TGF-β Induces Growth Arrest in Burkitt Lymphoma Cells via Transcriptional Repression of E2F-1

Lindsay C Spender,Gareth J Inman

doi:10.1074/jbc.m808080200

Lindsay C Spender, Gareth J Inman

Open Access

https://doi.org/10.1074/jbc.m808080200

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Abstract

Transforming growth factor-beta (TGF-beta) is a potent regulator of tissue homeostasis and can act as both a tumor suppressor and a tumor promoter. The ability to induce cell cycle arrest is a major component of the tumor suppressor function of TGF-beta. Lung, mammary, and skin epithelial cells exhibit a common minimal cytostatic program in response to TGF-beta signaling involving the repression of the growth-promoting factors c-MYC, Id1, Id2, and Id3. Loss of c-MYC expression is a pivotal event in this process, resulting in derepression of the cyclin-dependent kinase inhibitors CDKN1A (p21) and CDKN2B (p15) and ultimately leading to growth arrest. It is not clear, however, which responses are necessary for TGF-beta-mediated growth arrest in other cell types. Here, in human Burkitt lymphoma cells transformed by deregulated c-MYC expression, we demonstrate that efficient TGF-beta-induced cytostasis can occur despite both maintenance of c-MYC levels and a lack of p21 and p15 induction. TGF-beta treatment also results in induction, rather than repression, of Id1 and Id2 expression. In this context, growth arrest correlates with transcriptional repression of E2F-1, and overexpression of E2F-1 in Burkitt lymphoma cells largely overcomes the TGF-beta-mediated G(1) arrest phenotype. These data indicate that deregulation of c-MYC in lymphoma cells does not overcome the tumor suppressor function of TGF-beta and that repression of E2F-1 transcription is sufficient for the efficient induction of cytostasis.

Highlights

TGF-␤-mediated cytostasis is induced, at least in part, by Smad-dependent regulation of TGF-␤ target genes involved in cell cycle control
The key event in TGF-␤-mediated cytostatic responses of epithelial cells is the transcriptional regulation of c-myc and its target genes
G1 Arrest—To determine whether transcriptional regulation of formed by t(8;14) translocation of the c-myc gene, it was E2F-1 is sufficient for TGF-␤-mediated growth arrest in cells unknown whether TGF-␤ cell cycle arrest could occur by a with deregulated c-MYC expression, we constitutively re-ex- similar mechanism

Summary

EXPERIMENTAL PROCEDURES

Cell Lines, Reagents, and Treatments—CA46 Burkitt lymphoma cells were maintained in RPMI 1640. TGF-␤ induces efficient G1 cell cycle arrest in cells transformed by deregulated c-MYC expression. The percentages of CA46 cells in each phase of the cell cycle of a representative experiment are shown, counted at the times indicated. For TGF-␤ treatment time courses, cells were seeded at 3 ϫ 105/ml and cultured overnight prior to treatment with 1 ng/ml TGF-␤1 (Peprotech). Activation is inhibited by (v/v) fetal calf serum, 2 mM glutamine, penicillin, and streptoc-MYC binding to the promoter via its interaction with Sp1. Induces either apoptosis or cell cycle arrest in human Burkitt Plasmids—Promoter luciferase reporter constructs were lymphoma (BL) cells [16]. The mechanism of TGF-␤-mediated lowing primers: forward, 5Ј-tgtgcccatttgaaactaagg-3Ј, and cell cycle arrest induced in these cells is unknown. Bound immunocomplexes were detected by enhanced chemiluminescence (ECL; Amersham Biosciences)

Cell Cycle Analysis by Flow

Findings

In the context of deregulated