Brian W. Lee

In vivo detection of apoptosis

After several decades of debate, it is now widely acknowledged that apoptosis, also known as programmed cell death, is central to homoeostasis and normal development and physiology in all multicellular organisms, including humans. The dysregulation of apoptosis can lead to the destruction of normal tissues in a variety of disorders, including autoimmune and neurodegenerative diseases (too much apoptosis) or the growth of tumors (too little apoptosis). In addition, effective therapy of tumors requires the iatrogenic induction of programmed cell death by radiation, chemotherapy, or both. Given the central role of apoptosis, it would be desirable to have a noninvasive imaging method to serially detect and monitor this process in cancer patients undergoing conventional radiation and chemotherapy treatments as well as for the development and testing of new drugs. In this article, the latest modalities and contrast agents described in the literature for the imaging of apoptosis in vivo are reviewed. First, the most recent developments in the biochemical characterization of the many intracellular pathways involved in this complex process are discussed. Next, a variety of new radionuclide tracers, including radiolabeled annexin V and caspase inhibitors for PET and SPECT, are described. Finally, the use of MRI, MR spectroscopy, and ultrasound as possible alternative imaging modalities for the imaging of apoptosis is addressed.

Interactions of fluorochrome-labeled caspase inhibitors with apoptotic cells: A caution in data interpretation

Fluorochrome‐labeled inhibitors of caspases (FLICA, e.g., FAM‐VAD‐FMK, FITC‐VAD‐FMK) have been designed as affinity labels of the enzyme active center of caspases Their binding by apoptotic cells was interpreted as reflecting activation of caspases. We have recently observed, however, that their binding is more complex and may involve additional mechanisms. Our goal in this study was to clarify the ongoing utility of these probes.

Use of a Fluorescently Labeled Poly-Caspase Inhibitor for In Vivo Detection of Apoptosis Related to Vascular-Targeting Agent Arsenic Trioxide for Cancer Therapy

Arsenic trioxide (ATO, Trisenox) is a potent anti-vascular agent and significantly enhances hyperthermia and radiation response. To understand the mechanism of the anti-tumor effect in vivo we imaged the binding of a fluorescently-labeled poly-caspase inhibitor (FLIVO) in real time before and 3 h or 24 h after injection of 8 mg/kg ATO. FSaII tumors were grown in dorsal skin-fold window chambers or on the rear limb and we observed substantial polycaspase binding associated with vascular damage induced by ATO treatment at 3 and 24 h after ATO injection. Flow cytometric analysis of cells dissociated from the imaged tumor confirmed cellular uptake and binding of the FLIVO probe. Apoptosis appears to be a major mode of cell death induced by ATO in the tumor and the use of fluorescently tagged caspase inhibitors to assess cell death in live animals appears feasible to monitor and/or confirm anti-tumor effects of therapy.

Sequential activation of caspases and serine proteases (serpases) during apoptosis.

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TLCK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP- reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes. Key Words: Serpases, Caspases, Apoptosis, Enzymatic center, Chymotrypsin, FLICA, FLISP, Cell necrobiology

In Vitro and In Vivo Apoptosis Detection Using Membrane Permeant Fluorescent-Labeled Inhibitors of Caspases

Apoptosis detection methodology is an ever evolving science. The caspase family of cysteine proteases plays a central role in this environmentally conserved mechanism of regulated cell death. New methods that allow for the improved detection and monitoring of the apoptosis-associated proteases are key for further advancement of our understanding of apoptosis-mediated disease states such as cancer and Alzheimers disease. From the use of membrane permeant fluorescent-labeled inhibitors of caspases (FLICA) probe technology, we have demonstrated their successful use as tools in the detection of apoptosis activity within the in vitro and in vivo research setting. In this chapter, we provide detailed methods for performing in vitro apoptosis detection assays in whole living cells, using flow cytometry, and 96-well fluorescence plate reader analysis methods. Furthermore, novel flow cytometry-based cytotoxicity assay methods, which incorporate the FLICA probe for early apoptosis detection, are described. Inclusion of this sensitive apoptosis detection probe component into the flow-based cytotoxicity assay format results in an extremely sensitive cytotoxicity detection mechanism. Lastly, in this chapter, we describe the use of the FLICA probe for the in vivo detection of tumor cell apoptosis in mice and rats. These early stage in vivo-type assays show great potential for whole animal apoptosis detection research.

Col-F, a Fluorescent Probe for Ex Vivo Confocal Imaging of Collagen and Elastin in Animal Tissues

A new low‐molecular‐weight fluorescent probe, Col‐F, that exhibits affinity to collagen and elastin, was used successfully in imaging of extracellular matrix in freshly excised animal tissues. Col‐F readily penetrates between live cells into tissues and binds to fibers of collagen and elastin by a noncovalent mechanism. Fibers of collagen and elastin have been stained in a variety of tissues, including tendon, skeletal muscle, connective tissue, and arteries. Cells migrating in a Col‐F‐stained collagenous biomaterial were also imaged. No phototoxic effects were detected when live keratocytes were imaged in the in vitro culture in the presence of Col‐F. In conclusion, Col‐F provides a simple and convenient tool for fluorescence three‐dimensional imaging of intricate collagenous and elastic structures in live and fixed animal tissues, as well as in collagen‐containing biomaterials.