Cancer Letters

Cancer Letters

Volume 136, Issue 2, 1 March 1999, Pages 187-194
Cancer Letters

Identification and characterization of the putative retinoblastoma control element of the rat insulin-like growth factor binding protein-2 gene

https://doi.org/10.1016/S0304-3835(98)00321-8Get rights and content

Abstract

The authors previously identified a silencer of the rat IGFBP-2 gene. Sequence examination of the silencer has revealed that it contains the target sequence for the pRb (retinoblastoma) tumour suppressor gene, referred to as the retinoblastoma control element (RCE) which is frequently found in the regulatory element of cellular oncogenes and growth factors. The presence of RCE suggests that the IGFBP-2 gene may be regulated by the pRb tumour suppressor gene. An in vitro gel retardation assay has shown that the putative RCEs from the IGFBP-2 gene are complexed with multiple nuclear factors from the rat liver BRL-3A cells. These DNA–protein complexes were not detected with the nuclear extracts from the cells that were growth arrested at the G1/S border of the cell cycle by high cell density. Using specific antibodies, Sp1 was shown to be one of the components for the multiple DNA–protein complex while pRb does not appear to be directly involved in the formation of the complex.

Introduction

Insulin-like growth factors (IGFs) are metabolically active, mitogenic and differentiation-inducing polypeptide hormones that share structural homologies with proinsulin. IGFs belong to the category of 'progression' growth factors that influence DNA synthesis of cells that entered the cell cycle by 'competence' growth factors [1]. IGFs bind to specific receptors, designated as type 1 and type II receptors (IGF-R). In addition, IGFs are complexed with binding proteins (IGFBPs) in extracellular fluids. The affinity of the IGFBPs for the IGFs is higher than that demonstrated by the IGF receptor. Seven forms of IGFBPs have been purified from serum from human, rat or other mammalian species [2].

IGFBP2 was originally isolated from medium conditioned by the rat liver cell line BRL-3A [3]. In contrast to other IGFBPs, the physiological role of this protein is far from being elucidated. The IGFBP-2 locus was mapped to a 4-megabase pair interval near the insulin-dependent diabetes (IDDM) susceptibility gene (chromosome 2q34) suggesting that it may be a candidate for the diabetes susceptibility gene [4]. Its gene expression is rather interesting. It shows a strict tissue-specific and developmental stage expression pattern. It is frequently produced by tumour cells, suggesting that IGFBP-2 may be an important modulator in cell growth and tumours [5], [6], [7]. The molecular basis for this tissue-specific expression of IGFBP-2 remains elusive. Modulation of transcriptional activity is controlled by factors that either stimulate or repress transcription complex formation. The analysis of DNA–protein interaction suggests that regulation of the transcriptional activity is dependent on the presence of positive and negative regulatory elements. For many genes, the dominant control of specific expression is at the level of transcription and depends on binding of trans-acting factors to cis-regulatory DNA sequences, often located in the 5′-flanking region. One possibility of the strict tissue-specific expression of the IGFBP-2 gene is the presence of repressor proteins that selectively silence the IGFBP-2 gene. A second possibility is the existence of IGFBP-2 gene selective transactivators. The fact that the IGFBP-2 expression is enhanced in tumour cells may suggest that this gene is regulated by tumour-specific transcription factors including oncogenes and tumour suppressor genes.

Among the many genetic changes in neoplastic cells, inactivation of tumour suppressor genes plays an important role in the development of human cancer [8]. Retinoblastoma gene product (pRb) was the first tumour suppressor gene identified [9]. Studies have demonstrated that the pRb can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner through the retinoblastoma control element (RCE) which is present in genes responsible for cell growth and differentiation (Ref. [10] and references therein). Through sequence analysis, two putative retinoblastoma control elements (RCE) have been found in the promoter of the rat IGFBP-2 gene. This work was undertaken to explore the nature of these putative RCEs. Here we report the presence and characterization of cell-cycle dependent nuclear factors interacting with the putative RCEs of the rat IGFBP-2 gene.

Section snippets

Materials and methods

All the buffers and solutions were prepared and standard techniques were employed according to Sambrook et al. [11].

Cell culture and preparation of nuclear extracts

BRL-3A cells were grown as monolayers in 9-cm dishes at 37°C and 5% CO2 in minimal essential medium containing 10% foetal calf serum (FCS), 100 IU/ml penicillin and 100 mg/ml streptomycin (all from Gibco). Nuclear extracts were prepared by 'mini-prep' as described [12]. All the procedures were done on ice and centrifugation was performed in the cold room. Briefly, cells were

Identification of multiple nuclear factor(s) within the silencer domain

Previously we identified an element that has an inhibitory effect on transcription [12]. Given the background that sequence-specific factors play an important role in the modulation of transcriptional activity, we were interested in testing whether any nuclear factor(s) could interact within the area (−916 to −579, see Ref. [12]). Sequence examination of this region has revealed that it contains target sequences for pRb termed RCEs (retinoblastoma control elements) which have been found in the

Nuclear factor(s) interacting within the NRE

In this work we have identified and characterized the two elements (−778 to −752 and −701 to −687) located in the silencer domain of the rat IGFBP-2 gene [12]. They are complexed to multiple nuclear factors from rat liver BRL-3A cells (Fig. 2 and Table 2). Interestingly nuclear extracts prepared from the growth-arrested cells at the G1/S border of the cell cycle failed to form complexes with these elements (Fig. 4). This result might support the idea that the multiple factor(s) complexed with

Acknowledgements

The authors thank Drs. Jean-Luc Mary, Paul Robbins, Jonathan Horowitz, Ron Ninnis, Jean-Paul Giacobino, Alan Richardson and Didier de Chaffoy for discussions and Nicolas Ongenae, An Claeskens and Katja de Waepenaert for the preparation of the manuscript. This work was supported by the Schweizerische National Fond No. 32-31026.91 and 3206-037314.93.

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Present address: Klinikum für Schlafmedizin, CH-5330, Zurzach, Switzerland.

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