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]

Key Points• We report a first-in-human dose-escalation study in relapsed/refractory B-cell malignancies with the potent BTK inhibitor ONO/GS-4059.• ONO/GS-4059 induced clinically durable responses in relapsed/refractory B-cell malignancies without significant toxicities.We report the results of a multicenter phase 1 dose-escalation study of the selective Bruton tyrosine kinase (BTK) inhibitor ONO/GS-4059 in 90 patients with relapsed/ refractory B-cell malignancies. There were 9 dose-escalation cohorts ranging from 20 mg to 600 mg once daily with twice-daily regimens of 240 mg and 300 mg. Twenty-four of 25 evaluable chronic lymphocytic leukemia (CLL) patients (96%) responded to ONO/GS-4059, with a median treatment duration of 80 weeks; 21 CLL patients remain on treatment. Lymph node responses were rapid and associated with a concurrent lymphocytosis. Eleven of 12 evaluable patients with mantle cell lymphoma (92%) responded (median treatment duration, 40 weeks). Eleven of 31 non-germinal center B-cell diffuse large B-cell lymphoma patients (35%) responded but median treatment duration was 12 weeks due to development of progressive disease. ONO/GS-4059 was very well tolerated with 75% of adverse events (AEs) being Common Toxicity Criteria for Adverse Events version 4.0 grade 1 or grade 2. Grade 3/4 AEs were mainly hematologic and recovered spontaneously during therapy. One CLL patient experienced a grade 3 treatment-related bleeding event (spontaneous muscle hematoma) but no clinically significant diarrhea, cardiac dysrhythmias, or arthralgia were observed. No maximal tolerated dose (MTD) was reached in the CLL cohort. In the non-Hodgkin lymphoma cohort, 4 patients developed a doselimiting toxicity, yielding an MTD of 480 mg once daily. ONO/GS-4059 has significant activity in relapsed/refractory B-cell malignancies without major drug-related toxicity. The selectivity of ONO/GS-4059 should confer advantages in combination therapies. This trial was registered at www.clinicaltrials.gov as #NCT01659255. (Blood. 2016;127(4):411-419)

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

Addition ofcholera toxin (100 ng/ml) to quiescent cultures of Swiss 3T3 cells acts synergistically with serum (2-4%), insulin, phorbol esters, epidermal growth factor, and fibroblastderived growth factor to stimulate DNA synthesis. In the presence of insulin, cholera toxin caused a dose-dependent increase in cumulative [3H]thymidine incorporation into acid-insoluble material and in the intracellular cyclic AMP (cAMP) level. The dose-response curves for the two processes were similar. Furthermore, addition of 1-methyl-3-isobutylxanthine (15-500 FLM) or of 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (5-100 gLM), both of which are potent inhibitors ofcyclic nucleotide phosphodiesterase activity, stimulated DNA synthesis and increased cAMP levels in Swiss 3T3 cells. These compounds strikingly potentiated the effect of cholera toxin on DNA synthesis and on cAMP levels. When quiescent Swiss 3T3 cells were exposed to cholera toxin (100 ng/ ml) and insulin at 10 pg/ml (4-to 7-fold increase in cAMP level) or to these agents and 1-methyl-3-isobutyl xanthine at 50 gIM (35-fold increase in cAMP level), DNA (6)(7)(8)(9)(10)). An objection to many ofthese studies has been that these effects were elicited by high concentrations of analogues of cAMP and could be regarded as nonspecific (2, 4). Furthermore, a few other studies with 3T3 cells suggested that cAMP promotes proliferation, but this contention remained unproven. Thus, addition of cAMP enhanced the initiation of DNA synthesis produced by serum, but noncyclic purine nucleotides or nucleosides were also effective (11). In addition, cholera toxin, an irreversible activator of the adenylate cyclase of eukaryote cells (12) was reported to stimulate, rather than inhibit, cell division in 3T3 cells maintained in-the presence of serum (13).However, the mitogenic effect of cholera toxin was not mimicked by other agents that elevated cAMP levels of 3T3 cells (13). All these inconsistent findings preclude any definitive conclusion concerning the effect ofcAMP on the initiation of DNA synthesis of fibroblast cell lines.A complicating factor in most early studies concerning the effect of cyclic nucleotides on the initiation of DNA synthesis is that they have been conducted in cultures maintained in nutrient medium supplemented with unfractionated serum. The complex nature ofserum and its multiplicity ofactions have frequently rendered difficult the interpretations of experiments with cultured animal cells grown in medium containing serum. In recent years, certain combinations of insulin, epidermal growth factor (EGF), vasopressin, phorbol esters, and fibroblast-derived growth factor (FDGF), a cell-derived growth factor, have been shown to completely replace serum in stimulating DNA synthesis in quiescent cultures of 3T3 cells (1, 14-16). We used such mitogenic factors and defined culture conditions to assess the effect ofcAMP elevating agents on the stimulation of DNA synthesis in cultures of Swiss 3T3 cells. In contrast to previous reports (2-10), we found that incr...

Integrins are cell-surface glycoproteins found in different forms on all cells except erythrocytes. Integrins bind to cell adhesion molecules and to proteins found in the extracellular matrix. A tripeptidic sequence Arg-Gly-Asp (RGD) is often the primary site of recognition by integrins which are expressed on tumour cells and are responsible for tumour invasion and metastasis. A synthetic decapeptide designated alpha P2 containing two RGD sequences radiolabelled with technetium-99m was used to image malignant melanoma in vivo. Fourteen patients previously diagnosed with metastatic melanoma underwent gamma camera imaging 20-180 min following intravenous administration of the radiolabelled synthetic decapeptide alpha P2. Six out of eight (6/8) of the lymph node metastases (75%) and all other neoplastic sites (11 sites) were successfully imaged, with the exception of three sites in the mediastinal area which were not positively imaged. In two cases there was false positive uptake in the rounded pigmented areolar/nipple area. In three cases (seven sites) the peptide scan confirmed the absence of disease in suspected lesions (true-negative). The synthetic peptide was rapidly removed from the circulation by filtration through the kidneys and excretion in the urine. No toxicity or adverse events were recorded. Radiolabelled alpha P2 peptide, which binds specifically to adhesion molecules on tumours, can be used for the in vivo detection of neoplastic metastases.