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Cell Growth & Differentiation Vol. 10, 517-524, July 1999
© 1999 American Association for Cancer Research

Acquired Melanocyte Stimulating Hormone-inducible Chemotaxis following Macrophage Fusion with Cloudman S91 Melanoma Cells1

Michael Rachkovsky and John Pawelek2

Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06520


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Fusion of Cloudman S91 melanoma cells with macrophages results in hybrids with increased metastatic potential. Here, we report that such hybrids acquire new pathways for motility. Compared to parental melanoma cells and low metastatic hybrids, the metastatic hybrids showed far stronger responses to 3T3- and lung fibroblast-conditioned media, primary lung slices, fibronectin (FN), and a Mr 120,000 FN fragment and, unlike parental cells, were further stimulated by pretreatment with melanocyte-stimulating hormone/1-methyl-3-isobutylxanthine. Hybrid migration was due primarily to chemotaxis, with chemokinesis being a minor component. Thus, the metastatic hybrids acquired melanocyte-stimulating hormone-inducible motility, perhaps reflecting the FN fragment chemotaxis of macrophages. The results support a long-standing hypothesis that metastasis is initiated following hybridization between tumor-invading phagocytes and cells of the primary tumor.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Recently, we analyzed a number of traits in a panel of artificial fusion hybrids between normal mouse or human macrophages and a line of weakly metastatic Cloudman S91 cells (1 , 2) , along with a naturally occurring hybrid, PADA, formed in vivo within a Cloudman S91 tumor and a DBA/2J mouse tumor-infiltrating cell of unknown origin (3) . The majority of hybrids, including PADA, showed enhanced metastatic potential compared to parental Cloudman S91 cells (1) . Moreover, hybrids of highest metastatic potential also tended to be supermelanotic and more motile and to show enhanced responses to MSH3 /IBMX (1 , 2) . They also showed increased expression of mRNA for MC-1 receptors and increased MSH binding activity.4 Investigations into the molecular basis of these changes revealed that the metastatic hybrids appeared to have acquired a macrophage-like N-glycosylation system as at least one underlying cause of the multiple phenotypic changes (2) . The metastatic hybrids showed enhanced incorporation of [3H]glucosamine into total proteins as well as specific proteins LAMP-1 and tyrosinase that was further stimulated by MSH/IBMX. In addition, the metastatic hybrids showed increased expression of mRNA and enzymatic activity for ß-1,6-N-acetylglucosaminyltransferase V (EC 2.4.1.155), which catalyzes formation of ß-1,6 branches on the trimannosyl terminus of AspN-linked oligosaccharides, allowing for the addition of ß1-6GlcNAc-linked poly-N-acetylactosaminyl chains, a feature of a variety of malignant cells as well as normal granulocytes and monocytes (4, 5, 6, 7, 8, 9, 10, 11, 12 .5 Together, these results support a long-proposed hypothesis on hybridization as a source of metastatic cells within tumors. Within this concept, the increased metastasis of such hybrids would result from coexpression of traits of motility and homing from the phagocytic parent, coupled with deregulated growth from the neoplastic parent (reviewed in Refs. 1 and 2 ).

In previous studies, we also showed that, when parental melanoma cells were compared to metastatic and nonmetastatic hybrids, the metastatic hybrids displayed enhanced migration toward 3T3-CM in an in vitro assay system (1) , another common trait of metastatic cells (13, 14, 15, 16) . Here, we examined the motility characteristics of parental and hybrid cells in more detail, studying chemoattraction to different CM, FN, and a Mr 120,000 FN fragment (FN120), as well as the effects of pretreatment with MSH and IBMX on migration and morphology.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Motility of Hybrids with Different Metastatic Potentials.
Motility in the Costar (Corning, NY) Transwell system was compared for parental Cloudman S91 melanoma cells, three low metastatic and two high metastatic macrophage x melanoma polyethylene glycol (PEG) fusion hybrids, and PADA, a high metastatic, naturally occurring hybrid, with or without pretreatment with MSH/IBMX (Table 1)Citation . Regardless of metastatic potential, each of the hybrids showed elevated basal motility compared to the parental cells; however, this was most pronounced in the three high metastatic hybrids 95-H1, 95-H2, and PADA, and motility of these hybrids was strongly induced by MSH/IBMX. Low metastatic hybrid 95-H19 showed a small but significant (P < 0.02) stimulation of motility by MSH/IBMX. Ps for differences within each cell line between control and MSH/IBMX treatment are shown in Table 1Citation . Differences in motility between parental Cloudman S91 cells and low metastatic hybrids in the absence of MSH/IBMX were each significant to P <= 0.01 and for Cloudman S91 cells and high metastatic hybrids to P <= 0.001. The migration of high metastatic hybrids was significantly different from low metastatic hybrids (control, P < 0.05-P < 0.0001; with MSH/IBMX, P < 0.001-P < 0.0001). In summary of a number of experiments, MSH/IBMX pretreatment either had no effect or inhibited migration of parental Cloudman S91 cells, whereas it consistently stimulated migration of the metastatic hybrids with little or no effect on the low metastatic hybrids.


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Table 1 Metastatic potential in vivo vs. migration in vitro to the underside of a Costar Transwell apparatus of parental Cloudman melanoma cells, various macrophage x melanoma PEG fusion hybrids, and PADA, a naturally occurring hybrid, in response to 3T3-CM (33%, v/v) in the lower chambers

 
Photomicrographs of the underside of the upper chambers at 4 h postinitiation of the assay revealed, even in the absence of MSH/IBMX, dramatic differences in the number of migrated cells for metastatic hybrid 95-H1 in comparison to the parental Cloudman S91 cells (Fig. 1, a and b)Citation .



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Fig. 1. Migrated cells on the underside of the Costar Transwell separating membrane following 4 h exposure to 33% (v/v) 3T3-CM (a and b) or lung slices (c and d) in the lower chamber. Stained cells are visible along with the 12-µm pores of the polycarbonate membrane. a, 3T3-CM, parental S91 melanoma cells; b 3T3-CM, metastatic hybrid 95-H1; c, lung slices, parental S91 cells; d, lung slices, hybrid 95-H1 cells. Arrows (c and d), hematoxylin-stained areas where the lung tissue adhered to the underside of the upper chamber polycarbonate membrane. Migration for c and d is quantitated in Table 4Citation . Scale bar, 200 µm.

 
Kinetics.
Migration kinetics of parental Cloudman cells and metastatic hybrid 95-H1 were studied over 6 h in response to gradient conditions, with 3T3-CM in the bottom chamber and plain medium in the upper chamber, with or without pretreatment with MSH/IBMX (Fig. 2)Citation . As shown above, in the absence of MSH/IBMX, both the rate of migration and the total number of cells migrated were higher in the hybrid than in the parental cells. Pretreatment with MSH/IBMX markedly stimulated both the rate and extent of migration of the hybrid but had little effect on migration of parental cells.



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Fig. 2. Kinetics of migration of S91 melanoma cells and metastatic hybrid 95-H1 in a Costar Transwell system. Before the motility assay, cells were grown for 72 h in either plain culture medium or medium containing MSH/IBMX. Cells (5 x 104) in serum-free culture medium or medium containing MSH/IBMX were seeded into the upper chamber and 3T3-CM (33% v/v) was added to the lower chamber of the Costar Transwell system. Migrated cells on the underside of membrane filter were counted over 6 h at the times shown. Data points, means for triplicate assays; bars, SE. Where error bars are missing, the SE fell within the radius of the symbol. {square}, parental Cloudman S91 control; {blacksquare}, parental Cloudman S91 plus MSH/IBMX; {circ}, macrophage/melanoma hybrid 95-H1 control; •, macrophage/melanoma hybrid 95-H1 plus MSH/IBMX. The experiments were repeated with similar results.

 
Chemotaxis versus Chemokinesis.
Assays with chemoattractants in various combinations between the upper and lower chambers of the Costar Transwell system were used to distinguish between chemotactic and chemokinetic motilities of parental Cloudman S91 and hybrid 95-H1 cells (17, 18, 19) . With no MSH/IBMX treatment, in the absence of 3T3-CM, i.e., plain medium in both chambers, there was little migration for either cell line (Table 2A)Citation . Maximum migration for both parental Cloudman S91 cells and hybrid 95-H1 occurred with 3T3-CM in the bottom chamber and plain medium in the upper chamber, i.e., under conditions of a concentration gradient. When 3T3-CM was present in equal concentrations in the two chambers, migration to the underside was moderately increased over that in plain medium for hybrid 95-H1, an indication of chemokinetic motility, but little change in parental cell motility was seen. Following pretreatment with MSH/IBMX (Table 2B)Citation , migration of hybrid 95-H1 cells was enhanced to various degrees in all combinations of attractants, whereas migration of parental cells was either unaffected or in most experiments inhibited. The results demonstrated that, for both the parental and hybrid cell lines, chemotaxis was the major component, with chemokinesis being a minor component of the hybrids. MSH/IBMX treatment of hybrid 95-H1 cells stimulated predominantly the chemotactic component, although a small stimulation of chemokinetic motility was evident.


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Table 2 Migration of parental Cloudman S91 melanoma cells and metastatic macrophage x melanoma hybrid 95-H1 in response to 3T3-CM (100%, v/v) in various upper/lower chamber combinations, with and without prior treatment in culture for 72 h with MSH/IBMX

 
Responses to Lung-CM.
Motility responses to lung-CM were carried out under gradient conditions comparing parental Cloudman S91 cells to metastatic hybrid 95-H1 (Table 3)Citation . The hybrid showed enhanced motility compared to parental cells and was stimulated by pretreatment with MSH/IBMX. In contrast, parental Cloudman S91 cells were inhibited by pretreatment with MSH/IBMX throughout the range of doses.


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Table 3 Response of parental Cloudman S91 vs. metastatic hybrid 95H-1 to primary culture lung-CM with or without pretreatment with MSH/IBMX

 
Migration to Organ Slices.
Migration of cells was further studied in the Costar Transwell system, where 2–4-mm3 pieces of lung or liver from DBA/2J mice were placed in the lower chambers, such that they made contact with the underside of the upper chamber. Four h after initiation of the experiment, the membranes of the upper chambers were removed, fixed, stained, and mounted on slides, and the undersides were photographed by light microscopy. Areas of contact between the tissue and the underside of the upper chamber stained readily with hematoxylin. Neither hybrid 95-H1 nor parental S91 cells migrated to liver (data not shown), and only a few parental S91 cells migrated to lung (Fig. 1c)Citation . However, 95-H1 hybrids were strongly attracted to lung and were localized in or near the lung/filter adhesion areas (Fig. 1d)Citation . Furthermore, 95-H1 cells that were attracted to lung slices assumed a more bipolar morphology than did those that were attracted to 3T3-CM. Quantification of migrated cells to lung and liver pieces is shown in Table 4Citation .


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Table 4 Effects of lung and liver slices from DBA/2J mice on migration

 
FN and FN120 as Chemoattractants.
Human plasma FN and a Mr 120,000 NH2-terminal FN fragment (FN120) derived from it were tested as chemoattractants for hybrid and parental melanoma cells (Table 5)Citation . Parental S91 melanoma cells were weakly attracted by FN but not by FN120. MSH/IBMX inhibited the parental melanoma FN response. In contrast, hybrid 95-H1 cells were strongly attracted to both FN and FN120, and this was increased by pretreatment of 95-H1 cells in culture with MSH/IBMX. Inclusion of FN120 (100 µg/ml) in both the lower and upper chambers of the Costar Transwell apparatus resulted in no migration of 95-H1 to the underside, indicating that FN120 induced chemotaxis, i.e., directional migration along a FN120 concentration gradient, but not chemokinesis. Although 3T3-CM appeared to be a stronger chemoattractant than FN and FN120 in these experiments, direct comparisons of the potencies were not valid because we do not know the active component(s) of 3T3-CM, and the 3T3-CM concentrations used were relative, in this case, 33% (v/v), based on the dilution with DMEM.


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Table 5 Migration of parental S91 cells and hybrid 95-H1 to the underside of the upper chamber of a Costar Transwell system in response to 3T3-CM, human plasma FN, or a Mr 120,000 fragment (FN120) of human plasma FN, with or without pretreatment with MSH/IBMX for 72 h

 
Diffusion of Chemoattractants.
Regarding diffusion rates from the lower to upper chambers of the Transwell system, we have not yet determined the nature of the chemoattractants in the CM and did not attempt to measure the FN and FN120 diffusion rates. However, using spectrophotometric measurements of two model compounds under simulated motility assay conditions, equilibrium between chambers was reached in 45 min by phenol red (molecular mass = <103 Da) and 75 min by blue dextran (molecular mass = >106 Da), and it seemed reasonable to assume that the diffusion kinetics for chemoattractants, including FN and FN120, would fall into this time frame (data not shown).

Dendrite Number.
It has been shown that MSH and IBMX cause increased dendrite formation of Cloudman S91 melanoma cells (20, 21, 22) , a phenomenon that is likely to be associated with increased motility (23) . Here, it was shown that, whereas MSH/IBMX had little effect on dendrite number of the weakly metastatic parental Cloudman S91 melanoma cells used in these studies, the same treatment resulted in a 4-fold increase in dendrites per cell of both metastatic hybrid 95-H1 and of peritoneal macrophages from DBA/2J mice (Fig. 3Citation and Table 6Citation ).



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Fig. 3. Effects of MSH/IBMX on morphology and dendrite formation. Parental Cloudman S91 cells (a and b) and metastatic hybrid 95-H1 (c and d) were cultured in monolayer for 72 h with (b and d) or without (a and c) MSH/IBMX, as described in "Materials and Methods." The number of dendrites per cell from this experiment is shown in Table 6Citation . Scale bar, 100 µm.

 

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Table 6 Effects of MSH/IBMX on dendrite numbers per cell

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We show that highly metastatic macrophage x melanoma fusion hybrids express a motility system that, unlike that of the parental Cloudman S91 melanoma cells, is stimulated by MSH/IBMX. Although parental Cloudman S91 cells exhibited a certain level of chemotaxis when exposed to 3T3-CM, lung-CM, or FN concentration gradients, attraction to these agents was far stronger in the metastatic hybrids and, unlike parental cells, stimulated further by pretreatment with MSH/IBMX. In addition, parental Cloudman melanoma cells showed no response to FN120, whereas migration of hybrid 95-H1 was stimulated by FN120. Inclusion of chemoattractants in both upper and lower chambers of the Costar Transwell system revealed that chemotaxis was the principle component of both parental and hybrid motility toward 3T3-CM and lung-CM, with chemokinesis accounting for a smaller portion. Migration toward FN120, seen only in the metastatic hybrids, appeared to be solely through chemotaxis. Likewise, MSH/IBMX treatment stimulated predominantly chemotaxis in the metastatic hybrids. Increased motility of the metastatic hybrids paralleled the number of basal and MSH-/IBMX-inducible dendrites per cell.

Pretreatment of parental Cloudman S91 cells with MSH/IBMX either had no effect or, as was usually the case, was inhibitory to migration, whereas treatment of hybrids with elevated metastatic potential was strongly stimulated by MSH/IBMX, consistent with several previous reports suggesting that MSH and/or its cyclic AMP second messenger systems may be involved in melanoma metastasis. For example, highly metastatic subclones of the B16 melanoma were shown to have severalfold higher intracellular cyclic AMP levels than weakly metastatic clones (24) . MSH treatment of B16 melanoma cells in culture increased lung colonization abilities following i.v. injection of the cells into the tail (25 , 26) . MSH receptors were detected on human melanoma cells growing in culture and on human melanomas in situ (27 , 28) . Furthermore, the production of MSH itself has been correlated to the increased metastatic potential of mouse B16 melanoma cells (29 , 30) .

MSH receptors have also been shown to be expressed by monocytes and macrophages in humans and mice, and both MSH and the closely related adrenocorticorticotropic hormone peptides influence numerous functions in these cells (31, 32, 33, 34) , including stimulation of motility (35) . It is well established that MSH activation of melanogenesis throughout the vertebrates is mediated through binding to MC-1 receptors, eliciting up-regulation of G-protein linked, adenylate cyclase/cyclic AMP-dependent protein kinase systems (21 , 36, 37, 38, 39, 40) . It is, therefore, notable that many phenotypic traits of monocytes and macrophages are also regulated through elevated cyclic AMP (41, 42, 43, 44, 45, 46) , and at least in some cases, this occurs through MSH binding to MC-1 receptors (31) . In addition to expressing MC-1 receptors, macrophages, melanocytes, and melanoma cells synthesize proopiomelanocortin and produce MSH from it (29, 30, 31 , 47, 48, 49, 50) . Indeed, we show here that peritoneal macrophages from DBA/2J mice respond to MSH/IBMX with 4-fold increases in dendrite expression in primary culture (Table 6)Citation . Thus, the common expression of MSH-sensitive MC-1 receptors as well as the production of MSH by both macrophages and melanoma cells could help explain the accentuated responses to MSH by macrophage x melanoma hybrids with regard to motility, melanogenesis, and metastasis (1 , 2) .

Macrophages are among the most motile of all cells (23) , and it is, therefore, tempting to speculate that the metastatic hybrids acquired enhanced chemotaxis through the expression of this macrophage trait after fusion. The metastatic hybrids expressed enhanced chemotaxis toward FN and FN fragments, and it is well established that monocytes, macrophages, and many metastatic cancer cells are attracted by these and other extracellular matrix proteins (19 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71) . Norris and colleagues (53 , 60) suggested that FN fragments, released from sites of tissue injury by locally active proteases, attract circulating monocytes to these areas and that FN deposited in the tissue stroma acts as an adhesive matrix to promote monocyte accumulation, as shown by Bevilaqua et al. (72) . Therefore, metastasis of macrophage x melanoma hybrids to, for example, lung tissue in vivo, could reflect expression of this normal, developmentally determined monocytic capacity to respond chemotactically to circulating lung FN fragments and, once reaching the lung, then adhere to native lung matrix proteins.

It is interesting to note that although the Cloudman S91 melanoma was originally isolated as a soft tissue tumor at the base of the tail, subsequent studies showed that it had a high tendency for metastasis to the lung (73 , 74) . The Cloudman S91 subline used as a parent in our hybrid construction was only weakly metastatic, but when it did metastasize, like its ancestral tumor cells, it showed a clear preference for lungs (1) . Likewise, although we observed metastasis to many different organs by our macrophage x melanoma hybrids, lung tissue was the preferred target. These observations were paralleled in our in vitro motility studies showing a strong attraction to lung-CM as well as to cut pieces of lung by the hybrids. Lung tissue also elicited morphological changes in hybrid 95-H1 that were not seen with 3T3-CM (Fig. 1)Citation . Furthermore, we found that, although hybrid 95H-1 was attracted to lung tissue in vitro, it was not attracted to similar preparations of from liver, heart, or spleen (data not shown). We did not carry out histological studies of the binding of hybrids to lung tissue per se and, therefore, do not know the type of cells, e.g., lung endothelium, that were adhered to in vivo.

Although we do not yet know the mechanisms underlying enhanced motility in the metastatic hybrids, our evidence regarding increased melanogenesis in these same hybrids suggests that expression of macrophage-associated glycosylation pathways may be involved (2) . The possible role of protein glycosylation as a basis for increased migratory activity of the metastatic hybrids is currently under investigation in our laboratory.

Our work has focused primarily on artificially constructed hybrids; however, we emphasize that the PADA cell line (3) , a naturally occurring hybrid between a Cloudman S91 tumor cell and a tumor-invading host cell, exhibits all of the traits distinguishing it from parental Cloudman cells that we have identified in the artificial hybrids (1 , 2) , namely, enhanced metastatic potential, melanogenesis, motility, and MSH responsiveness. Because these traits are characteristic of macrophages and metastatic melanoma cells alike and in light of the fact that primary tumors are generally infiltrated with phagocytes and intratumoral hybrids are known to be generated, our results on enhanced chemotaxis of such hybrids provide support for the hybrid model as a mechanism for metastatic transformation. Although formal proof of this model in human metastasis remains elusive, our results indicate that it deserves serious consideration as a potential initiating event in the metastatic transformation of melanoma and other primary tumor cells.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells and Culture.
Cells were maintained in Ham’s F-10 or DMEM nutrient media with 10% horse or fetal bovine serum, in a gassed, humidified incubator (5% CO2 in air, 37°C). The parental Cloudman S91 cell line, PS1-HGPRT--1 (hypoxanthine-, aminopterin-, and thymidine-sensitive and G418-resistant) and peritoneal macrophages, used as fusion partners as well as for the isolation and maintenance of macrophage x melanoma hybrids, were as described previously (1 , 75) .

Migration Assay.
To assess motility, we seeded cells (5 x 104 per 0.5 ml of culture medium without serum) into Costar Transwell cell culture chamber inserts (12-µm pore diameter) and placed them into wells containing 1.5 ml of DMEM nutrient medium without serum and with chemoattractants as noted, in a gassed, humidified incubator. At various times up to 6 h, the inserts were withdrawn, cells on the upper surface were removed with a cotton swab, and the cells on the underside were fixed with methanol and stained with hematoxylin. The membrane filters were cut with a scalpel and mounted on slides. Migration was expressed as the total cells counted in 10 microscopic fields with a light microscope at x430 magnification.

Treatment with MSH/IBMX.
Prior to some migration assays, cells were exposed in culture for 72 h to ß-MSH (2 x 10-7 M) in conjunction with the cyclic nucleotide phosphodiesterase inhibitor IBMX (10-4 M) to potentiate the MSH-mediated elevation of cyclic AMP (75) . In experiments with MSH-/IBMX-pretreated cells, MSH and IBMX were also included in both the upper and lower chambers of the Costar Transwell motility assay system.

Chemoattractants.
FN and a Mr 120,000 NH2-terminal FN fragment (FN120) from human plasma (76 , 77) , were from Life Technologies, Inc. The production of CM from 3T3 Swiss albino cell cultures was as described previously (1) . For preparation of lung-CM, DBA/2J mice were euthanized, and their lungs were removed, gently rinsed three times with DMEM, minced into <=1-mm3 slices with scissors, and placed into Corning 100-mm tissue culture Petri dishes in 30 ml of DMEM with 10% fetal bovine serum, in a gassed (95% air-5% CO2), humidified 37°C incubator. After 24 h, larger slices of lungs were removed, and the medium was replaced every 2 days. After 7–10 days, adherent cells were removed by trypsinization and plated in 175-cm2 flasks. Primary mouse lung cells thus obtained as well as 3T3 Swiss albino cells were grown in 175-cm2 flasks to {approx}75% confluence. For production of CM, cells were exposed to DMEM without serum for 24 h. In another procedure, minced slices of mouse lung, liver, spleen, and heart were prepared and rinsed three times in DMEM without serum, and 30 ml of fresh DMEM were added to Petri dishes and conditioned for 24 h. In all cases, CM were centrifuged at 3000 x g for 10 min, and the supernatants were frozen and stored at -70°C.

Organ Slices.
One piece of lung or liver ({approx}2–4 mm3) was placed in a well of a Costar Transwell system described above containing 1.5 ml DMEM without serum directly before the beginning of the motility assay and was kept there for the duration of the 4-h experiment. Cells migrating to the underside of the 12-µm pore membrane filter were counted in 10 consecutive microscopic fields in the area along the periphery of contact between the underside of the membrane and the piece of the organ. Adhering material from the organ piece in this area of the filter was stained by hematoxylin after the organ slices were removed.

Dendrites.
The number of dendrites per cell was determined by counting dendrites on a minimum of 30 cells in phase contrast photographs of monolayer cultures. Results were confirmed independently by two observers.


    Acknowledgments
 
We are grateful to Dr. Ashok Chakraborty for data on macrophage dendrite numbers; Jeffry Oestreicher for determining diffusion rates in the two-chambered system; Dr. Ruth Halaban for providing the PADA cell line; and Stefano Sodi, James Platt, Dr. Jean Bolognia, Dr. Brooks Low, and Dr. David Bermudes for helpful discussions.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from Vion Pharmaceuticals, Inc. (New Haven, CT). Back

2 To whom requests for reprints should be addressed, at Department of Dermatology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: (203) 785-4411; Fax: (203) 785-7637; E-mail: john.pawelek{at}yale.edu Back

3 The abbreviations used are: MSH, melanocyte-stimulating hormone; IBMX, 1-methyl-3-isobutylxanthine; MC-1, melanocortin-1; CM, conditioned media; FN, fibronectin. Back

4 A. Chakraborty, Y. Funasaka, M. Ichihashi, S. Sodi, and J. Pawelek. Macrophage fusion up-regulates expression of genes for the melanocortin-1 receptor but not for melanogenic proteins in Cloudman S91 melanoma cells, submitted for publication. Back

5 A. Chakraborty, Y. Ikeda, E. Miyoshi, M. Ichihashi, N. Taniguchi, and J. Pawelek. Hybridization with macrophages up-regulates expression of the N-acetylglucosaminyltransferase V gene, ß1–6 branching, and metastasis in Cloudman S91 mouse melanoma cells, submitted for publication. Back

Received for publication 1/27/99. Revision received 4/23/99. Accepted for publication 4/26/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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