High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment:Soft Agar Assay
The perinucleolar compartment (PNC) is a non-membrane-bound nuclear subdomain that is associated with but structurally distinct from the nucleolus. The PNC is a heritable trait, in which the number of PNCs per cell in daughter cells often mimics that of their mother cells. The PNC is heterogeneous in shape, is stable through interphase, disassembles during mitosis, and reassembles in early G1. more ..
The perinucleolar compartment (PNC) is a non-membrane-bound nuclear subdomain that is associated with but structurally distinct from the nucleolus. The PNC is a heritable trait, in which the number of PNCs per cell in daughter cells often mimics that of their mother cells. The PNC is heterogeneous in shape, is stable through interphase, disassembles during mitosis, and reassembles in early G1. The PNC is concentrated with newly synthesized RNA polymerase III (pol III) RNAs (MRP RNA, RNAse P H1 RNA, hY RNAs[hY1, 2, 5], Alu RNA, and SRP [7SL] RNA) and RNA binding proteins (nucleolin, PTB, CUG-BP, KSRP, Raver1, Raver2, and Rod). Continuous transcription by pol III is necessary for the structural integrity of the PNC and some protein components have been shown to bind RNAs within the PNC implicating involvement of PNCs in pol III RNA metabolism. Chemical biology studies have demonstrated that PNC maintenance is dependent on the integrity of DNA, specifically DNA base pairing, and cell biology investigations demonstrated that the PNC is nucleated upon a DNA locus, indicating that the components of the PNC are most likely directly interacting with the DNA locus.
In collaboration between Sui Huang from Northwestern University and the NCGC, a high content assay was developed and screened. The hits were then tested in follow-up assays. In vitro cellular transformation detection assays are semi-quantitative and measure the morphological transformation of cell colonies modulated by chemicals. This transformation is associated with certain phenotypic changes such as loss of contact inhibition (cells can grow over one another) and anchorage independence (cells form colonies in soft agar). Anchorage independence can be described in the light of primary fibroblasts and many other cell lines (e.g. BALB/c3T3, NIH-3T3, etc.) that must attach to a solid surface before they can divide. They fail to grow when suspended in a viscous fluid or gel (e.g. agar or agarose), however when these cell lines are transformed, they are able to grow in a viscous fluid or gel and become anchorage-independent. This process, by which these phenotypic changes occur, is assumed to be closely related, and hence a good predictor, of in vivo carcinogenesis.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH082371
Assay Submitter (PI): Sui Huang, Northwestern University, Feinberg School of Medicine, Cell and Molecular Biology, Chicago, Illinois.
PC3M cells were gently suspended in 0.35% agar in serum/antibiotic free RPMI, supplemented with 10% FBS, 1% penicillin-streptomycin, and compounds whose concentrations was based on the EC10 and EC20. Then, 5000 cells/mL per well were seeded onto 1:1 mixture of 1% agar RPMI containing supplements into 6-well plates. After the top layer had dried, the agarose was overlaid with 3 mL RPMI +10% FBS and drug concentration at EC10 or EC20. The overlaying media was changed twice per week. After 14 and 21 days, the number and size of colonies were visualized and measured, using 40x and 20x objectives. Triplicate wells were used for every treatment condition and the same field was used for analysis and then averaged.
Data Table (Concise)