Monitoring The Apoptosis Related Caspase Activation With Optical Imaging

Apoptosis or programmed cell death is an elementary process of life that maintains homeostasis of tissues and organs in concert with proliferation, growth, and differentiation. It is a physiological form of cell death responsible for the deletion of unrepairably damaged or mutated cells which have lost their functions. Apoptosis is an active, genetic controlled process. Many vital functions, for example, termination of inflammatory reactions, wound healing, physiological tissue renewal, and elimination of cells with irreparable damage of the genomic DNA, depend on intact apoptosis.Caspases are a conserved family of cysteine proteases. These proteases bear an active-site cysteine and cleave substrates after aspartic acid residues. Because caspases play a central role in mediating the initiation and propagation of the apoptotic cascade, the ability to real-time image the activation of these zymogens would provide a significant advantage for rapid and dynamic screening as well as validation of experimental therapeutic agents.In this study, we applied advanced bioengineering techniques to construct fluorescence resonance energy transfer (FRET)-based indicators for detection of the caspases activation, which are composed of an enhanced cyan fluorescent protein, a caspases-sensitive linker, and an enhanced yellow or red fluorescent protein (DsRed). Despite its considerable promise, however, greater responsivity of fluorescence to the proteolysis has been desired for a better understanding of spatio-temporal pattern of the activation of caspases during apoptosis.Main results of the study are as follows:1) Five FRET probes, which were constructed by bioengineering techniques, were identified by restriction enzyme map and DNA sequence analysis. These FRET probes were named CYx or CDx according to the particular acceptor (EYFP or DsRed). In this study, CYx include CY3, CY8, CY9, each with a linker of the caspase-3 cleavage sequence (CCS, DEVD), the caspase-8 cleavage sequence (CCS, IETD) and the caspase-9 cleavage sequence (CCS, LEHD) respectively. CDx include CD2 and CD3, each with a linker of the caspase-2 cleavage sequence (CCS, VDVAD) and the caspase-3 cleavage sequence (CCS, DEVD) respectively.2) To identify the sensitivity and specificity of CD2 and CD3 to caspase activation, the recombinant FRET probes (CD2 and CD3) were purified for in vitro analysis. Then the properties of CD2 and CD3 were analyzed using CD2 or CD3 expressing HeLa cells by western blot and spectroscopic measurements. Both in vitro and in vivo results indicate that the CD2 and CD3 are recognized uniquely by the activated caspase-2 and caspase-3, respectively. Using a three-filter imaging system, the values of RFRET were obtained from the ROI in different cells and were plotted as a function of time. These results demonstrate that the caspase-2 activation profile is significantly different from that of caspase-3. During cisplatin-induced apoptosis, the activation of caspase-2 enhanced gradually, taking at least 30 min (n=11), while the activation of caspase-3 rapidly reached its maximum within a shorter period because ratio changes were completed in less than 10 min. We also observed that the activation of caspase-2 and caspase-3 adhered to a strictly temporal order. This result demonstrates that, in the mitochondrial apoptotic pathway, caspase-2 activation occurs upstream of caspase-3 in cisplatin-induced apoptosis, with classical properties of initiator caspase.3) In comparison with the preceding FRET pairs, DsRed as an acceptor is brighter and insensitive to H+. So CD3 is insensitive to the change of H+ concentration during apoptosis. On the other hand, DsRed will not be significantly bleached when illuminated with strong light from a mercury lamp and is thus insensitive to the environment. Therefore, CD3 is much more reliable in studying the pathway of apoptosis. Furthermore, to provide more information about TRAIL-induced apoptosis, three groups of cells (group 1: apoptotic cells with caspase-3 activation, group 2: apoptotic cells with no caspase-3 activation, group 3: non-apoptotic cells) were identified by the number method of counting cells, combined with analysis of morphological characteristics, Hoechst 33258 staining and FRET analysis. The total percentage of apoptotic cells was 56.08% of TRAIL-treated HeLa cells, and the percentage of apoptotic cells via caspase-3-dependent pathway was 21.50% of TRAIL-treated HeLa cells. Thus, the remained apoptotic cells proceeded from caspase-3-independent pathways. This result shows that caspase-3-dependent pathways and caspase-3-independent pathways coincidently proceed from the apoptosis induced solely by TRAIL. To provide more evidence that caspase-3-dependent apoptotic pathways are not unique in TRAIL-treated HeLa cells, we quantitatively analyzed the activation of caspase-3 by a CE system. The percent of cleaved CD3 was 25.11%. This result is similar to the 21.50% apoptotic rate via caspase-3-dependent pathways, but very different from the total apoptotic rate of 56.08% in TRAIL-treated HeLa cells. As the cleaved CD3 is directly proportional to the apoptotic cells in the CE system, and thus the percent of cleaved CD3 can indirectly represent the apoptotic rate via caspase-3-dependent pathway by CE analysis. Therefore, through the utilization of CE analysis, we reconfirmed the TRAIL-induced apoptotic rate of caspase-3 activation pathways as revealed by FRET analysis.