We sought to determine whether nucleolin, a bcl-2 mRNAbinding protein, has a role in the regulation of bcl-2 mRNA stability in MCF-7 and MDA-MB-231 breast cancer cells. Furthermore, we examined the efficacy of the aptamer AS1411 in targeting nucleolin and inducing bcl-2 mRNA instability and cytotoxicity in these cells. AS1411 at 5 Mmol/L inhibited the growth of MCF-7 and MDA-MB-231 cells, whereas 20 Mmol/L AS1411 had no effect on the growth rate or viability of normal MCF-10A mammary epithelial cells. This selectivity of AS1411 was related to a greater uptake of AS1411 into the cytoplasm of MCF-7 cells compared with MCF-10A cells and to a 4-fold higher level of cytoplasmic nucleolin in MCF-7 cells. Stable siRNA knockdown of nucleolin in MCF-7 cells reduced nucleolin and bcl-2 protein levels and decreased the halflife of bcl-2 mRNA from 11 to 5 hours. Similarly, AS1411 (10 Mmol/L) decreased the half-life of bcl-2 mRNA in MCF-7 and MDA-MB-231 cells to 1.0 and 1.2 hours, respectively. In contrast, AS1411 had no effect on the stability of bcl-2 mRNA in normal MCF-10A cells. AS1411 also inhibited the binding of nucleolin to the instability element AU-rich element 1 of bcl-2 mRNA in a cell-free system and in MCF-7 cells. Together, the results suggest that AS1411 acts as a molecular decoy by competing with bcl-2 mRNA for binding to cytoplasmic nucleolin in these breast cancer cell lines. This interferes with the stabilization of bcl-2 mRNA by nucleolin and may be one mechanism by which AS1411 induces tumor cell death. [Cancer Res 2008;68(7):2358-65]

bcl-2 mRNA contains an AU-rich element (ARE) that functions in regulating bcl-2 stability. Our earlier studies indicated that taxol-or okadaic acid-induced bcl-2 mRNA destabilization in HL-60 cells is associated with decreased binding of trans-acting factors to the ARE. To identify factors that play a role in the regulation of bcl-2 mRNA stability, bcl-2 ARE-binding proteins were purified from HL-60 cells. Three polypeptides of 100, 70, and 32 kDa were isolated from a bcl-2 ARE affinity matrix. Matrix-assisted laser desorption ionization mass spectroscopy analysis identified these proteins as full-length nucleolin and proteolytic fragments of nucleolin. RNA gel shifts assays indicated that recombinant nucleolin (residues 284 -707) binds specifically to bcl-2 ARE RNA. In addition, recombinant nucleolin decreases the rate of decay of mRNA in HL-60 cell extracts in an ARE-dependent manner. Taxol or okadaic acid treatment of HL-60 cells results in proteolysis of nucleolin in a similar time frame as drug-induced bcl-2 mRNA down-regulation. These findings suggest that nucleolin functions as a bcl-2-stabilizing factor and that taxol and okadaic acid treatment induces apoptosis in HL-60 cells through a process that involves down-regulation of nucleolin and destabilization of bcl-2 mRNA.

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AS1411 is a DNA aptamer that is in phase II clinical trials for relapsed or refractory acute myeloid leukemia and for renal cell carcinoma. AS1411 binds to nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of some tumor cells compared with normal cells. Studies were performed to determine whether cell surface nucleolin is a receptor for AS1411 in the acute myeloid leukemia cell line MV4-11. Biotinylation of MV4-11 cell surface proteins followed by immunoblotting of the biotinylated proteins showed that full-length (106 kDa) and truncated forms of nucleolin were present on the cell surface. In contrast, K-562 cells, which are 4-fold less sensitive than MV4-11 cells to AS1411, showed no full-length nucleolin and lesser amounts of the truncated forms of nucleolin on the cell surface. Incubation of MV4-11 cells with

Introduction CML is a clonal disorder of pluripotent hematopoietic stem cells characterized by the Philadelphia (Ph) chromosome, which results from the reciprocal translocation between the long arms of chromosomes 9 and 22. 1-4 This hybrid B-cell receptor (BCR)-ABL1 gene encodes for a fusion protein Bcr-Abl1 with a constitutive tyrosine kinase activity. 3,4 Despite high rates of clinical responses in early chronic phase CML (CML-CP) to the Bcr-Abl1 kinase inhibitor imatinib, 5-8 development of resistance is a major problem in late CML-CP and in the treatment of blast crisis CML (CML-BC). 9-12 Although Bcr-Abl1-independent mechanisms also exist, 13-15 resistance in CML-CP is usually associated with the expression of mutant Bcr-Abl1 proteins, including T315I and Y253F/H mutations against which the second generation ABL tyrosine kinase inhibitors (TKI) such as nilotinib and/or dasatinib show limited effect. 15-17 Nonetheless , BCR-ABL1 mutations may not account for all cases of drug resistance in CML (CP and BC); indeed, alternative Bcr-Abl1-dependent mechanisms including alterations of sphingolipid metabolism and signaling, 18 might account for TKI resistance. Sphingolipids, ceramide and sphingosine 1-phosphate (S1P) included, are a family of membrane lipids with important roles in the regulation of the fluidity and subdomain structure of membranes. 19-21 Ceramide can be hydrolyzed by ceramidases to release sphingosine, which is phosphorylated by sphingosine kinases-1 or-2 (SK-1 or SK-2) to generate S1P. 20 Ceramide plays proapoptotic roles 21 whereas S1P mediates proliferation and/or resistance to apoptosis 22,23 generally via G-protein-coupled S1P1-5 receptor signaling. 24 However, receptor-independent intracellular functions of S1P were also reported. 25 Recently, alteration of the balance between the proapoptotic ceramide and antiapoptotic S1P via up-regulation of SK-1 was shown to mediate imatinib resistance in K562 CML-BC patient-derived cells by an unknown mechanism. 18 Here, we report the identification of a novel mechanism by which SK-1/S1P mediates imatinib resistance by regulation of the PP2A-dependent and SHP-1-mediated Bcr-Abl1 dephosphoryla-tion and stability selectively via receptor 2 (S1P2) signaling in CML (CP and BC). In addition, our data suggest that targeting the SK-1/S1P2 signaling axis provides a novel strategy to modulate wild-type (wt) or mutant (T315I or Y253H) Bcr-Abl1 stability by restoring PP2A function, and attenuate drug resistance both in cell culture and in mice bearing 32D/T315I-Bcr-Abl1 allografts. Human CML cell lines K562, LAMA4, and their imatinib-resistant derivatives K562/IMA-0.1,-1,-3, or LAMA4/IMA, were maintained as described. 18 The Bcr-Abl-expressing 32Dcl3 cells, 32D-p210 Bcr-Abl (wt), 32D-p210 Bcr-Abl (Y253H) and (T315I) were maintained in RPMI containing 15% FBS, 2mM L-glutamine, and penicillin and streptomycin (P/S; 100 ng/mL each). MEFs (wt and SK-1 /) were maintained in DMEM with 10% FBS and P/S. Human CD34 primary cells from CML patients and normal donor were obt...

The antiapoptotic Bcl-2 protein is overexpressed in a variety of cancers, particularly leukemias. In some cell types this is the result of enhanced stability of bcl-2 mRNA, which is controlled by elements in its 3-untranslated region. Nucleolin is one of the proteins that binds to bcl-2 mRNA, thereby increasing its halflife. Here, we examined the site on the bcl-2 3-untranslated region that is bound by nucleolin as well as the protein binding domains important for bcl-2 mRNA recognition. RNase footprinting and RNA fragment binding assays demonstrated that nucleolin binds to a 40-nucleotide region at the 5 end of the 136-nucleotide bcl-2 AU-rich element (ARE bcl-2 ). The first two RNA binding domains of nucleolin were sufficient for high affinity binding to ARE bcl-2 . In RNA decay assays, ARE bcl-2 transcripts were protected from exosomal decay by the addition of nucleolin. AUF1 has been shown to recruit the exosome to mRNAs. When MV-4-11 cell extracts were immunodepleted of AUF1, the rate of decay of ARE bcl-2 transcripts was reduced, indicating that nucleolin and AUF1 have opposing roles in bcl-2 mRNA turnover. When the function of nucleolin in MV-4-11 cells was impaired by treatment with the nucleolin-targeting aptamer AS1411, association of AUF1 with bcl-2 mRNA was increased. This suggests that the degradation of bcl-2 mRNA induced by AS1411 results from both interference with nucleolin protection of bcl-2 mRNA and recruitment of the exosome by AUF1. Based on our findings, we propose a model that illustrates the opposing roles of nucleolin and AUF1 in regulating bcl-2 mRNA stability.Bcl-2, the prototype for its family, is an antiapoptotic protein. Its overexpression has been implicated in multiple cancers and associated with resistance to chemotherapy, making it an important prognostic factor, particularly in hematological malignancies. The Bcl-2 protein is often highly expressed in chronic lymphocytic leukemia (CLL), 5 even though there is no evidence of gene rearrangements that are known to up-regulate bcl-2 transcription. Recently, Otake et al.(1) reported that Bcl-2 overexpression in CLL is related to bcl-2 mRNA stabilization.It is becoming increasingly clear that mRNA stability is an important control point in the regulation of gene expression. In mammalian cells, regulation of mRNA turnover can dramatically alter the abundance of a particular mRNA without changes in transcription. One of the best characterized regulatory elements present in the 3Ј-untranslated region (3Ј-UTR) of mRNAs is the AU-rich element (ARE) (for a review, see Ref.2). These elements are usually composed of AUUUA sequences embedded in a U-rich stretch, and they act as potent mRNA-destabilizing sequences, targeting mRNAs for rapid decay. The bcl-2 mRNA contains an ARE in the 3Ј-UTR that plays a role in regulating its stability (3, 4). The ARE bcl-2 is a sequence of 136 nucleotides (nucleotides 921-1057) just downstream from the stop codon, containing two AUUUA pentamers and a UUAUUUAUU nonamer, which has also been shown to destabili...

The spontaneously hypertensive rat (SHR) is generally considered to be a suitable genetic model for the study of attention deficit hyperactivity disorder (ADHD), since it displays hyperactivity, impulsivity, poorly sustained attention, and deficits in learning and memory processes. Converging evidence suggests a primary role of disturbance in the dopaminergic neurotransmission in ADHD patients and in SHR, and in addition, some studies have also demonstrated alterations in adenosinergic neurotransmission in SHR. In the present study, adult female Wistar (WIS) and SHR rats received caffeine (1-10 mg/kg i.p.) 30 min before training, immediately after training, or 30 min before a test session in the spatial version of the Morris water maze. The effect of caffeine administration on WIS and SHR blood pressure was also measured. SHR needed significantly more trials in the training session to acquire the spatial information, but they displayed a similar profile to that of WIS rats in the test session (48 h later), demonstrating a selective deficit in spatial learning. Pre-training administration of caffeine (1-10 mg/kg i.p.) improved this spatial learning deficit in SHR, but did not alter the WIS performance. In contrast, post-training administration of caffeine (3 mg/kg i.p.) did not alter the SHR test performance, but increased memory retention in WIS rats. No dose of caffeine tested altered the mean blood pressure of WIS or SHR. These results demonstrate a selective spatial learning deficit in SHR which can be attenuated by pre-training administration of caffeine. In addition, the present findings indicate that the spatial learning deficit in SHR is not directly related to hypertension.

Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3′-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes.