LONDON ¿ The question regarding the extent to which activation of particular proteins correlates with a poor prognosis in certain human diseases is a fascinating one. It may soon be possible to plan pharmaceutical treatment of diseases ranging from cancer to diabetes on the basis of tests showing which proteins are inappropriately activated in a patient¿s cells.
One requirement for such a scenario is a means to detect activation of specific proteins; once that is available, scientists also need to be able to use such tests on stored tissue samples so they can examine the degree of protein activation in a population of patients whose fate is known, correlating cellular biochemistry with clinical outcome.
A team of researchers at the Imperial Cancer Research Fund (ICRF), one of the U.K.¿s larger cancer charities, has now solved the first part of this requirement for one such protein, and is planning a study that will address the second part.
Tony Ng, of the ICRF¿s Protein Phosphorylation Laboratory, and his colleagues from the Cell Biophysics and the Hedley Atkins/ICRF Breast Pathology Laboratories, report their results in a paper in the March 26 issue of Science, titled ¿Imaging Protein Kinase C-alpha Activation in Cells.¿
Peter Parker, principal scientist at ICRF and senior author of the paper, told BioWorld International, ¿In human disease, there are probably many examples where a genetic change in a cell type within a tissue has a knock-on effect, and triggers particular groups of signal transduction pathways, leading to the development or propagation of disease. It may be valuable to target these pathways therapeutically.¿
Parker and his colleagues are planning a large study to examine levels of activation of particular signaling proteins in samples of human breast tumors. They will then compare the categories of activation they find to see if there is any correlation between the outcome of treatment experienced by the patient or between the grade of tumor at the time of diagnosis. Parker added: ¿If there is a correlation between patients who fare badly and a high level of protein activation, that would clearly be a motivating force to persuade drug companies to develop selective inhibitors to this particular target.¿
The target which has been the subject of their studies reported in Science is the signaling protein called protein kinase C-alpha. This protein is activated by directly binding to a second messenger molecule at cell membranes. Historically, researchers have estimated activation of protein kinase C-alpha by finding out to what extent it is membrane-bound or present in the cytoplasm of the cell.
One limitation to this type of study is that the protein can be membrane-bound before it is activated; furthermore, this method cannot be used to study patients¿ stored tissue samples.
Parker¿s group reports in Science that it has identified a modification to protein kinase C-alpha which is associated with the molecule¿s activation. This modification is a phosphorylation. The team also developed a fluorescent antibody which selectively binds to the phosphorylated part of the molecule. Although this meant they could visualize the presence of activated protein kinase C-alpha, they wanted to refine the specificity of the technique.
¿The problem with antibody reagents such as this one is that they are defined by very specific epitopes on the target protein,¿ Parker said. ¿These epitopes are small and potentially not unique. One way around this problem is to develop a two-site assay in which you ask if the protein is there, and, simultaneously, whether the modification is present on the protein.¿
They therefore exploited a physical phenomenon called fluorescence resonance energy transfer (FRET). This involves exciting a small molecule called a fluorophore ¿ such as the fluorescent tagged antibody, for example ¿ with a laser. The fluorophore responds by emitting energy at a different wavelength. For FRET to occur, the energy emitted by this fluorophore needs to be within the range that excites a second fluorophore in close proximity to the first; this, in turn, will emit energy at a third wavelength.
Various technical problems with this method mean that the results from it are most informative when only the kinetics of energy emission by the first fluorophore are measured. This measurement can be achieved with a technique known as fluorescence lifetime imaging microscopy, or FLIM.
Data presented in the Science paper show that the FLIM measurements on live and fixed cells can be employed to follow protein kinase C-alpha activation.
The team also investigated the activation of protein kinase C-alpha in fixed tissue samples. They write: ¿The results demonstrate that protein kinase C-alpha is activated in situ in a significant number of human breast tumors. Notably, whereas some tumors displayed an up-regulation of protein kinase C-alpha, protein kinase C content per se did not correlate with activation.¿
Parker added: ¿These results confirm that, for signal transduction proteins, we need to know activation status rather than concentration. Activation implies that the pathway is switched on, in whatever pathological situation you are looking at. If it is not switched on, it is unlikely to be a valid therapeutic target. Concentration per se does not tell you whether the pathway is on or off.¿
Comment on the significance of the study will have to wait until many more tumor tissue samples have been tested, Parker said. Another objective, he added, would be to apply the same technique to other related proteins. For example, one subset of human breast tumors has high levels of expression of epidermal growth factor receptors, which act as tyrosine kinases. Research programs have already begun to examine whether inhibitors of tyrosine kinases can have an impact in the treatment of these tumors. The approaches developed at ICRF could bring more information to bear on these studies.