Bioconjugate Chem. 1994, 5, 591-601
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Multivalent Melanotropic Peptide and Fluorescent Macromolecular Conjugates: New Reagents for Characterization of Melanotropin Receptors? Shubh D. Sharma,* Michael E. Granberry,s Jinwen Jiang,l Stanley P. L. Leong,§ Mac E. Hadley,lJ' and Victor J. Hruby*" Departments of Chemistry, Surgery, Anatomy, and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721. Received April 8, 1994@
Radioreceptor binding studies have documented the presence of melanotropin receptors on some but not all of the various human melanoma cell lines that have been studied. Using a newly developed class of multivalent fluorescent melanotropin-macromolecular conjugates, we have demonstrated for the first time the presence of specific melanotropin receptors on all of the melanoma cell lines, both mouse and human, melanotic as well as amelanotic, that were investigated. The conjugates developed by us consisted of multiple copies of both a potent melanotropin analogue and a fluorophore, both arranged in a pendent fashion on a biologically inert macromolecule. While the multivalency of these conjugates may have established stronger binding with the melanotropin receptors on the cell surface (perhaps by establishing simultaneous multiple interactions), the presence of multiple copies of the fluorophore also greatly increased the level of detection in fluorescence labeling experiments. Membrane receptor-hormone-associated phenomena, such as capping and internalization of the receptor-ligand complex, also were observed. The details of these methods are described using B-16 mouse melanoma cells as a model system. The demonstration of MSH receptors as a common marker for melanoma suggests that this methodology might be employed for early clinical detection and anatomical localization of melanoma. These results also offer the possibility that substitution of the fluorophore in these conjugates by a chemical agent of (chemo-)therapeutic relevance may provide a powerful tool for site specific (tumor) targeting and cytotoxicity.
INTRODUCTION
noma cells may also possess receptors for melanotropic peptides. Radioreceptor binding assays and autoradioga-Melanocyte stimulating hormone (MSH, a-melanotropin),l a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His- raphy techniques have been used to identify melanotropin receptors in mouse (6-12) and several human Phe-Arg-Trp-Gly-Lys-Pro-Val-NHz, is the physiologically melanoma cell lines (13-16). These studies, however, relevant hormone that controls skin pigmentation of the revealed extremely varied results. Interestingly, the skin in most vertebrates (1). This systemic hormone is demonstration of the presence of MSH receptors was derived from the pars intermedia of the pituitary gland. found to be dependent on the type of radiolabeled Although adult humans do not possess a pars intermedia melanotropin analogue used in these studies. For ex(I),there is ample evidence that a melanotropin [either ample, Libert et al. (13) using radioiodinated a-MSH a-MSH or ACTH, (2)] increases melanin pigmentation showed that specific binding could be demonstrated only of the skin under certain pathological conditions (3). in four out of 10 human melanoma cell lines studied. The Injections of a-MSH and its potent analogues into use of radioiodinated [Nle4-~-Phe71-a-MSH, on the other humans increase melanin pigmentation of the skin (3hand, established the presence of MSH-receptors in six 5). These observations suggest that human epidermal out of the same 10 cell lines (13). In spite of this melanocytes possess melanotropin receptors. dependence on the radioligand, investigations by various It has long been speculated that, like mouse melanoma other workers also have substantiated the finding that cells and human epidermal melanocytes, human melanot all human melanoma cell lines possesses melanotropin receptors (7, 13, 16). We have utilized [Nle4-~-Phe71' This work was supported by grants from the U S . Public a-MSH, a more stable, potent, and enzymatically resisHealth Service DK 17420 (V.J.H.) and the National Science tant analogue of a-MSH developed in our laboratory Foundation (V.J.H.). (17-20), to synthesize macromolecular conjugates (21). * Author to whom correspondence should be addressed a t the These conjugates consisted of a biocompatible carrier Department of Chemistry, University of Arizona, Tucson, AZ macromolecule to which multiple copies of this peptide 85721. Department of Chemistry. as well as multiple copies of a reporter group (a fluoroDepartment of Surgery. phore) were covalently attached. The fluorescent melDepartment of Anatomy. anotropin macromolecular conjugate was used t o visuDepartment of Molecular and Cellular Biology. alize melanotropin receptors on mouse and human Abstract published in Advance ACS Abstracts, September melanoma cells.
*
@
1, 1994.
Abbreviations: a-MSH, a-melanocyte stimulating hormone; [Nle4-~-Phe71-a-MSH,[norleucine*, D-phenylala~~ine~l-a-melanocyte stimulating hormone; FITC, fluorescein isothiocyanate; PVA, poly(viny1 alcohol); DTT, dithiothreitol; 2-ME, 2-mercaptoethanol; DMF, Nfl-dimethylformamide; DMAP, (Nfl-dimethy1amino)pyridine; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 1043-1802/94/2905-0591$04.50/0
EXPERIMENTAL PROCEDURES
Fluorescein isothiocyanate-isomer I (FITC), (N&dimethy1amino)pyridine (DMAP), and poly(viny1alcohol) (average molecular weight 110 000) were obtained from Aldrich Chemical Co. (St. Louis, MO). Dithiothrietol
0 1994 American Chemical Society
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Sioconjugafe Chem., Vol. 5, No. 6,1994
(DTT), mercaptoethanol, 4-(p-maleimidophenyl)butyric acid N-hydroxysuccinimide ester, and 3-(2'-pyridyldithio)propionic acid N-hydroxysuccinimide ester were obtained from Sigma Chemical Co. (St. Louis, MO). Tissue culture media and fetal calf serum were obtained from GIBCO. Protected amino acid derivatives were obtained from Bachem California (Torrence, CAI. The binding buffer for fluorescence labeling experiments was a low ionic strength (15 mM) buffer made isotonic by the addition of glucose and sucrose (22). The composition of this buffer was as follows: triethanolamine (0.015 M), glycine (0.24 MI, sucrose (0.009 M), glucose (0.025 M), potassium acetate (0.004 M), and calcium chloride (0.0003 M). Acetic acid was used to adjust the pH of the buffer to 5, and if necessary the osmolarity was adjusted to 0.310 osmol using sucrose. The concentrations of all the conjugates were calculated from their initial amounts and measurements of the volumes of the preparations after dialysis. Cell Cultures. All cells were grown in Falcon 75-cm2 tissue culture flasks at 37 "C in a humidified atmosphere of 5% COZand 95% air. Mouse and Human Melanoma. The B16/F10 mouse melanoma cell line was originally provided by Dr. A. Overjera of the Frederick Cancer Center, Frederick, MD. The human melanotic cell line A375P was obtained through the American Type Culture Collection (ATCC). The other human melanoma cells were obtained from Arizona Cancer Center Tissue Culture Core Facility, University of Arizona, Tucson, AZ. The cell lines were maintained in monolayer cultures and were grown in Ham's F-10 medium with NaHC03 (1.2 g/L) supplemented with 10% horse serum and 2% fetal calf serum (both heat-inactivated at 56 "C for 30 min) and 1% penicillin-streptomycin (100 units/mL, 100 pg/mL, respectively). Cells were subcultured weekly and were maintained in a monolayer culture for only 10 passages to avoid phenotypic drift that is often observed in longterm cultures. Other Cell Lines. A human small cell lung cancer cell line (NCI-N592) was obtained from Dr. Tom Davis, Department of Pharmacology, University of Arizona. The NCI-N592 line grows as floating aggregates in Ham's F-10 medium supplemented with 10% heat-inactivated fetal calf serum and 1% penicillin-streptomycin. The human breast cancer MCF-7 cell line was obtained from Dr. D. Blask, Department of Anatomy, University of Arizona. The mammary cancer cells were maintained in monolayer culture and grown in Ham's F-10 medium supplemented with 10%heat-inactivated fetal calf serum and 1%penicillin-streptomycin. Peptide Synthesis. The three peptides used in this study, namely Na-desacetyl-Na-(3'-mercaptopropionyl)[Nle4-~-Phe7]-a-MSH [I], [W-(3'-mercaptopropiony1)-Lysl3]dynorphinl-13-amide [II], and Na-(3'-mercaptopropionyl)substance-P [III], were synthesized by solid-phase methods of peptide synthesis (23)on a p-methylbenzhydrylamine resin (substitution 0.51-0.72 mequiv of amine/g of resin) using a VEGA 250 semiautomated peptide synthesizer. A 4-fold excess of appropriate Nu-Boc-protected amino acid was used at each coupling step. Couplings were performed by using diisopropylcarbodiimide-N-hydroxybenzotriazole (DIC-HOBt) as coupling reagent and were monitored by the Kaiser test (24)in all cases except when the incoming amino acid was coupled to the proline residue. In this instance the Chloranil test (25) was performed to ascertain the progress of the coupling. A mixture of trifluoroacetic acid-dichloromethane-anisole (TFA-DCM-anisole, 50:48:2) was used to deblock the Boc group after each coupling step. The following three
Sharma et al.
fully protected peptide-resins corresponding to peptides 1-111were synthesized: Na-Boc-Ser(Bzl)-Tyr(BzlCl&Ser-
(Bzl)-Nle-Glu(OBzl)-His(Bom)-D-Phe-Arg(Tos)-Trp-GlyLys(ZC1)-Pro-Val-resin[Ivl,Na-Boc-Tyr(BzlClz)-Gly-GlyPhe-Leu-Arg(Tos)-Arg(Tos)-Ile-Arg(Tos)-Pro-Lys(ZC1)Leu-Lys(N'-Fmoc)-resin [VI, and Na-Boc-Arg(Tos)-Pro-
Lys(ZC1)-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-resin [VI]. The Na-Boc groups from IV and VI and N6-Fmocgroup on the thirteenth amino acid residue in V were cleaved, respectively, by the treatment of IV and V with TFADCM-anisole (50:48:2) and VI with 20% piperidine in N-methylpyrrolidinone for 20 min. To each of the resulting peptide resins 3'-(S-(p-methylbenzyl)thio)propionic acid was individually coupled using DIC-HOBt. The dried peptide resins were individually treated with HFthioanisole for 45 min at 0 "C and the resulting crude peptides purified by HPLC and characterized by fast atom bombardment mass spectrometry and amino acid analysis. Analytical HPLC was performed on a Cls column (Vydac 218TP104,25 cm x 4.6 mm). Thin layer chromatography (TLC) was performed on Baker 250-nm analytical silica gel glass plates in the following solvent systems: (A) 1-butanoUacetic acid/pyridine/water (5:5:1:4 v/v), (B) 2-propanol/25% aqueous ammonidwater (3:l:l v/v), (C) ethyl acetate/pyridine/acetic acidwater ( 5 5 :1:3 v/v), and (D) 1-butanol/acetic acidwater (4:1:5). The peptides were visualized by ninhydrin and iodine vapor. Analytical data for peptides: I: mass (M) = 1692.6 (calcd 1692.97); a z 3=~ -50.6" (c 0.41, 10% aqueousAcOH); HPLC K' = 7.02 (gradient of 10%-40% acetonitrile in 0.1% aqueousTFA completed in 30 min at 1.5 mL/min); TLC Rfvalues = 0.33 (A), 0.61 (B), 0.0 (C), 0.02 (D). 11: mass = 1691.5(calcd 1691.13);a Z 3 =~ -28.1' (c 0.33, 10% aqueous AcoH); HPLC R = 5.17 (gradient of 15%-45% acetonitrile in 0.1% aqueous TFA completed in 30 min a t 1.5 mumin); TLC Rfvalues = 0.71 (A), 0.80 (B), 0.54 (C), 0.40 (D). 111: mass = 1434.9 (calcd 1434.78); a23D = -37.2' (c 0.41, 10% aqueous AcoH); HPLC R = 5.6 (gradient of 10%-40% acetonitrile in 0.1% aqueous TFA completed in 30 min at 1.5 mumin); TLC Rf values = 0.53 (A), 0.75 (B), 0.64 (C), 0.36 (D). Synthesis of Fluorescent-Macromolecular-Pep tide Conjugates. Three types of macromolecular conjugates were synthesized: (A) conjugates containing multiple copies of fluorescein on the one hand and multiple copies of one of the three peptides synthesized above on the other (Scheme 1);(B) conjugates bound only to fluorescein; and (C) conjugates consisting only of the peptide component. The conjugates with only one of the two components present were synthesized for use in control experiments. [Fluorescein/,-PVA MI]. Ten mg (9.1 x mol) of poly(viny1 alcohol) (PVA, average MW 110 000) was dissolved in 4 mL of HEPES buffer (50 mM, pH 7.5). It was mixed with a mixture of fluorescein isothiocyanate (FITC, 1.77 mg, 4.55 x mol) and (Nfl-dimethylamino)pyridine (DMAP, 0.5 mg, 4.55 x mol) dissolved in 1mL of DMF. The resultant clear solution was mixed by shaking in the dark for 12-18 h. The reaction mixture was then placed in a dialysis bag (MW cutoff 10 000) and dialyzed extensively against 50 mM HEPES (pH 7.5) to remove the unconjugated FITC. The final volume of the resulting dialysate was measured. Spectrophotometric measurements at l. 492 for FITC ( E = 78 000) (26)indicated substitution of 12-18 FITC molecules per molecule of PVA in different preparations. This preparation was dialyzed extensively against the binding buffer for its use in the receptor binding experiments described below. PVA-[(Maleimidophenyl)butyrate], [VIII]. Twenty-
Bioconjugate Cbem., Vol. 5, No. 6,1994 593
Melanotropic Peptide Multivalent Fluorescent Conjugates
Scheme 1. Synthetic Routes for the Fluorescent Macromolecular Conjugates in which the Ligand Is Either Linked to the Polymer through a Thioether W ,XVI, XVIII] or a Disulfide Bond [XVJ
I
m e t i c route for
disulfide-linked conjugate
+CH,-CH + AH
Sunthetic route for
Polyvinyl alcohol [PVAI
thioether-linked conjugate
\ \
I
[FITCI,
-
1
-
PVA [-C-(CH2)z-S-S0
[ FITC,
-
PVA .I S-S[XVI
Ligand 1,
[FITCI,
-
0
R
PVA - [ - C - ( C H & G N >
Ligand, ]
HSLigand is : HS-(CH2),-CO-[N1e4,D-Phe7]-a-MSH, [I];
0
-
[ FITC,
PVA
-
[ XIV, XVI
S-Ligand
In
S -S - Ligand, ]
- XVIII 1
[L~S(N~(CO-CH~CH~-SH)J’~]-D~~O~~~~~,.,~-NH,, (111; HS-(CH,),-CO-[Nle”]-Substance-P, (1111; and
HS-CHz-CHz-OH
five mg (2.27 x moles) of PVA (average MW 110 000) was dissolved in 9 mL of HEPES buffer (50 mM, pH 7.5). The solution was added to a mixture of 4-(p-maleimidopheny1)butyric acid N-hydroxysuccinimide ester (4.02 mg, 1.13 x mol) and DMAP (1.38 mg, 1.13 x mol) dissolved in 1 mL of DMF. The resultant clear solution was mixed by shaking for 12-18 h. The reaction mixture was then placed in a dialysis bag (MW cutoff 10 000) and dialyzed extensively against 50 mM HEPES (pH 7.5) to remove the unconjugated maleimide and the resulting volume of the dialysate measured. Spectrophotometric measurements at A 255 (measured E = 215) (27) indicated a substitution of 10- 16 maleimidophenyl moieties per molecule of PVA in various trials. Alternatively, the substitution was measured spectrophotometrically by reacting this product with thiophenol (&fold molar excess) for 5 h, followed by extensive dialysis to remove unreacted thiophenol [A 269 ( E = 700) (2811, and the same substitution levels were obtained. PVA-[(Pyridyldithio)propionate], [IX].Twenty-five mg (2.27 x mol) of PVA (average molecular weight 110 000) was dissolved in 9 mL of HEPES buffer (50 mM, pH 7.5). It was mixed with a mixture of 3-(2’pyridyldithio)propionic acid N-hydroxysuccinimide ester (3.53 mg, 1.13 x mol) and DMAP (1.38 mg, 1.13 x mol) dissolved in 1mL of DMF. The resultant clear solution was mixed by shaking for 12-18 h. The reaction mixture was then placed in a dialysis bag (MW cutoff 10 000) and dialyzed extensively against 50 mM HEPES (pH 7.5) to remove the unconjugated reagent and the resultant volume of the dialysate measured. Spectrophotometric measurements done at A 255 (measured E = 215) (29)indicated substitution of 10-16 pyridyl moieties per molecule of PVA in different trials. Alternatively, the substitution was also measured spectrophotometri-
cally by reacting this product with mercaptoethanol (5fold molar excess) for 5 h, followed by direct measurements at A 343 ( E = 8080) for the 2-thiopyridine (30)that is released in this reaction. The same values for levels of substitution were obtained. [FITClm-PVA-[(Maleimidophenyl)butyrateln [XI. A sample of 10 mg (9.1 x mol) of VI11 (ca. 4 mL in 50 mM HEPES buffer, pH 7.5) was added to a mixture of FITC (1.77 mg, 4.55 x mol) and DMAP (0.5 mg, 4.55 x moles) dissolved in 1 mL of DMF. The resultant clear solution was mixed in the dark by shaking for 1218 h. The reaction mixture was then placed in a dialysis bag (MW cutoff 10 000) and dialyzed extensively against 50 mM HEPES (pH 7.5) to remove the unconjugated FITC. Spectrophotometric measurements done as above at A 492 ( E = 78 000) (26) indicated substitution 11-16 FITC moieties per molecule of PVA. [FITClm-PVA-[(Pyridyldithio)propionateln [XI]. A sample of 10 mg (9.1 x mol) of M (ca. 4 mL in 50 mM HEPES buffer pH 7.5) was added to a mixture of FITC (1.77 mg, 4.55 x mol) and DMAP (0.5 mg, 4.55 x mol) dissolved in 1 mL of DMF. The resultant clear solution was treated in the same manner as described under the synthesis of X to give a similar substitution value. Synthesis of PVA-[-S-MSHI,, [XI11 and P V A I - S S MSHI,, [XIIII. Treatment of 2 mg of the derivatized polymer VI11 or M in 1.5-2.0 mL of 50 mM HEPES (pH 7.5) with a 2-4-fold molar excess of I dissolved in DMF (0.3 mL) was accomplished at room temperature for 2-5 h. The conjugate was dialyzed extensively, and the degree of ligand substitution calculated spectrophotometrically at A 280 nm ( E = 4840) to be between 10 and 16 MSH molecules per molecule of the polymer. These
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594 Bioconjugate Chem., Vol. 5, No. 6,1994
Table 1. Results Demonstrating Specific Fluorescence Labeling of Melanotropin Receptors on Various Melanoma Cell *Des by Fluorescent MSH-Macromolecular ComDosites malignant cell types human normal lung breast cell types mouse melanoma cancer cancer melanoma ~- mouse B-l@ JHb LH164ga LR165@ LR1714" WCb KE" A375Fsc NCI-N417 MCF-7 Spleen Liver conjugate FITC-PVA [VI11 FITC-PVA-S-MSH BIVl ++ ++ ++ ++ ++ ++ FITC-PVA-SS-MSH [xvl ++ ++ ++ ++ FITC-PVA-SS-MSH Lxv] + DTT FITC-PVA-S-MSH BIVl + DTT ++ ++ ++ FITC-PVA-ME [XVIII] FITC-PVA-S-DYN MI FITC-PVA-S-SP M I ]
++
++
++
a Melanotic cell lines. b Amelanotic cell lines. c Similar positive responses were obtained with all other human melanoma cells that were assayed ( > 17 cell lines). ++, indicates strong fluorescence labeling. -, indicates no fluorescence labeling.
preparations were dialyzed against the binding buffer for their use in the binding experiments with the cultured cells. Synthesis of [FITCIm-PVA-[-S-MSH/, [XTVI, [FITCIm- PVA-[-SS-MSHl, [xv],[FITC/,- PVA-[-Sdynorphin], [XVI], [FITC/,-PVA-[-S-substance PI, [XVII], and [FITC/,-PVA-[-ME], [XVIII]. Treatment of 2 mg of the derivatized polymer X or XI in 1.5-2.0 mL of 50 mM HEPES (pH 7.5) with a 2-4-fold molar excess of I, 11,111,or mercaptoethanol (ME) dissolved in DMF (0.3 mL) was accomplished as described above under XI1 and XIII. The conjugate in each case was dialyzed extensively and the degree of ligand substitution calculated spectrophotometrically. The substitution in each case was found to range between 10 and 16 molecules of the ligand per molecule of the polymer. All these preparations were dialyzed against the binding buffer for this compatibility in the binding experiments with the cultured cells. Fluorescence Labeling of Melanotropin Receptors. One million cells of each type (Table 1) per test tube were treated individually with conjugates VI1 and W - X V I I I a t various concentrations (0.5 mg/mL was the best concentration) for various lengths of time (15 min incubations were found to be best) at room temperature in the dark. The cells were then washed in the test tube with the binding buffer (3 x 1 mL). The final cell pellet was resuspended in 0.5 mL of buffer. About 30 pL of the resulting cell suspension was dropped onto a glass slide and mixed with the same volume of glycerin. Samples were examined under an fluorescence microscope for visualization of cellular fluorescence. The specimens were first photographed by phase contrast and then under fluorescence conditions. RESULTS
Conjugate Synthesis. PVA of average molecular weight 110 000 has 2500 hydroxy groups that could be derivatized for the synthesis of multivalent conjugates (Scheme 1). However, the limiting factor in these conjugation experiments turned out to be the water solubility of the resulting products at physiologically acceptable pH values. In general, all attempts to make more highly substituted conjugates, both in terms of the number of FITC and the number of ligand molecules per PVA monomer, were met with limited success. From a practical standpoint, introduction of 10- 16 ligand molecules and almost the same number of the FITC molecules per polymer molecule yielded conjugates that were soluble in buffers of low ionic strength, pH 6. When transferred to buffers at physiologicalionic strength, e.g., phosphate-buffered saline (150 mM), the conjugates
slowly precipitated. Once precipitated these conjugates would not go back into solution even in the binding buffer of low ionic strength. For these reasons all the intermediates as well as the final conjugates were stored as solutions. The preparations were observed to be stable for up to 2 months when stored a t 4 "C. Conjugate Binding to Mouse and Human Melanoma Cells. Binding of the conjugates with cell membrane receptors was established within 15 min (Figures 1-4). Binding was extremely strong so that all the incubations and washing protocols could be performed at room temperature. In addition, the binding buffer of pH 5 was utilized to remove any nonspecific binding. This pH had no effect on the specific binding that existed in all the positive controls. This strong binding probably resulted due to the fact that a single assembly of the conjugate is able to interact with more than one receptor on the cell membrane. This phenomenon of cooperative affinity that results in stronger binding properties associated with multivalent ligands has been documented previously in the literature (31, 32). Certain classes of polyvalent antibodies that also are capable of establishing multiple interactions also exhibit much higher binding affinities as the result of cooperative affinity (33). Specificityof Binding. Cleaving the Disulfide Linkage (by Treatment with 2-ME or DTT). The disulfidelinked melanotropin conjugate offered the best possibility of a control experiment to establish the specificity of the fluorescence labeling by the MSH-containing conjugates. Agents such as dithiothreitol (DTT) are able to reduce the S-S bond and therefore release hormone. FITCPVA-MSH (XV) was incubated with DTT (final concentration: 50 mM) for 1h at room temperature with constant shaking. The DTT- or 2-ME-treated conjugate when incubated with B16/F10 mouse melanoma cells failed to label these cells (Table 1). This experiment indicates the specific labeling of melanotropin receptors by FITC-PVA-MSH. When a similar amount of DTT was included in the binding experiment using the thioether linked MSH macromolecule XTV (which is stable to DTT treatment) there was no effect on the fluorescence labeling (Table 1). This ruled out receptor inactivation by DTT during the labeling experiment in the earlier case. Preincubation in the Presence of Unconjugated Free Ligand ("Swamp Out"). It was interesting to note that the binding established between the MSH-containing macromolecules X W or XV with the melanoma cells was so strong that the presence of even millimolar amounts of free ligand [Nle4-~-Phe7]-a-MSH as competitor for the receptors caused no effect on the fluorescence intensity. However, when the cells were pretreated with the free
Melanotropic Peptide Multivalent Fluorescent Conjugates
Bioconjugate Chem., Vol. 5, No. 6,1994 595
Figure 1. Fluorescence labeling of mouse B16F10 melanoma cells by fluorescent melanotropin macromolecular conjugate (FITCPVA-S-MSH, Xnr). Top: phase contrast micrograph. Bottom: fluorescent micrograph. Arrows illustrate the capping phenomenoa exhibited by cells.
ligand (1 x M) for 1h before their exposure to the fluorescent macromolecular ligand XIV, a very dramatic loss of the fluorescence intensity was observed (data not shown). This suggests that either the receptor is blocked (occupied) or it is internalized. Though we do not have direct evidence for which is the case, the results do strongly indicate that the MSH conjugates are highly specific for the melanotropin receptors that interact with [N1e4-o-Phe7]-a-MSH. Binding Studies Using FITC-PVA NII]. This conjugate that lacked any melanotropic ligand failed to bind to any type of cells used under all binding conditions (Table 1).This negative control established the biological inactivity of the fluorescent macromolecular carrier molecule (Figure 5). Other Peptide Conjugates (e.g.,Dynorphin, SubstanceP, and Mercaptoethanol). These three ligands were chosen to act as negative controls to prove the point that
the binding seen with the MSH conjugate is specific for melanotropin receptors. All three of these nonmelanotropic ligands containing fluorescent conjugates (XVI, XVII, and XVIII) failed to label B-16 mouse melanoma cells (Table 1). Cellular Specificity (Other Cell Types). A few cell types of nonmelanogenetic origin (Table 1)were included in this study to act as negative controls for the demonstration of melanoma-specific melanotropin receptors. As is evident from the results, human small cell lung cancer (NCLN592) and breast cancer cells (MCF-7) did not exhibit any fluorescent labeling after incubation with the fluorescent MSH conjugate (Table 1). Normal mouse liver, kidney, and spleen cells also failed to exhibit fluorescence following incubation with the conjugate. Homogeneity of Conjugate Binding to Melanoma Cells and CappingDnternalization of the Ligand. During the fluorescence labeling experiments it was
596 Bioconjugate Chem., Vol. 5, No. 6,1994
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Figure 2. B-16/F-10 mouse melanoma cells showing capping (or internalization) of MSH receptors visualized by specific binding of FITC -PVA-SS-MSH [xvl. Top: phase contrast micrograph. Bottom: fluorescent micrograph.
evident that all the cells in each and every melanoma cell line were uniformly labeled by the melanotropin conjugates XI1 or XI11 (Figures 1-5). This suggests cellular homogeneity of all the melanoma cell lines with respect to melanotropin receptor expression. Some of the cells (in the case of both mouse and some human melanomas) that were oriented favorably (microscopically) t o the observer exhibited capping of the receptors (Figures 1, 2, and 4). In certain instances the fluorescence appeared to be located intracellularly suggesting internalization of the receptor-ligand (macromolecule) complex (Figures 2 and 4). These observations are consistent with earlier experiments with fluorescence labeling of MSH-receptors using fluorescent tobacco mosaic virus-MSH peptide conjugates (32, 34). The capping and internalization phenomena were further examined using the disulfide-linked melanotropin macromolecular conjugate X V . In a fluorescence labeling experiment using this ligand a portion of the labeled cells
was incubated for 30 min in the binding buffer at pH 7.2 that also contained 50 mM DTT. As DTT cleaved the disulfide linkage, thereby removing the MSH molecules from the polymer, cell membranes were expected t o lose the fluorescence. In the case of cells showing intracellular fluorescence, no loss of the fluorescence was observed. This clearly was indicative of the internalized receptor ligand complex. DISCUSSION
The studies reported here using fluorescent microscopic visualization methods demonstrate specificity of the fluorescent melanotropin-PVA macromolecular conjugate (MSH-PVA-FITC) for melanotropin receptors associated with melanoma cells (Table 1). The specificity of receptor-mediated binding was demonstrated by the following observations: (1)The positive control, mouse B16/F10 melanoma cells (known to express MSH receptors), strongly bound the conjugate. (2) The negative
Melanotropic Peptide Multivalent Fluorescent Conjugates
L
Bioconjugafe Chem., Vol. 5, No. 6,1994 597
I
an amelanotic human melanoma cell line (JH) showing cellular homogeneity of MSH receptors. Top: phase contrast micrograph. Bottom: fluorescent micrograph.
Figure 3. Binding of FITC-PVA-S-MSH
[XIV] to
control, MCF-7 human breast cancer cells and NCI-N592 small cell lung cancer cells (both assumed not to express MSH receptors), failed to bind to the conjugate. In addition, a number of other cell lines of nonmelanocytic origin did not bind to the conjugate (data not shown). 3) Incubation of FITC-PVA alone (no hormone attached), or incubation of a fluorescent dynorphin or substance-P conjugate, did not result in binding of the conjugates to the cells (Table 1). (4) Pretreatment of XV, a disulfide linked melanotropin-conjugate (MSH-SS-PVA-FITC), with reducing agents (dithiothreitol or 2-mercaptoethanol), resulted in cleavage of the bond between the PVA backbone and the hormone, and this treatment resulted in a conjugate (now lacking ligand) that did not bind to receptors. (5) If binding is specific to melanotropin receptors, one would expect that the presence of the conjugated analog would compete with the unbound ligand ([Nle4-~-Phe71-a-MSH) for receptor binding. In competition experiments preincubation of melanoma cells
with micromolar concentrations of [Nle4-~-Phe71-a-MSH effectively blocked the binding of the fluorescent conjugate XlV to the binding sites on the melanoma cell membrane (data not shown). Receptor internalization is one of the processes by which cells recycle (or degrade) receptors. Internalization has been observed for several different types of receptors (e.g.,35-37). The "capping" phenomenon is believed to indicate receptor internalization (endocytosis). Similar receptor aggregation has been observed for some other polypeptide hormones (e.g.,38-40). For example, Jarett and Smith (38) found insulin receptor aggregation on adipocyte plasma membranes by using a ferritin-insulin conjugate in conjunction with transmission electron microscopy. De Priester et aZ. (39) reported capping of concanavalin (Con A) receptors on Dictyostelium cell membranes after incubation of cells with fluorescent Con A. This capping phenomenon was also observed using our
598 Bioconjugate Chem., Vol. 5, No. 6,1994
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Figure 4. Capping of MSH receptors observed upon specific binding of FITC-PVA-S-MSH [XIVIwith MSH receptors in a human melanoma cell line (LR 1649). TOD: Dhase contrast micromaDh. - - Bottom: fluorescent micrograph. Arrows illustrate the capping phenomena exhibited by cells. L
A
fluorescent conjugate (Figures 1, 3 and 4) and was observed on the surface of some cells of all human melanoma cell lines (Figure 4). The results suggest that capping may be a phenomenon involving one or more focal aggregates of receptors. These observations suggest that MSH receptors become internalized. These observations thus provide further evidence for the presence of melanotropin receptors on the cell surface of human melanoma cells. The observation of melanotropinreceptor internalization was first reported by Varga et al. (41). By using a fluorescent-labeled P-MSH, the authors found that the hormone was internalized a t a physiological temperature in Cloudman (murine) melanoma cells. Later, Lerner et al. (42), using ferritinlabeled P-MSH, found that some of the internalized hormone may be further translocated to premelanosomes. Recently, Orlow et al. (43) demonstrated that receptor internalization resulted in translocation of the hormone
t o internal binding sites within the cells. In these latter experiments the authors used 1251-labeledP-melanotropin (P-MSH) and sucrose density centrifugation techniques to determine the internal binding sites for MSH. GarciaBorron et al. also have provided data that [1251]-[Nle4-~Phe7]-a-MSH is internalized in B16/F10 mouse melanoma cells (10). In our experiments, we did not investigate the time course for the formation and the duration of capping on the cell surface. This future study should provide more information as to the kinetics of the capping phenomenon and, therefore, may help us to better understand the metabolism or recycling of melanoma melanotropin receptors. Our unique and relatively simple methods for the visualization of ligand-receptor membrane capping may prove to be a useful tool for the study of other cell types and their specific hormone receptors. Our results are consistent, in part, with previous work
Melanotropic Peptide Multivalent Fluorescent Conjugates A
Figure 5. FITC-PVA-S-MSH
[XIV] binds to human melanotic melanoma cell line (A375P). A Phase contrast micrograph. B: Fluorescent micrograph. C: FITC-PVA MI],a conjugate that lacks [Nle4-~-Phe7]-a-MSH does not bind to A375P cells. Bar = 100 pm.
reported by a number of other investigators. Ghanem et al. (15)found that three out of eight human melanoma cell lines displayed specific binding of [1251]-a-MSHto the cells. Siegrist et al. (7)reported that 10 out of 12 human melanoma cell lines showed specific binding sites for [1251]-a-MSH. Using a n in situ binding technique to demonstrate melanotropin receptors in surgical specimens of human melanoma, Tatro et al. (16)reported that among the melanoma specimens from 11patients, three of them showed a high level of specific binding, five a low level binding, and another three no detectable binding. Although the radioligand binding technique allows indirect estimation of binding sites for radiolabeled agents in target cells or tissues, the results generated from the binding assay are in general based upon the mass binding of the radioactive probe to a population of cells. This technique is unable to provide information about the receptor status of a n individual single cell. In
Bioconjugate Chem., Vol. 5, No. 6, 1994 599
addition, these previous radioligand technologies may not be sensitive enough for those cells with a low number of receptors or low numbers of cells actually expressing receptors at the time (cell cycle) of the experiment. Hence, it appears that our visualization methods may offer a unique advantage over other presently used methods to study the homogeneity or heterogeneity of melanotropin receptor distribution. Several previous reports have indicated that melanotropin receptors may also exist in some tissues other than cutaneous tissue. Tatro and Reichlin (12) showed that MSH receptors were widely distributed in vivo in the tissues of rodents including glandular organs, adipose tissues, and bladder. However, in our experiments, the melanotropin-macromolecular conjugates failed to bind to a number of nonmelanotic cells, such as human small cell lung cancer cells, endometrial carcinoma (data not shown), breast cancer MCF-7 cells and some normal mouse cells, such as spleen cells as well as liver cells (Table 1). Similar results were reported by Siegrist et al. (7)where melanotropin receptors were‘found to be specifically present in human and mouse melanoma cells, but not in five other human neuroblastoma or glioma cell lines. The discrepancy between our results and others as to melanotropin receptor distribution in different tissues remains to be clarified. It has been suggested by one research group that MSH receptors are predominantly expressed in the G2 phase of the cell cycle (41,44).Other investigations have been unable to confirm such G2 restriction (45,46). In the present studies we noted that our melanotropin conjugates were bound to every individual mouse or human melanoma cell. Since the cells were not synchronized, it is assumed that binding (30min incubation) to the cells occurred during each phase of the cell cycle. It was also reported earlier that at least some of the responses to MSH (increased cyclic AMP production and tyrosinase activity) occur in the G2 phase of the cell cycle (47), the reasons being that the receptors for MSH are most active at this time in the cycle. In fact, it is claimed that melanoma cells “regulate their response to MSH by the discontinuous appearance of receptors for the hormonal signal” (48).Although our results do not restrict the ability of cells to bind MSH to the G2 phase of the cycle, the data provides no information on the functional ability (to transduce signals and effect second messenger formation) of melanotropin receptors throughout the cycle. SUMNLARY AND PERSPECTIVES
We have designed a new class of multivalent peptide hormone macromolecular composites which may serve as powerful diagnostic, imaging, and therapeutic tools. Composites have been synthesized in which multiple copies of a biospecific ligand (e.g., a hormone) were covalently attached to a biologically compatible but inert polyfunctional macromolecule. In addition, the conjugation of multiple copies of a fluorophore directly to the macromolecule provided a n enhanced visual means of detection of ligand-macromolecular conjugates bound to target cells in in vitro binding assays. A fluorescent melanotropin macromolecular conjugate has been synthesized and used to demonstrate the presence of specific melanotropin receptors on various human melanoma cell lines. Most importantly, every cell of every melanoma cell line possessed melanotropin receptors as visualized by fluorescence microscopy. Cells of nonmelanocyte origin did not exhibit such receptors. These cell specific melanotropin receptors may serve as cell surface markers for melanoma. Fluorescent melanotropin conjugates
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should be useful in determining w h e t h e r all (primary and metastatic) tumors possess s u c h receptors. These recept o r s may provide targets for the identification, localiza-
Sharma et al.
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