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Dansylated Estramustine, a Fluorescent Probe for Studies Of Proc. Natl. Acad. Sci. USA Vol. 82, pp. 8483-8487, December 1985 Cell Biology Dansylated estramustine, a fluorescent probe for studies of estramustine uptake and identification of intracellular targets (antimitotic drug/microtubules/microtubule-associated proteins) MARK E. STEARNS*t, DONALD P. JENKINS*, AND KENNETH D. TEWt *Department of Anatomy, Georgetown University Schools of Medicine and Dentistry, Washington, DC 20007; and tDepartments of Medicine and Pharmacology, Lombardi Cancer Center, Georgetown University, Washington, DC 20007 Communicated by Keith R. Porter, July 8, 1985 ABSTRACT Fluorescence-microscopic studies with of DnsEM was measured by their effect on the colony- dansylated estramustine (DnsEM) has permitted investigation forming ability of DU 145 human prostatic carcinoma cells. of the mechanism of estramustine (EM) uptake in live human The results presented here show that DnsEM is as cytotoxic prostatic tumor cells (DU 145). DnsEM appeared to enter cells to cells as EM and can be used as a direct probe for analysis rapidly at the peripheral cell margins. A progressive increase of EM's mechanism of action in live cells. in fluorescence was observed until the perinuclear material and cytoplasm were labeled brightly and the nucleoplasm was METHODS labeled faintly. Light microscopy showed that DnsEM is assimilated first in preexisting vesicles and then in numerous Dansylation of EM. The synthesis of DnsEM involved the newly created vesicles that accumulate in the cytoplasm and dansylation reaction as first introduced by Gray (9) and around the nucleus. Colony-forming assays showed EM and modified by Jenkins (10). Fifty milligrams of 5- DnsEM to be equally cytotoxic to cultured DU 145 cells. dimethylaminonaphthalene-1-sulfonyl chloride (DnsCl; Cellular uptake and subsequent manifestation of cytotoxicity Pierce) and 20 mg of EM (Leo, Helsingborg, Sweden) were are presumably dependent upon these vesicles. However, after dissolved in 1.0 ml of either absolute ethanol or acetone uptake of DnsEM, its diffusion into the cytoplasm was ob- (anhydrous, spectral grade). To the DnsCl/EM/acetone served. solution, 1.0 ml buffer solution [0.1 M sodium bicar- bonate/0.1 sodium carbonate, 1:1 (vol/vol)] was added. The Estramustine [EM; estradiol-3-bis(2-chloroethyl)carbamate] pH of the buffer was 9.2 (compared to a pH 10.00 standard); is an effective antineoplastic agent for treatment of prostatic this increased to 10.5 upon dilution with ethanol and to 10.7 tumors (1). Chemically, EM consists of estradiol covalently upon dilution with acetone (10). The final concentrations of linked to nitrogen mustard by a carbamate ester bond (see the reactants were 93 mM DnsCl and 17 mM EM [DnsCl Fig. 1). The cytotoxicity of the compound apparently de- molarity 5.5 times that of EM (9)]. The reaction occurred in pends on the combined effects ofthe hydrophobic steroid and total darkness, at 230C. hydrophilic mustard moieties, since neither steroid nor ni- Isolation of the Product. After 3 hr, 1.0 ml of the trogen mustard produce similar pharmacologic effects them- bicarbonate/carbonate buffer was added to the reaction selves (2-5). Initially, the specificity ofantitumor activity was mixture, which resulted in precipitation and the formation of shown to result from the presence of high concentrations of an oil phase. The reaction mixture was then left at 23°C in an EM-binding protein (EMBP) of 46 kDa in prostate tissue darkness for an additional 24 hr, during which time the oil (6). EMBP apparently modulates the accumulation of EM in phase precipitated; the precipitate was yellow, soluble in prostate tumors, with resultant increased therapeutic effica- ethanol or acetone, and insoluble in either water or 0.1 M cy. Nevertheless, the precise mechanism by which EM sodium bicarbonate. The product was isolated by centrifu- induces cytotoxicity remains unknown. gation (1200 X g) and washed in a Buchner funnel on More recent immunofluorescence studies have revealed Whatman no. 40 filter paper with 0.1 M sodium bicarbonate that prostatic binding protein is present in numerous cyto- until no visible fluorescence remained in the filtrate. The plasmic vesicles in rat ventral prostate secretory cells (7). product was dried in a stream of nitrogen to a powder and Since it is thought that prostatic binding protein and EMBP used for TLC, spectroscopy, in vitro incorporation experi- may be the same or similar proteins (7), one possibility is that ments, and cytotoxicity assays. EM accumulation in secretory vesicles is somehow toxic to Chemical Analysis of the Product. The absorbance spec- the cell. However, the cellular targets of EM remain to be trum of the product in ethanol was obtained with a Beckman identified and there is no evidence that EM remains in DU 7 spectrophotometer and compared to known spectral vesicles after initial drug uptake. By using cytological meth- characteristics of dansyl compounds (9). For NMR, the ods, we have shown that EM has hitherto unexpected effects products, EM, and DnsCl were dissolved in dimethyl on cellular structure and integrity. In related studies, EM has sulfoxide. 2H20 was used to substitute and identify the 17,B been shown to disrupt microtubule complexes and their hydroxyl group of the EM molecule. Elemental analysis of related function in cultured fish epithelial pigment cells, as the product was carried out as a quantitative microanalysis well as in DU 145 tumor cells (8). by Galbraith Laboratories (Knoxville, TN). Here we report the use of a fluorescent drug probe to Control Dansylation of Prostatic Tumor Cells. DU 145 determine the sites of the incorporation and action of EM. prostatic tumor cells in monolayer growth on glass coverslips Conjugation of EM and dansyl chloride (DnsCl) permitted were fixed in 2.0% glutaraldehyde and then in acetone visualization of drug uptake into live cells by phase-contrast (-20°C). In some studies, the cells were dansylated after and epifluorescence microscopy. The cytotoxicity ofEM and Abbreviations: Dns, dansyl; EM, estramustine; EMBP, estra- The publication costs of this article were defrayed in part by page charge mustine-binding protein; MAP, microtubule-associated protein. payment. This article must therefore be hereby marked "advertisement" tTo whom reprint requests should be sent at: Fox Chase Cancer in accordance with 18 U.S.C. §1734 solely to indicate this fact. Center, 7701 Burholme Street, Philadelphia, PA 19111. 8483 Downloaded by guest on September 23, 2021 8484 Cell Biology: Steams et al. Proc. Natl. Acad. Sci. USA 82 (1985) fixation. The cells were first washed in 0.1 M sodium bicarbonate and then suspended in the dansylation reaction mixture at 230C. The reaction mixture was identical to that N(CH3)2 used in the dansylation of the EM molecule, except that EM was omitted. After 1 hr, the cells were removed from the reaction mixture, washed in 0.1 M sodium bicarbonate until (CICH2C2H)NCOO no visible fluorescence was present in the filtrate, and ESTRAMUSTINE microscopically observed without dehydration or drying, in DANSYL CHLORIDE the bicarbonate solution. Control Products. Several products were used for control I experiments in this study. DnsCl was used as obtained from Pierce. It is contaminated at :2% by dansic acid, presumably 13)2 because of hydrolysis of the acid chloride. No attempt was made to recrystallize the chloride form. Dansic acid was obtained as a crystalline powder from the reaction of DnsCl with the aqueous reaction mixture buffer. Other yellow fluorescent by-products ofthe dansylation reaction (9, 10, 12) were isolated by precipitation in either water or acetone from dansylation reaction mixtures that did not contain EM. These products were tested for incorporation into live DU 145 cells in culture. Preparation of Ceils and Uptake of DnsEM. DU 145 (Amer- (CICH2CH2 )NCOOIJ ican Type Culture Collection HTB 81) human prostatic DANSYL- ESTRAMUSTINE carcinoma cells were maintained in monolayer culture in RPMI 1640 medium (M.A. Bioproducts, Walkersville, MD) FIG. 1. Chemical formulae of EM, DnsCl, and the proposed supplemented with 10% heat-inactivated fetal bovine serum, dansylated derivative of the drug. The only side group reactive to penicillin (100 units/ml), and streptomycin (100 ,ug/ml). DnsCl is the ionized form of the hydroxyl group in the 17,B position of the EM molecule. Elemental analysis and NMR studies are Cultures were maintained at 370C under 5% CO2 and were consistent with the structure ofthe dansylated product as the sodium routinely passaged at S x 104 cells per ml, either for tissue salt. culture flask or coverslip experiments. For experimental studies, the cells were cultured on carbon-coated, glow-discharged coverslips. Cells were shown). Over the period of a week, in the mixed solvent of washed twice with Dulbecco's phosphate-buffered saline (pH the reaction, DnsEM crystallized. Absorbance spectropho- 7.3) at 230C or with fresh medium and were incubated with tometry of the precipitate showed a hypochromic shift in the phosphate-buffered saline or medium containing EM or benzenoid maximum, consistent with the reaction of the analogues of EM. Live cells were photographed under sulfonyl chloride with a reactive group other than water (data epifluorescence microscopy (Zeiss). not shown). NMR ofthe product suggested substitution ofthe Stock solutions of NaCN, 2,4-dinitrophenol, and Ca2+ 17/3 hydroxyl position of the estradiol moiety (data not were made in phosphate-buffered saline, and the appropriate shown); deuterium substitution on the EM molecule was used volume of stock solution was added to experimental solu- to determine the identity of the 17,3 spectral position. Ele- tions. Ca2+/EGTA buffers were made according to methods mental analysis data for the product were as follows [calcu- of Steams (11). lated for C35H42O5N2SCl2Na (found)]: C, 59.3 (58.4); H, 6.0 In Vitro Cytotoxicity Assay. The cytotoxicity ofthe DnsEM (6.6); N, 3.9 (3.7); S, 4.5 (3.3); Cl, 10.0 (13.9).
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