A Cellular Viability Assay to Monitor Drug Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 290

folding is the underlying mechanism of several human disorders
including enzyme deficiencies of mitochondrial fatty acid oxidation,
cystic fibrosis, and phenylketonuria ( 1, 2 ). The folding-prone
mutant protein may suffer from accelerated cellular degradation
and impaired assembly into enzymatically active enzyme com-
plexes. There is a growing interest in identifying strategies to fight
loss of vital protein function that are based on enhancing the fold-
ing and assembly of folding-impaired proteins ( 3 ). One strategy
has been to increase the cellular level of molecular chaperones, a
group of protein that ensures cellular protein homeostasis by
assisting and enhancing the protein folding process. Celastrol is a
natural substance that has been used for many years in traditional
Chinese medicine where it has been administered to individuals
with inflammatory diseases. Subsequent biological studies have
shown that celastrol is able to induce the transcription of molecu-
lar chaperones in several types of cells ( 4, 5 ) but that celastrol also
may exert toxic effects in some cell types. A thorough investiga-
tion of the toxic and folding-enhancing effects of a drug requires
that a relatively large number of different drug concentrations
and incubation periods are analyzed in a cellular context. It is
therefore vital to use an assay that can accurately evaluate the
viability of cultured cells exposed to a drug in a fairly high sample
throughput manner. Here, a protocol for testing the viability of
cells cultured in 96-wells microplates is described that is based on
the MTT cellular reduction assay originally developed by
Mosmann ( 6 ). This colorimetric assay is based on quantification
of dark-colored formazan produced by the reduction of the tetra-
zolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide) by metabolically active cells. Cells with
decreased viability are considered to be less metabolically active
and hence will reduce less MTT. The magnitude of a decrease in
formazan production in cells exposed to certain drug is taken as a
measurement of the cytotoxicity of the drug. The described pro-
tocol addresses a putative toxic effect of celastrol on the viability
of lymphoblastoid cells. This cell type is chosen because it can
readily be derived from a simple blood sample from, for example,
patients with an inherited “loss of function” protein misfolding
disorder. A patient-derived cell line expresses the disease-associ-
ated and mutated folding-prone protein in a genetically relevant
context and can constitute a good cell model for the testing of
putative folding-enhancing substances. However, the described
protocol can be used for the evaluation of other chemical sub-
stances and with small adjustments for other cell types including
adherent cells. The simplicity of the MTT protocol is reflected by
the absence of washing steps and the fact that all steps following
cell seeding are performed in a microplate. This combined with
the broad availability of microplate readers have made MTT type
assays widely used.