Monitoring Activation of Protein Kinase C-alpha via HT Imaging (Z’ > 0.5)
An essential process of drug discovery is testing candidate pharmaceuticals on cells for potential therapeutic or toxic effects. Many drug discovery companies have instituted high throughput (HT) screening programs to test novel chemicals yet those methods are not readily applicable to certain applications. Assays that rely on analysis of cell images (for example, assays that visualize proteins that migrate to the cell membrane) are extremely difficult to integrate into HT screening programs because thousands of images are acquired and must be analyzed for relevant information. Visual inspection and grading of the images is extremely labor intensive, requires days to weeks to complete, and semi-quantitative at best. Expensive software and instrumentation are also typically required. We discuss here an economical new reagent and software tool that visualizes an important cell regulatory protein and enables automatic quantitative analysis of cellular images acquired with HT imaging systems.
Protein kinase C (PKC) represents a family of serine/threonine kinases that help regulate cell division and migration, processes that when improperly controlled lead to tumor growth and metastasis. PKC-alpha is the most prevalent isoform of PKC. When activated, PKC-alpha migrates from the cytoplasm to the plasma membrane where it interacts with regulators of the cell cycle and cell motility pathways. Modulators of PKC have shown promise as potential anti-tumor drugs. Therefore, they are a subject of intense interest in the oncology field.
Vala Sciences has developed a set of reagents that enable the direct observation of PKC-alpha activation, based on PKC-alpha migration to the plasma membrane (PKC-alpha activation kit, Cat # K001). The kit includes reagents to visualize PKC-alpha for cells cultures in 96-well dishes and Vala Sciences’ CyteSeer® image analysis software which quantifies the intensity of cell images (obtained via digital photomicroscopy) at the plasma membrane. CyteSeer® functions on images obtained with any fluorescence-based microscope and is compatible with a wide variety of computer platforms. While the software is optimized to be used with Vala Sciences’ PKC-alpha kit reagents, it can also be used to quantify membrane activation events as visualized by a wide variety of staining and fluorescent cellular imaging techniques.
Figure 1
HeLa cells stained for PKC-alpha. Upper panel, control cells; middle panel, cells exposed to 100 nM PMA, lower panel, cells treated with PMA with membranes identified by Vala Sciences CyteSeer® image analysis software.
Assay of PKC-alpha activity via cell image-based HTS
HeLa cells were cultured in glass coverslip-bottomed 96-well dishes and exposed to concentrations of PMA, a well known tumor-promoting PKC-alpha agonist. As expected, HeLa cells maintained in control media (with 0.1 % DMSO) displayed PKC-alpha staining that was located primarily in the cytoplasm (Figure 1). In contrast, for cells exposed to 100 nM PMA, PKC-alpha was found prominently at the plasma membrane. A unique algorithm, designed by Vala Sciences (patent pending), identified cell borders, allowing membrane fluorescence to be selectively identified and quantified.
Figure 2
PKC-alpha activation in HeLa cells detected via HTS techniques. A) Well display. Cells were exposed to increasing concentrations of PMA, from left, to right, with all wells in a single column receiving the same dose. Column 1 was the control (DMSO only). Wells exhibiting low responsiveness are coded in “cool” colors (dark blue is lowest) with increasing response represented by “warm” colors (dark red is highest). Note that well A12 was used for calibration purposes and had photobleached. B) Concentration-dependent activation of PKCα in HeLa cells by PMA. Each symbol represents the mean ± SD for 4 wells. The Z’ statistic (0.88), which compares data variability to the range of the data, is exceptionally high. Assays with a Z’ score above 0.6 are considered excellent candidates for HT screening, (Zhang et al., 1999)
To illustrate the ability of the PKC-alpha kit to generate quantitative data, HeLa cells were treated with varying concentrations of PMA, ranging from 0.75 to 800 nM. The cells were then imaged, and the images automatically analyzed using CyteSeer® (Figure 2). Based on the results obtained with Vala Sciences’ reagents and CyteSeer®, PMA activated PKC-alpha with an EC50 of approximately 20 nM, which is consistent with published results. Similar results have also been obtained with UMR106 cells (rat, osteosarcoma) and IEC18 cells (rat, intestinal epithelium, data not shown).
Potential Applications for the Technology
The PKC-alpha Reagents can be used to define the degree to which compounds activate PKC-alpha. The reagents can identify potential antagonists of PKC-alpha by conducting screens in the presence of PKC agonists (e.g., phorbol esters or bryostatin). Also, since PKC-alpha is “downstream” of many hormone-G-protein coupled receptor pathways, this system could be used to screen chemicals for their antagonistic action against such pathways. Candidate chemicals might block PKC-alpha activation by acting as receptor antagonists (e.g., preventing hormone binding to the receptor), or by interfering with the coupling mechanism between the receptor and PKC-alpha.
Conclusions
We envision a variety of practical uses for Vala Sciences CyteSeer® Software and the PKC-alpha reagents. The software will allow automatic analysis of assays where activation or inhibition of a cellular protein is demonstrated by migration to the plasma membrane. Because no manual visual inspection is required to analyze the images, the use of CyteSeer® will result in efficient high throughput screens of cell image-based experiments. While CyteSeer® is uniquely suited for images acquired by high throughput screening instruments, its abilities can also be applied in laboratories that are generating fewer images per experiment.
References
Hanauske, A. –R., Sundell, K., Lahn M. 2004. The role of protein kinase C-alpha (PKC-α) in cancer and its modulation by the novel PKC-α-specific inhibitor aprinocarsen. Current Pharmaceutical Design 10:1923-1936.
Korfee, S., Gauler, T., Hepp, R., Pottgen, C., Eberhardt, W. 2004. New targeted treatments in lung cancer – overview of clinical trials. Lung Cancer 45 Suppl. 2: S199-S208
Lahn, M., Sundell, K., Gleave, M., Ladan, F., Su, C., Li, S., Ma, D., Paterson, B. M., Bumol, T. F. 2004. Protein kinase C- alpha in prostate cancer. BMU International 93:1076-1081.
Leontieva, O. V., Black, J. D. 2004. Identification of two distinct pathways of protein kinase Calpha down-regulation in intestinal epithelial cells. J. Biol. chem. 279:5788-5801.
Mapelli E., Banfi, P., Sala, E., Sensi, M., Supino, R., Zunino, F., et al. 1994. Effect of protein kinase C inhibitors on invasiveness of human melanoma clones expressing different levels of protein kinase C isoenzymes. Int. J. Cancer 57:281-286.
Michie, A. M., Nakagawa R. 2005. The link between PKCα regulation and cellular transformation. Immunology Letters 96:155-162.
Radeff, M. J., Tingh, A. T. K., Stern, P. H. 2004. Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase C α and interleukin-6 in UMR-106 osteoblastic cells. Cellular Signalling 16:105-114.
Sharma, G. D., Ottino, P., Bazan, N. G., Bazan, H. E. P., 2004. EGF and HGF, but not KGF, modulate PKCα translocation to the plasma membrane through 15(S)-HETE synthesis. J. Biol. Chem. (epublication ahead of print).
Zhang et al., 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomolecular Scr. 4:67-73
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