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The early growth response 1 (EGR-1) gene product is a transcription factor with roles in differentiation and growth. We

EGR-1, a transcription factor with important functions in the regulation of growth and differentiation, is highly expressed in brain. Previous studies have shown that EGR-1 suppresses the transformed phenotype. However, the expression and role of EGR-1 in human glioblastoma cells are not yet determined. In this study, we found that the basal expression of the EGR-1 protein is undetectable, but is inducible in four human glioblastoma cell lines. To determine EGR-1 functions, we reexpressed EGR-1 in human glioblastoma U251 cells and found that the secretion of transforming growth factor-␤1 (TGF-␤1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin ( Egr-1 (also known as NGFI-A (1), TIS8, Krox-24, and Zif268) (33) is a member of the immediate-early gene family that encodes a nuclear phosphoprotein. EGR-1 contains three zinc finger motifs that bind and regulate transcription through GCrich elements (GCEs) 1 with a consensus sequence of 5Ј-GCG(T/ G)GGGCG-3Ј (2-4). The promoter regions of many genes, including several growth factors and cytokines, are regulated by EGR-1 (for a review, see Ref. 5). The Egr-1 gene is rapidly and transiently induced by growth factors and other signals and is functionally implicated in cell proliferation and in differentiation processes (6 -8).Egr-1 is broadly expressed during development and in the tissues of adults of many species. It can be found in epithelial tissues, heart, thymus, and central and peripheral nervous systems. The basal expression of the EGR-1 protein in adult rat and mouse brain is high (for a review, see Ref. 9). At a functional level, several in vitro and in vivo studies initially characterized Egr-1 as having a role in the control of cell growth, proliferation, differentiation, and development. However, whether EGR-1 is a participant in disparate activities or is a more central factor regulating coordinate expression of a characteristic phenotype is unclear. Our previous studies have shown that stable re-expression of EGR-1 inhibits transformation in model cells and in several human tumor cell lines (10,11,38,39). The re-expression of Egr-1 in fibrosarcoma HT-1080, glioblastoma U251, and U373 cells leads to decreased DNA synthesis, growth, and tumorigenicity (39). The mechanism of the EGR-1 suppressive function has been studied in detail in fibrosarcoma HT-1080 cells (40 -42). The EGR-1 protein specifically binds the GCE sites of the human TGF-␤1 promoter, transactivates the TGF-␤1 gene, and enhances the expression and secretion of functional TGF-␤1 in the Egr-1-expressing fibrosarcoma cell line, leading to inhibition of cell proliferation and restoration of anchorage-dependent growth (40).TGF-␤1 is the prototype of a large family of cytokines that control cell proliferation, differentiation, adhesion, and extracellular matrix metabolism. TGF-␤1 has been shown to induce fibronectin (FN) expression at both the mRNA and protein levels (12, 24) and promotes net matrix deposition by increasing the expression of specific ECM components such as FN and collagen ...

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The human ®brosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFb1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The eect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by dierent pathways, although each additively increased the level of p21 WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting eects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFb1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival eect of Egr-1 was dominant over the apoptotic eect of p53.