, Sajani S Lakka & Jasti S Rao

  2. Department of Pharmaceutical Sciences, Texas Technical University Health Sciences Center, Amarillo, TX, USA

    Kalkunte S Srivenugopal

  3. Department of Neurosurgery, University of Illinois, Peoria, IL, USA

    Dzung H Dinh, William C Olivero & Jasti S Rao

  4. Department of Neuropathology, University of Illinois, Peoria, IL, USA

    Meena Gujrati

  5. Zymogenetics, Inc., Seattle, WA, USA

    Donald C Foster

  6. University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA

    Walter Kisiel

  7. Department of Neurosurgery, University of Texas, MD Anderson Cancer Center, Houston, TX, USA

    Francis Ali-Osman

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  1. Santhi D Konduri
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  2. Kalkunte S Srivenugopal
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  3. Niranjan Yanamandra
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  4. Dzung H Dinh
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  5. William C Olivero
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  6. Meena Gujrati
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  7. Donald C Foster
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  8. Walter Kisiel
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  9. Francis Ali-Osman
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  10. Shakuntala Kondraganti
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  11. Sajani S Lakka
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  12. Jasti S Rao
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Correspondence to Jasti S Rao.

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Konduri, S., Srivenugopal, K., Yanamandra, N. et al. Promoter methylation and silencing of the tissue factor pathway inhibitor-2 (TFPI-2), in human glioma cells. Oncogene 22, 4509–4516 (2003). https://doi.org/10.1038/sj.onc.1206695

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