LH-RH analogue carrying a cytotoxic radical is internalized by rat pituitary cells in vitro

Peptides, Vol. 15, No. 2, pp. 359-366, 1994 Copyright © 1994ElsevierScienceLtd Printed in the USA. All rightsreserved 0196-9781/94 $6.00 + .00

Pergamon 0196-9 781 (93)E00 22 -J

LH-RH Analogue Carrying a Cytotoxic Radical Is Internalized by Rat Pituitary Cells In Vitro B A L A Z S S Z O K E , 1 J U D I T H O R V A T H , 2 G A B O R H A L M O S , Z O L T A N Rt~KASI, K A T E G R O O T , A T T I L A N A G Y A N D A N D R E W V. S C H A L L Y 3

Endocrine, Polypeptide, and Cancer Institute, Veterans Administration Medical Center, New Orleans, LA 70146 and Section of Experimental Medicine, Department of Medicine, Tulane University Medical School, New Orleans, LA 70112 R e c e i v e d 26 A u g u s t , 1993 SZOKE, B., J. HORVATH, G. HALMOS, Z. Rt~KASI, K. GROOT, A. NAGY AND A. V. SCHALLY. LH-RH analogue carrying a cytotoxic radical is internalized by rat pituitary cells in vitro. PEPTIDES 15(2) 359-366, 1994.--The binding and internalization ofa cytotoxic analogue ofluteinizing hormone-releasing hormone (LH-RH), T-98 (agonist [D-Lysr]LH-RH linked to glutaryl-2-(hydroxymethyl)anthraquinone), by rat anterior pituitary cells was investigated. Analogue T-98 was bound to pituitary membrane binding sites for LH-RH with a high affinity (Kd= 1.2 nM) and was 17 times more potent in releasing luteinizing hormone (LH) from superfused rat pituitary cells than LH-RH. The labeling of this cytotoxic LH-RH analogue was carried out both with radioactive (~25I)and nonradioactive iodine. Monoiodination of the Tyr~residue of T-98 did not significantly affect its binding affinity but greatly decreased its LH-releasing activity to about 3% of the original value. Di-iodination in the same position lowered binding affinity twenty-threefold and further diminished LH-releasing potency. [~25I]T-98was found to bind very strongly to polystyrene, which precluded the use of regular tissue culture plasticware in our experiments. In pituitary cells cultured in glass vials, binding and internalization of [~2S1]T-98were observed, which were time and temperature dependent, and which could be inhibited by excess unlabeled analogue. No enzymatic degradation of labeled T-98 was detected in the culture medium during the incubation. Our results indicate that T-98 is internalized by pituitary gonadotropes through receptor-mediated endocytosis. Because this new class of compounds was designed as anticancer drugs, our findings also suggest that this cytotoxic LH-RH agonist may also be internalized by LH-RH receptors present in breast, prostate, ovarian, and other tumors. Drug targeting

Cytotoxic peptides

Receptor-mediated endocytosis

A major drawback of cancer chemotherapy is that most antineoplastic agents also have nonselective toxic effects on normal cells. A new approach that is being developed to overcome this problem is the targeting, which exploits the selectivity of carrier molecules having specific binding sites in t u m o r tissues ( 15,41). Chemotherapeutic compounds and toxins can be covalently attached to various carriers, including hormones, for which receptors are present on cancer cells and antibodies that preferentially recognize tumor cells (15,41). Such conjugates are expected to deliver cytotoxic agents more selectively to target cells. For these conjugates to act as targeted chemotherapeutic agents, certain criteria have to be met. The conjugate must preserve the binding ability of the carrier to the specific binding sites. The hybrid molecule must also either retain the cytotoxic activity of its drug component, or a cytotoxic metabolite has to be formed at or within the target cell. The cytotoxic conjugate may exert its effect after internalization or simply by binding to the cell surface, without entering the cell (47). The release of the cytotoxic radical is likely to occur following endocytosis of the hybrid

molecules if the carrier or the chemical bond of conjugation is sensitive to enzymatic cleavage within the cell. Various conjugates of cytotoxic compounds with analogues of LH-RH have been synthesized in our laboratory (2,3,23) to take advantage of the presence of receptors for LH-RH in breast, prostatic, endometrial, and ovarian cancers (11,13,14,43). These hybrid compounds, in addition to being targeted chemotherapeutic agents, may also exert antitumor activities based on the inhibition of the pituitary-gonadal axis or direct inhibitory effect of their LH-RH analogue moiety (16,40,41). One of these cytotoxic hormone analogues, T-98, consisting of glutaryl-2-(hydroxymethyl)anthraquinone coupled to the ~a m i n o group of [D°Lys6]LH-RH, was found to bind with high affinity to membranes from h u m a n breast cancers (23) and mouse M X T m a m m a r y tumor (31) and inhibited [3H]thymidine incorporation into DNA in cultures of h u m a n breast and prostate cancer cell lines. In vivo, T-98 significantly suppressed the growth of estrogen-independent M X T m a m m a r y tumors in mice (44) and D u n n i n g 3327H prostate cancer in rats (37).

1On leave from 1st Institute of Biochemistry, Semmelweis University Medical School, Budapest, Hungary. 2 On leave from Department of Anatomy, University Medical School, Prcs, Hungary. 3 Requests for reprints should be addressed to Andrew V. Schally.

359

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SZOKE ET AL.

To obtain an insight into the mechanism of action of T-98, we decided to investigate if this cytotoxic LH-RH analogue is internalized by cells that have functionally active LH-RH receptors. In the first stage of this project, we studied the internalization of T-98 by rat pituitary gonadotropes. METHOD

lodination ~/ T-98 Cytotoxic LH-RH analogue T-98 was synthesized in our laboratory by coupling 2-(hydroxymethyl)anthraquinone HMAQ to the free e-amino group in [D-Lys6]LH-RH through the glutaric acid spacer (4). Nonradioactive iodination of this compound was carried out using chloramine-T (CT) as oxidizer (17). T-98 (1.8 mg, 1 #mol) was dissolved in 500 #1 dimethylformamide, and 4.5 ml 0.05 M sodium phosphate buffer (pH 7.6) and 220 ~zl 10 mM Nal (2.2 umol) was added. To this solution, four 50~zl portions of 10 m M CT (total of 2 #mol) in phosphate buffer were added at 10-s intervals with stirring. The reaction was stopped with 10 umol sodium metabisulfite and dilution with HPLC eluents (see below), 15 s after the last addition of CT. This procedure resulted in 74% conversion of T-98 to monoand di-iodinated derivatives with a product ratio of about 2:1. The reaction mixture was separated on an Aquapore RP-300 (250 × 7 mm, Brownlee Labs, Santa Clara, CA) reversed phase HPLC column, using linear gradients of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in 70% aqueous acetonitrile (solvent B). The fractions collected were lyophilized. UV spectra of reaction products were acquired during analytical HPLC runs with the help of a Beckman System Gold HPLC equipped with model 168 diode array detector. Radioiodinations of T-98 were routinely performed also by the CT method. Using a 12 × 75 mm glass test tube, 70 #1 0.5 M phosphate buffer (pH 7.6) and 1.5 mCi (1 Ci - 37 GBq) NaJ251 (Amersham, Arlington Heights, IL) was added to a solution of 10 ug T-98 in 20 ~1 0.02 M acetic acid. The reaction was initiated by the addition of 10 ug CT in 10 ~1 phosphate buffer and continued with stirring for 45 s at room temperature. Iodination was stopped with a tenfold molar excess of sodium metabisulfite and the reaction mixture was immediately applied to a C18 HPLC column (W-Porex 5C18, 250 × 4.6 mm, Phenomenex, Torrance, CA). Separation was carried out with the same solvents as for nonradioactive iodinations using the gradient shown on Fig. 1. The effluent was monitored by a UV detector at 257 nm and a flow-through radioactivity detector constructed in our laboratory from a ratemeter (SML-2, Technical Associates, Canoga Park, CA). Fractions corresponding to the mono- or diiodinated analogue were neutralized by adding an equal volume of 0.25 Mphosphate buffer (pH 7.6) containing 1% bovine serum albumin (BSA) and 60 ~g/ml bacitracin and were stored at -70°C. Under these conditions, no significant tracer degradation was detected by HPLC for up to 2 weeks of storage. Iodogen (Pierce, Rockford, IL) was also used successfully instead of CT as an oxidizer. Labeling was performed for 3 min at room temperature in glass tubes coated with 10 ~g Iodogen. At the end of the reaction, the solution was removed from the tube and applied on HPLC. Specific activity of [~25I]T-98 was calculated from the radioactivity and the mass of the HPLCpurified tracer. The mass was determined from the UV peak based on calibration with unlabeled material at 257 rim. At this wavelength, which is the absorption maximum of the cytotoxic group, the iodination does not affect absorptivity. Specific activity values thus obtained ranged between 1600 and 2200 Ci/mmol and were found to be identical to the specific activity of iodide125 used for labeling.

Adsorption of [125I]T-98 on Different Surfaces The borosilicate glass, polypropylene, and polystyrene tubes tested (all 12 × 75 mm) were purchased from Baxter Scientific Products (McGaw, IL). The coating of glass tubes or vials with poly-D-lysine (mol.wt.: 58,000, Sigma, St. Louis, MO) was carried out with a 0.1 mg/ml aqueous solution for 20 min at room temperature followed by rinsing twice with distilled water. Some of the polystyrene tubes were preincubated with 1% BSA in 10 mM Tris-HC1 (pH 7.5) for 1 h at 37°C and used without rinsing (BSA coated). Approximately 200,000 cpm [~25I]T-98or [IzsI][DTrp6]LH-RH in 1 ml of RPMI- 1640 medium containing 0.1% or 1% BSA were incubated in different tubes for 1 h at 37°C in a CO2 incubator. After incubation, the tubes were placed on ice, and unbound tracer aspirated. The tubes were rinsed twice with ice-cold medium and counted in a gamma counter. Acid wash of the tubes involved incubation with 1 ml ice-cold solution containing 0.2 M acetic acid and 0.5 M NaC1 for 5 min on ice, followed by removal of the solution and rinse with I ml of the same solution. Radioactivity in the tubes was counted again, and finally the tubes were incubated with 1 ml of 0.5 M NaOH overnight at 37°C, aspirated, washed once with the same solution, and counted for the remaining radioactivity.

Receptor Binding Assay Pituitary membrane fraction was prepared as described (19). [D-Trp6]LH-RH was iodinated by the CT method (12). Displacement binding assays were carried out in 12 × 75-mm glass tubes in a total volume of 150 ~1 binding buffer (10 m M TrisHCI, pH 7.5, 1 m M dithiothreitol, 0.1% BSA) for 90 min at 4°C. Membrane homogenates containing 40-70 #g protein were incubated in duplicates with 70,000 cpm (~0.15 nM) [~zsI][DTrp6]LH-RH as radioligand and with increasing concentrations ( 10 ~2-10-6 M) of nonradioactive peptides. Separation of bound and unbound ligand was achieved by centrifugation as described (4). At the end of incubations, 125 ul aliquots of suspension were transferred onto the top of 1 ml of ice-cold binding buffer containing 1.5% BSA in siliconized polypropylene microcentrifuge tubes (Sigma). Tubes were centrifuged at 12,000 × g for 3 min at 4°C (Beckman J2-21M). Supernatants were aspirated; the bottoms of the tubes containing the pellet were cut off and counted in a gamma counter. Final binding parameter estimates were calculated by the computer program of McPherson (29).

Pituitary Cell Superfusion Anterior pituitary cells from two male Sprague-Dawley rats (200-250 g, Charles River, Wilmington, MA) were used in each of the two chambers of the superfusion apparatus, which was similar to that described previously (7). The rats were decapitated, the anterior pituitaries removed, cut into small pieces, and incubated with 0.5% collagenase CLS2 (Worthington, Freehold, N J) for 50 min in a metabolic shaker. The dispersed cells were transferred into the superfusion chamber and mixed with 0.8 ml of swollen Sephadex G- 10. The cells were perfused overnight with Medium 199, containing 0.25% BSA, at a flow rate of 20 ml/h. The collection of 3-min fractions was started the next morning. The solutions of the materials to be tested were prepared from stock solutions immediately before use. The cells were stimulated with 3-min pulses of 500 pM and 5 n M LHRH, 20 and 200 pM T-98, or 200 pM and 2 n M mono-[~ZSl]T98. Di-[~251]T-98 was tested only at 2 nM. The concentrations of the iodinated hormones were calculated from the radioactivities and specific activities measured. The fractions were stored frozen until analyzed by radioimmunoassay for LH. The LH-

CYTOTOXIC LH-RH ANALOGUE releasing potencies ofT-98 and mono-[~25I]T-98 were calculated from four data points at each concentration.

Radioimmunoassay Rat LH levels in aliquots of superfusion or cell culture medium were determined by double-antibody radioimmunoassay. The antiserum (a-rLH S-10, AFP-571487), the reference hormone (rLH RP-3, AFP 7187 B), and the antigen for iodination (rLH-I-9, AFP 10250 C) were provided by the National Hormone and Pituitary Program (NIDDK, NHPP). Because some of the samples contained substantial amounts of [~25I]T-98, several standard curves were prepared with or without the inclusion of different amounts of the radioactive LH-RH analogue. No significant interference of [~25I]T-98 with the determination of LH was observed.

Pituitary Cell Culture Cells were prepared from anterior pituitaries of male SpragueDawley rats (200-250 g, Charles River) by previously described collagenase-dispersion procedures (42,46). Because polystyrene culture plates could not be used for the internalization experiments due to the strong adsorption of tracer, cells were cultured in 26-mm diameter glass shell vials (Kimble #60965D-7, VWR Scientific, Harahan, LA). Vials were treated with 0.3 M HCI in 70% ethanol for 1 h, rinsed with water, and sterilized in autoclave before seeding approximately 700,000 cells (2 ml) in each vial. Cells were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD) for 2-4 days before internalization experiments were carried out. To facilitate attachment of cells, vials were treated with poly-D-lysine in some experiments, although acceptable attachment of pituitary cells was also observed without coating.

Measurement of Internalization The method of Haigler et al. (18) was used to differentiate between receptor-bound and internalized analogue. Pituitary cells cultured in glass shell vials were washed twice with the incubation medium (0.25% BSA, 25 m M Hepes in DMEM without serum). Medium (1 ml) was added and the cells were equilibrated to the temperature of the incubation (37°C or 4°C). Binding reaction was started by adding 200,000 cpm [~25I]T-98 or the same amount of tracer plus 1 ~.M unlabeled analogue in 125 ul to replicate vials. Cells were incubated for different time periods either at 37°C in a CO2 incubator or at 4°C. At the end of the incubation, vials were immersed into ice/water and the medium was collected for LH determination and tracer degradation analysis. Cells were rapidly washed with 2 × 1 ml icecold incubation medium and then treated for 5 min with 1 ml of chilled 0.2 M acetic acid, 0.5 M NaCI (pH 2.5) at 4°C. Acid wash was transferred to test tubes, cells were rinsed with 0.75 ml of the same solution, and the washes were combined. Subsequently, cells were dissolved in 1 ml 0.2 M NaOH by overnight incubation, transferred to tubes, and combined with a 0.75-ml rinse. Radioactivity of acid wash (receptor-bound tracer) and alkali-solubilized cells (internalized tracer) was quantitated in a gamma counter.

[1251]T-98 Degradation During Internalization Breakdown of[ ~25I]T-98during incubation with pituitary cells was measured by HPLC analysis of the culture medium. Cell cultures were incubated with labeled T-98 as described above, and medium was collected and immediately frozen at the end

361 of incubation. Samples were thawed on ice just before applying a 250-#1 aliquot (~45,000 cpm) directly onto a 250 × 4.6 mm W-Porex 5C 18 large pore size column (Phenomenex) fitted with an in-line filter and a 30 × 2.1 mm Aquapore RP-300 guard cartridge (Brownlee). The elution was carried out with a linear gradient from 40-60% B in 20 min, using the same solvents as for tracer purification, followed by a washing step from 60-100% B in 8 min to remove BSA from the column. Fractions (1 ml) were collected and counted for radioactivity in a gamma counter. The extent of degradation was calculated by expressing the amount of radioactivity present in the intact [~25I]T-98 peak as the percentage of total radioactivity recovered during the run. R E S U L T S

Preparation of ~251-Labeled T-98 Before attempting to radiolabel T-98, several trial reactions were carried out with nonradioactive iodide to test the stability of the cytotoxic moiety (HMAQ-G) and to optimize iodination conditions. It was found that CT could be effectively used to label T-98, as long as the reactive, oxidized form of iodine was not present in large excess at any time in the reaction mixture. Otherwise an extensive oxidation, iodination, and degradation of the analogue occurred as detected by analytical HPLC. An excess of CT also caused some side reactions, but to a much smaller extent. Accordingly, preparative, nonradioactive iodination of T-98 was performed with incremental addition of the oxidizers. Radioiodination of T-98 was carried out by two different methods using either Iodogen or CT as oxidizers. Both procedures yielded the same products, although the use of CT resulted in more favorable product ratios under the conditions applied. HPLC separation of a typical CT iodination reaction mixture is shown in Fig. 1. Three radioactive peaks were routinely obtained in addition to free iodide-125. The major radioactive peptide peak (peak No. 5 in Fig. 1) was identified as [mono-125ITyrS]T-98, whereas the smaller peak eluting later (peak No. 6)

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mixture of T-98. Solid line, radioactivity;dotted line, UV absorption at 257 nm; dashed line, %Bgradient. Peak 1, free iodide-125; peak 2, chloramine-T; peak 3, unlabeledT-98; peak 4, side product; peak 5, [3J25iodoTyrS]T-98;peak 6, [3,5-diJ25iodo-TyrS]T-98.Separation was carried out on a W-Porex 5C 18 analyticalcolumn using0.1% TFA in water as solvent A and 0.1% TFA in 70% aqueous acetonitrile as solvent B. Eluent composition was 20% B at injection time and increased from 44% to 60% B between 10 and 34 min after injection (see dashed line).

362

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FIG. 2. Adsorption and desorption of[~Z~l]T-98 in tubes made of different materials. Approximately 200,000 cpm tracer in 1 ml medium containing 0. I% BSA was incubated in polystyrene (bar 1), BSA-coated polystyrene (bar 2), polypropylene (bar 4), glass (bar 5), or polyD-Lys-coated glass (bar 6) tubes at 37°C for 1 h. BSA-coated polystyrene tubes were also incubated with the same amount of tracer in medium containing 1% BSA (bar 3). The bars represent the amount of radioactivity remaining in empty tubes after aspirating the medium and rinsing the tubes twice with ice-cold medium (after medium wash), after subsequent incubation of the tubes with 0.2 M acetic acid, 0.5 M NaCI at 4°C for 5 min, followed by a rinse with the same solution (after acid wash), and after an overnight incubation with 0.5 M NaOH at 37°C and a rinse (after NaOH wash). The counts were corrected for differences in counting efficiency due to different tubes. Average _+ SD of quadruplicate tubes are shown.

Binding o[ T-98 and Its lodinated Derivatives to Rat Pituitary Membranes The affinity of T-98 to pituitary L H - R H receptors was determined in competitive binding experiments. [~25I][D-Trp6]LHRH was used as radioligand because of its proven suitability to characterize binding of various analogues to L H - R H receptors (12). Figure 3 shows that T-98 effectively displaced the tracer from the binding sites on pituitary membranes. Equilibrium dissociation constant calculated using the Ligand program indicated high binding affinity for T-98 (Kd = 1.2 + 0.3 nM, Table 1). The specificity is demonstrated by the observation that although buserelin completely displaced the radioligand, none of the structurally or functionally unrelated peptides tested could inhibit the binding at l0 6 M concentration (Fig. 3). The binding characteristics of the iodination products of T98 were also determined in competitive binding experiments. For this purpose, nonradioactive iodinated derivatives of T-98 were used to displace the radioligand, [~251][D-Trp6]LH-RH, from pituitary membranes (Fig. 3). This approach proved to be preferable to saturation binding analysis using the radiolabeled T98 analogues because of their extremely high, nonspecific binding. Computer analysis of the data showed high binding affinity of I-T-98 that was similar to that of the unlabeled peptide (Table 1). Di-iodinated T-98, however, showed significantly lower affinity ( ~ 2 0 - 2 5 times smaller) for L H - R H receptors, which precluded its use in the binding and internalization studies (Table 1).

LH-Releasing PotentT q/" T-98 and Its lodinated Derivatives was determined to be [diJ251-TyrS]T-98, based on elution position. In nonradioactive iodination experiments, the products with the same retention times as those of peaks No. 5 and 6 showed the characteristic U V spectral shifts of mono- and diiodinated tyrosine, respectively. Specific activities were calculated from the radioactivities and the areas of U V peaks on the H P L C chromatograms. For this purpose, UV detection both during the separation and the H P L C calibration with unlabeled T-98 was carried out at the absorption maximum (257 nm) of the cytotoxic anthraquinone moiety. At this wavelength the iodination does not affect the absorptivity of the analogue. Specific activity values obtained in this fashion were found to be the same (peak No. 5) or twice as large (peak No. 6) as that of the iodide-125 used for labeling. The third radioactive product (peak No. 4), eluted on the down slope of unlabeled T-98 (peak No. 3), most likely represents an oxidation product.

The LH-releasing activities of unlabeled T-98 as well as its iodination products were assessed in rat pituitary superfusion system (Table 2). T-98 showed LH-releasing potency approxi-

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Figure 2 shows that the labeled analogue bound very strongly to polystyrene surfaces under the conditions of the internalization assay. Adsorption on polypropylene was less powerful but still substantial, whereas practically no binding was observed on borosilicate glass. The BSA decreased adsorption of [J2SI]T-98 either when it was preadsorbed onto polystyrene tubes or when its concentration in the incubation buffer was increased. Coating glass with polyD-lysine, a method used for improving cell attachment, did not significantly change the favorable nonadsorptive characteristics of glass. Part of the adsorbed radioactivity could be removed selectively by 0.2 M acetic acid treatment and the remainder by 0.5

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FIG. 3. Displacement of [~zsI][D-Trp6]LH-RH from rat pituitary membrane receptors by increasing amounts of unlabeled T-98 (©), I-T-98 (V), I2-T-98 ([~), o r 10 -6 M buserelin (0). Other unrelated peptides like epidermal growth factor (EGF) (V), human growth hormone-releasing hormone (hGH-RH) (e), thyrotropin-releasing hormone (TRH) (m), and gastrin releasing peptide (GRP) (A) did not displace the tracer in 10 6 M concentration. Incubations were carried out as described in the Method section. Data points are the mean _+SE of duplicate determinations from two separate experiments.

CYTOTOXIC LH-RH ANALOGUE

363

TABLE 1 EQUILIBRIUM DISSOCIATION CONSTANTS OF T-98 AND ITS IODINATED DERIVATIVESMEASURED IN RAT PITUITARY CELL MEMBRANES

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mately 17 times higher than LH-RH. The activity of the monoiodinated T-98 was only 50% of the potency of L H - R H , and diiodination resulted in a further decrease in the activity (< 10%).

Internalization of[roll T-98 by Rat Pituitary Cells" In the internalization experiment carried out at 37°C, the highest specific acid-dissociable binding attributed to the receptor-bound tracer was measured at the earliest time point tested (6 min) (Fig. 4). Longer incubations resulted in a decrease in the amounts of surface-bound analogue, although the binding remained significantly higher than that found in control vials without cells at any time point tested. Acid-resistant binding assumed to be due to the internalized analogue was small and similar to the control values at 6 min, but increased continuously in time and finally exceeded the extent of surface binding at about 45 min. At 2 h, about 70% of total cell-associated radioactivity was found to be acid resistant. An increase in the concentration of LH released into the medium during the incubation was also simultaneously observed. Figure 5 shows the specific binding of [~25I]T-98 to pituitary cells at 4°C. The incubation was carried out under conditions identical to those of the internalization experiments at 37°C, except for the temperature. Acid-dissociable radioactivity at-

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binding (B) was determined with cell monolayers under identical conditions as in Fig. 5 except that the reaction was at 37°C. Open symbols (V, D) represent binding measured in control vials without cells. The LH released into the medium by cells incubated with approximately 0.5 nM tracer only (in vials used to estimate total binding) is shown by the dashed line. Results from one of two experiments yielding similar results are shown (mean +_ SE).

tributed to the receptor-bound tracer increased in a time-dependent fashion, reaching binding equilibrium by the end of the incubation (2 h). This binding occurred on cells, as demonstrated by the negligible radioactivity measured in the control vials without cells (Fig. 5), and it was specific because it could be displaced with an excess of unlabeled analogue. On the contrary, acid-resistant binding was very low and did not increase with prolonged time of incubation. At 2 h, only 15% of the total cellassociated radioactivity was found to be acid resistant. In one of the internalization experiments, the stability of [JESI]T-98 during the incubation with pituitary cells was also

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TABLE 2 RELATIVE LH-RELEASINGPOTENCIES OF T-98 AND ITS IODINATED DERIVATIVES AS MEASURED IN RAT PITUITARY SUPERFUSION ASSAY Compound LHRH T-98 [3J 2Siodo-TyrS]T-98 [3,5-di-125iodo-TyrS]T-98

Relative LH-ReleasingPotency 1.0 17.1 (10.4-28.2) 0.5 (0.2-1.1) <0.1 *

Potencies were calculated by the factorial analysis of Bliss and Marks (38). The 95% confidence limits are given in parentheses. * Determined in one concentration only.

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Incubotion time (rain) FIG. 5. Time course of [t25I]T-98 binding and internalization in rat pituitary cells at 4°C. Specific acid-dissociable (v) and acid-resistant binding

(B) was determined with cell monolayers cultured in glass vials as described in the Method section. Open symbols (V, F1) represent binding measured in control vials without cells. Results from one of two experiments yielding similar results are shown (mean _+ SE).

364

SZ(~KE ET AL.

determined. HPLC analysis of the incubation medium showed that most of the tracer (~95%) remained intact during the 2-h incubation. A small extent of degradation observed was slightly different for vials containing cells compared to controls without cells, but did not depend on the temperature or the presence or absence of a one thousandfold excess of unlabeled analogue (Fig. 6).

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~ DISCUSSION

In the course of our investigation of the mechanism of action of LH-RH analogues containing cytotoxic radicals (2,3,23,31,37,41,44), we decided to examine initially the internalization by the pituitary gonadotropes before focusing on various tumors that have LH-RH receptors. We selected the agonistic analogue T-98 containing HMAQ because of its high binding affinity to LH-RH binding sites in breast cancer tissue (23) and its ability to suppress the growth of certain tumors containing receptors for LH-RH (37,44). To study the internalization of this cytotoxic LH-RH analogue, the protocol of Haigler et al. (18) was adapted. This quick biochemical method provides quantitative results and has already been successfully used to study the receptor-mediated internalization of a large number of peptides and proteins, e.g., somatostatin (48), bombesin (49), insulin (39), epidermal growth factor (18), insulinlike growth factor (IGF) I and II (8), and transferrin (30). The method selected requires the use of radioiodinated ligand. We successfully labeled T-98 using two different iodination methods without causing damage to the cytotoxic group. Our findings, shown on Fig. 1, demonstrate the homogeneity of the purified tracers and support their identification as mono- and di-iodinated derivatives. Our first internalization experiments with [~25I]T-98 were carried out with cells cultured in polystyrene multiwell plates. In these studies, very high acid-dissociable radioactivity, supposedly receptor-bound, as well as acid-resistant, presumably internalized, radioactivity was found. This binding was time dependent and part of it was considered specific because it could be displaced with an excess of unlabeled analogue. Surprisingly, however, similar values were also observed in control wells that did not contain cells. This finding nullified the validity of these results and prompted us to study the adsorption of [~25I]T-98 on different materials. The results, which showed that the tracer was bound very stongly to polystyrene, less intensively to polypropylene, and practically not to glass (Fig. 2), clearly precluded the use of polystyrene multiwell plates and indicated that glass tubes and culture dishes have to be used in our experiments with [~251]T-98. The same pattern, i.e., decreasing adsorption with decreasing hydrophobicity of the surface, was also found with a less hydrophobic LH-RH tracer ([~251][D-Trpr]LH-RH). However, in the case of this tracer, which does not contain the anthraquinone group, the extent of binding was much smaller, and amounted to only about one-tenth of that of [JZSl]T-98 (data not shown). These results suggest that the very strong binding of [~251]T-98 to different surfaces is due to hydrophobic interactions and can be mainly attributed to the very apolar anthraquinone moiety of the cytotoxic group. It is important to emphasize that 50-80% of the total adsorbed tracer could be displaced from polystyrene by a one thousandfold excess of unlabeled compound, i.e., the binding to tissue culture plates appeared to be specific by this criterion. The amount of radioactive analogue desorbed from culture plates by acid and NaOH treatment was high (7-10% of total activity added), and, therefore, receptor-bound or internalized radioactivities originating from the cells were completely masked and could not be quan-

4o

V ~

2o

-lo o n," 0

Incubation Temperature Cells "Cold" T - 9 8

N/A

120'

120'

120'

120'

4°C

37°C +

37°C +

37*C

10-SM

-

+

FIG. 6. Stability of [J25I]T-98 tracer during internalization experiment. Approximately 200,000 cpm [~251]T-98was incubated with or without monolayers of rat pituitary cell (700,000 cells/vial) for 2 h at 4°C or 37°C in the presenceor absenceof 10 6M unlabeledanalogue.Incubation media were analyzed by HPLC as described in the Method section. Bars • • represent radioactivity found ~resent in [125 I]T-98peak and expressed as percent of total radioactivityrecovered during run. Each value is the average _+SE of two determinations. *p < 0.05; **p < 0.01 vs. control; Duncan's test [F(4, 8) - 38.63, p = 2.82E-05, one-way ANOVA].

titated. Our findings stress the importance of investigating the tracer adsorption and of selecting carefully optimized experimental conditions in binding studies with radioactive compounds. Binding characteristics of T-98 and its iodinated derivatives were determined using in vitro radioreceptor assay. T-98 was bound to pituitary membrane receptors with a high affinity (Kd = 1.2 _+ 0.3 nM). Similar Kd values (0.3-2.4 nM) were reported for [D-Lys6]LH-RH (28), the carrier peptide ofT-98, indicating that T-98 preserved the full binding ability of its carrier. No significant difference was detected in receptor binding affinities between unlabeled T-98 and its monoiodinated derivative, although di-iodinated T-98 showed significantly lower affinity for LH-RH receptors. This is in agreement with other reports in which no decrease in receptor binding affinity was observed as a result of monoiodination of the Tyr~ residue of LH-RH (12) or its superagonist analogues (6). These results, together with previously reported data showing high-affinity binding of T-98 to breast cancer membranes (31), support the view that T-98 can be used as a targeted anticancer agent for LH-RH receptorbearing tumors. In the superfusion experiments T-98 was found to be a potent LH-RH agonist, as previously reported (23). This is in accord with the high binding affinity measured, and with the well-established observations that hydrophobic D-residues in position 6 (an anthraquinone moiety in the case ofT-98) greatly increase the LH-releasing activity ofLH-RH agonists [for review see (27)]. Furthermore, this finding suggests that this targeted cytotoxic compound can exert antitumor effects characteristic of certain LH-RH agonists also on the basis of inhibition of pituitarygonadal axis [reviewed in (27,40,41)] and direct action on cancer cells (14,16,41). The incorporation of one iodine atom into the Tyr 5 residue of the molecule, however, dramatically reduces its biological activity to about 3% of its original potency. Incorporation of a second iodine further decreases the already very low LH-releasing activity. Similar effects of iodination were also

CYTOTOXIC LH-RH ANALOGUE

365

observed with LH-RH and some of its analogues, where monoiodination generally caused a tenfold decrease, whereas di-iodination resulted in a one hundredfold decrease in original LHreleasing activity [(45), B.S., unpublished data]. The method used to measure the internalization of our cytotoxic LH-RH analogue is based upon the finding that a short incubation with an acidic buffer (pH 2.5-3.0) at 4°C dissociates receptor-bound hormone molecules from the cell surface, and internalized molecules are not affected (1,18). This acid-stripping method as well as quantitative electron microscopic autoradiography were used by Wynn et al. (50) to investigate the internalization of agonist and antagonist analogues of LH-RH. They concluded that a more reliable estimate of internalization can be obtained with the acid-dissociation technique than with the autoradiographic method in case of those analogues that do not have a free amino group. In our experiments, both surface binding and acid-resistant binding were sensitive to temperature and to the presence of excess unlabeled hormone, suggesting that they reflect binding to specific receptors and receptor-mediated internalization, respectively. Luteinizing hormone release into the medium during the incubation period indicates the functional integrity of the gonadotrope cells. The fact that the acid-resistant binding remained very low throughout the entire incubation at 4°C agrees well with other reports suggesting that no internalization occurs at this temperature (20,21,25,26). These data also prove the effectiveness of the acid treatment in removing surface-bound tracer, because more than 85% of total cell-associated radioactivity was recovered in the acidic wash. These results are in good agreement with previous findings and the proposed model of internalization of LH-RH agonist [reviewed in (32)]. Association kinetics were reported to be very fast for LH-RH agonists on pituitary cells. Naor et al. (34) found that binding of [~251]buserelin reached equilibrium in approximately 10 min at 37°C, whereas Wynn et al. (50) observed an association t~2 of 7 min for [D-Lys6]LH-RH at 23°C. The reduction in surface binding that was observed after 6 min at 37°C in our experiments is explained by receptor downregulation as a result of internalization. The time course of internalization of T-98 is virtually identical to that measured with two other LH-RH agonists using a similar method (50). Several studies, both biochemical and morphological, have shown that agonist analogues of LH-RH are internalized by receptors on pituitary gonadotropes

(5,9,10,20-22,24-26,33,36,50), In some of these studies, analogues with a bulky label, such as ferritin (22,25), rhodamine (20,33), or colloidal gold (24-26), attached to the side chain of D-Lys6 of the molecule were used. Because T-98 has the anthraquinone residue in the same position, our results further confirm the observation that bulky side chains in position 6 of LH-RH agonists do not prevent their uptake by receptor-mediated endocytosis. The fact that a small degradation of[~251]T-98 observed during incubation with pituitary cells did not depend on the temperature or on the excess concentration of unlabeled analogue indicates that this slight breakdown is not due to an enzymatic process, which agrees well with previous data showing that LH-RH is not degraded by pituitary cells (35). It appears that the presence of cells slightly increased the rate of tracer radiolysis, the mechanism of which is not clear. These results also provide indirect evidence that acid-resistant radioactivity did not represent the uptake of degradation products because the internalization depended on the temperature and was abolished by excessive amounts of unlabeled analogue. In summary, the binding and internalization of an LH-RH analogue carrying a cytotoxic anthraquinone radical was studied in rat pituitary cells. It was demonstrated that this hybrid molecule showed high-affinity binding to LH-RH receptors, acted as a potent LH-RH agonist by evoking LH-release, and subsequently was internalized by receptor-mediated endocytosis. Our results also suggest that this cytotoxic LH-RH analogue and related hybrid molecules may also be internalized by LH-RH receptors present in breast, prostatic, ovarian, endometrial, and other tumors if they are functionally similar to pituitary receptors. These phenomena are subject of ongoing investigations aimed at improvement of present methods for cancer therapy. ACKNOWLEDGEMENTS We thank Katalin Halmos for expert experimental assistance and the National Hormone and Pituitary Program, National Institute of Diabetes and Digestiveand Kidney Diseases; for the gifts of materials used in radioimmunoassay. The work described in this paper was supported by the National Institutesof Health Grants CA 40003 and by the Medical Research Service of the Veterans Affairs (to A.V.S.). It is a pleasure to acknowledge usefuldiscussionsand valuableadvice from ProfessorJuergen Engel, Asta Medica/Frankfurt. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the offacal views of the National Cancer Institute.

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