PC3M Cytotoxicity Assay for Compounds that Inhibit the Perinuclear Compartment: SAR
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 ..
BioActive Compound: 1
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.
Extensive in vitro studies have shown that the PNC is unique to tumor cells and preferentially forms in tumor cells derived from solid tissues. Examination of cancer cell lines from various origins and malignant capacities has shown that PNC prevalence correlates with the malignancy of tumors and has the potential to be developed as a pan-cancer prognostic marker. In vivo investigations using human breast tissue samples demonstrated that PNC prevalence was 0% in normal breast epithelium, increases in parallel with disease progression (as determined by staging), and reaches nearly 100% in distant metastases, demonstrating that PNC prevalence associates with the malignancy of breast cancer in vivo. Multivariate analysis further showed that a high PNC prevalence is associated with poor patient outcomes independent of current prognostic factors for stage I breast cancer patients. These studies demonstrate that the presence of the PNC reflects an advanced transformation state of cancer cells that are capable of metastasis.
Although the PNC is associated with a DNA locus and a handful of components have been identified, much remains to be investigated regarding the structure and function of the PNC and its mechanistic link to cancer. We have previously shown that PNC prevalence reduction is a valid strategy to identify compounds that can be used to study the structure and function of the PNC. It is necessary, however, to create a screening platform that is able to test a large number of compounds in an automated fashion. Here we report the development and robustness of an automated whole-cell phenotypic image-based screening assay that is capable of identifying such compounds.
We conducted a followup assay to measure the effect of the lead candidates on cell health by measuring ATP levels (ATPLite). The highly metastatic PC3M-GFP reporter cell line was used in this study.
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.
1. Cells at a density of 75-90% confluency in T175 flasks were harvested using 7 ml TrypLE Express cell dissociation reagent. After dissociation (5 minutes room temperature incubation) 10 ml of complete media was added to the TrypLE/cell suspension. The liquid was transferred to a 50 ml Conical tube and cells were pelleted at 1000 RPM for 5 min. Supernatant was removed and cells were resuspended to a final density of 150-200 cells/ul in complete media.
2. Cells were plated in 5 ul volume (2000 cells/well) into 1536 well White solid bottom plates and allowed to recover and adhere at 37 C in a humidified 5% CO2 incubator for 4 hours.
3. After 4 hour incubation, compound libraries (23 nl of 12.5 uM in columns 5-48) were transferred to the plates in duplicate using a 1536 well pintool. Camptothecin positive control (59 uM final) was added to column 2 and 1:3 dilutions of camptothecin was added to column 3. DMSO negative controls were present in columns 1 and 4.
4. After a 24 hour incubation in the 37 C incubator, all wells were treated with 3 ul of ATPLite reagent using a multidrop combi dispenser (ThermoFisher). Bubbles that formed during dispensing were removed by spinning the plates for 1 minutre at 1500 RPM on a table top centrifuge.
5. Luminescencent signal was detected on a Viewlux CCD based imager (PerkinElmer) with a clear filter and a 30 second integration time.
The PC3M cell line stably expressing the PTB-GFP was provided by Sui Huang of Northwestern University. The media and cell culture reagents were purchased from Invitrogen (Carlsbad, CA), ATPLite came from PerkinElmer.
Compounds are considered "active" if their IC50 is below 20 uM; compounds are considered "inactive" (cytotoxic) if their IC50 is above 20 uM. All compounds that are active are assigned an activity score of 70, all compounds that are inactive are assigned an activity score of 10. This assay measures levels of ATP and converts it inoto light. Though this is a counterscreen, "active" is desireable outcome.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)