Expression of Melanoma-Associated Antigens by Normal and Neurofibroma Schwann Cells1

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(CANCER RESEARCH 46, 5887-5892, November 1986] Expression of Melanoma-associated Antigens by Normal and Neurofibroma Schwann Cells1

Alonzo H. Ross,2 David Pleasure, Kenneth Sonnenfeld, Barbara Atkinson, Barbara Kreider,3 Donna M. Jackson, Ingrid Taff, Elio Scarpini, Robert P. Lisak, and Hilary Koprowski The Wistar Institute of Anatomy and Biology [A. H. R., D. M. J., H. K.]; The Children's Hospital of Philadelphia, (D. P., B. K.J; Hospital of the University of Pennsylvania [B. A., R. P. L.J Philadelphia, Pennsylvania 19104; Mt. Sinai School of Medicine, New York, New York 10029 Â¡K.S., I. TJ; and University of Milan School of Medicine, Milan, Italy [E. S.J

ABSTRACT reported that the staining pattern of human peripheral nerves and neurofibromas with anti-NGF MAbs suggested that normal The cell surface antigen distribution on traumatic neuroma Schwann Schwann cells and Schwann-like cells in neurofibromas ex cells and neurofibroma Schwann-like cells was characterized using mono pressed the NGF receptor (8) and that Schwann-like cells clonal antibodies that define melanoma-associated antigens. Immunoflu- orescence staining of cultured cells, immunoprecipitation of radioiodi- specifically bind NGF (9). nated antigens from cells placed in short-term cultures, and immunoper- To better understand the growth and immunological prop oxidase staining of frozen tissue sections revealed most of the melanoma- erties of normal and neurofibroma Schwannian cells and their associated antigens tested on traumatic neuroma and neurofibroma correlation, if any, with tumor progression, we have analyzed Schwann cells and on fetal and adult femoral nerve. The cross-reactivity the antigenic profiles of these cells using anti-melanoma MAbs. of the antibodies with neural cells may reflect the common neural crest We report here that traumatic neuroma Schwann cells and embryological origin of Schwann cells and ntelanocytes. Cell sorter neurofibroma Schwann-like cells express many of the antigens analysis of neurofibroma cells using a monoclonal antibody directed detected with anti-melanoma MAbs. These anti-melanoma against the melanoma nerve growth factor receptor resulted in cell MAbs also exhibited reactivity in femoral nerve frozen tissue cultures highly enriched for Schwann-like cells which may bear the sections. The cross-reacting antigens detected on the Schwann genetic defect responsible for neurofibromatosis. The antigen detected by cells are similar to the corresponding melanoma-associated this monoclonal antibody is the neurofibroma nerve growth factor recep antigens as judged by SDS-PAGE. These results define new tor and the antibody was a potent inhibitor of nerve growth factor binding to neurofibroma cells. cross-reactivities of the anti-melanoma MAbs with peripheral nerves which are relevant to future therapeutic and diagnostic applications of the MAbs, identify tumor-associated antigens INTRODUCTION present on neurofibroma cells, provide a means by cell sorting Schwann cells are the major glial elements in the peripheral of preparing pure cultures of neurofibroma Schwann-like cells, nervous system and are embryologically derived from the neural and demonstrate that two tumors of similar embryological crest (1). In adults, these cells surround all peripheral nerve origin, neurofibroma and melanoma, express a similar antigenic fibers and, except in the case of injury or disease, have a very profile. low rate of division (2). Neurofibroma is a relatively common autosomal dominant disorder most often involving benign der mal lesions containing Schwann-like cells, fibroblasts, mast MATERIALS AND METHODS cells, and neuronal processes (3). The primary proliferating cell MAbs. The production and characterization of the anti-melanoma is thought to be the Schwann-like cell (4). MAbs have been described (6, 8, 10). MAbs P3X63Ag8, ME31.3, Little is known of the molecular or cellular properties of the ME19-19, ME20.4, ME75-29, ME061, ME13-17, and ME491 are Schwann-like cells, as compared with those of melanoma cells IgGl isotype and MAbs MENu4B and ME37-7 are IgG2a isotype. (5). A large number of anti-melanoma MAbs4 are available that Table 1 summarizes the antigens detected and cross-reactivities of the have been used to characterize the distribution of melanoma- MAbs. The proteoglycan antigen identified in this study with MAb associated antigens among normal and transformed tissues (6, ME31.3 has also been extensively studied by other groups using MAbs 7). This information is important for applications of MAbs to 9.2.27 (15), 155.8 (16), 225.28S (17), and B5 (18). The M, 97,000 therapy and diagnosis as well as to the determination of the glycoprotein identified in this study with MAb ME061 has been exten melanoma-associated antigen function and role in the trans sively studied using MAbs 96.5 (19, 20) and L235 (18) and has been shown to be structurally related to transfemn (21). formed phenotype. Many MAbs do not bind to normal tissue Tissue Samples. Neurofibromas removed for therapeutic purposes melanocytes but do bind to nevi, the benign melanocytic lesions, and femoral nerves removed postmortem from adults with no apparent and to neural-related tumors such as astrocytomas and neuro- neuropathies were quick frozen. Fragments of traumatic neuromas were blastomas. This binding pattern may be related to the neural obtained at the time of delayed secondary nerve repair. Fetal femoral crest embryological origin of melanoma (1). We previously nerves were obtained from therapeutic abortions at 19 or 22 wk gesta tion. The fetal neural samples were rapidly frozen by immersion in Received 4/29/86; revised 7/31/86; accepted 8/5/86. isopentane cooled with liquid nitrogen. The costs of publication of this article were defrayed in part by the payment Cell Cultures. The derivation of melanoma cell lines WM-9 and 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. A875 has been described (22, 23). Single cell suspension were prepared 1This investigation was supported by USPHS Grants CA-25874, NS-21716, from freshly excised neurofibromas and traumatic neuromas. Tissue CA-21124.CA-10815, RR-05540. NS-08075, NS-11037, and HD-08536, and by samples were minced in Hanks' balanced salt solution with 1 mM generous support from the National Neurofibromatosis Foundation, the Muscular Dystrophy Association, and the Dysautonomia Foundation. EDTA and lacking magnesium and calcium, then transferred to RPMI 2To whom requests for reprints should be addressed. 1640 medium containing 15% fetal calf serum-25 mM 4-(2-hydroxy- 3 Research Postdoctoral Fellow of the National Neurofibromatosis Founda ethyl)-l-piperazineethanesulfonic acid, dispase, 1.25 units/ml (crude; tion. Boehringer)-0.05% (w/v) collagenase (Worthington)-0.1 % (w/v) hya- 4 The abbreviations used are: MAb. monoclonal antibody; NGF, nerve growth luronidase (Sigma), and incubated overnight at 37Â°Cin 5% CO2/95% factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MEM, minimal essential medium: PBS, phosphate-buffered saline; gp260. gpl 20, air. The next day the medium was removed and replaced with RPMI a glycoprotein with a molecular weight of 260,000 or 120,000, respectively. 1640 without serum containing dispase, 1.25 units/ml and 0.05% 5887

Table 1 Properties of anti-melanoma MAbs and corresponding antigens withMAbME31.3andME19-19MENu4BME20.4ME75-29ME061AntigenM, Cross-reactivity

tissuesNevus, adult 260,000 glycoprotein + M, sweat gland, basal keratinocytes, 500,000proteoglycanM, connective tissue in colon, capillary en- dotheliumNevusNevus,

glycoproteinNGF105,000, 130,000

receptor, M, 75,000 glycopro nervesNevusNevusTumorsAstrocytomaAstrocytomaPheochromocytoma,adrenal medulla, peripheral neuroblastoma, teinM, melanomaAstrocytoma,

120,000glycoproteinM, sarcomaAstrocytoma,carcinoma,

97,000 glycoproteinNormal carcinoma, lymphoma,Ref.5-7,5-685-Â«5-711-12

ME491 M, 30,000 to 60,000 glycoprotein Nevus, some epithelial and hematopoietic Astrocytoma, some carcinomas 13 cells

ME37-7 and HLA-DR Nevus, B-lymphocytes, macrophages Lymphoma, some carcinomas 14 ME13-17

collagenase. After 30 min at 37Â°C,the suspension was pipetted vigor Cells Neuroma Neurofibroma Melanoma ously, then spun down and the pellet washed three times with RPMI (M^j-Ã§^r^cN^cor^cN^tOÃ¬ OÃ OÃ• 1640 medium containing 15% (v/v) fetal calf serum. The cells were inÃ¶Â»-'r^LnÃ¶^-"r^ir>Ã¶ MAb f^rgrocor^cNcncor^cNJ seeded on poly-L-lysine coated glass coverslips. u i i)i jji in in n i ni ni in ut Radioiodination, Immunoprecipitation, and Electrophoresis. Cells Mr (x IO'3) were labeled by the lactoperoxidase procedure and extracted with solubilizing buffer [0.5% Nonidet P-40:140 mM NaCl:10 mM NaF:10 mM Tris:5 mM EDTA:aprotinin (100 kallikrein IU/ml):l mM phenyl- â€” gp260 methylsulfonyl fluoride (pH 7.5)] (24). Immunoprecipitations were I,,' Â» carried out using 100 n\ of hybridoma culture supernatant per sample. 200 â€” â€” gp120 Antigen:mouse immunoglobulin complexes were bound with 50 n\ goat anti-mouse immunoglobulin agarose (Sigma) and washed three times with solubilizing buffer. The samples were analyzed by SDS-PAGE â€” â€” NGF-R using a 10% gel (25). Autoradiography was carried out using a Cronex Lightning-Plus intensifying screen (DuPont). Immunofluorescence and Immunoperoxidase Microscopy. Cells to be 43 â€” analyzed by immunofluorescence microscopy were incubated without prior fixation with undiluted hybridoma culture supernatant, washed with MEM, overlaid with rhodamine-conjugated goat anti-rabbit im HLA-DR munoglobulin (Cappel), washed with MEM, fixed with ice-cold 5% acetic acid (v/v) in ethanol, washed five times with MEM, and washed 25.7 â€” once with water. Preparation of frozen sections and immunoperoxidase staining have been described (14, 26). Fluorescence-activated Cell Sorting. Cells to be analyzed were re leased from the substratum with dispase, 1.25 units/ml and 0.05% (w/ v) collagenase. The cells were washed with Dulbecco's PBS (lacking 1 2 3 4 5 6 7 8 9 10 11 12 Ca2+ and Mg2*) and resuspended at 5 x IO6 cells/ml in hybridoma Fig. l. Immunoprecipitation of radioiodinated antigens with anti-melanoma culture supernatant diluted 1:2 with PBS for I h at 4Â°C.The cells were MAbs. Short-term cultured neuroma (lanes I to 4) or neurofibroma (lanes 5 to iV)cells or melanoma line WM-9 cells (lanes 9 to 12) were surface labeled with pelleted, washed three times with PBS, resuspended at 5 x IO6 cells/ 'â€¢"'Ibythe lactoperoxidase method and extracted with solubilizing buffer. The ml in fluorescein-conjugated rabbit anti-mouse immunoglobulin (60 specific antigens were immunoprecipitated with MAb ME31.3 (anti-gp260, pro fig/ml) and incubated for l h at 4Â°C.The cells were washed in the same teoglycan; lanes I, 5, and 9), ME37-7 (anti-HLA-DR; lanes 2, 6, and JO), ME75- manner and resuspended in PBS at 1 x IO7cells/ml. An aliquot of the 29 (anti-gpl20; lanes 3, 7, and //), or ME20.4 (anti-NGF receptor (A); lanes 4, 8, and 12) and analyzed by SDS-PAGE and autoradiography. cells was analyzed by flow cytometry (Ortho Cytofluorograf 50 HH). The cutoff for positive staining was determined using cells stained in parallel with P3X63Ag8, a nonspecific control MAb. The remainder results for MAbs ME31.3, ME37-7, ME75-29, and ME20.4. of the cells were sorted on a Becton Dickinson FACS IV retaining the ME31.3 is specific for the melanoma-associated chondroitin 50% most fluorescent of the positive cells for cell culture. sulfate proteoglycan and its core protein gp260 (27). Both species were detected in the traumatic neuroma, neurofibroma, RESULTS and melanoma samples (lanes 1, 5, and 9). The gp260 compo nent is marked on the gel, and the proteoglycan component is Detection of Melanoma-associated Antigens on Traumatic visible as a broad band in the stacking gel (delineated by arrows). Neuroma Schwann and Neurofibroma Cells. Cells from a trau Additional bands in lanes I to 4 are due to higher background matic neuroma and from a dermal neurofibroma were placed immunoprecipitation for the traumatic neuroma. The ME37-7 into short-term culture for 1 day and then radioiodinated by antigen, HLA-DR, was detected only in the melanoma sample the lactoperoxidase method. For comparison, the melanoma (lane 10). The ME75-29 antigen, gpl20, was detected in the cell line WM-9 was also iodinated. The cells were solubilized, neuroma, neurofibroma, and melanoma samples (lanes 3, 7, and specific antigens were immunoprecipitated with anti-mel and 11). The ME20.4 antigen, the NGF receptor, was also anoma MAbs and analyzed by SDS-PAGE. Fig. 1 shows the detected in all three samples (lanes 4, 8, and 12). Table 2 5888

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1986 American Association for Cancer Research. SCHWANN CELL SURFACE MARKERS Table 2 ImmunoprecÂ¡pilotionsfrom Schwannian cells using anti-melanoma the neurofibroma NGF receptor in binding studies with 125I- MAbs labeled NGF (Fig. 3). ME20.4 ascites fluid at a 10"6 dilution testedMAbME31.3 No. cultures positive/no, cultures strongly inhibited I25l-labeled NGF binding. Two other MAbs, neuroma ME 19-19 and ME 13-17, which bind to other proteins on the Schwanncells2/2 cells2/2 neurofibroma cell surface, did not inhibit ' 25I-labeled NGF binding at these dilutions. MENu4BME20.4 1/1 1/1 I/I 2/2 The fraction of neurofibroma cells expressing melanoma- ME75-29 2/2 2/2 associated antigens was determined by flow cytometry. In an ME061 0/1 1/1 (weak) ME491ME37-7Traumatic 0/1 0/1 initial study, we analyzed 11 cultures of dermal and plexiform 0/2Neurofibroma 0/2 neurofibroma cultures for NGF receptor expression (MAb ME20.4). Between 16 and 66% of the cells were positive for NGF receptor [39.1 Â±16.4% (SD)]. We also analyzed four Mr (x IO'3) dermal neurofibroma cultures with MAbs ME20.4, ME491, ME31.3, and ME13-17 (Table 3). ME20.4 and ME491 con sistently stained a large percentage of neurofibroma cells, 200 whereas ME31.3 stained a smaller fraction. ME 13-17 reactivity was variable, with staining of some cultures but not others. In controls with WM-9 melanoma cells, the ME20.4, ME491, 92.5 and ME13-17 antigens were uneffected by the protease treat ment used to prepare the cells for flow cytometry, but the ME31.3 antigen was greatly reduced. The identity of the melanoma-associated antigen-expressing 68 cells in the neurofibroma cultures was determined by inumino fluorescence microscopy. Fig. 4, A to C shows that the cells 43 stained with MAbs ME37-7 and ME20.4 are the long spindle- shaped cells with oval nuclei (Schwann-like cells). The fibro- blastic cells did not fluoresce detectably. The Schwann-like cells but not the fibroblasts were positive for MAbs ME31.3 (5 of 6 cultures), ME37-7 (6 of 6 cultures), ME20.4 (5 of 6 cultures), ME75-29 (3 of 6 cultures), MENu4B (3 of 6 cultures), and 25.7

3 4 Fig. 2. Detection of ME491 antigen by Western blotting. Membrane extracts from either a neurofibroma (lanes I and }) or melanoma cell line A87S (lanes 2 Fig. 3. Inhibition of MAbs of '"I-labeled NGF binding to dissociated neuro and 4) were applied to an SDS-polyacrylamide gel and then electroblotted onto fibroma cells. Dissociated neurofibroma cells were incubated for 90 min at 37'C nitrocellulose, and incubated with either control MAb P3X63Ag8 (lanes I and 2) or MAb ME491 (lanes 3 and 4). The antigens were then visualized with an '"I- in binding buffer |RPMI 1640 containing 25 itiM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid and BSA, 5 mg/mlj containing '"1-labeled NGF (5 ng/ labeled second antibody and autoradiography. ml) and several dilutions of MAb-containing ascites [ME20.4 (â€¢).ME19-19 (O). and ME37-7 (A)]. Triplicate samples were centrifuged for 30 s to separate bound from free '"I-labeled NGF, and bound '"I-labeled NGF was measured in a summarizes the immunoprecipitation experiments with these gamma counter. Nonspecific binding (approximately 30% of total binding) was and other anti-melanoma MAbs. determined in parallel samples to which unlabeled NGF (10 fig/ml) was added and has already been subtracted from the data shown. Binding in the absence of The ME491 antigen present in cultured neurofibroma cells MAb was I600cpm/1.4 x IO4cells. was identified by Western blotting (Fig. 2, lane 3). The neurofibroma antigen which binds MAb ME491 has a molecular Table 3 Flow cytometry analysis of neurofibroma cells weight similar to that of the melanoma antigen but appears to Cells from each of four neurofibroma cultures were incubated with anti- be less heterogeneous. Longer autoradiographic exposures of melanoma MAb followed by a fluorescent-stained antibody and analyzed by flow the same blot revealed that the difference was not due to the cytometry for the percentage of cells expressing the melanoma-associated anti gens. The percentage of positive cells with control MAb P3X63Ag8 (2%) was greater intensity of the melanoma ME491 band (lane 4). The subtracted from all values. blot shown is representative of three experiments made with % of cells positive for melanoma-associated antigen the detergent extract of a single neurofibroma culture. The MAb ME491 antigen was not detected by immunoprecipitation (Ta (range)ME31.3ME20.4 Mean Â±SD ble 2) probably because most of the antigen is intracellular and, Â±2 (0-5) hence, not accessible to cell surface lactoperoxidase iodination 50 Â±22(19-72) (13). ME491 56 Â±9 (47-69) The protein bound by MAb ME20.4 was demonstrated to be ME13-172 13 Â±13(2-31) 5889

_ (.*.

Fig. 4. Phase contrast and indirect immunofluorescence microscopy. Schwann-like cells as visualized in cells cultured from a traumatic neuroma (A and /'). a dermal neurofibroma (C and D), or Schwann-like cells enriched from a dermal neurofibroma by cell sorting using anti-NGF receptor MAb (E and F) were analyzed for melanoma-associated antigen expression. Cells were stained with MAb ME37-7 (A to D) or MAb ME20.4 (E and F). Bars, 20 ion (A to D) or 70 urn (E and F).

5890

ME061 (1 of 6 cultures). The Schwann-like cells did not stain The antigens detected by MAbs ME31.3, MENu4B, ME20.4, with nonspecific control MAbs (not shown) and no staining ME75-29, and ME061 by immunoprecipitation were appar was observed for MAb ME491 (0 of 6 cultures). The positive ently identical from melanoma and neurofibroma as judged by results with ME491 using flow cytometry (Table 3) may reflect SDS-PAGE. The neurofibroma ME491 antigen detected by greater accessibility to the antigen following protease treatment Western blotting was less heterogeneous than the correspond to release the cells from the substratum. ing melanoma antigen. Since this antigen is heavily glycosylated Cell Sorting of Neurofibroma Cell Cultures. To prepare pure (13, 28), the difference may be the result of more homogeneous cultures of the NGF receptor-positive cells (ME20.4), a small glycosylation in the neurofibroma. The ME20.4 antigen is fraction of the neurofibroma culture was analyzed by flow indeed the NGF receptor in neurofibroma cells since MAb cytometry. The rest of the cells were then sorted, collecting the ME20.4 completely blocked '"I-labeled NGF binding (Fig. 3). 50% most fluorescent of the positive cells. An essential prereq We have also detected many of the melanoma-associated uisite for this procedure was the preparation of single cell antigens on frozen sections of adult and fetal peripheral nerves. suspensions from the neurofibroma cultures using the dispase Although the exact assignment of the melanoma-associated procedure since the presence of aggregates resulted in poor antigen-bearing cells awaits more detailed histolÃ³gica! analysis, yields and impure cultures. The viability of the sorted cells was possibly including electron microscopy, it is clear that normal 99% as judged by trypan blue exclusion immediately after elements of the peripheral nervous system express many of the sorting and about 50% of these cells, when placed into culture, melanoma-associated antigens. Preliminary studies using anti- adhered and grew, resulting in cultures consisting of about 98% NGF receptor MAb indicate that cells of the perineurium which Schwannian cells (Fig. 4, E and F). are probably of neural crest embryological origin, consistently Immunoperoxidase Staining of Normal Peripheral Nerve and show strong staining. Schwann cells appeared to stain in some Neurofibromas. The expression of the melanoma-associated cases but not in others,5 perhaps reflecting heterogeneity of antigens in vivo was assayed by immunoperoxidase staining of frozen tissue sections (Table 4). MAbs MENu4B, ME75-29, Schwann cells in different nerves. and ME061 bound to the femoral nerve fiber but not to neu- The cross-reactivity of these MAbs with normal tissue does rofibromas. MAbs ME31.3, ME20.4, ME491, and ME37-7 not rule out their use for melanoma immunotherapy. The anti- bound to both nerve fibers and neurofibroma Schwann-like carcinoma MAb CO 17-1A, for example, has been used in cells. ME31.3 and ME37-7 appeared to bind to a subset of the clinical trials for patients with colon and pancreatic carcinomas neurofibroma Schwann-like cells, in agreement with flow cy and has resulted in remissions for some patients (29). Radioim- tometry measurements (Table 3). All of the MAbs except for aging studies have demonstrated that CO 17-1A MAb localizes ME31.3 and ME37-7 also bound to fetal femoral nerve. at the tumor in patients even though some epithelial cells in normal colon express the antigen (30, 31). This paradoxical result is apparently due to greater accessibility of the MAb to DISCUSSION antigen in tumor than in normal colon. Using a combination of methods, we have detected mela The melanoma-associated antigen for which we have the most noma-associated antigens on Schwann cells from traumatic information is the NGF receptor which is not detected in frozen neuromas and neurofibroma Schwann-like cells. Because these sections of normal melanocytes but is easily detected in sections cultures contain a heterogeneous mixture of cells, we verified of nevi and melanomas (8). Human neonatal melanocytes in that the antigens are indeed associated with the morphologically short-term culture under conditions inducing cell division ex identified Schwannian cells by immunofluorescence microscopy press low levels of the receptor (5, 6). Both Schwann cells and of the cultured cells or by immunoperoxidase staining of frozen nerve soma in neonatal monkey dorsal root ganglia also show tissue sections. With some MAbs, the antigens were detected strong staining.6 In the present study, fetal and adult peripheral by one method but not by another, perhaps reflecting differing nerves were strongly stained with anti-NGF receptor MAb, sensitivities of the two methods, differing abilities of MAbs to perhaps reflecting the related neural crest embryological origin function in different assays, or differing sensitivities of the of melanocytes, Schwann cells, and sympathetic and sensory melanoma-associated antigens to proteases used to release the neurons. Schwann cells and melanocytes may derive from a cells prior to some assays. In some cases, these cultures were common bipotent progenitor cell (32). It has been shown that sorted with anti-NGF receptor MAb to obtain nearly pure the expression of other tissue-specific genes is the result of a cultures of Schwann-like cells. The continued detection of the complex interaction between multiple fra/w-acting factors (33). NGF receptor after sorting demonstrates that the Schwann-like We suggest that the expression of the NGF receptor requires cells actually express the NGF receptor and do not simply at least two factors, the first of which is normally expressed in absorb receptor synthesized and released by some other cell neural crest-derived cells and is necessary but not sufficient for type. expression of NGF receptor. The proposed second factor re Table 4 Detection of melanoma-associated antigens by immunoperoxidase quired for NGF receptor expression is induced in stimulated staining of neurofibroma frozen sections melanocytic cells capable of undergoing cell division in culture ofAdult staining or in nevic or melanoma lesions but not in normal tissue MAbME31.3 femoral femoral Neurofibroma melanocytes, which rarely divide. In sensory nerves and some nerveÂ± nerve Schwann-likecells+ Schwann cells, this second factor or a substitute factor is (few cells) expressed. This model suggests many experiments using the MENu4BME20.4 recently cloned NGF receptor gene (34) to identify factors ME75-29 " M. A. Bothwell and E. Scarpini, unpublished observations. ME061 Â± * N. MaraÃ±o,B. Dietzschold, J. J. Earley, G. Schatteman. S. Thompson, P. ME49I Â± Grob, A. H. Ross, M. Bothwell. B. F. Atkinson, and H. Koprowski, Purification ME37-7ImmunoperoxidaseÂ±Fetal + (focal) and amino terminal sequencing of human melanoma nerve growth factor receptor. " ++, strong brown color; -I-,brown color. Â±,slight brown color; â€”,nostaining. J. Neurochem., in press. 5891

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1986 American Association for Cancer Research. SCHWANN CELL SURFACE MARKERS responsible for the tissue-specific expression of the NGF recep Koprowski, H. Expression of DR antigen in freshly frozen human tumors. Hybridoma, /: 161-168, 1982. tor and its normal expression in melanomas. 15. Bumol., T. F., and Reisfeld, R. A. Unique glycoprotein-proteoglycan complex defined by monoclonal antibody on human melanoma cells. Proc. Nati. Acad. Sci. USA, 79:1245-1249, 1982. ACKNOWLEDGMENTS 16. Harper, J. R., Bumol, T. F., and Reisfeld, R. A. Characterization of mono clonal antibody 155.8 and partial characterization of its proteoglycan antigen on human melanoma cells. J. luminimi., 132: 2096-2104, 1984. We are very grateful to Drs. Allan Rubinstein (Department of Neu 17. Wilson, B. S., Imai, K., Natali, P. G., and Ferrane, S. Distribution and rology, Mt. Sinai Medical School) and A. Lee Osterman (Department molecular characterization of a cell-surface and a cytoplasmic antigen de of Orthopedic Surgery, University of Pennsylvania) for their help in tectable in human melanoma cells with monoclonal antibodies. Int. J. Cancer, obtaining the tissue samples for this study. Marina Aquino provided 28: 293-300, 1981. 18. Houghton, A. N., Eisinger, M., Albino, A. P., Cairncross, J. G., and Old, L. excellent technical assistance. We thank Jeffrey Faust for running the J. Surface antigens of melanocytes and melanomas. Markers of melanocyte cell sorter, Marina Hoffman for editorial assistance, and Meenhard differentiation and melanoma subsets. J. Exp. Med., 156: 1755-1766, 1982. Herlyn for helpful discussions and preparation of hybridoma culture 19. Brown, J. P., Wright, P. W., Hart, C. E., Woodbury, R. G., Hellstrom, K. supernatants. E., and Hellstrom, I. Protein antigens of normal and malignant human cells identified by immunoprecipitation with monoclonal antibodies. J. 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5892

Alonzo H. Ross, David Pleasure, Kenneth Sonnenfeld, et al.

Cancer Res 1986;46:5887-5892.

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