Quantitative characterization of mitosis-blocked tetraploid cells using high content analysis.
A range of cellular evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. Cancer research would benefit if these cellular properties could instead be measured by a single, quantitative, automated assay method, such as high content analysis (HCA). Thus, nocodazole-treated cells were fluorescently labeled for different cell cycle-associated properties, including DNA content, retinoblastoma (Rb) and histone H3 phosphorylation, p53 and p21(WAF1) expression, nuclear and cell sizes, and cell morphology, and automatically imaged, analyzed, and correlated using HCA. HCA verified that nocodazole-induced mitosis block resulted in tetraploid cells. Rb and histone H3 were maximally hyperphosphorylated by 24 h of nocodazole treatment, accompanied by cell and nuclear size decreases and cellular rounding. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and histone H3 dephosphorylation accompanied by cellular respreading. This was accompanied by increased p53 and p21(WAF1) expression levels. The range of effects accompanying nocodazole-induced block of mitosis and the resulting tetraploid cells' reversal to a pseudo-G1 state can be quantitatively measured by HCA in an automated manner, recommending this assay method for the large-scale biology challenges of modern cancer drug discovery.
Grove
Linnette E
LE
Cellomics, Inc., Pittsburgh, PA, USA.
Ghosh
Richik N
RN
D000970:Antineoplastic Agents / Q000138:chemical synthesis; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000032:analysis / Q000378:metabolism; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D006657:Histones / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000295:instrumentation / Q000379:methods; D008938:Mitosis / Q000187:drug effects* / Q000235:genetics; D008940:Mitotic Index; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D011123:Polyploidy; D016160:Retinoblastoma Protein / Q000032:analysis / Q000378:metabolism; D013678:Technology, Pharmaceutical / Q000295:instrumentation / Q000379:methods; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000378:metabolism
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004273:DNA, Neoplasm; D006657:Histones; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D015739:Nocodazole