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Labeling of Sulfhydryl Groups in Intact Mammalian Cells with Coumarins Janina Baranowska-Kortylewicz and Amin I. Kassis' Department of Radiology, Nuclear Medicine Division, Harvard Medical School, Shields Warren Radiation Laboratory, 50 Binney Street, Boston, Massachusetts 02115. Received October 27, 1992
4-(Bromomethyl)-6,7-dimethoxy-2-oxo-Wbenzopyran (1) and its 3-[l25I1iododerivative (2)were reacted with sulfhydryl groups of proteins in viable Chinese hamster V79 lung fibroblasts. Pretreatment of cells with dithiothreitol (DTT) as the reducing agent was necessary to give detectable levels of fluorescence. In the absence of DTT, V79 cells incorporated about 1 X 10-3 pCi/cell of the radioiodinated, nocarrier-added derivative; following incubation of cells with 320pM DTT the uptake was nearly doubled (1.87X pCi/cell).
checked by the trypan blue dye exclusion assay. Routinely about 70% of the cells were viable. For determination of membrane-bound 1251, cell pellets were suspended in 0.001 M potassium phosphate buffer, pH 7.0,containing 0.25 M sucrose and 0.001 M EDTA, allowed to swell for about 10 min, and disrupted in a Dounce homogenizer with a loosely fitting Teflon pestle. The slurry was centrifuged at 2000g for 20 min to remove unbroken cells and nuclei. The pellet was then homogenized and centrifuged a second time. The supernatants from the two centrifugations were combined and centrifuged at lOOOOg for 30 min to collect cell membranes (6). The radioactivity associated with membrane pellets was determined in a y counter. Control cells were first treated with 320 pM DTT and then incubated with 0.01% L-cysteine in PBS for 20 min. After washing with medium and PBS, cells were resuspended in Ca+2-and Mg+2-free PBS and reacted with 2 at 0-4 OC for 1 h. Following this treatment cells were processed as described above. EXPERIMENTAL PROCEDURES Coupling of 3-[ 12SI]Iodo-4-(bromomethyl)-6,7dimethoxy-2-oxo-2H-benzopyran(2) to V79 Lung All chemicals were reagent grade. Sodium [1251]iodide Fibroblasts through N-Succinimidyl 3 42-PyM NaOH was (specific activity 2200 Ci/mmol) in ridy1dithio)propionate(SPDP). The cell-surfaceamino purchased from NEN (Du Pont, Billerica, MA). Radiogroups of V79 cells were reacted with 25 pmol SPDP/107 iodination of coumarin has been described previously (5). Direct Coupling of 3-[12~I]Iodo-4-(bromomethyl)- cells per mL of PBS at 0-4 OC for 20 min on a rotating mixer. Cells were washed twice with medium containing 6,7-dimethoxy-2-0~0-2H-benzopyran (2) to Sulfhydryl 10% fetal calf serum to remove any free SPDP and twice Groups on Cell Membranes. Exponentially growing with PBS by pelleting (9OOg,10 min). They were then monolayers of Chinese hamster V79 fibroblasts were suspended in PBS (lo7cells/mL) and reacted with 0,3.2, released from plates by 0.13'% trypsin, washed with Ca+232, or 320 pM DTT (see above). The optical density of and Mg+z-freePBS,' and suspended in the same buffer (1 supernatants at 343 nm was used as an approximate X 107 cells/mL). A solution of DTT (0.1M) in Ca+2- and measure of the total number of free sulfhydryl groups on Mg+z-freePBS was added to the cell suspension in a final the cell surface (7,8).For 0 and 3.2 pM DTT the change concentration of 0, 3.2, 32, or 320 pM. After a l-h in absorbance was below 0.005 AU and thus outside the incubation at 0-4 OC on a hematological roller shaker, the limits of experimental error. Cells washed with PBS were cells were washed three times with PBS by pelleting then reacted with 2 and treated as described above. (9OOg,10min) and resuspended in PBS containing 0.1pCi Fluorescence Microscopy. V79 cells grown overnight of 2 (2200 Ci/mmol) per 107 cells. The reaction was as monolayers on glass slides were incubated with 320pM continued at 0-4 OC for 1 h on a rotating mixer. The cells DTT in Ca+2-and Mg+2-freePBS, washed with the same were pelleted at 600g for 10 min. Aliquots of supernatant buffer, incubated with 0.1 pM 1 in saline for 5-10 min, were counted in a y counter. Cell pellets were washed washed with medium and PBS, and then examined under three times in Dulbecco's Modified Eagle Medium conthe fluorescence microscope. taining 10% fetal calf serum and their radioactive content was determined. The viability of radiolabeled cells was RESULTS AND DISCUSSION
Several derivatives of coumarin have been used in histopathology as fluorescent probes to label thiol groups in tissue sections ( I , 2). Some of these reagents have also been examined for their ability to bind to living cells (3). Only 3-(4-maleimidylphenyl)-4-methyl-7-(dimethylamino)coumarin stained viable cells (3, 4). Since we have (1) found that 4-(bromomethyl)-6,7-dimethoxycoumarin selectively reacts with free sulfhydryl groups in soluble proteins, it was of interest to determine whether this reactivity would also be expressed toward sulfhydryl groups in intact mammalian cells. Such an agent could also be useful in examining the radiotoxic effects of Augerelectron-emitting radioisotopes, such as 1251,when localized on the cell surface. Chinese hamster V79 lung fibroblasts were incubated with fluorescent compound 1 or the nocarrier-added, radioiodinated derivative 2,and the ability of these compounds to label thiol groups in intact cells was examined.
* Author to whom correspondence should be addressed.
'Abbreviations: PBS,phosphate-buffered saline, pH 7.4; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; SPDP, N-succinimidyl3-(2-pyridyldithio)propionate.
The labeling of sulfhydryl groups on the cell membrane was examined to determine the potential value of 1 and 2 as fluorescent probes in flow cytometry, in assessing the cell-membrane thiol content, and as carriers of Auger-
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I
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Concentration D l T (pM)
Figure 1. Uptake of no-carrier-addedradioiodinated coumarin 2 in Chinese hamster V79 lung fibroblasts exposed to variable concentrations of DTT (mean and standard error of mean are shown; n = 3). Cells were treated with DTT, washed with PBS, and reacted with 0.1 pCi/107 cells.
electron-emittingradionuclides for examining their cellular radiotoxicity. Both coumarin9 were found to label the sulfhydryl groups of intact mammalian cells. The incorporation of radioiodide as measured in disrupted and pCi/cell fractionated cells indicated that about 1 X reacted with the proteins of the V79 lung fibroblast membranes in the absence of DTT; when cells pretreated with 320 pM DTT were exposed to 2 the incorporation pCi/cell) (Figure 1). The was nearly doubled (1.8 X average labeling of the cell surface was 1.07 X [standard deviation (SD) 0.40 X pCi/cell for cells not treated with DTT and 1.20 X (SD 0.36 X 1.46 X 10-3 (SD 0.42 X lO-3), and 1.87 X (SD 0.34 X pCi/cell for cells treated with 3.2, 32, and 320 pM DTT, respectively (mean and standard deviation were determined from data obtained in three experiments). All was found to be associated with cell membranes. of the 1251 Control cells that were first treated with 320 pM DTT and then incubated in PBS containing 0.01 % L-cysteine incorporated only 2.73% (SD 1.05) of the total added 2. The radioactivity associated with cell membranes was 4 X pCi/cell (SD 1 X lo4), about 50% less that in untreated cells, indicating that there are free sulfhydryl groups on the cell surface even prior to DTT treatment. Since the labeling with the no-carrier-added material resulted in a relatively low degree of substitution, surface proteins of viable V79 cells were derivatized with SPDP to increase the number of available thiol groups. V79 cells that were first reacted with SPDP and then reduced with DTT incorporated over 30% of the total added radioactivity (3.5 X pCi/cell; SD 0.5 X but only 40% of this 1251 was bound to the cell membranes. The average incorporation into SPDP-treated cells without DTT was about 1X 10-3 pCi/cell, 20% of which was located on the cell surface. A no-carrier-added reagent was used in all experiments involving DTT reduction as well as in these with SPDP/DTT; therefore the labeling of intracellular proteins in cells derivatized with SPDP cannot be attributed to the concentration of the tracer. A more likely explanation seems to be the damaging effect of SPDP/ DTT treatment on cell membranes. Fluorescencemicroscopy confirmed binding of 1to V79 cells (Figure 2) and demonstrated that pretreatment of the cell surface with excess DTT was necessary to achieve detectable labeling levels. Cysteine,which also reacts with
Figure 2. Fluorescent stainingof cell membranes;UV excitation, original magnification X40. Live Chinese hamster V79 lung fibroblasts were treated with 320 p M DTT and exposed to 0.1 pM 1 for 10 min. Cells incubated with 1 alone were invisible under fluorescence microscopy (data not shown).
free sulfhydryl groups on the cell surface (9),effectively blocked binding of radiolabeled coumarin and its fluorescent analog, indicating that only sulfhydryl groups were derivatized. The viability of V79 fibroblasts following treatment with DTT and 1 or 2 was reduced from over 95 % to 70%, as indicated by the trypan blue dye exclusion estimate. The coumarin derivative tested by Olive et al. (3) had a mean lethal dose (MLD; approximate concentration required to reduce survival to 37 % ) of 8 pg/mL; the concentration of no-carrier-added 2 was several orders of magnitude lower than the MLD. Similarly the concentration of 1was about 200 times lower than the MLD. Therefore it appears that the reduction of the viability of fibroblasts to 70 % resulted from the overall handling of cells (reaction with DTT, repeated washing, etc.). The estimated number of sulfhydryl groupsradiolabeled with 2 is about 300/cell; the DTT pretreatment increases this value to 600. Earlier estimates put the number of sulfhydryl groups at 2.75 X lo5, 19 X lo5, and 370 X lo5/ cell in platelets, lymphocytes, and Ehrlich ascites tumor, respectively (9). Since it is unlikely that V79 cells have an extremely small number of sulfhydryl groups on the cell surface, it appears that our method may have grossly underestimated the total expected number of cellular thiols. It is also possible that since cellular binding is reported to be directly dependent on the coumarin:cell ratio (3), only the most accessible sulfhydryl groups are radiolabeled at the trace levels of 2. ACKNOWLEDGMENT
This research was supported by DOE Grant DE-FGO286ER60460 and NIH Grant R 0 1 CA 15523. The authors wish to thank Dr. Zbigniew P. Kortylewicz for helpful discussions and Mr. Robert M. Berman for his excellent technical assistance. LITERATURE CITED (1) Sippel,T. 0.(1981)New fluorochromesfor thiols: maleimide and iodoacetamide derivativesof 3-phenylcoumarin. J. Histochem. Cytochem. 29,314-316. (2) Sippel, T. 0. (1981) Microfluorometric analysis of protein thiol groups with coumarinylphenylmaleimide.J. Histochem. Cytochem. 29,1377-1381. (3) Olive, P. L., Biaglow, J. E., Varnes, M. E., and Durand, R. E. (1983) Characterization of the uptake and toxicity of a fluorescent thiol reagent. Cytometry 3,349-353.
Technical Notes
(4) Durand, R. E., and Olive, P. E. (1983) Flow cytometry techniques for studying cellular thiols. Radiat. Res. 95,456470. (5) Baranowska-Kortylewicz,J., and Kortylewicz, Z. P. (1991) Radioiodination of 7-methoxy and 6,7-dimethoxy-4-bromomethylcoumarins. J . Labelled Compds. Radiopharm. 29, 1301-1307. (6) Morre, D. J., and Morre, D. M. (1989) Preparation of mammalian plasma membranes by aqueous two-phase partition. Biotechniques 7,946-958.
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(7) Grassetti, D. R., and Murray, J. F. (1967) Determination of sulfhydryl groups with 2,2‘- and 4,4’-dithiodipyridine. Arch. Biochem. Biophys. 119,4149. (8) Pedersen, A. O., and Jacobsen, J. (1980) Reactivity of the thiol group in human and bovine albumin at pH 3-9, aa measured by exchange with 2,2’-dithiodipyridine. Eur. J. Biochem. 106,291-295. (9) Mehrishi, J. N., and Grassetti, D. R. (1969) Sulfhydrylgroups on the surface of intact Ehrlich ascites tumour cells, human blood platelets and lymphocytes. Nature 224, 563-564.
