Thymidine phosphorylase inhibits the expression of proapoptotic protein BNIP3

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Abstract

An angiogenic factor, thymidine phosphorylase (TP), confers resistance to apoptosis induced by hypoxia. We investigated the molecular basis for the suppressive effect of TP on hypoxia-induced apoptosis using Jurkat cells transfected with TP cDNA, Jurkat/TP, and a mock transfectant, Jurkat/CV. TP and 2-deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, suppressed hypoxia-induced apoptosis. They also inhibited the upregulation of hypoxia-inducible factor (HIF) 1α and the proapoptotic factor, BNIP3, and caspase 3 activation induced by hypoxia. Introduction of siRNA against BNIP3 in Jurkat cells decreased the proportion of apoptotic cells under hypoxic condition. These findings suggest that the suppression of BNIP3 expression by TP prevents, at least in part, hypoxia-induced apoptosis. Expression levels of TP are elevated in many malignant solid tumors and thus 2-deoxy-d-ribose generated by TP in these tumors might play an important role in tumor progression by preventing hypoxia-induced apoptosis.

Section snippets

Materials and methods

Antibodies. The monoclonal anti-HIF-1α antibody was purchased from Transduction Laboratories (Lexington, KY). The monoclonal anti-actin (C-2) antibody was from Santa Cruz Biochemistry (Santa Cruz, CA) and monoclonal anti-BNIP3 (ab10433) were from Abcam Ltd. (Cambridge, UK).

Cell lines and induction of hypoxia. Human leukemia Jurkat cells and human colon cancer SW-620 cells were maintained in RPMI1640 containing 10% fetal calf serum. Hypoxia was induced with a Gas Pak Pouch Anaerobic System

The effect of TP on hypoxia-induced apoptosis in Jurkat cells

We examined the TP expression levels in Jurkat cells transfected with TP cDNA by immunoblotting using an anti-TP antibody. Jurkat/TP overexpressed TP, while Jurkat cells transfected with a control vector, Jurkat/CV, did not express a detectable amount of TP (Fig. 1A). We have previously shown that TP and 2-deoxy-d-ribose confer resistance to hypoxia-induced apoptosis on KB-3-1 cells [11]. To examine whether the similar effect of TP is observed in Jurkat cells, we measured hypoxia-induced

Discussion

Previous studies demonstrated that TP confers resistance to apoptosis induced by hypoxia and that the enzymatic activity of TP is required. 2-Deoxy-d-ribose, a degradation product of thymidine generated by TP activity can also prevent hypoxia-induced apoptosis in human KB epidermoid carcinoma cells [11] suggesting that it may be a downstream mediator of the TP function. 2-Deoxy-d-ribose prevented the hypoxia-induced activation of caspase 3 and 9 in HL-60 cells [12]. TP-overexpressing Jurkat/TP

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      Although TP is not a classical growth factor as it lacks a cell surface receptor [6], site-directed mutagenesis of active site residues and small molecule inhibitors have demonstrated that TP promotes angiogenesis via its catalytic activity [7,8]. The dephosphorylated product of thymidine catabolism, 2dDR, has chemotactic activity in vitro and angiogenic activity in vivo [7–10], and can confer resistance to hypoxia-induced apoptosis [11–14], glycate proteins altering their activity [15,16], and support alternate metabolic pathways [17,18]. These processes have the potential to sustain tumor growth and survival under nutrient deficient conditions and may underpin the aggressive and resilient nature of some cancers.

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    These authors contributed equally to this work.

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