Anal. Chem. 2005, 77, 2281-2287
Accelerated Articles
Selective Determination of the Doxorubicin Content of Individual Acidic Organelles in Impure Subcellular Fractions Yun Chen, Richard J. Walsh, and Edgar A. Arriaga*
Department of Chemistry, University of Minnesota, 207 pleasant Street S.E., Minneapolis, Minnesota 55455
Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laserinduced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/ C2 cell lines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) × 10-4 cm2 V-1 s-1 while those of the other organelles spread from (-0.6 to -3.5) × 10-4 cm2 V-1 s-1. In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 ( 0.5 and 10.6 ( 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating * To whom correspondence should be addressed. Phone: 612-624-8024. E-mail:
[email protected]. 10.1021/ac0480996 CCC: $30.25 Published on Web 03/18/2005
© 2005 American Chemical Society
acidic organelle maturation and the role of these organelles in drug resistance.
Doxorubicin (DOX), a clinically important antitumor agent,1 is capable of halting malignant cell proliferation by stabilizing a topoisomerase IIR-DNA complex in the nucleus of the cell.2 Unfortunately, some tumor cells may develop multidrug resistance (MDR), a phenomenon described as the tumor’s resistance not only to the drugs being administered but also to a host of other structurally and mechanistically diverse drugs to which the tumor has not yet been exposed.3 Differences between drug-resistant and drug-sensitive tumor cells include changes in the type and amount of cellular lipids and in the expression of proteins4 such as membrane pumps. These latter proteins include P-glycoprotein (Pgp) and MDR-associated protein (MRP),3 which are expressed in the plasma membrane and in the Golgi apparatus. There have also been an increasing number of reports of MDR cells that do not overexpress Pgp4-6 or use MRP for the intracellular elimination of anthracyclines.7 Some of these studies have also reported that MDR cells contain more acidic organelles than their corre(1) Hortobagyi, G. N. Drugs 1997, 54 ( Suppl 4), 1-7. (2) Bodley, A.; Liu, L. F.; Israel, M.; Seshadri, R.; Koseki, Y.; Giuliani, F. C.; Kirschenbaum, S.; Silber, R.; Potmesil, M. Cancer Res 1989, 49, 59695978. (3) Altan, N.; Chen, Y.; Schindler, M.; Simon, S. M. J. Exp. Med. 1998, 187, 1583-1598. (4) Taylor, C. W.; Dalton, W. S.; Parrish, P. R.; Gleason, M. C.; Bellamy, W. T.; Thompson, F. H.; Roe, D. J.; Trent, J. M. Br. J. Cancer 1991, 63, 923929. (5) Dalton, W. S.; Cress, A. E.; Alberts, D. S.; Trent, J. M. Cancer Res. 1988, 48, 1882-1888. (6) Harker, W. G.; Slade, D. L.; Dalton, W. S.; Meltzer, P. S.; Trent, J. M. Cancer Res. 1989, 49, 4542-4549. (7) Leier, I.; Jedlitschky, G.; Buchholz, U.; Cole, S. P.; Deeley, R. G.; Keppler, D. J. Biol. Chem. 1994, 269, 27807-27810.
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sponding drug-sensitive cell line8 or have a higher pH gradient between the cytoplasm and the acidic organelles.9 These observations have led to the formulation of a protonation, sequestration, and secretion (PSS) model as an additional mechanism for drug resistance that works separately from the Pgp and MRP drugefflux pumps.10 This model postulates that acidic organelles in some MDR cells have a lower than normal pH that results in the protonation of weak base antitumor drugs, thereby sequestering them from the nucleoplasm and cytosol. The drugs are subsequently secreted from the cell through the normal pathways of vesicular traffic and secretion. While confocal imaging has shown different accumulations of DOX in the acidic organelles of several drug-resistant and drug-sensitive cell lines,3 no direct measurement of the actual DOX content of these acidic organelles that would directly link the DOX accumulation in these subcellular compartments to MDR phenotype has been reported.11 Acidic organelles have been actively investigated in whole cells using flow cytometry12 and microscopy.12,13 These studies provide useful information on the average properties of the entire organelle population within the cell.14,15 Another relevant technique is capillary electrophoresis with laser-induced fluorescence (CE-LIF), which has been proven to be effective for separating and detecting individual particles and organelles.16-20 This technique has been applied to studies of microspheres,16 liposomes,17 and mitochondria,18 with these particles detected as individual spikes as they migrate separately due to their different electrophoretic mobilities.21 We have previously used CE-LIF to detect individual acidic organelles, with selective labeling carried out by the endocytosis of fluorescently labeled microspheres.19 In this report, we extend the use of the CE-LIF analysis of individual organelles to the determination of the DOX content of individual acidic organelles from human leukemia CEM/C2 and CCRF-CEM cell lines, commonly used to test anticancer drug treatments. This was accomplished by separately detecting nanosphere- and DOX-containing organelles with an LIF dual-channel detector in two fluorescent emission ranges. Not only are we able to measure the DOX content in individual acidic organelles, other concomitant DOX-containing organelles did not interfere with the analysis. (8) Sognier, M. A.; Zhang, Y.; Eberle, R. L.; Sweet, K. M.; Altenberg, G. A.; Belli, J. A. Biochem. Pharmacol. 1994, 48, 391-401. (9) Belhoussine, R.; Morjani, H.; Sharonov, S.; Ploton, D.; Manfait, M. Int. J. Cancer 1999, 81, 81-89. (10) Schindler, M.; Grabski, S.; Hoff, E.; Simon, S. M. Biochemistry 1996, 35, 2811-2817. (11) Holtzman, E. Lysosomes; Plenum Press: New York, 1989. (12) Newman, K. D.; Elamanchili, P.; Kwon, G. S.; Samuel, J. J. Biomed. Mater. Res. 2002, 60, 480-486. (13) Zucker, R. M.; Hunter, E. S., 3rd; Rogers, J. M. Methods 1999, 18, 473480. (14) Beyer, U.; Rothern-Rutishauser, B.; Unger, C.; Wunderli-Allenspach, H.; Kratz, F. Pharm. Res. 2001, 18, 29-38. (15) White, J. G.; Amos, W. B.; Fordham, M. J. Cell Biol. 1987, 105, 41-48. (16) Duffy, C. F.; McEathron, A. A.; Arriaga, E. A. Electrophoresis 2002, 23, 2040-2047. (17) Duffy, C. F.; Gafoor, S.; Richards, D. P.; Admadzadeh, H.; O’Kennedy, R.; Arriaga, E. A. Anal. Chem. 2001, 73, 1855-1861. (18) Duffy, C. F.; Fuller, K. M.; Malvey, M. W.; O’Kennedy, R.; Arriaga, E. A. Anal. Chem. 2002, 74, 171-176. (19) Fuller, K. M.; Arriaga, E. A. Anal. Chem. 2003, 75, 2123-2130. (20) Anderson, A. B.; Xiong, G.; Arriaga, E. A. J. Am. Chem. Soc. 2004, 126, 9168-9169. (21) Radko, S. P.; Chrambach, A. Electrophoresis 2002, 23, 1957-1972.
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EXPERIMENTAL SECTION Materials and Reagents. Doxorubicin was purchased from Bedford Laboratories (Bedford, OH). Polystyrene latex Fluoresbrite YG spheres (50-nm and 1-µm diameter) were purchased from Polysciences, Inc. (Warrington, PA). Fluorescein was purchased from Molecular Probes (Eugene, OR). Sucrose and D-(+)-mannitol were purchased from Fisher Scientific (Pittsburgh, PA). Ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl) piperazineN′-(2-ethanesulfonic acid) (HEPES), and sodium citrate were purchased from Sigma (St. Louis, MO). Sodium borate was from EM Science (Gibbstown, NJ). Sodium dodecyl sulfate (SDS) was from Bio-Rad Laboratories (Hercules, CA). A fractionation buffer composed of 210 mM D-(+)-mannitol, 70 mM sucrose, 5 mM HEPES, and 5 mM EDTA, adjusted to pH 7.4 with potassium hydroxide (Fisher Scientific), was used for cell washing and disruption. The CE buffer for individual acidic organelle analysis was 250 mM sucrose and 10 mM HEPES free acid adjusted to pH 7.4 with potassium hydroxide. The buffers used for DOX fluorescence spectral measurements were 10 mM SDS, 10 mM borate, pH 9.4 (BS buffer), and 10 mM sodium citrate, pH 5.0 (citrate buffer) and were adjusted with hydrochloric acid (Fisher). All buffers were filtered through a 0.22-µm Nalgene filter and stored at room temperature for up to a month. Cell Culture. Nonadherent CCRF-CEM and CEM/C2 cells (ATTC, Manassas, VA) were cultured in 90% RPMI-1640 medium, 10% fetal bovine serum (Sigma) at 37 °C, and 5% CO2, and were split by addition of new medium every 2-3 days. When needed, supplementation of the cell medium with 50-nm-diameter nanospheres at 2 × 1011/mL or 25 µM DOX was performed 48 and 12 h prior to cell disruption and fractionation. Controls included cells without any treatment, cell cultures treated only with nanospheres, and cell cultures treated only with DOX. Confocal Microscopy. After incubation with nanospheres for 48 h, cells were washed with fresh serum-free medium twice by centrifugation, and 0.2 mL of this suspension (3 × 105 cells/mL) was placed on a poly(D-lysine)-coated coverslip bottom Petri dish (BD Biosciences, Bedford, MA), for 1 min prior to the addition of 2 mL of fresh serum-free medium. The medium was then removed and replaced with 25 µM DOX in serum-free medium at 37 °C and incubated for 15 min. Confocal images (40 layers, 0.35 µm thick) were collected on an Olympus IX81 inverted microscope (Melville, NY) outfitted with an Olympus IX2-DSU disk scanning unit and a 60× (NA 1.45) oil immersion objective. Excitation wavelengths were supplied by an Exfo (Addison, TX) XCite 120 metal halide lamp source coupled to the microscope via a liquid light guide. Olympus also supplied a GFP excitation cube (excitation 460-480 nm; dichroic 485 nm; emission 495-540 nm) used for imaging nanosphere localization and a RFP excitation cube (excitation 535-555 nm; dichroic 560 nm; emission 570-620 nm) used for imaging DOX. Imaging was done with a C9100-01 CCD camera (Hamamatsu, Bridgewater, NJ). Spectral cross-talk was not observed using these excitation cubes and the standard camera settings. CImaging Simple PCI 5.3 (Compix Inc., Cranberry Township, PA) software was used to control the microscope and camera and to collect and process the images. Cell Disruption and Fractionation. Following the nanosphere and DOX treatment, the cells were pelleted at 1480g for 5 min and washed 2 times in fractionation buffer.19 Cells were
counted with a Fuchs-Rosenthal hemacytometer (Hausser Scientific, Horsham, PA). The samples were disrupted using 40 strokes in a Dounce homogenizer with (0.5-2.5) × 10-3 in. clearance (Kontes, Vineland, NJ) on ice. Comparison of the number of cells under bright-field microscopy indicates that ∼95% of the cells were disrupted with this procedure. Whole cells, nuclei, and large cell debris were removed by centrifugation at 600g for 5 min, and the supernatant was removed and centrifuged a second time. Acidic organelles as well as other organelles (e.g., mitochondria) were pelleted from the supernatant by centrifugation at 14000g for 20 min and resuspended in 300 µL of CE buffer.22,23 All samples were stored on ice until analyzed. Capillary Electrophoresis. The design and setup of the electrophoresis system with postcolumn LIF detection used for this study incorporates a sheath flow cuvette design previously described.18,24 Briefly, the 488-nm line from an argon ion laser (Melles Griot, Irvine, CA) was used for fluorescence excitation. To reduce scattering at 488 nm caused by interactions between the laser beam and organelles or air bubbles, a 495-nm LP filter (Omega Optical) was placed in front of the interference filter. The dual-channel optical configuration of the detector was carried out with a 585-nm long-pass dichroic mirror (CP-AR-585, CVI Laser, Albuquerque, NM), a 635DF55 (607.5-662.5 nm) band-pass filter, and a 510WB40 (517.5-552.5 nm) band-pass filter (Omega Optical). The dichroic mirror transmitted DOX fluorescence at wavelengths of >585 nm that then was filtered with a 635DF55 filter. Nanosphere fluorescence was reflected by the dichroic mirror at wavelengths of 5.1 × 10-20 mol of DOX are not shown in (A) and (B), respectively.
integrated over the transmission range of the DOX channel (608663 nm). To estimate the DOX content of an individual acidic organelle (m) based on the peak area of an organelle (a) in an electropherogram, the factors, s, F, d, and h are used in the following formula:
m ) (sdh/F) a ) (7.96 × 10-18)a
(1)
Using this formula, DOX content per acidic organelle varies over 2 orders of magnitude from 1.6 × 10-22 to 9.7 × 10-20 mol in CEM/ C2 cells and 1.6 × 10-22 to 8.4 × 10-20 mol in CCRF/CEM cells. Histograms of the DOX content of individual acidic organelles calculated using eq 1 are shown in Figure 5A and 5B for organelle fractions isolated from CEM/C2 and CCRF-CEM cells, respectively. The average DOX content per acidic organelle in CEM/ C2 and CCRF-CEM cell lines is 11.1 ( 0.5 and 10.6 ( 0.4 zmol, respectively. In addition, there are no striking differences between the overall DOX content per acidic organelle distribution for these two cell lines (Figure 5). Together, these results suggest that similar factors are determining the distribution of this drug in both cell lines. On the other hand, as mentioned in the Classification of Detected Organelles, differences in DOX content may originate from differences in organelle size, the pH within individual acidic organelles, or leakage of DOX from the organelle over time. A wide range in acidic organelles size is reasonable since a span in volume (i.e., DOX content) over 2 orders of magnitude corresponds just to a 5-fold difference in diameter. As acidic organelles mature, they fuse with each other, increasing their average size. The different accumulations of DOX in individual acidic organelles are also affected by differences in pH between acidic organelles. At physiological pH (pH 7.4; DOX pKa ) 8.4)41,42 ∼9% of the doxorubicin molecules are neutral. At this pH, the DOX molecule is capable of passively diffusing across the lysosomal membrane, entering the lumen of the acidic organelle, where it then becomes charged and sequestrated. Loss of DOX from acidic organelles upon cell disruption may also explain the wide ranges in DOX content observed in Figure 5. It is also possible that loss of the transmembrane pH gradient due to ATPase inhibition or Donnantype equilibrium may lead to DOX leakage. We have seen that
DOX leakage from the organelle fractions, monitored spectrofluorometrically over time (data not shown), is not detected if the preparations are kept on ice. The low temperature likely aids in keeping pH gradients stable for a longer period of time. The relevance of size, pH within individual acidic organelles, and leakage in defining the wide range of DOX content will be the topic of future studies. As mentioned earlier, since CEM/C2 multidrug resistance arises from a mutation in topoisomerase I,28 acidic organelle DOX sequestration was not expected to be different when comparing this cell line with the CCRF-CEM cell line. Surprisingly, the total amounts of DOX in acidic organelles (summation of individual DOX content) is (1.5 ( 0.2) × 10-18 mol for CEM/C2 cells and (2.2 ( 0.9) × 10-19 mol for CCRF-CEM cells, respectively, suggesting that the PSS model may be a secondary process conferring added drug resistance on the CEM/C2 cell line. Given that “other organelles” containing DOX may be mitochondria, it is also possible to estimate their DOX content and compare those values with those of previous reports.20 Using a modified eq 1 where h ) 1, we find that “other organelles” have medians 29 and 19 zmol/organelle for CEM/C2 and CCRF-CEM cells, respectively. These values are low compared to the respective 51 and 54 zmol/mitochondrion values that have been previously reported.20 Whether these large differences between studies result from “other organelles” not being truly mitochondria or differences in sample preparation or treatment bring up the importance of further refining analytical methods for subcellular analysis and requires further investigation. CONCLUDING REMARKS Using a dual CE-LIF, it is feasible to determine the DOX content in acidic organelles in crude organelle fractions that likely contain acidic organelles and mitochondria. The findings reveal that the DOX content of individual acidic organelles is highly heterogeneous, and this may be due to size and pH variations as well as changes predicted to occur during their maturation process. It is also possible that DOX may be leaking from organelles and that the values reported here are therefore conservative. If this is the case, this report is underestimating the relevance that DOX sequestration by these organelles (i.e., PSS model) may play in decreasing the availability of anthracycline to cellular targets. Further developments in the labeling schemes for acidic organelles and the combination of CE-LIF with pharmacological treatments (e.g., ATPase inhibitors) will likely promote the use of these approaches to more quantitatively describe biological process such as multidrug resistance and acidic organelle maturation. ACKNOWLEDGMENT This work was supported by the National Institute of Health (R01 GM61969). E.A.A. acknowledges an NIH Career Award (1K02-AG21453). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 23, 2004. Accepted February 17, 2005. AC0480996 Analytical Chemistry, Vol. 77, No. 8, April 15, 2005
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