How multifunctional signals combine to specify unique cell fates during pattern formation is not well understood. Here, we demonstrate that together with the transcription factor Lozenge, the nuclear effectors of the EGFR and Notch signaling pathways directly regulate D-Pax2 transcription in cone cells of the Drosophila eye disc. Moreover, the specificity of D-Pax2 expression can be altered upon genetic manipulation of these inputs. Thus, a relatively small number of temporally and spatially controlled signals received by a set of pluripotent cells can create the unique combinations of activated transcription factors required to regulate target genes and ultimately specify distinct cell fates within this group. We expect that similar mechanisms may specify pattern formation in vertebrate developmental systems that involve intercellular communication.

Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin and CCI-779, have shown preclinical potential as therapy for multiple myeloma. By inhibiting expression of cell cycle proteins, these agents induce G 1 arrest. However, by also inhibiting an mTOR-dependent serine phosphorylation of insulin receptor substrate-1

In vitro studies indicate the therapeutic potential of mTOR inhibitors in treating multiple myeloma. To provide further support for this potential, we used the rapamycin analog CCI-779 in a myeloma xenograft model. CCI-779, given as 10 intraperitoneal injections, induced significant dose-dependent, antitumor responses against subcutaneous growth of 8226, OPM-2, and U266 cell lines. Effective doses of CCI-779 were associated with modest toxicity, inducing only transient thrombocytopenia and leukopenia. Immunohistochemical studies demonstrated the antitumor responses were associated with inhibited proliferation and angiogenesis, induction of apoptosis, and reduction in tumor cell size. Although CCI-779-mediated inhibition of the p70 mTOR substrate was equal in 8226 and OPM-2 tumor nodules, OPM-2 tumor growth was considerably more sensitive to inhibition of proliferation, angiogenesis, and induction of apoptosis. Furthermore, the OPM-2 tumors from treated mice were more likely to show downregulated expression of cyclin D1 and c-myc and up-regulated p27 expression.Because earlier work suggested heightened AKT activity in OPM-2 tumors might induce hypersensitivity to mTOR inhibition, we directly tested this by stably transfecting a constitutively active AKT allele into U266 cells. The in vivo growth of the latter cells was remarkably more sensitive to CCI-779 than the growth of control U266 cells. IntroductionThe phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) signaling pathway is important for the survival and growth of multiple myeloma (MM) cells and is an attractive target for antitumor therapy. [1][2][3] An important downstream target of PI3-K/AKT is the mammalian target of rapamycin (mTOR), which mediates phosphorylation of p70S6 kinase (p70) and 4E-BP1, 4 proteins responsible for the translation and expression of D-type cyclins and c-myc. 5,6 By preventing these phosphorylation events, mTOR inhibitors down-regulate such expression and induce G 1 cell cycle arrest. 7 In addition, these drugs up-regulate expression of the p27 CDK inhibitor, which may also contribute to G 1 arrest. 8 The in vitro sensitivity of MM cells to the antitumor effects to mTOR inhibitors frequently correlates with heightened AKT activity. [9][10][11] Rapamycin is a classical mTOR inhibitor. The poor solubility that compromised rapamycin as an intravenous agent led to the development of a more soluble ester analog of rapamycin, CCI-779. 12 We have shown in vitro anti-MM activity of rapamycin and CCI-779. 9,11,13 Exposure to these mTOR inhibitors prevents the proliferation of PTEN-and RAS-mutated myeloma cell lines and of interleukin-6 (IL-6)-stimulated proliferation of nonmutated myeloma clones. To provide a further preclinical rationale for the development of mTOR inhibitors in patients, we initiated the current study testing the effects of CCI-779 in vivo against human MM tumor growth in a murine xenograft model. Our results confirm that CCI-779 is effective in vivo against myeloma cells and demonstrate inhibited proliferation, angiogenes...

Mammalian target of rapamycin (mTOR) inhibitors curtail cap-dependent translation. However, they can also induce post-translational modifications of proteins. We assessed both effects to understand the mechanism by which mTOR inhibitors like rapamycin sensitize multiple myeloma cells to dexamethasone-induced apoptosis. Sensitization was achieved in multiple myeloma cells irrespective of their PTEN or p53 status, enhanced by activation of AKT, and associated with stimulation of both intrinsic and extrinsic pathways of apoptosis. The sensitizing effect was not due to posttranslational modifications of the RAFTK kinase, Jun kinase, p38 mitogen-activated protein kinase, or BAD. Sensitization was also not associated with a rapamycin-mediated increase in glucocorticoid receptor reporter expression. However, when cap-dependent translation was prevented by transfection with a mutant 4E-BP1 construct, which is resistant to mTORinduced phosphorylation, cells responded to dexamethasone with enhanced apoptosis, mirroring the effect of coexposure to rapamycin. Thus, sensitization is mediated by inhibition of cap-dependent translation. A high-throughput screening for translational efficiency identified several antiapoptotic proteins whose translation was inhibited by rapamycin. Immunoblot assay confirmed rapamycin-induced down-regulated expressions of XIAP, CIAP1, HSP-27, and BAG-3, which may play a role in the sensitization to apoptosis. Studies in a xenograft model showed synergistic in vivo antimyeloma effects when dexamethasone was combined with the mTOR inhibitor CCI-779. Synergistic effects were associated with an enhanced multiple myeloma cell apoptosis in vivo. This study supports the strategy of combining dexamethasone with mTOR inhibitors in multiple myeloma and identifies a mechanism by which the synergistic effect is achieved. (Cancer Res 2006; 66(4): 2305-13)

Lozenge (Lz) is a multifunctional transcription factor that is activated in a pool of pluripotent cells at the beginning of a wave of morphogenesis during Drosophila eye development. Lozenge belongs to the Runx class of transcription factors that includes the mammalian proteins AML1, Runx 2, and Runx 3. These proteins allow a tissue-specific precursor population of cells to attain multiple terminally differentiated fates. We investigated the transcriptional control of lz to determine the mechanism by which precursor populations achieve their identity. We have identified a 251-bp region in the second intron of the lz gene that functions as a minimal eye-specific enhancer. We provide evidence that Sine oculis and Glass are the two major activators of Lz expression during eye development. This work establishes a bridge between early eye specification genes and late cell-specific transcription factors required for terminal determination of cone cells in the eye.

Studies investigating the association between maternal adiposity and risk of pre-eclampsia showed contradictory results. Therefore, we performed a meta-analysis of prospective cohort studies to estimate the effect of maternal adiposity on pre-eclampsia. We reviewed 1,286 abstracts and finally included 29 prospective cohort studies with 1,980,761 participants and 67,075 pre-eclampsia events. We pooled data with a random-effects model, and obtained risk estimates for five predetermined bodyweight groups: low, normal-weight (reference), overweight, obese and severely obese. In the cohort studies that unadjusted for pre-eclampsia risk factors, the pooled unadjusted relative risks (RR) with 95% confidence intervals (95%CI) for pre-eclampsia of overweight, obese and severely obese women were 1.58 (95% CI 1.44-1.72, P < 0.001), 2.68 (95% CI 2.39-3.01, P < 0.001) and 3.12 (95% CI 2.24-4.36, P < 0.001), respectively. In those cohorts that adjusted for pre-eclampsia risk factors, the pooled unadjusted RRs for pre-eclampsia of overweight, obese and severely obese women were 1.70 (95% CI 1.60-1.81, P < 0.001), 2.93 (95% CI 2.58-3.33, P < 0.001) and 4.14 (95% CI 3.61-4.75, P < 0.001), respectively. Sensitivity analysis showed maternal adiposity was associated with increased risk of pre-eclampsia in both nulliparous and multiparas women. In conclusion, overweight or obese pregnant women have a substantially increased risk of pre-eclampsia, and maternal adiposity is an independent risk factor of pre-eclampsia.

Ribosomal protein L34 (RPL34) was reported to be involved in the regulation of cell proliferation of prokaryotes, plant and animal cells. In the present study, we analyze the expression and function of RPL34 in NSCLC. Immunohistochemical analysis, qPCR and Western blot were used to detect the expression of RPL34 in NSCLC tissues and cells lines. Flow cytometry was used to detect cell activity of NSCLC cell line H1299 under lentivirus-mediated RNAi on RPL34. Cell proliferation and colony formation assays were used to analyze the role of RPL34 in NSCLC cell proliferation. We found that expression of ribosomal protein RPL34 was significantly up-regulated in NSCLC tissues compared to adjacent normal tissues. Lentivirus-mediated shRNA knockdown of RPL34 in NSCLC cell line H1299 resulted in a strong decrease of proliferation, and a moderate but significant increase of apoptosis and S-phase arrest. These data indicate that over-expressed RPL34 may promote malignant proliferation of NSCLC cells, thus playing an important role in development and progress of NSCLC.

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