Biomedical Engineering Reference
In-Depth Information
However, large numbers of agents are currently under development or approved for
routine use, and it is likely that drugs to target all the potentially oncogenic signaling
pathways will become available within the next few years.
Although the major signaling pathways are well understood in terms of their key
components and the fundamental biochemical processes involved, the regulation of
cell signaling in normal and diseased tissues in vivo is highly complex and context
sensitive. Complexity is particularly evident in cancers, where more than one
signaling element is often affected and there is considerable cellular heterogeneity
within the malignant cell population and its interactions with the surrounding stroma.
This complexity explains why drugs that are highly effective in tissue culture are often
much less active when used to treat cancer patients, and is a major challenge at the
interface between the development of a drug and its effective use in the clinic.
The development of phosphospecific antibodies directed toward activated signal-
ing proteins greatly facilitates the study of signal transduction in biological samples,
including material obtained from cancer patients. Continuing improvement in anti-
body production techniques has resulted in the availability of a wide range of high-
affinity and target-specific monoclonal antibodies that are suitable for flow cytometry
applications. Single-cell measurement of activated signaling proteins using flow
cytometry has a number of important advantages, relative to bulk assays:
1. It addresses cellular heterogeneity of signaling activity and allows measure-
ments to be made in subpopulations of cells defined using standard phenotypic
markers.
2. Activity can be measured using relatively small numbers of cells.
3. Signaling pathways function in highly regulated networks, and this can be
appreciated with flow cytometry through the combined use of multiple phos-
phospecific antibodies.
4. Whole blood samples can be processed for signaling analysis by flow cyto-
metry, which maintains the pharmacodynamic effect of agents that diffuse from
the target during cell isolation procedures.
7.2.3 Cell Signaling: Assay Basics
As discussed in Chapter 15, these techniques have been successfully applied to patient
samples and have identified novel and medically important alterations in cell
signaling in both leukemia and inflammatory diseases. Although powerful, this
approach has been relatively slow to gain widespread use due to the technical
complexity of the assays, relative to more established flow cytometry methods such
as immunophenotyping. The measurement of intracellular antigens requires fixation
and permeabilization, and the optimumconditions canvary for different epitopes [13].
Importantly, the phosphorylation states of signaling proteins are necessarily highly
dynamic, and can changewithin time frames of less than 1 min in response to pathway
stimulation or by the action of phosphatases. These transient states can be stabilized
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