Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 (Ink4c) , a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.

Effects of 15-deoxy-Delta(12, 14) prostaglandin J(2) and ciglitazone on human cancer cell cycle progression and death: The role of PPAR gamma

The role of PPAR-gamma in ciglitazone and 15-d PGJ(2)-induced apoptosis and cell cycle arrest of Jurkat (before and after PPAR gamma gene silencing), U937 (express high levels of PPAR gamma) and HeLa (that express very low levels of PPAR gamma) cells was investigated. PPAR gamma gene silencing, per se, induced a G2/M cell arrest, loss of membrane integrity and DNA fragmentation of Jurkat cells, indicating that PPAR gamma is important for this cell survival and proliferation. Ciglitazone-induced apoptosis was abolished after knockdown of PPAR gamma suggesting a PPAR gamma-dependent pro-apoptotic effect. However, ciglitazone treatment was toxic for U937 and HeLa cells regardless of the presence of PPAR gamma. This treatment did not change the cell cycle distribution corroborating with a PPAR gamma-independent mechanism. On the other hand, 15-d PGJ(2) induced apoptosis of the three cancer cell lines regardless of the expression of PPAR gamma. These results suggest that PPAR gamma plays an important role for death of malignant T lymphocytes (Jurkat cells) and PPAR gamma agonists exert their effects through PPAR gamma-dependent and -independent mechanisms depending on the drug and the cell type. (C) 2007 Elsevier B.V. All rights reserved.

Quantitative structure-activity studies on a series of migrastatin analogs as inhibitors of cancer cell metastasis

Migrastatin, a macrolide natural product, and its structurally related analogs are potent inhibitors of cancer cell metastasis, invasion and migration. In the present work, a specialized fragment-based method was employed to develop QSAR models for a series of migrastatin and isomigrastatin analogs. Significant correlation coefficients were obtained (best model, q(2) = 0.76 and r(2) = 0.91) indicating that the QSAR models possess high internal consistency. The best model was then used to predict the potency of an external test set, and the predicted values were in good agreement with the experimental results (R(2) (pred) = 0.85). The final model and the corresponding contribution maps, combined with molecular modeling studies, provided important insights into the key structural features for the anticancer activity of this family of synthetic compounds based on natural products.

Stem cell Researches in Brazil: Present and Future Challenges

A bill allowing researches with human embryonic stem cells has been approved by the Brazilian Congress, originally in 2005 and definitively by the Supreme Court in 2008. However, several years before, investigations in Brazil with adult stem cells in vitro in animal models as well as clinical trials, were started and are currently underway. Here, we will summarize the main findings and the challenges of going from bench to bed, focusing on heart, diabetes, cancer, craniofacial, and neuromuscular disorders. We also call attention to the importance of publishing negative results on experimental trials in scientific journals and websites. They are of great value to investigators in the field and may avoid the repeating of unsuccessful experiments. In addition, they could be referred to patients seeking information, aiming to protect them against financial and psychological harm.

Cell death and lumen formation in spheroids of MCF-7 cells

3D (three-dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F-actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF-7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.

Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy and RET/PTC rearrangements represent key genetic events frequently associated to this cancer, enhancing proliferation and dedifferentiation by activation of the RET/PTC-RAS-BRAF-mitogen-activated protein kinase (MAPK) pathway. Recently, let-7 microRNA was found to reduce RAS levels in lung cancer, acting as a tumor suppressor gene. Here, we report that RET/PTC3 oncogenic activation in PCCL3 rat thyroid cells markedly reduces let-7f expression. Moreover, stable transfection of let-7 microRNA in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation. As a result, let-7f was capable of reducing TPC-1 cell growth, and this might be explained, at least in part, by decreased messenger RNA (mRNA) expression of cell cycle stimulators such as MYC and CCND1 (cyclin D1) and increased P21 cell cycle inhibitor mRNA. In addition, let-7 enhanced transcriptional expression of molecular markers of thyroid differentiation such as TITF1 and TG. Thus, reduced expression of let-7f might be an essential molecular event in RET/PTC malignant transformation. Moreover, let-7f effects on thyroid growth and differentiation might attenuate neoplastic process of RET/PTC papillary thyroid oncogenesis through impairment of MAPK signaling pathway activation. This is the first functional demonstration of an association of let-7 with thyroid cancer cell growth and differentiation.

The geodiamolide H, derived from Brazilian sponge Geodia corticostylifera, regulates actin cytoskeleton, migration and invasion of breast cancer cells cultured in three-dimensional environment

We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera, on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted HsS78T malignant phenotype to polarized spheroid-like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time-lapse microscopy showed that the peptide inhibited migration of these cells in a dose-dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide.