| I.
Testing Cell Lines Exposed to Drugs or Other
Chemicals Evaluating
New Drug Candidates
Pharmaceutical companies and biotech companies
spend much time, effort, and money screening
and assessing drug candidates. This process
could be made much more efficient and less
costly with tools that can quickly and accurately
assess issues such as (1) Sites and modes
of action of drugs, (2) Drugs with high
specificity versus drugs with side effects,
and (3) Beneficial as well as detrimental
interactions with other drugs. PM technology
provides an ideal tool and can be used in
all these ways to evaluate potential new
drugs.
The experimental approach is very analogous
to testing cells with genetic changes. In
the case of genetic changes, a gene can
be "knocked out" which usually
means that a protein is not made (i.e.,
also "knocked out") and consequently
some cellular function is blocked. Most
drugs are targeted to "knock out"
the function of a specific protein, so adding
a drug to a cell should result in phenotypic
effects that are very similar to "knocking"
out the gene.
Drugs can be added to cells prior to inoculation
into PMs. By looking at the phenotypes altered
by the drug one can determine the physiological
functions in the cell that are affected.
This information will indicate: (1) the
site and/or mode of action of a drug, (2)
whether the drug is specifically hitting
one target or whether it is interfering
also with other cellular processes and therefore
likely to cause side-effects, (3) potentially
beneficial as well as detrimental interactions
with other drugs (many of the phenotypes
in the PMs test for increased sensitivity
or resistance to existing drugs).
More information on this is provided in
the section on Drug
Discovery Using PMs.
Toxicological Testing
Toxicological testing based on the use
of cell lines is gradually replacing animal
testing as a more cost-effective and humane
approach. Using PMs, testing would be performed
essentially as described in the preceding
paragraph for drug testing. One simply adds
a chemical to the cells and inoculates into
PMs. Interference with an aspect of cell
physiology will be manifested as an altered
response in the PMs. The information from
PMs will indicate toxicity levels as well
as mechanisms of toxicity. Some chemical
agents may be toxic only under specific
growth conditions or only in combination
with other toxic chemicals. Since PMs contain
thousands of different testing conditions,
they provide toxicological information that
is much more thorough and comprehensive.
II. Testing Cell Lines with Genetic
Differences
Determining Functions
of Important Genes (Functional Genomics)
Cells have on the order of 2,000 to 120,000
genes. Even in the simplest and most studied
microbial cells, only about half of the
genes have a known function. Through a variety
of genetic and biochemical techniques, scientists
are identifying many genes as being "especially
interesting". For example, from studies
of hereditary human genetics, these genes
may be implicated in an important disease
or syndrome. Alternatively, they may be
involved in cancer or microbially-induced
or chronic diseases and thereby be potential
targets for new drugs. Genes of great importance
(both biologically and commercially) are
also being identified in other animals,
plants, and microorganisms. However, with
current technology it is very difficult,
expensive, and not-at-all straightforward
to determine the function of these important
genes.
The method most commonly proposed for determining
the function of unknown genes relies on
the use of nucleic acid- based microarrays.
DNA microarrays are used to measure mRNA
levels under several growth conditions,
and then the data are analyzed to see if
the mRNA levels of the unknown gene go up
and down in correlation with the mRNA levels
of a known gene. The hope is that by grouping
genes that are regulated in the same manner,
biologists will be able to discern genes
that are members of the same functional
pathways. This approach is rather lengthy,
expensive, and complex and it relies on
making numerous assumptions that may be
incorrect.
PMs make it possible to go directly from
a gene of interest to a cellular function.
The experimental approach is to simply "knock
out" the gene of interest in a cell
line to create an isogenic pair of strains.
The biologist simply inoculates the isogenic
cell lines into the PMs and looks for one
or more phenotypic differences. Alternatively
one can do a "knock in" genetic
construction in which a gene of interest
is added to a cell line. Here again, the
isogenic cell lines are assayed in PMs and
one looks for discernable phenotypic differences.
By analyzing isogenic strains and using
PMs in large scale, high-throughput studies
we can start from a genomic map and generate
a virtual phenotypic map. PM technology
is fast, inexpensive, and simple, and it
does not make assumptions about gene transcription,
translation, or post-translational modifications.
Several published examples of using PM
technology to determine gene function can
be found in the PM Bibliography.
Finding Genes that
Code for New Drug Targets
PM technology can be used in comparisons
of cell lines to find new drug targets.
For example, in antimicrobial R&D, pairs
of pathogenic and non-pathogenic microbial
strains can be compared. Pathogenic strains
are known to contain additional genes such
as drug resistance genes and pathogenicity
islands. These extra genes code for proteins
that convey additional phenotypes to the
microbe and may be useful as drug targets.
Another example is the search for new anti-cancer
agents. Cancerous cells can be compared
to non-cancerous cells in PMs to look for
phenotypic sites of potential vulnerability.
Improvement of Cell Lines
A major objective of many animal and plant
genetic projects is the improvement of targeted
cell lines or seed lines. After genetic
manipulation, cell lines must be evaluated
to see if they picked up the desired phenotypic
traits and also to see if they picked up
any undesired secondary phenotypic traits.
PM technology will clearly be a very useful
tool in these types of developments.
Testing Cell Cultures
for Phenotypic Stability
Cell lines can and do change when they
are subcultured. This is due, at least in
part, to unstable genetic constructions
and to selective pressures that biologists
unknowingly apply to cultured cells. Cell
line stability is an important issue in
basic research (e.g. cancer research) and
in important medical applications such as
vaccine and recombinant protein production.
It is essential to know when and in what
ways cell lines are changing.
III. Direct Testing of Cell Lines
Optimizing Growth Conditions
for Cultured Cells
There is a great deal of interest in basic
research and applied development in optimizing
conditions for growing cells, especially
animal and plant cells. In basic research
it is important to understand the growth
requirements of cells so that they can be
handled properly and cultured rapidly. In
commercial ventures, cells are cultured
in vitro for many applications including
cell transplantation and gene therapy, tissue
replacement, vaccine production, and recombinant
protein production. Many of the PMs contain
biochemicals that may act as nutrients for
certain cell types. A stimulatory effect
on the cells will be detected as an increase
in respiration and therefore an increase
in color from the redox dye. This will point
the scientist toward nutrients that improve
the growth and health of the cells.
Optimizing Conditions
for Sporulation and Germination
Many plants and microorganisms can go through
stages in their life cycle where they form
seeds or spores and later germinate. The
conditions that trigger these changes are
often very difficult to discern, requiring
a very precise combination of culture conditions.
Knowing optimal conditions for sporulation
and germination can have important economic
benefits. Sporulation is important in basic
research and genetic and physiological studies,
for example with actinomycetes, yeasts,
and filamentous fungi. Germination is important
in agriculture for cultivation of mushrooms
and plants. PM's will offer biologists a
very simple and straightforward means to
streamline this testing.
Optimizing Production
of Secondary Metabolites Such as Antibiotics
Secondary metabolites such as antibiotics
and pigments typically are produced under
very specific growth conditions, often involving
some special limitation of growth. PMs can
provide thousands of growth conditions and
allow a very rapid and easy way to look
for optimal production conditions.
Finding Useful Enzyme
Activities
Many of the phenotypes that are measured
with PMs indicate the presence of enzymatic
activities that may have potential commercial
use. Examples are enzymes involved in catabolism
of carbon, nitrogen, phosphorus, and sulfur,
and enzymes that protect cells against toxic
chemicals. Therefore PMs can be useful for
screening microorganism collections to look
for these activities.
Other Cell-Based Assays
PMs provide an easy and highly efficient
means for testing cells under thousands
of diverse growth conditions. It provides
a flexible and versatile format for many
other types of cell-based experiments and
assays. The limits are defined by the creativity
and imagination of the scientist using them. |
