Isolation, identification and characterization of DNA expressed sequences (ESTs) from glycocorticoid-regulated genes in the normal tumor phenotypic reversal of rat glioma ST1 cells

Author(s)

Cleber Giovane Vedoy

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2000-11-17

Examining board members

Mari Cleide Sogayar; Emmanuel Dias Neto; Hamza Fahmi Ali El Dorry; Arnaldo Zaha; Bianca Zingales

• Advisor: Mari Cleide Sogayar

Abstract

Treatment of the C6 rat glioma cell variant, ST1 cells, with glucocorticoids, leads to a complete reversion of their transformed phenotype and loss of tumorigenic potential. In this work, two subtracted and equalized cDNA libraries were constructed, based on suppressive PCR, in order to identify glucocorticoid-regulated genes related to the ST1 phenotypic reversion. Analysis of both subtracted cDNA libraries revealed 7 differentially expressed genes, some of which are well known negative growth regulators: thrombospondin-1, cyclin G, tyrosine phosphatase CL 100 and NRP/B. In addition, a cDNA clone similar to TN-X, but exibiting a different transcript size was also isolated. Isolation and characterization of a full-length cDNA corresponding to this transcript, revealed that it is composed of fibronectin type III repeats and one single fibrinogen domain. Glucocorticoid induction of this transcript may suggest a different role of TN-X variants in controling cell proliferation. Expression analysis of the C6-derived glucocorticoid-resistant, P7 variant, and of the human glioblastoma U87 cells, revealed very low or undectable levels of these transcripts, further suggesting that their expression is related to the ST1 cells\' phenotypic reversion. Prevalence of rare and differentially expressed transcripts in these libraries suggests that they are likely to be useful in automated systems (DNA microarrays) to rapidly validate differentially expressed clones and to reveal a panel of candidate genes that mediate the anti-proliferative and anti-tumor effects of glucocorticoids. (AU)