Editorial Article
Tuesday June 01, 2010
by Caitlin Smith
Apoptosis– programmed cell death – is a cell’s way of politely kicking the bucket when its time has come. A vital part of nearly all normal processes, apoptosis is so important to the healthy functioning of the organism that when it goes awry, it initiates cancer cells that can take out the entire organism – hence, the considerable interest in apoptosis research among humans.
“The biggest challenges in apoptosis research probably involve understanding the role of apoptosis in various diseases,” says Michael Hambleton, technical specialist at DiaPharma. “Scientists are studying the molecular apoptotic pathways and dysregulation of those pathways using a variety of research tools. A major challenge will be to determine the usefulness of apoptosis kits as diagnostic tools and to advance kits toward clinical utility as an FDA-regulated product.” Luckily, tools for studying apoptosis are getting more advanced as more components in the pathway are identified.
Biomarker tools
Debra Meyer, professor in the department of biochemistry at the University of Pretoria, studies HIV-associated/influenced apoptosis using flow cytometry. “We have recently also initiated a project where we're trying to corroborate flow cytometry findings using apoptosis-guided mass spectrometry metabonomics,” says Meyer. “We are still in the early stages of this work but have already detected (via biofluid-mass spectrometry) several biomarkers implicated in apoptosis.” Meyer’s group uses metabonomics – the study of metabolic responses to drugs and diseases – to identify and quantify apoptotic markers.
Meyer notes that a problem in apoptosis research is the difficulties in the in vitro induction of programmed cell death. “In the in vivo system, apoptosis mediators or regulators can have a bi-functional role (increase or decrease apoptosis) and not all the mechanisms or pathways in which these operate are known, which is problematic when using them in in vitro studies,” she says. “Difficulties in detecting apoptosis in cell types where the frequency of occurrence is low is another problem. A problem for detecting apoptosis using flow cytometry is the overlap between different types of cell death (early/late apoptosis vs. necrosis).” Meyer says that the more sensitive flow instruments and dyes being developed will likely help to distinguish between different forms of cell death. Meyer hopes that in the long run, progress in “infectious disease metabonomics should allow for the detection of novel metabolites which may assist in the elucidation of apoptotic pathways.”
More specific tools
Labeled protein substrates for the myriad of proteases involved in apoptosis are increasingly in demand. “OncoImmunin sells cell-permeable fluorogenic substrates for measurement of caspase as well as other protease activities in live cells,” says Beverly Packard, co-founder of OncoImmunin. “OI's substrates cross all membranes by passive diffusion, thereby enabling measurement of protease activities in their native environments. Once a substrate comes in contact with a protease that recognizes its sequence, the substrate is cleaved and fluorescence is measured in the physiologic environment in which the protease is active in nature. Also, OI's probes can be made in multiple colors, enabling multiplexing, e.g., multiple caspase activities in the same live cell.”
OncoImmunin’s probes can measure the activity of cytotoxic lymphocytes into target cells. For example, the serine protease granzyme B is made in cytotoxic lymphyocytes, where it is inactive due to the low pH of the storage granules. “It is measured immediately upon delivery into target cell cytoplasms where the pH is neutral,” says Packard. “This is followed by cleavage of procaspase 3 by granzyme B in the target cell cytoplasm and the subsequent induction of apoptosis. All proteolytic events can be quantitated at the live single-cell level by flow cytometry and imaged by confocal microscopy.”
Promega recently released an assay for in vitro multiplexed measurements of viability and apoptosis. “Both measures are performed in the same cell sample well in a sequential add-mix-measure, add-mix-measure protocol,” says Pam Guthmiller, marketing manager at Promega. “The multiplex of a viability assay with a caspase-3/7 assay allows additional information to be learned about potency, mechanism and kinetics of cell death, in addition to mitigating false results by having a second measure in the same well.”
Promega has also developed a new in vivo bioluminescence caspase-3/7 substrate designed for in vivo animal imaging of apoptosis in cancer models. “The advancements in in vivo imaging with ability to not only image the treatment of a tumor in a mouse model, but [also] confirm the mechanism of action (i.e. apoptosis) of a potential drug compound in vivo will help speed up this portion of the drug development process [by] providing more biologically relevant information,” says Guthmiller.
Another approach is Roche’s xCELLigence system, which offers a non-invasive method based on impedance measurements for real-time cellular analysis, for example in label-free studies, and in cell invasion or migration assays. “xCELLigence gives us the technology where we can monitor the cellular activity as far as morphology, shape, and viability in real time,” says Steven Hurwitz, marketing manager at Roche Diagnostics. “We use a special plate with an array of gold electrodes adhered to the bottom of the wells. This is a normal tissue culture plate, so as we plate the cells, they lay down, attach, change shape, grow, proliferate, or even vice-versa, we can get a real-time kinetic profile of what’s happening with that cell or population of cells.” New 384-well plates will be released this fall for high-throughput screening. Roche also offers a wide variety of specific endpoint assays to zero in on different parts of the apoptotic pathway.
Hurwitz says that some of the biggest challenges in this field lie within the complexity of apoptotic pathways. “The more we understand apoptosis and the players involved in the pathways, the more targets there are available for addressing cancer.” Hurwitz says that a future direction of apoptosis research is “understanding the pathway as a whole, and then targeting which part of the pathway is going to be relevant for disrupting a cancer event – and then coming up with a therapeutic based on that.”
From tools to treatments
A common hope among bench scientists who study apoptosis is that their results will contribute to advancing cancer treatments. “The most exciting developments in the area of apoptosis are its potential for monitoring disease states,” says Hambleton. “Dysregulation of apoptosis is recognized in a multitude of disease states and increased apoptosis in the blood could be an early indication of disease.”
DiaPharma offers their M30 Apoptosense® ELISA and M30 CytoDeath™ ELISA kits for research use, to measure epithelial cell apoptosis specifically. “The assays are based on the ability of the M30 monoclonal antibody to recognize a neo-epitope on cytokeratin 18 (CK18) that becomes exposed after cleavage by caspases during apoptosis,” says Hambleton. “These conventional, reagent-complete ELISAs give clear results and early detection of apoptosis, as a result of specific cleavage of one of the earliest apoptosis markers.” The M30 Apoptosense® ELISA has proven useful for measuring treatment response in the oncology field, and in liver disease as well.
Fluofarma offers a new approach to studying apoptosis called DCffer® (Drug Comparison by efficacy and EC50 readout), which is “a very unique method to test a potential anticancer molecule against different human cancer cell lines by simultaneously detecting 3 key parameters at two time points: proliferation inhibition, cytolysis and apoptosis,” says Bruno Brisson, chief business officer at Fluofarma. “The DCffer® approach is based on the development and the comparison of multi-parametric functional signatures obtained on tumoral cellular lines in culture over time. This is a powerful method allowing the determination of the intracellular mechanism of action of anti-cancer drug candidates and especially the study of apoptosis.”