Defining Epigenetic Drivers of Medulloblastoma - Post 2 - Screening
Creators: name: Armstrong, Stephen
Publisher: name: Zenodo
Year: 2019
Subjects: epigenetics
medulloblastoma

description: <strong>Update #02 - Screening Established Medulloblastoma Cell Lines</strong> The first part of my project involves screening medulloblastoma cell lines that successfully proliferate in vitro with SGC’s epigenetic probe library. The idea of this work is to identify common epigenetic targets that influence medulloblastoma proliferation and could potentially be used as prognostic or therapeutic targets. <strong>Cell Lines: </strong> I have screened four medulloblastoma cell lines so far: These include D425, D458, Med8A, and ONS-76. D425 is a Group 3 MB cell line established from a primary tumor that has mutated p53, diploid status, MYC amplification, and easily engrafts in mice. D458 is a cell line from a spinal metastasis from the same patient as D425, and together these two cell lines represent a primary and metastatic matched pair. D458 is tetraploid, and interestingly, has wildtype p53. Both cell lines grow as suspension cultures and are robust <em>in vitro</em>. D283 is a Group 3 established suspension culture that grows weakly in vitro, and lacks MYC amplification. Med8A is an adherent Group 3 established cell line with wildtype p53, and MYC amplification. It’s adherent nature and fast growth makes it ideal for drug screening, since it can reach full confluence. ONS-76 is also an established adherent cell line like Med8A, except it belongs to the SHH subgroup of medulloblastoma, and has a tetraploid status. an SHH MB cell line with tetraploidy (1). All cell lines are cultured in D-MEM medium with 10% Fetal Bovine Serum, and 1% Penicillin/Streptomycin added. Medium is changed every three days, and cells are sub-cultured every three to six days depending on the seeding number used. <strong>Probe Preparation: </strong> Each epigenetic probe is dissolved in DMSO to achieve a concentration of 10 mM, and sequestered into 96 well plates designated “Master Mix”. Master mixes are frozen at -80 C, and only thawed once to make Working Mix plates. When thawed, each Master Mix plate has enough volume to dilute 1:10 in DMSO to make “Working Mix” plates at 1 mM. Working Mix plates are used for screening experiments and are thawed a minimal amount of times during experiments. <strong>Screening: </strong> Cells are plated in 96 well plates in triplicate, in a total volume of 100 μL per well. They are incubated for 24hrs. The following day, 2 μM of each probe is added in a volume of 100 μL to each well for a final concentration of 1 μM. According to SGC’s probe brochure, 1 μM is a concentration sufficient to observe an effect <em>in vitro</em>. The probe library I am using currently contains 39 probes and a DMSO control. Cells are maintained in culture with 1 μM probe until the DMSO control reaches a confluence of 100%. During this time, cells are treated with 1 uM of probe every three days for maintenance. Cells usually reach confluence by two weeks. <strong>Analysis: </strong> Cell plates are analyzed using the IncuCyte machine, which is an automatic plate analyzer capable of taking four images of each well (4 images x 3 replicates = 12 images/ probe). Confluence is measured by training the IncuCyte software to analyze each image, and assign a percent value to each section. Percent confluence/probe is compared to that of the DMSO control to generate a heat map. Red represents probes that result in a significant reduction in cell confluence compared to the DMSO control, while gray and pink represent probes that do not show significant difference in confluence compared to the DMSO control. <strong>Results &amp; Discussion:</strong> Three probes show consistent results across three or more cell lines (Figure 1), which I will briefly discuss in this update: LLY-283 &amp; GSK591 are sister probes with the same specific target PRMT5. PRMT5 is an arginine methyltransferase responsible for catalyzing the methylation of arginine residues, and is involved in gene regulation (2). PRMT5 is upregulated in multiple cancers including medulloblastoma patient tissue samples, making it the most interesting target so far. BAY-299 has two specific targets including BRD1, as well as a core transcription factor component, TAF1. BRD1 part of the BET protein family. BRD1 is part of the MOZ/MORF acetyltransferase complex and can acetylate histones H3 and H4, thereby regulating gene expression. (3). JQ1 also targets BRD1, but also the rest of the BET family, making it less specific. TAF1 is a major subunit of the TATA box TFIID core transcription factor, responsible for expression of constitutively expressed genes (4). I will be studying the effects of BAY-299 and LLY-283 in medulloblastoma in greater depth in future updates. The next step will be to confirm the effects of these probes using a dose-response assay (covered in next update). <strong>References: </strong> Ivanov, D., et al. 2016. Journal of Biotechnology. DOI: 10.1016/j.jbiotec.2016.07.028 Stopa, N., et al. 2015. Cell Mol Life Sci. DOI: 10.1007/s00018-015-1847-9 Cai, Q., et al. 2018. Mol Cell Research, 16(4): 610-622 Curran, E., et al. 2018. Scientific Reports, 8(4630). DOI:10.1038/s41598-018-22879-5 <strong>Figure 1 – </strong>Heat map of the four medulloblastoma cell lines screened using the epigenetic probe library. Red squares indicate probes that have significantly decreased proliferation of cells compared to the DMSO control, while blue squares indicate probes that are associated with a significant increase in proliferation over the DMSO control. (see attached file "Figure 1-2.png")