Alison J. Butt

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells.

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-xL protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G1 arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G1 arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.

The IGF axis and programmed cell death.

Insulin‐like growth factors (IGF) are mitogenic peptides that have been implicated as positive regulators of cellular proliferation. In recent years, several studies have suggested an additional role for the IGF axis in the regulation of apoptosis. Signalling through the IGF receptor has been shown to have a potent survival function and protect cells from a variety of apoptotic stimuli. The actions of IGF are regulated by a family of high‐affinity IGF binding proteins (IGFBP), which sequester the IGF from the IGF receptor. However, there is some evidence that one of these binding proteins, IGFBP‐3, may have its own pro‐apoptotic effects that are independent of its ability to modulate IGF bioavailability. In addition, it has been suggested that the tumour suppressor p53, a crucial mediator of apoptosis in response to cellular stress, may elicit several of its apoptotic effects through manipulation of components of the IGF axis. This review summarizes what is currently known about the role of the IGF system in the regulation of apoptosis, highlighting its implications in the context of tumorigenesis.

Functional Activation of Nedd2/ICH-1 (Caspase-2) Is an Early Process in Apoptosis

The ICE/CED-3 family of proteases (caspases) play a central role in the execution phase of apoptosis. These proteases are synthesised as precursor molecules that require processing at specific aspartate residues to produce the two subunits that comprise the active enzyme. The activation of some of these proteases has been shown to occur during apoptosis. Here we show that Nedd2/ICH-1 (caspase-2) is activated during apoptosis induced by a variety of apoptotic stimuli. This activation occurs very early upon treatment of cells with apoptotic agents and appears to precede the activation of CPP32 (caspase-3). The activation of Nedd2 was not seen in cells that are resistant to apoptosis. These observations suggest that Nedd2 is an early effector in the pathway leading to cell death. Our observations also lend weight to the hypothesis that a group of caspases containing long prodomains are the first to be activated in response to apoptotic signals and that they lie upstream of a second class of caspases such as CPP32 containing short or no prodomains.

Growth Inhibition by Insulin-like Growth Factor-binding Protein-3 in T47D Breast Cancer Cells Requires Transforming Growth Factor-β (TGF-β) and the Type II TGF-β Receptor

This study explores the relationship between anti-proliferative signaling by transforming growth factor-β (TGF-β) and insulin-like growth factor-binding protein-3 (IGFBP-3) in human breast cancer cells. In MCF-7 cells, the expression of recombinant IGFBP-3 inhibited proliferation and sensitized the cells to further inhibition by TGF-β1. To investigate the mechanism, we used T47D cells that lack type II TGF-β receptor (TGF-βRII) and are insensitive to TGF-β1. After introducing the TGF-βRII by transfection, the basal proliferation rate was significantly decreased. Exogenous TGF-β1 caused no further growth inhibition, but immunoneutralization of endogenous TGF-β1 restored the proliferation rate almost to the control level. The addition of IGFBP-3 did not inhibit the proliferation of control cells but caused dose-dependent inhibition in TGF-βRII-expressing cells when exogenous TGF-β1 was also present. Similarly, receptor-expressing cells showed dose-dependent sensitivity to exogenous TGF-β1 only in the presence of exogenous IGFBP-3. This indicates that in these cells, anti-proliferative signaling by exogenous IGFBP-3 requires both the TGF-βRII and exogenous TGF-β1. To investigate this synergism, the phosphorylation of TGF-β signaling intermediates, Smad2 and Smad3, was measured. Phosphorylation of each Smad was stimulated by TGF-β1 and, independently, by IGFBP-3 with the two agents together showing a cumulative effect. These data suggest that IGFBP-3 inhibitory signaling requires an active TGF-β signaling pathway and implicate Smad2 and Smad3 in IGFBP-3 signal transduction.