Regulation of USP37 Expression by REST-Associated G9a-Dependent Histone Methylation.

The deubiquitylase (DUB) USP37 is a component of the ubiquitin system and controls cell proliferation by regulating the stability of the cyclin-dependent kinase inhibitor 1B, (CDKN1B/p27Kip1). The expression of USP37 is downregulated in human medulloblastoma tumor specimens. In the current study, we show that USP37 prevents medulloblastoma growth in mouse orthotopic models, suggesting that it has tumor-suppressive properties in this neural cancer. Here, we also report on the mechanism underlying USP37 loss in medulloblastoma. Previously, we observed that the expression of USP37 is transcriptionally repressed by the RE1 silencing transcription factor (REST), which requires chromatin remodeling factors for its activity. Genetic and pharmacologic approaches were employed to identify a specific role for G9a, a histone methyltransferase (HMT), in promoting methylation of histone H3 lysine-9 (H3K9) mono- and dimethylation, and surprisingly trimethylation, at the USP37 promoter to repress its gene expression. G9a inhibition also blocked the tumorigenic potential of medulloblastoma cells in vivo Using isogenic low- and high-REST medulloblastoma cells, we further showed a REST-dependent elevation in G9a activity, which further increased mono- and trimethylation of histone H3K9, accompanied by downregulation of USP37 expression. Together, these findings reveal a role for REST-associated G9a and histone H3K9 methylation in the repression of USP37 expression in medulloblastoma.Implications: Reactivation of USP37 by G9a inhibition has the potential for therapeutic applications in REST-expressing medulloblastomas. Mol Cancer Res; 15(8); 1073-84. ©2017 AACR.

Chromatin remodelling at the topoisomerase II-beta promoter is associated with enhanced sensitivity to etoposide in human neuroblastoma cell lines.

Etoposide, an inhibitor of topoisomerase II, promotes DNA damage and apoptosis of cancer cells and is a component of standard therapy for neuroblastoma. Resistance to etoposide has been observed in neural tumour cells expressing lower levels of topoisomerase II. In the present study, we have examined the contribution of epigenetic modulation of gene expression in the potentiation of etoposide-mediated cytotoxicity in neuroblastoma cells. Specifically, we studied the effects of histone deacetylase inhibition with valproic acid on topoisomerase II gene expression and apoptosis in response to etoposide. Using human neuroblastoma cell lines SK-N-AS and SK-N-SH, we show that although the combination of valproic acid and etoposide promoted a reduction in growth compared to either drug alone in both cells, the effect was substantially enhanced in SK-N-AS compared to SK-N-SH cells. An increase in histone H3 acetylation and p21 expression was observed in both cell lines, however, upregulation of topoisomerase II-beta gene expression and an increase in PARP cleavage was observed in SK-N-AS cells only. Furthermore, chromatin immunoprecipitation assays revealed an increase in acetylation of histone H3 at the cognate topoisomerase II-beta gene after treatment with valproic acid in SK-N-AS cells. These results suggest a potential epigenetic mechanism of regulation of the topoisomerase II-beta gene and a possible role for its increased expression in the sensitivity of SK-N-AS neuroblastoma cells to etoposide.

Reactivation of death receptor 4 (DR4) expression sensitizes medulloblastoma cell lines to TRAIL.

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.

Histone acetylation resulting in resistance to methotrexate in choroid plexus cells.

Choroid plexus carcinomas are rare tumors that typically occur in young children. Prognosis is poor, and very little information is available to optimize treatment protocols. We used a cell culture model to evaluate whether combining chemotherapeutic agents such as methotrexate with histone deacetylase inhibitors (HDACI) such as valproic acid and MS-275 could improve efficacy. Valproic acid increased the cytotoxicity of radiation and of all the chemotherapeutic agents in Z310 and SV11 mouse choroid plexus cell lines, with the exception of methotrexate. Both HDACIs made choroid plexus cells resistant to this folate antagonist. Searching for a molecular explanation, we found that thymidylate synthase was up regulated when the cells were incubated with HDACI. We also confirmed this finding in human choroid plexus carcinoma cells. Methotrexate should not be combined with HDACI in the treatment of choroid plexus carcinoma.

Dielectric cell separation of fine needle aspirates from tumor xenografts.

As an approach to isolating tumor cells from fine needle biopsy specimens, we investigated a dielectric cell preparation method using an in vivo xenographic tumor model. Cultured human MDA-MB-435 tumor cells were grown as solid tumors in nude mice and fine needle aspiration biopsies were conducted. Biopsied cells were suspended in sucrose medium and collected on slides patterned with microelectrode arrays (electrosmears) energized by electrical signals in the range 10 to 960 kHz. The unlabeled cells adhered to characteristic regions of the slides in accordance with their morphology as a result of dielectric forces. Tumor cells were trapped between 40 and 60 kHz and were separated according to whether they were mitotic, large and complex, or small. Damaged tumor cells were captured at between 60 and 120 kHz; granulocytes between 70 and 90 kHz; lymphocytes between 85 and 105 kHz; healthy erythrocytes between 140 and 180 kHz, and damaged erythrocytes above 180 kHz. Using intrinsic cell characteristics, the electrosmear presented cell subpopulations from fine needle aspiration biopsy specimens in a manner that is compatible with automated slide-based analysis systems. The approach has the potential to facilitate the analysis of the role of cell subpopulations in disease.

Valproic acid induces p21 and topoisomerase-II (alpha/beta) expression and synergistically enhances etoposide cytotoxicity in human glioblastoma cell lines.

OBJECT: Etoposide, a topoisomerase-II inhibitor promotes DNA damage and apoptosis of cancer cells. In this study, we have examined the ability of the histone deacetylase inhibitor, valproic acid (VPA) to modulate gene expression and sensitize glioblastoma cell lines to the cytotoxic effects of etoposide in vitro. METHODS: The effect of VPA and etoposide alone or a combination of the two drugs on the growth of three different glioblastoma cell lines (U87, LN18, and U251) were measured by MTT assays. Drug treated cells were analyzed for their cell cycle profile, gene expression, differentiation status, and induction of apoptosis by flow-cytometry, western blotting, immunofluorescence assays, and caspase activity measurements. RESULTS: We observed that while VPA and etoposide independently inhibited the growth of U87, U251, and LN18 cells, exposure of tumor cells to both drugs significantly enhanced the cytotoxicity of etoposide in all cell lines. VPA promoted a G(1) accumulation of U87, while an increase in the G(2)/M population of U251 and LN18 cells was observed upon exposure to the drug. Treatment with etoposide resulted in a G(2)/M arrest of U87, U251, and LN18 cells, whereas, exposure to both drugs increased the fraction of cells with a G2/M and sub-G1 DNA content. Further, VPA and not etoposide, promoted acetylation of histone H4 and induced the expression of the cyclin-dependent kinase inhibitor (CDKI), p21/WAF1. VPA also up-regulated the expression of the alpha and beta isoforms of topoisomerase-II, as well as the glial differentiation marker, glial fibrillary acidic protein. Finally, a significant increase in caspase-3 activity and apoptosis was observed in the presence of both VPA and etoposide compared to either agent alone. CONCLUSION: 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.

Dielectrophoretic segregation of different human cell types on microscope slides.

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.