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Basic Science Investigations |
1 Department of Radiopharmaceutical Chemistry, German Cancer Research Center, Heidelberg, Germany
2 Department of Nuclear Medicine, University of Heidelberg, Heidelberg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: apoptosis • caspase substrates • Jurkat cells • Tat • radioiodine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Scintigraphic imaging with radiolabeled annexin V, a human 36-kDa protein, is currently the most promising technique to detect apoptosis in vivo (5). This agent allows the imaging of an early event in apoptosis, involving the externalization of phosphatidylserine from the inner to the outer leaflet of the plasma membrane (6). The high affinity of annexin V for cells with exposed phophatidylserine (7) is the basis for detecting apoptosis in vivo (5).
Two 99mTc-annexin V compounds have been studied in humans, differing in the bifunctional chelators (8–10). In one case, annexin V was derivatized with the monodentate 1-imino-4-mercaptobutyryl side chain (99mTc-i-AnxV) (11) and, in the second case, the protein was modified with a bis(mercaptoacetyl)diaminopentanoyl group (99mTc-BTAP-AnxV) (10). Recently, a 99mTc-annexin V derivative has been described in which the protein was modified with hydrazinonicotinamide (99mTc-HYNIC-annexin V) (5,12). The investigation of 99mTc-HYNIC-annexin V for human application revealed favorable biodistribution characteristics (11). In addition, 99mTc-HYNIC-annexin V is easily obtained by a kit-formulated preparation (5).
An alternative target in the apoptotic pathway that is potentially useful for the in vivo detection of apoptosis is the appearance of activated caspases (13–15). All known caspases have a cysteine located in the active site and cleave peptide bonds on the carboxyl side of an aspartate residue (termed as P1). Caspases are divided into two classes based on the length of their N-terminal prodomains. Caspases-1, -2, -4, -5, -8, and -10 have long prodomains, whereas caspases-3, -6, -7, and -9 have short prodomains. The caspases with long prodomains play a relevant biologic role in the cleavage of downstream caspases. Downstream caspases (short prodomain) are the foot soldiers of apoptosis, operating at the downstream end of the cascade to cleave substrates (16). Recognition of at least 4 amino acids counted from the N-terminus of the cleavage site is a necessary requirement for efficient catalysis. The preferred tetrapeptide recognition motif differs significantly among caspases, which is an explanation for the diversity of their biologic functions (17). During the execution phase of apoptosis, caspase-3 is responsible for the proteolysis of a large number of substrates either wholly or in part. These substrates contain a DXXD motif similar to that originally described for PARP (poly(adenosine diphosphate-ribose) polymerase) (18).
In an early investigation, radioiodinated Z-VAD-fmk, a pan-caspase inhibitor used as an experimental tool in apoptosis research, was tested with apoptotic Morris hepatoma cells (MH3924Atk8), resulting in a distinct uptake preference but an insufficient cell uptake (19). Enzyme inhibitors have the intrinsic disadvantage of binding-site saturation. Substrates that leave the active enzyme site after cleavage lead to a signal enhancement. One prerequisite, however, which has to be met, is the retention of the cleaved imaging agent.
To obtain information about the uptake and retention in apoptotic and normal control cells, we evaluated ten radioiodinated peptides with nonadherently growing Jurkat cells. The peptide sequence used here was selective for downstream caspases such as caspase-3. One part of the cells was triggered for apoptosis and the other fraction was used as control cells. Before performing these experiments, both cell suspensions were characterized using flow cytometry to determine the apoptotic cell fraction. The kinetics of the cell uptake was measured as a function of time. Proof of caspase fragmentation was given by a competitive caspase-3 assay and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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-detection (Bioscan). Mass spectroscopy was performed on the MALDI time-of-flight mass spectrometers MALDI III (Kratos-Shimadzu) and Reflex II (Bruker Daltonik). Flow cytometry data were recorded on a Dako Galaxy flow cytometer. The fluorescence measurements were performed with a fluorescence plate reader (Fluoroscan Ascent; Labsystems, Merlin Diagnostika). Radioactive uptake measurements were performed on a Cobra II auto -counter (Canberra Packard).
Chemistry
Peptide Syntheses.
The peptides summarized in Table 1 were prepared by solid-phase peptide synthesis using N-
-Fmoc–protected amino acids (Calbiochem-Novabiochem GmbH) and standard O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) coupling chemistry. All peptides were synthesized as C-terminal amides and purified by HPLC at a flow rate of 3.7 mL/min using 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B) as eluants by a linear gradient of 0% B to 40% B over 30 min. The purity of the products was confirmed by analytic HPLC, and the identity of each peptide was confirmed by MS.
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99mTc Labeling of HYNIC-Annexin V.
HYNIC-annexin V (Theseus Imaging) was labeled with 99mTc using stannous chloride as a reducing agent and tricine as a coligand. Quality control was performed after 15 min using instant thin-layer chromatography (Pall Corp.) and 0.9% NaCl for development. The radiochemical yields were >95%.
Biology
Cell Cultures.
Exponentially growing human leukemia cells (Jurkat J6) were obtained from the Deutsches Krebsforschungszentrum Heidelberg. The culture medium consisted of RPMI 1640 medium supplemented with 1% 200 mmol/L glutamine and 10% fetal calf serum (all from PAN Biotech GmbH). One day before the experiments were performed, the cells were split 1:4 to achieve logarithmic-phase growth at the time point of the experiment.
Annexin V/Propidium Iodide Assay.
Apoptosis was induced in 106 cells per milliliter using camptothecin concentrations of 1, 10, and 100 µmol/L (Sigma-Aldrich). Samples of the camptothecin-treated cells were harvested after 2, 3, 4, 5, and 6 h, centrifuged for 5 min at 800 rpm, washed with cold PBS, and suspended in 100 µL cold annexin-binding buffer (10 mmol/L N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid), 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1.8 mmol/L CaCl2, pH 7.4). After the addition of 1.5 µL annexin V-Alexa Fluor 488 (Molecular Probes) and an incubation period of 15 min, the suspensions were mixed with 300 µL cold annexin-binding buffer. Flow cytometry measurements were performed with mixtures of 100 µL cell suspension, 900 µL cold annexin-binding buffer, and 10 µL propidium iodide solution (250 µg/mL). The fluorescence emissions were adjusted to 530 (FL-I) and >575 nm (FL-III) and measured in triplicate.
After indicated time periods following induction, apoptosis was monitored with annexin V-Alexa Fluor 488/propidium iodide by flow cytometry (two determinations) and Z-DEVD-R110 (EnzChek Caspase-3 Assay Kit; Molecular Probes) by fluorescence-intensity measurements (three determinations).
Caspase Assay.
Apoptotic cells produced as described were taken from the incubation solution after 2, 3, 4, 5, and 6 h and centrifuged for 5 min at 800 rpm. The pellets were washed with 1 mL cold PBS and suspended on ice for approximately 30 min in 50 µL lysis buffer (10 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid [EDTA], 0.01% Triton X-100, pH 7.5). After 30 min on ice, the lysate was centrifuged for 5 min at 5,000 rpm to remove cellular debris. Fifty microliters of the supernatant obtained from each sample were stored at –80°C before analysis. For fluorescence measurements, the supernatants were transferred to a microtiter plate covered with 50 µL substrate working solution (20 mmol/L PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], 4 mmol/L EDTA, 10 mmol/L dithiothreitol, 0.2% CHAPS [3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate], 50 µmol/L Z-DEVD-R110, pH 7.4). The plates were incubated at room temperature for 60 min, and the fluorescence was measured at 538 nm (excitation, 485 nm).
Cell Uptake Measurements.
The cell uptake measurements were performed with staurosporine-treated cells and Jurkat cell controls. Before commencing the cell uptake measurements, control cells and staurosporine-treated cells were examined by flow cytometry for apoptotic cell fractions. The cell counts were 106 cells/100 µL annexin- binding buffer (99mTc-HYNIC-annexin V experiments) and 5 x 106 cells/350 µL RPMI 1640 medium (radioiodinated peptide experiments). Treated and control cells were incubated at 37°C with 0.16 MBq 99mTc-HYNIC-annexin V (0.371 µg annexin/mL) and 0.2 MBq of the respective radioiodinated peptide. The amount of unlabeled annexin V corresponded to the annexin V concentration used for flow cytometry. The specific activities of the peptides were no carrier added. After 10, 15, 20, 30, and 60 min, the tubes were briefly vortexed and 10-µL samples of the control and apoptotic cells were layered on top of a 400-µL microcentrifuge tube (Roth) containing 350 µL of a 75:25 mixture of silicon oil, density = 1.05 (Aldrich), and mineral oil, density = 0.872 (Acros) (21). Instantaneous centrifugation at 15,000 rpm for 2 min afforded the complete separation of cellular radioactivity from the medium. After freezing the tubes with liquid nitrogen, the bottom tips containing the cell pellet were cut off. The cell pellets and the supernatants were counted in a -counter. The cell uptake was calculated as % uptake = 100 x (cpm in pellet)/(cpm in pellet + cpm in supernatant). The experiments were performed in triplicate.
Proof of Caspase-Induced Peptide Cleavage (Competition Experiment).
Stauroporine-treated cells were harvested 3 h after induction, centrifuged for 5 min at 800 rpm, washed in 1 mL cold PBS, suspended in 50 µL lysis buffer, and kept on ice for approximately 30 min. The lysate was centrifuged at 5,000 rpm for 5 min to remove cellular debris. Fifty microliters of the supernatant obtained from each sample were frozen at –80°C until the onset of the competition experiment. Twenty-five microliters of 100 µmol/L Z-DEVD-R110 substrate solution and 25 µL of the synthesized peptide solutions were transferred to the microtiter plate, resulting in final concentrations of 0, 100, 200, and 400 µmol/L. The thawed caspase-3 containing supernatants were added to each well. After an incubation period of 90 min at room temperature, the fluorescence was measured at 538 nm (excitation, 485 nm).
Proof of Caspase-Induced Peptide Cleavage (MALDI-MS Experiment).
Human caspase-3 solution (1.7 µL, 17 units; Calbiochem-Novabiochem GmbH) was mixed with 1 µL of a 1 mmol/L solution of peptide 7 dissolved in caspase reaction buffer. After 1 h at 30°C, the reaction mixture was analyzed by MALDI-MS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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In Figure 1 apoptosis induction of Jurkat cells is shown as a function of time and drug concentration. The apoptotic cell fraction monitored by flow cytometry peaked 5 h after addition of 10–100 mmol/L campthothecin (Fig. 1A). The decline in apoptotic cells at later times reflects the increase in dead cells. Similar results were obtained using the caspase-3 assay with Z-DEVD-R110. The curves in Figure 1B show an almost identical curvature using the same campthothecin concentrations.
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The uptake of radiolabeled peptides 1–4 as a function of time is shown in Figure 4. In general, the absolute uptake values of peptides 1–4 proved to be rather low, ranging between 0.3% and 4.0% of the total dose. Differences between apoptotic and control cells were not observed within the first half of the experiments. At later times, the control cells preferentially accumulated peptide 1 and, to a lesser degree, peptides 2, 3, and 4.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The dynamics of apoptosis required the determination of the optimal time period after which uptake measurements are best performed. Time-course measurements of externalized phosphatidylserine and caspase-3 activation in the apoptotic cell fractions were performed (Fig. 1). Because of the coherent occurrence of these effects, the rapid performance of flow cytometry assay was favored, which simplified the characterization of the cells before uptake measurements.
Besides camptothecin, we used staurosporine for the initiation of apoptosis. Camptothecin is a quinoline-based and staurosporine is an indole-based alkaloid. Whereas the former is a DNA topoisomerase I inhibitor, the latter represents an inhibitor of kinase proteins. As shown in Figure 2, staurosporine proved to be more effective in stimulating apoptosis. The cell uptake measurements with the series of radioiodinated peptides were, therefore, performed with staurosporine.
The flow cytometry data correlated well with cell uptake experiments performed with 99mTc-HYNIC-annexin V in staurosporine-treated and control cells (Fig. 3). The high binding values of 99mTc-HYNIC-annexin V at the beginning of the experiment and the steady course over 1 h are due to the unhindered access of this agent to externalized phosphatidylserine-binding sites on apoptotic cell membranes. The slight, but steady, increase of radioactivity may reflect unspecific binding or diffusion of its metabolite into the cells.
Using the same technique, two radiolabeled peptides comprising the DEVDG sequence (1 and 2) were evaluated for their differential uptake in control and apoptotic cells. DEVDG was used because it is a sequence accepted by caspase-3 (23). To examine the influence of the label position, 3-131I-iodotyrosine was placed either on the N- or at the C-terminal end. In addition, the influence of the free carboxylic acid groups on the uptake behavior of DEVDG was examined by replacing aspartic acid (D) and glutamic acid (E) with asparagine (N) and glutamine (Q), yielding NQVNG (3 and 4). The results of the cell uptake studies are summarized in Figures 4A and 4B.
Throughout the experiments, the early uptake values of radiolabeled peptides 1–4 were similar in control and apoptotic cells. Beyond 30 min, control cells started to increase peptide uptake. The divergent uptake characteristic might be related to the compromised cell membrane structure during apoptosis, which impeded cell membrane transport of these peptides.
An additional observation was that the position of 3-131I-iodotyrosine exhibits an uptake-directing role. The comparison of peptides 2 and 4 with N-terminal bound 3-131I-iodotyrosine and peptides 1 and 3 with the corresponding C-terminal position reveals that the C-terminal bound 3-131I-iodotyrosine favored the cell uptake to some degree.
From these results it becomes evident that the DEVDG or NQVNG sequences alone are insufficient for gaining uptake differences between apoptotic and control cells opposite to the observed ones. The main reason for this shortcoming is probably the lack of cell membrane permeability of these peptides. Therefore, cell membrane transporter sequences were taken into consideration. Two groups of cell-penetrating peptides have been described. MAP (model amphipathic peptide [X-KLALKLALKALKAALKLA-amide]) (24) and transportan (25) belong to the first group, whereas Tat (26) and penetratin (27) belong to the second group. The difference in cargo delivery efficiency between these 2 groups of cell-penetrating peptides is explained by the ability of the amphipathic transportan–MAP group of peptides to enter and exit cells, in contrast to the Tat/penetratin group of peptides, which are trapped in the cells (28).
Due to the trapping characteristic of Tat, the DEVDG sequence was conjugated to Tat49–57 (RKKRRQRRR), including the label D-3-131I-iodotyrosine (y). Tat48–60 (GRKKRRQRRRPPQ) encompasses the whole basic region and nuclear localization signal of the HIV transactivating factor protein Tat (29). The sequence lacking N-terminal glycine and C-terminal proline-proline-glutamine was taken up by Jurkat cells (26) and was, therefore, selected for these experiments. Penetratin, which was also considered for this purpose, contains methionine and is, consequently, less appropriate because of its sensitivity against chloramine-T.
The yDEVDG sequence was coupled to the N-terminal (5) as well as to the C-terminal (7) end of Tat49–57. The same synthesis was performed with the inverse sequence of the Tat fragment Tat57–49 (6 and 8). When incubating apoptotic and control cells with Tat conjugates 7 and 8, a general higher cellular uptake was observed as compared with peptides 5 and 6, indicating yDEVDG on the C-terminus of Tat to be more favorable for cell uptake (Fig. 5). With peptides 7 and 8, the uptake preference was, for the first time, reversed, meaning that apoptotic cells retained more radioactivity than the control cells. This observation met our working hypothesis that the interaction of a radiolabeled peptide with an activated caspase would lead to a retention advantage over cells not containing the activated enzyme. The prerequisite for this assumption is the recognition of the offered peptides as caspase substrates (vide infra).
The uptake values of the apoptotic cells were well above those of the controls. The decrease in cellular radioactivity indicates the efflux of either the intact peptide or a fragment carrying the label. In contrast to the results obtained with radiolabeled (30) and fluorescence tagged Tat49–57 and Tat57–49 sequences (26), the Tat sequence order seems to play a minor role here with respect to the uptake and retention characteristics of Jurkat cells.
The Tat conjugates 7 and 8 were also transformed to tat49–57-yDEVDG (9) and tat57–49-yDEVDG (10) by replacing L- with D-amino acids in the Tat sequence. As observed with peptides 1–6, this modification resulted again in a favored uptake into normal Jurkat cells (Fig. 5).
Although the cell membrane of apoptotic cells does not give preference to facilitated transport, the differences observed with peptides 7 and 8 can be related to the interaction with the activated enzymes. To obtain experimental proof for this assumption, the caspase-3 substrate properties of YDEVDG-NH2 (2) and Tat49–57yDEVDG-NH2 (7) were investigated with two different approaches.
Assuming that the peptides 2 and 7 are caspase-3 substrates, Z-DEVD-R110 could be used to monitor the degree of enzyme competition by measuring the depletion of the fluorescence signal. The result of this experiment shown in Figure 6 demonstrates the suppression of R110 formation as a consequence of increasing amounts of the two peptides, which identifies especially peptide 7 as a superior caspase-3 substrate.
An additional experiment applied MALDI-MS to show caspase-mediated fragmentation products. Commercially available caspase-3 (Fig. 7) and apoptotic cell lysate containing natural caspase-3 (data not shown) were incubated with peptide 7, indicating the expected fragmentation. MALDI-MS detectable fragmentation of peptide 2 was not detected under these conditions, a result correlating with the less-effective competitive characteristics of this compound shown in Figure 6.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: Michael Eisenhut, PhD, Deutsches Krebsforschungszentrum, Abt. Radiopharmazeutische Chemie–E030, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
E-mail m.eisenhut{at}dkfz.de
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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