Differentiation and Mutation Changes in Mammalian Cells of Shared Lineage Determined With Assay Method that Measures Multiple Energy-Producing Pathways
News Mar 31, 2011
In a paper published in the journal PLoS ONE, researchers from Biolog, Inc., and the Broad Institute of Harvard & MIT describe the successful application of redox-based assays to study the growth properties of mammalian cells.
The technology, termed Phenotype MicroArray™ (PM), comprises a panel of cell assays providing a colorimetric readout of diverse energy-producing pathways in any type of animal cell. Energy is needed to drive virtually all cellular processes, including growth, differentiation, stress responses, and responses to drugs and other small molecules.
To generate energy, cells have multiple enzymatic pathways for producing NADH, which can then be converted to ATP and other useful forms of energy. Scientists at Biolog, Inc. developed new redox dye chemistries that yield a purple color as cells generate NADH.
Over a few hours, the assay measures the rate at which cells are generating NADH without harming the cells. Data from seven different cancer cell lines shows that each utilizes different sets of energy-producing pathways. Most energy-producing pathways in cells use mitochondria to produce NADH, and the authors show that small molecules that poison mitochondria also block color formation in those pathway assays.
Bridget Wagner and Paul Clemons, scientists at the Broad Institute, used PM technology to compare metabolism in fat-storing adipocytes. Mammals have two types of adipocytes: brown adipocytes generate heat to maintain body temperature, and have been shown to be developmentally related to muscle cells. More abundant white adipocytes store excess calories in the form of lipids.
Wagner and Clemons used PM technology to analyze energy pathways in these cell types and their precursor "preadipocytes," and found numerous metabolic differences. Such differences can be exploited in attempts to selectively culture stem or precursor cells, or mature, differentiated cell forms.
According to Barry Bochner, CEO & CSO at Biolog, "The PM assay is really measuring a flow of electrons. The electrons originate from the substrates that the cell is metabolizing, are passed along to NAD or NADP, and then to the redox dye. Although this is a metabolic assay, it is quite different from other metabolic assay approaches such as metabolomics, which measures pool levels of metabolic intermediates, but is difficult to use for measuring pathway activity or flux. Measurement of pathway flux is typically done with radioactive tracers, which is complex, expensive, and requires that all pathways are known to correctly interpret the data. PM assays can elucidate unknown pathways and be performed by any laboratory."
A major advantage of PM technology is its simplicity. Even small differences can be accurately detected. An example described in the paper is the metabolic differentiation of the HepG2/C3A cell line from its precursor cell line, HepG2. Furthermore, it is useful in a wide range of studies. In cancer research, PM technology can be used to study the Warburg effect and the relationship between changes in oncogenes and changes in metabolism.
In diabetes and obesity, shifts in energy metabolism are important for when and how cells burn or store calories. Energy metabolism changes are also fundamental to understanding chemical toxicity, mitochondrial dysfunction and aging. Additionally, PM technology can be a productive tool for optimizing industrial bioprocesses where cells use energy to grow and produce a desired product.
Phenotype MicroArray technology, initially developed with SBIR funding from NIH, is proving to be an important profiling technology. It allows scientists to study the growth properties and culture condition responses of bacterial, fungal, and even human cells. As such it is becoming a core technology for many cellular studies.