Our study demonstrates that VPA sensitizes U87, U251, and LN18 cells to the cytotoxic effects of etoposide in vitro by inducing differentiation and up-regulating the expression of p21/WAF1 and both isoforms of topoisomerase-II.

A new method for preparing cells for microscopic examination is presented in which cell mixtures are fractionated by dielectrophoretic forces and simultaneously collected into characteristic zones on slides. The method traps cells directly from the suspending medium onto the slide, reducing cell loss. Furthermore, it exploits differences in the dielectric properties of the cells, which sensitively reflect their morphology. Because different cell types are trapped in characteristic zones on the slide, the technique represents an advance over existing methods for slide preparation, such as centrifugation and smears where cells are randomly distributed. In particular, the new method should aid in the detection of rare and anomalous cell subpopulations that might otherwise go unnoticed against a high background of normal cells. As well as being suitable for traditional microscopic examination and automated slide scanning approaches, it is compatible with histochemical and immunochemical techniques, as well as emerging molecular and proteomic methods. This paper describes the rationale and design of this so-called electrosmear instrumentation and shows experimental results that verify the theory and applicability of the method with model cell lines and normal peripheral blood subpopulations. KeywordsSlide preparation; Dielectrophoresis; Cell separation; Cell discrimination; Microelectrodes Advances in cytological slide preparation techniques have facilitated the identification and characterization of different cell types and have improved the ease and accuracy of disease diagnosis. In particular, numerous staining procedures, including chemical dye-, 1 fluorescent-, 2 enzyme-linked-, 3 immunosensitive-, 4 and specific molecular-methods 5 have been developed. While these techniques allow target cells to be differentiated from other cell types, specimen screening efficiency depends on how well the pathologist discriminates target components such as bacteria, precancerous lesions or cancerous cells from background cells. In diseases characterized by the presence of extremely small numbers of marker cells against a background of large numbers of normal cells, identifying the randomly distributed target cells is tedious, time consuming, and prone to error. Recently, a number of instruments have been introduced that automatically scan slides and attempt to identify putative marker cells for later review by a pathologist. 6 These instruments are based on machine vision through a high quality microscope and they are expensive and relatively slow.

The RE1 Silencing Transcription Factor (REST) is a repressor of neuronal differentiation and its elevated expression in neural cells blocks neuronal differentiation. In the present study, we demonstrate a role for REST in the control of proliferation of medulloblastoma cells. REST expression decreased the levels of CDKNIB/p27, a cyclin-dependent kinase inhibitor and a brake of cell proliferation in these cells. The reciprocal relationship between REST and p27 was validated in human tumor samples. REST knockdown in medulloblastoma cells derepessed a novel REST-target gene encoding the deubiquitylase ubiquitin-specific peptidase 37 (USP37). Ectopically expressed wild type USP37 formed a complex with p27, promoted its deubiquitination and stabilization and blocked cell proliferation. Knockdown of REST and USP37 prevented p27 stabilization and blocked the diminution in proliferative potential that normally accompanied REST loss. Unexpectedly, wild type USP37 expression also induced the expression of REST-target neuronal differentiation genes even though REST levels were unaffected. In contrast, a mutant of USP37 carrying a site-directed change in a conserved cysteine failed to rescue REST-mediated p27 destabilization, maintenance of cell proliferation and blockade to neuronal differentiation. Consistent with these findings, a significant correlation between USP37 and p27 was observed in patient tumors. Collectively, these findings provide a novel connection between REST and the proteasomal machinery in the control of p27 and cell proliferation in medulloblastoma cells.

Object Apoptosis, a key cellular response to therapeutic agents is often inactivated in tumor cells. In this study, we evaluated the expression of the tumor necrosis family of death receptors, DR4 and DR5, in medulloblastoma tumor samples and cell lines to determine if epigenetic modulation of gene expression could sensitize tumor cell lines to TRAIL-mediated apoptosis. Methods Human medulloblastoma samples and cell lines were analyzed for DR4 and DR5 expression by quantitative PCR and immunofluorescence assays. Cell lines with downregulated expression of one or both genes were treated with the histone deacetylase inhibitor, MS-275, and the expression of DR4 and DR5 measured by quantitative PCR, Western blotting, flow cytometry and chromatin immunoprecipitation assays. Induction of apoptosis in the presence of MS-275 was evaluated by TUNEL assay and its ability to augment TRAIL-mediated cytotoxicity was determined by MTT assays, Western blotting and flow cytometry. Results Compared to normal cerebellum, DR4, but not DR5 expression was consistently downregulated in medulloblastoma tumor samples and in Daoy and D283 cell lines. Interestingly, MS-275 decreased cell growth and induced apoptosis in Daoy and D283 cells. In Daoy cells, this coincided with increased histone H3 and H4 acetylation at the DR4 promoter and enhanced DR4 gene and protein expression as well as elevated Caspase-8 activity. The involvement of DR4 in the cellular response to MS-275 was further confirmed by the observation that knockdown of DR4 and FADD abrogated apoptosis. Further, addition of TRAIL to MS-275 treated cells resulted in an enhancement of apoptosis, suggesting that the upregulated death receptors were functional. Conclusion Our study provides an understanding of the role of DR4 in apoptosis of medulloblastoma cell lines and suggests a potential contribution of aberrant histone deacetylation to the resistance of medulloblastoma cells to therapeutic death.