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RESEARCH PROFILES Measuring drugs in individual organelles very tiny,” he says. Over time, however, The effects of a drug depend on where it organelles were first labeled with comacidic organelles tend to fuse together, goes in a cell. To get a better idea of how mercially available fluorescent nanoforming larger and larger acidic organspheres. The nanospheres were specific much of a drug accumulates in particuelles. To detect DOX, the acidic organlar subcellular compartments, Edgar Ar- for acidic organelles and therefore did not label other organelles, such as mito- elle may need to be really large so that riaga and colleagues at the University of it contains a significant amount of the chondria. Minnesota have developed a CE laserdrug, speculates Arriaga. Another induced fluorescence (LIF) methpossibility is that some of the acidod with dual-channel detection. ic organelles will lose their pH The technique can measure the gradient with time. “If they lose properties of a particular organtheir pH gradient, the drug is elle even in the presence of other going to escape,” he says. types of organelles. Now that the researchers have In the April 15 issue of Analytthe ability to measure exactly how ical Chemistry (pp 2281–2287), much DOX accumulates in indithe researchers used the CE-LIF vidual acidic organelles, the next approach to detect the amount of step is to compare the behavior of the anticancer drug doxorubicin a drug-resistant cell line with that (DOX) in individual acidic orof a cell line that is sensitive to ganelles. The results could shed DOX, to determine whether one light on how acidic organelles ma20 µm has the ability to sequester DOX ture and their role in multidrug while the other does not. “It may resistance. seem simple, but it is complicated DOX is a drug commonly because that involves needing to used for treating leukemia and Composite confocal image of human leukemia cells know exactly how many acidic orother types of cancer. Some tu(CEM/C2) incubated with 50-nm-diam nanospheres (green) ganelles there are in a cell,” says mors, however, have developed and 25-µM DOX (red). Arriaga. resistance to DOX. Researchers The researchers also hope to eventuAll of the acidic organelles successfulhave speculated that acidic organelles ally look at changes in proteins in acidic ly encapsulated the fluorescent nanosequester DOX, making it less available organelles as the organelles mature. “We spheres by endocytosis after a 48-h into the cell. Until now, however, no one would like to investigate some features cubation period. The researchers then could measure exactly how much DOX that might be responsible for changes in accumulates in these individual subcellular exposed the cells to DOX for 15 min. their ability to accumulate doxorubicin,” Because nanospheres emit at a shorter compartments. says Arriaga. One area that they hope to wavelength (green) than DOX (red), Drugs that are sequestered by acidic investigate is the expression or lack of Arriaga and colleagues could easily disorganelles are usually weak bases, says tinguish the acidic organelles with DOX expression of ATPases, which are responArriaga. “When these drugs are in the sible for pumping protons in and out of from those without DOX by monitorcytoplasm, they are neutral and someacidic organelles. “It might be that some what hydrophobic, so they can cross the ing two different spectral ranges. Dualacidic organelles have the ability to mainchannel detection also allowed them to acidic organelle membrane. But once tain the pH gradient for longer times determine how much DOX was taken they hit the acidic organelle, they are than others,” says Arriaga. up by other organelles, such as mitoexposed to low pH and become protoBut future work depends on the adchondria, which had no detectable nated,” he says. When the drugs bevancement of proteomics technologies. nanospheres. come protonated, they cannot escape “That is one of the bottlenecks right Most of the acidic organelles quickly the acidic organelles and therefore are now for anybody who is interested in took up and concentrated DOX in the not as effective, he adds. nuclear region; however, a small fraction subcellular analysis and working with To determine whether the sequestravery small samples. The sensitivity and tion of DOX by acidic organelles is rele- of them did not take up DOX during the limits of detection of present provant to drug resistance, Arriaga and col- the 15-min treatment. Arriaga believes teomics strategies are still not there,” that one reason has to do with the size leagues measured the DOX content in says Arriaga. a of the acidic organelles. “When an individual acidic organelles from two —Britt E. Erickson acidic organelle is formed, it is usually human leukemia cell lines. The acidic 160 A
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