Identification of cyclin D3 as a direct target of E2A using DamID.

The transcription factor E2A can promote precursor B cell expansion, promote G(1) cell cycle progression, and induce the expressions of multiple G(1)-phase cyclins. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated the 1-kb promoter of cyclin D3, which contains two E boxes. However, deletion of the E boxes did not disrupt the transactivation by E2A, raising the possibility of indirect activation via another transcription factor or binding of E2A to non-E-box DNA elements. To distinguish between these two possibilities, promoter occupancy was examined using the DamID approach. A fusion construct composed of E2A and the Escherichia coli DNA adenosine methyltransferase (E47Dam) was subcloned in lentivirus vectors and used to transduce precursor B-cell and myeloid progenitor cell lines. In both cell types, specific adenosine methylation was identified at the cyclin D3 promoter. Chromatin immunoprecipitation analysis confirmed the DamID findings and localized the binding to within 1 kb of the two E boxes. The methylation by E47Dam was not disrupted by mutations in the E2A portion that block DNA binding. We conclude that E2A can be recruited to the cyclin D3 promoter independently of E boxes or E2A DNA binding activity.

The impact of endoscopic ultrasonography on the management of suspected pancreatic cancer--a comprehensive longitudinal continuous evaluation.

OBJECTIVES: Between 1997 and 2001, a single-center chart review demonstrated significant impact of endoscopic ultrasonography (EUS) in evaluating suspected pancreatic cancer (PCA). Repeating and comparing this review with that from 2001 to 2004 was performed to determine whether increased use of EUS results in more patients being accurately chosen for curative versus palliative procedures, and for surgical versus nonsurgical oncotherapy. METHODS: The complete systematic review was made up of electronic files from the gastroenterology, oncology, and pathology departments of patients presenting with suspected PCA. Results were compared with those obtained in 1997-2001. RESULTS: From 2001 to 2004, 72 patients had PCA. Seven tumor types were identified. Forty-seven percent (34/72) of patients with suspected PCA were preoperatively staged by EUS; 24% (17/72) of all patients underwent surgery. Comparatively, from 1997 to 2001, only 32% (20/62) of patients were evaluated by EUS (P = 0.056) and 45% (28/62) of all patients underwent surgery (P < 0.01). The EUS detected a tumor in 32 of 34 cases. The EUS-guided fine-needle aspiration cytology identified PCA in 14 of 18 cases. F-18-deoxyglucose-positron emission tomography and magnetic resonance imaging were not used. Endoscopic retrograde cholangiopancreatography was performed in 29% (21/72) of patients, with 15 stents inserted. CONCLUSIONS: Increased EUS use for diagnosing and staging PCA resulted in fewer patients undergoing futile surgery. The EUS plays a pivotal role in the management of patients with PCA.

The mTOR inhibitor CCI-779 induces apoptosis and inhibits growth in preclinical models of primary adult human ALL.

Acute lymphoblastic leukemia (ALL) in adult patients is often resistant to current therapy, making the development of novel therapeutic agents paramount. We investigated whether mTOR inhibitors (MTIs), a class of signal transduction inhibitors, would be effective in primary human ALL. Lymphoblasts from adult patients with precursor B ALL were cultured on bone marrow stroma and were treated with CCI-779, a second generation MTI. Treated cells showed a dramatic decrease in cell proliferation and an increase in apoptotic cells, compared to untreated cells. We also assessed the effect of CCI-779 in a NOD/SCID xenograft model. We treated a total of 68 mice generated from the same patient samples with CCI-779 after establishment of disease. Animals treated with CCI-779 showed a decrease in peripheral-blood blasts and in splenomegaly. In dramatic contrast, untreated animals continued to show expansion of human ALL. We performed immunoblots to validate the inhibition of the mTOR signaling intermediate phospho-S6 in human ALL, finding down-regulation of this target in xenografted human ALL exposed to CCI-779. We conclude that MTIs can inhibit the growth of adult human ALL and deserve close examination as therapeutic agents against a disease that is often not curable with current therapy.

Cell division rates of primary human precursor B cells in culture reflect in vivo rates.

Bone marrow stroma-based cultures provide a powerful model for studying cell division and apoptosis of primary human precursor B cells. Studies using this model are elucidating the mechanisms by which stromal cells inhibit apoptosis of cultured normal precursor B cells and have demonstrated that the apoptotic rate of cultured leukemic precursor B cells can predict clinical outcome in acute lymphoblastic leukemia. In contrast to apoptosis, cell division in this model has not been well characterized. In this study, we quantified the rates of cell division in cultured primary human normal and leukemic precursor B cells by labeling precursor B cells with the fluorescent dye carboxyfluorescein diacetate, succinimyl ester. Based on the rate of decreasing fluorescent signal over 3 weeks, normal CD19(+), CD10(+) precursor B cells divided once every 90.5 hours, a number that correlates well with the known in vivo rate of 65.5 hours. The division rates were similar among different cultures and constant throughout the 3 weeks of culture, suggesting that the variable expansions of precursor B cells seen among different samples and culture durations are not secondary to different cell division rates. Unlike normal cells, cultured leukemic B cells had a heterogeneous division rate that ranged from once every 26-240 hours. These rates correlated well with their respective in vivo proliferation index. These findings indicate that the stroma-based cultures faithfully replicate in vivo cell division rates and can be used to elucidate the pathways that regulate cell division of primary human precursor B cells.