Taxol- and okadaic acid-induced destabilization of bcl-2 mRNA is associated with decreased binding of proteins to a bcl-2 instability element

Abstract

The observation that overexpression of the anti-apoptotic protein Bcl-2 is associated with both cancer development and anti-cancer drug resistance suggests that factors which regulate bcl-2 expression may be important therapeutic targets. We report here that taxol or okadaic acid (OA) treatment of HL-60 cells reduced bcl-2 mRNA steady state levels to 50% of control cell levels in 20–24 hr of treatment. The 3′-untranslated region of bcl-2 mRNA contains four potential A+U-rich elements (AREs), which are associated with mRNA destabilization. RNA gel mobility shift assays revealed that HL-60 cell extracts contain proteins that bind to RNA transcripts containing the first bcl-2 ARE (ARE 1). ARE 1 binding activity was substantially reduced in extracts of cells treated for 20 hr with taxol or OA and was abolished after 32 hr of treatment. UV-induced RNA cross-linking assays revealed that untreated HL-60 cell extracts contain approximately eight proteins, ranging in size from 32 to 100 kDa, that bind to ARE 1 RNA. Following 20 hr of taxol or OA treatment, RNA cross-linking to ∼70 and ∼38 kDa proteins was greatly reduced, and cross-linking to four proteins of 45–60 kDa sizes was progressively reduced with 10–34 hr of OA or taxol treatment. Collectively, these results suggest a novel action of taxol and OA on bcl-2 expression, which involves bcl-2 mRNA downregulation through inactivation of bcl-2 mRNA stabilizing factors.

Introduction

Bcl-2 was one of the first proto-oncogenes that was observed to promote carcinogenesis by prolonging cell survival rather than increasing cell replication [1]. Overexpression of Bcl-2 is thought to be an important component in the development of B-cell lymphomas that contain a t(14;18) translocation, which moves the bcl-2 gene into the IgH locus near a transcriptional enhancer [2]. Overexpression of bcl-2 also is seen in other cancers where translocation and enhanced transcription of bcl-2 has not occurred [3]. Moreover, high Bcl-2 expression in some malignant cells is an obstacle to chemotherapeutic treatment, since it interferes with drug-induced apoptosis [4], [5], [6]. Accordingly, factors that regulate the expression of bcl-2 may be important therapeutic targets for reversing both the malignant phenotype and anti-cancer drug resistance.

While inhibitors of Bcl-2 protein function have been studied extensively, comparatively little is known about factors that regulate bcl-2 transcription or mRNA stability. Previous studies have shown, however, that taxol-induced apoptosis of OV2008 ovarian cancer cells is associated with downregulation of bcl-2 mRNA as well as Bcl-2 protein [7]. Subsequently, Riordan et al.[8] found that apoptosis of HL-60 cells induced by OA is preceded by decreases in bcl-2 mRNA and protein levels, while actin and Bcl-X_L protein levels are unaffected. Interestingly, both Liu and Priest [7] and Riordan et al.[8] found that decreased bcl-2 mRNA levels were due to mRNA destabilization rather than decreased transcription. This is an interesting observation since it suggests that trans-acting factors that regulate the stability of bcl-2 mRNA can play an important role in the response of cancer cells to apoptotic stimuli.

Cis-elements that regulate mRNA stability have been identified in numerous cytokine [9], [10] and oncogene [11], [12] mRNAs. Prominent among these is the ARE that is found in the 3′-UTR of mRNAs that have short half lives [13]. Although there is little sequence similarity between AREs, they typically contain multiple copies of an AUUUA pentamer within an A+U-rich region. AREs have been divided into three classes, based upon their sequence features and the kinetics of mRNA decay [14]. The 3′-UTR of bcl-2 mRNA contains a conserved ARE [15] that has the features of a Class I ARE [14]. The bcl-2 ARE consists of a A+U-rich region containing two pentamers and a cluster of three overlapping pentamers. Similar to the Class I AREs found in c-fos[16] and c-myc[17] mRNAs, the bcl-2 ARE can confer instability on an intrinsically stable mRNA in transfected cells [15]. The observation that a reporter-bcl-2–ARE fusion mRNA was further destabilized by treatment of transfected cells with C₂-ceramide suggests that the ARE may play a role in the regulation of bcl-2 expression by apoptotic agents [15]. Donnini et al.[18] subsequently reported that multiple proteins in Jurkat cell extracts bind to the bcl-2 ARE in vitro and that the pattern of ARE-binding proteins changes following treatment of cells with UV-C irradiation. Recently, it was reported [19] that the ARE-binding protein AUF1 binds to the bcl-2 ARE in vitro and in vivo. It was further found that UV-C irradiation of Jurkat cells was associated with increased complex formation between a bcl-2 ARE riboprobe and the p45 isoform of AUF1 present in cell extracts, suggesting that AUF1 plays a role in UV-C-induced downregulation of bcl-2 mRNA.

Since induction of DNA damage by UV-C treatment may produce different effects on bcl-2 mRNA regulating factors than those induced by taxol or OA, it remains unclear how taxol or OA modulate bcl-2 mRNA stability. In particular, it is not known if taxol produces similar affects on bcl-2 mRNA and its regulating factors as OA. Also, it is not clear whether taxol- or OA-induced bcl-2 mRNA downregulation involves inactivation of bcl-2 mRNA stabilizing factors such as HuR or activation of destabilizing factors such as AUF1. To address these questions, we have examined the effects of taxol and OA on bcl-2 mRNA stability in HL-60 cells, where the level of bcl-2 mRNA is elevated relative to normal B cells and Jurkat T cells [20]. RNA gel mobility shift assays were employed to probe for proteins that bind to the first of the four potential AREs in bcl-2 mRNA. Additionally, we have examined the effects of taxol and OA on ARE-binding proteins in HL-60 cells to better understand the relationship between trans-acting factors and bcl-2 mRNA stability in specific cancer cells.