Mitogen Accumulation in von Reckhnghausen Neurofibromatosis Nancy Ratner, PhD," Michael A. Lieberman, PhD,? Vincent M. Riccardi, MD,S and Dingming Hong" Most, but not all, patients with von Recklinghausen neurofibromatosis develop tumors (neurofibromas) that contain large numbers of Schwann cells and fibroblasts. To begin to understand the molecular events that contribute to cell proliferation in these benign tumors, we have analyzed extracts of neurofibromas to determine whether they contain mitogens for Schwann cells or fibroblasts, or both. Schwann cell and fibroblast mitogens are present in neurofibroma extracts. All the neurofibromas analyzed contain a Schwann cell mitogen similar to a neuronal cell surface molecule known to stimulate Schwann cell proliferation during normal development; this mitogen also stimulates fibroblast proliferation. Basic fibroblast growth factor is present in 60% of tumors evaluated. Accumulation of mitogenic substances may contribute to the growth of neurofibromas. Ratner N, Lieberman MA, Riccardi VM, Hong D. Mitogen accumulation in von Recklinghausen neurofibromatosis. Ann Neurol 1990;27:298-303 von Recklinghausen neurofibromatosis (NF1) is inherited as an autosomal dominant trait with high penetrance of the mutated gene and is among the most common human inherited diseases 11,23. Patients with NF1 develop benign neurocutaneous tumors on cranial and spinal nerve roots; major nerves of the trunk, neck, and limbs; the sympathetic chain and ganglia; the dermis and adjacent tissues; and the visceral sympathetic plexuses 13, 43. While patients with NF1 all appear to have mutations within the same centromeric region of chromosome 17 15-71, neurofibroma formation occurs only in some patients and at discrete locations 131. Thus, the NF1 mutation alone cannot explain tumor formation, which must be triggered by secondary generic changes or epigenetic influences at tumor sites. Neurofibromas are characterized histologically as nonencapsulated masses containing abundant extracellular collagenous and mucinous matrices, and varying numbers of Schwann cells, fibroblasts, perineurial cells, and mast cells 181. Up to 80% of the cells in neurofibromas 181 are Schwann cells, as defined using immunological criteria. Like normal Schwann cells, these cells do not proliferate in response to serum mitogens and can respond to specific Schwann cell growth factors 19-12}. Fibroblasts, which make up the majority of the remaining cells in neurofibromas, also appear untransformed, since they do not exhibit increased sensitivity to mutagens or x-rays and respond
~~~~~~
Materials and Methods Tumor samples, including both dermal and plexiform neurofibromas, were obtained incidental to therapeutic surgery or at autopsy from patients who met the criteria for NF1 [I]. Tumors were frozen in pieces no larger than 2 inches in diameter, either on dry ice or in liquid nitrogen, and were stored at - 80°C for up to 2 years before analysis. No differences were obtained in data derived from dermal and plexiform neurofibromas. Normal peripheral nerves were obtained from biopsies of non-NF1 individuals and stored at
- 80°C.
~~
From the *Department of Anatomy and Cell Biology and ?Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati Medical Center, Cincinnati, OH, and SNeurofibromatosisProgram, Baylor College of Medicine, Houton, Tx.
to serum mitogens, becoming contact inhibited at high density 19, 101. One possible explanation for increased cellular proliferation in neurofibromas is that aberrantly produced growth factors stimulate proliferation of Schwann cells and fibroblasts. Neurons are candidate growth factor producers, since they are known to stimulate Schwann cell proliferation 113-161 and contain the fibroblast mitogen (and competence factor) basic fibroblast growth factor (bFGF) 1171. In addition, crude extracts of neurofibromas have been reported to stimulate the proliferation of Schwann cells from these tumors 1181. Insulin-like growth factor I, a fibroblast progression factor, has been demonstrated in neurofibromas using immunocytochemistry 1191. The present series of investigations was designed to define mitogens present in neurofibromas that may drive proliferation of tumor cells.
~~
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Received May 12, 1989, and in revised form Aug 24. Accepted for publication Aug 25, 1989.
Address correspondence to Dr Department of Anatomy and Cell Biology, University of Cincinnati Medical Center, Cincinnati. OH 45267-0521.
298 Copyright 0 1990 by the American Neurological Association
Primary Schwann cell cultures were prepared from neonatal rat sciatic nerves, as described previously 1201. Swiss 3T3-NR6 cells were grown as described [20). Measurement of the stimulation of DNA synthesis in both neonatal rat Schwann cells [20) by autoradiography and NR6 cells 120) by trichloroacetic acid precipitation has been described previously. Frozen tumor specimens (5 gm; n = 18), stored at -8O"C, were minced into small (1-2 mm) pieces and thawed rapidly in 35 ml ice-cold buffer (0.16 M sucrose, 1 mM MgC12, 10 mM Tris HC1, p H = 7.4, containing 1 mM phenylmethylsulfonylfluoride and 60 unitdm1 aprotinin). Tumors were disrupted by polytron homogenization (2 bursts of 30 seconds each). Two methods were used to generate samples for analysis. In method I both soluble (S1) and particulate (Pl) fractions were generated for further study. The initial homogenate was clarified by centrifugation (10 minutes at 2,000 rpm), and the resultant supernatant was further clarified by a high-speed centrifugation (1 hour at 100,000 g ) , and designated S1. The insoluble material from the first spin (PI, tumor particulate) was separately extracted in the same buffer containing 0.5 M NaCI, 1 mM EGTA (35 ml) for 1 hour on ice and clarified by centrifugation for 1 hour at 100,000 g . Heparin purification of these samples was achieved by the addition of heparin-Sepharose (Pharmacia) (1 mVgm weight tumor) to extracts, followed by 1 hour of mixing at 4°C. Resin was washed with at least 10-column volumes 0.5 M NaCl in the same buffer, then eluted with 2column volumes of buffer containing 0.8 M NaC1. Alternatively (method 11) tumor homogenates were brought to 0.5 M NaCl by the addition of 5 M NaCI, maintained for 1 hour at 4"C, and clarified by centrifugation (1 hour at 100,000 g). In this procedure particulate-associated mitogens would be expected to be released during the high salt incubation, so that both soluble and particulateassociated mitogens should be present in the supernatant. The supernatant was diluted 4 times with water before being loaded onto a heparin-Sepharose column (1 mVgm weight tumor). After loading, the column was washed with 20 mM Tris HC1, p H = 7.4, 1 mM EDTA, 0.1 mM dithiothreitol until the Azso reached baseline and eluted with a 0.7 to 2.0 M NaCl gradient in the same buffer. All samples were dialyzed exhaustively against phosphate-buffered saline before addition to neonatal rat Schwann cells; the removal of salt was not necessary before addition to 3T3-NR6 cells. Western blotting was performed as previously described [20). The IgG fraction of an antiserum was generated against residues 1-24 of bovine pituitary bFGF [21).
Results In order to determine if mitogens are present in neurofibromas, extracts from 14 neurofibromas were tested for their ability to stimulate proliferation of Schwann cells and fibroblasts. Extracts of particulate fractions obtained by method I from all the neurofibromas stimulated incorporation of L3H1thymidine into quiescent primary Schwann cells cultured in the absence of serum (Fig 1). Extracts of neurofibromas were mitogenic for Schwann cells over the same concentrations of protein as are extracts of membranes from neonatal rat
70
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0 0 neurofibroma 0-0 neurofibroma A-A neurofibroma
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neurofibroma neurofibroma neurofibroma
10 100 [EXTRACT], ug/ml
?f3
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Fig 1. Extraction of mitogenic activity from particulate fractions of neurofibromas.Neurofibromas were disrupted by polytron homogenization and extracts of particulate fractions were prepared by method I (see Materials and Methods) and dialyzed against phosphate-buflered saline; 3 t o 100 pl was then added t o Schwann cells in vitro in a final volume of 250 pl. Prolijeration was assayed by autoradiography;300 to 1,000 cells were counted per sample. A representative sampling of tumor extracts with d i f f i n g speci$c activity is shown; all 18 samples tested using this paradigm were mitogenicfor Schwann cells. As noted in the text, soluble fractions were also mitogenicfor Schwann cells. For comparison with mitogen extracted under identical conditions from neonatal rat membranes, a representative preparation of this mitogen is included in the figure.
brain membranes, a source of highly enriched neuronal membranes, which contain a neuronal cell surface mitogen that stimulates Schwann cell proliferation El 3, 14, 16, 201. To test whether neurofibromas contain mitogens that can stimulate fibroblast proliferation, neurofibroma extracts were assayed on NR6-3T3 fibroblasts, which proliferate in response to a variety of mesodermal cell mitogens. Crude extracts obtained by method I1 from 2 neurofibromas exhibited 4,500 and 4,800 units of mitogenic activity per gram tumor, respectively (not shown). To characterize this activity, the crude extract was fractionated on a heparin-Sepharose column. All activity bound to the column, and in 5 of 7 tumors tested, 2 peaks of mitogenic activity eluted from heparin (Fig 2); the other 2 tumors contained only detectable amounts of the lower-affinity mitogen(s). The 2 peaks of mitogenic activity for NR6-3T3 cells purified by heparin-Sepharose chromatography were tested separately for their ability to stimulate Schwann cell proliferation. Only the low-affinity peak stimulated the incorporation of { 3H]thymidine into Schwann cells. We have previously demonstrated that a neuronal cell surface molecule mitogenic for Schwann cells binds to heparin-Sepharose and elutes at low (0.6-0.8 M) salt concentrations {20} and can stimulate proliferation of NR6-3T3 cells (R. Malhotra and N. Ratner, unpublished observations), suggesting simiRatner et al: Mitogen Accumulation in NF1 2%
0.0
A,
A
2.4 -/
I/ 1
n
b 0.7
I
3
5
7
9 1 1 13 15 FRACTION NUMBER
Fig 2. Heparin-Sepharose chromatography of neurof broma tumor extract (solubleand particulate fractions). Neurofbromas were disrupted by polytron homogenizations and 0.5 M NaCl was added directly to homogenates. After clarification by centrifugation (method II, see Materials and Methods), samples were loaded onto heparin-sepharose columns and eluted using a salt gradient. One-$ aliquots of 0.7-mifractions were assayed for their ability to stimulate {3H}thymidineincorporation on NRG-3T3 cells as described {17}. L & ordinate, Units of mitogenic activity, where 2 units of stimulation are defined as counts per minute (cpm) incorporated in the presence of 10% calf serum. Two peaks of heparin-bindingNR6-3T3 cell mitogen were obtained (n = 5). The two peaks were pooled separately and dialyzed against phosphate-buflered saline, and 3 to 100 $ was added to Schwann cell proliferation assays. Only the h e r affinitypeak contained Schwann cell mitogen. The higheraffinitypeak eluted at 1.3 to 1.5 M NaCl, suggesting identity with basic fibroblast grozuth factor (bFGF).Inset, Western blots of the pooled sample of the 1.3- to 1.5-M NaClpeak. Samples of the 1.3- to 1.5-M NaClpeak were concentrated by centrifugation in Amicon Y M 10 centricons (Amicon Co, Danvers, M A ) , and resolved on sodium dodeqlsulfate gels. ( I = neurofbroma, 1.3- to 1.5-M peak; 2 = 25 ng bFGF (CollaborativeResearch, Bedford, MA); 3 = 25 ng acidic FGF (aFGF; Collaborative Research)).Lanes 1 and 2 were probed with anti-bFGF antiserum; lane 3 was probed with anti-FGF antiserum. (a = migration of aFGF; b = migration of bFGF).
larity of this mitogen with that present in neurofibromas. Several other heparin-binding mitogens were not present in this fraction. Protein from this fraction did not cross-react with either anti-acidic or basic FGF antibodies (not shown). Platelet-derived growth factor (PDGF) also binds with low affinity to heparin 122). We tested the ability of antibodies that block PDGFstimulated cell proliferation to block the activity of neurofibroma-derived mitogen; no inhibition of NR6 cell proliferation was observed (not shown). Mitogenic activity eluted from heparin-Sepharose by 1.2 to 1.5 M NaCl contained material that cross-
300 Annals of Neurology Vol 27 No 3 March 1990
17
19
21
23
reacted with a monospecific polyclonal antiserum raised against amino acids 1-24 of bFGF 121) in solid-phase enzyme-linked immunosorbent assay (not shown) and co-migrated with bFGF in Western blots (Fig 2, inset). N o acidic FGF was detected in Western blots. These data indicate that neurofibromas contain at least 2 fibroblast mitogens, bFGF and molecule(s) that elute from heparin-Sepharose at 0.7 M NaC1. Mitogenic activity is present in both the particulate (Pl) and the soluble S1 fraction of neurofibromas. While all neurofibromas contain mitogenic activity for Schwann cells, the proportion of activity in the soluble fraction varies among tumors from a low of 0% to a high of 93% of the total recovered activity (Table). S i x of 7 tumors contained more than 24% of the total activity in the soluble fraction. Since saturation of Schwann cell proliferation was rarely achieved in the Schwann cell bioassay, estimates of soluble mitogenic activity were corroborated in 3 cases by adding heparin-purified mitogen from soluble and particulate fractions in parallel to NR6-3T3 cells. Using this more sensitive assay, saturation was achieved for both normal nerve and tumor samples. The results corroborated those obtained in Schwann cell proliferation assays; in 3 tumor samples, the percentage of activity in the soluble fraction was 0, 43.3, and 82.5, while no detectable activity was obtained in the soluble fraction from normal human nerve. These results are consistent with earlier observations showing that essentially all of the mitogen associated with normal neurons is associated with membrane fractions C13); our data suggest that, in contrast, a variable amount of mitogenic activity in neurofibromas is soluble.
Discussion Although NF1 is inherited as an autosomal dominant mutation, the development of neurofibromas must be
Solubility of Schwann Cell Mitogen in Neurofibromasa
Tumor N o . 1
2
3
4
5
6
7
Normal H u m a n Nerve
24.0
52.7
39.3
63.0
93.2
55.5
0
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~~~
% soluble mitogedtotal mitogen
“Heparin-binding mitogen from soluble and particulate fractions of neurofibromas or normal human nerve were purified separately using method I (see Materials and Methods). Samples were dialyzed against phosphate-buffered saline and 3 to 100 pl was added to Schwann cell proliferation assays. The halfmaximal percent labeled nuclei were calculated for soluble and particulate fractions from each sample. The data are expressed as the percentage of labeled nuclei (LI) in the soluble sample using the formula LI (soluble)/LI (soluble + particulate) x 100.
a multistep process since not all patients with a defective NF1 gene form tumors. Mutations in the NF1 gene therefore confer only susceptibility to tumor formation. Since we have detected substantial levels of Schwann cell and fibroblast mitogen in neurofibromas, it is possible that mitogens cause cell proliferation in neurofibromas, and that mitogen accumulation in tumors reflects an obligate step in the multistep process of neurofibroma formation. Mitogen accumulation in neurofibromas might occur as a direct consequence of a first “hit,” a defect in the NF1 gene. In this case, all peripheral nerves from NF1 patients would be expected to contain abnormally high levels of mitogen. Me have been unable to obtain the NF1 peripheral nerves necessary to investigate this possibility. Therefore, we cannot rule out constitutive overproduction of mitogen, but since neurofibromas do not occur on all peripheral nerves in all patients with a defect in the NF1 gene, the simplest hypothesis to explain the data is that mitogen accumulation in tumors is a consequence of a second hit at nascent tumor sites. The proximate cause of neurofibroma formation may be genetic second hits in tumor cells. An argument against a genetic second hit comes from analysis of chromosomal structure in neurofibromas. At least a portion of genetic second hits should result in detectable chromosomal deletions or translocations, but such rearrangements have not been detected in DNA from 30 neurofibromas examined (B. Seizinger, personal communication, 1989). Epigenetic changes at tumor sites are also possible second hits. Several environmental factors have been proposed to modulate neurofibroma formation. Injury is a suggested tumor promoter in NF1, and mice with bovine papilloma virus integrated into their genome develop tumors preferentially at wound sites 123). Mast cell degranulation products have been postulated to facilitate neurofibroma formation 1241. Changes in sex steroid levels, as brought about by puberty or pregnancy, may also augment neurofibroma development; several reports of dramatic tumor enlargement associated with puberty and pregnancy have appeared 125, 261, estrogen binding sites are present in neuro-
fibromas 1271, and it is possible that maternal inheritance of the NF1 gene can affect the severity of the disease 1281. If epigenetic changes cause tumor formation, proliferation of neurofibroma cells might occur only because of excessive mitogen accumulation, and not because of intrinsic abnormality in Schwann cells or fibroblasts. Specific amplification of normal cells by abnormal mitogen production is known to occur in atherosclerotic plaques, in which smooth-muscle cells are driven to proliferate in response to PDGF secreted by endothelial cell, fibroblasts, and mast cells 129, 30). This hypothesis is consistent with analysis of the response of Schwann cells and fibroblasts placed into tissue culture to appropriate mitogens 19- 12). However, some possible differences have been reported between neurofibroma-derived cells and their normal counterparts 112, 311, and no complete studies using matched human control subjects have been carried out, so it remains possible that Schwann cells are abnormal in NF1, either as a function of the NF1 mutation or a second hit. Schwann cells have previously been shown to respond to only one mitogen under the serum-free culture conditions used here. This mitogen is a neuronal cell surface protein 113, 14) associated with a heparan sulfate proteoglycan in the neuronal membrane 120, 32). The data shown in Figures 1 and 2 suggest that neurofibromas contain a molecule similar to the neuronal mitogen, based on ability to stimulate Schwann cell proliferation in the absence of serum and on affinity for heparin-Sepharose. The only other mitogen described to date that will vigorously stimulate Schwann cells is glial growth factor 133); significant amounts of gllal growth factor-like activity were not present in 4 of 4 neurofibromas assayed 134). Schwann cells are refractory to a variety of other mitogens and extracts of normal and tumor tissues do not contain Schwann cell mitogen 113, 34, 35). If the neurofibroma Schwann cell mitogen is similar to the mitogen found in neuronal membranes, the few neurons in neurofibrornas cannot account for the large amount of mitogen present in these tumors. We have shown that neurofibroma mitogen is found both in
Ratner et ak Mitogen Accumulation in NF1
301
the soluble and particulate fractions prepared from tumors. Since soluble fractions from normal neurons contain little, if any, mitogenic activity, it is hypothesized that during neurofibroma formation neuronal mitogen dissociates from the plasma membrane and stimulates the proliferation of Schwann cells (and fibroblasts) distant from z o n a l processes. Heparin has been shown to dissociate adult axolemmal mitogen from neuronal membranes 1361. Mast cell degranulation, causing release of heparin, might play a role in dissociation of mitogen from neurons. The finding of a bFGF-like factor in neurofibromas is consistent with the finding of bFGF-like molecules in a variety of tumors, such as gliomas 1371, rhabdomyosarcomas 1381, and hepatomas C391. It is not clear if the primary role of FGF in these tumors is as an angiogenic factor 1401, or to promote the proliferation of fibroblasts and endothelial cells. The cells responsible for bFGF production in neurofibromas are not known, but neurons represent a potential source of this mitogen since immunocytochemical localization demonstrated bFGF in neurons C171. Since not all neurofibromas contained detectable levels of bFGF, accumulation of this mitogen may be secondary to development of neurofibromas. This is consistent with the finding that some neurofibromas take the form of “onion bulbs” C411 in which concentric rings of Schwann cells accumulate around neurons, and fibroblast overproduction is not observed. The demonstration of mitogens in neurofibromas predicts that the regulation of mitogen production is important in tumor formation and that, if mitogen production can be blocked, tumor formation will be reduced. Such working models will be useful for the development of treatments for NF1.
This work was supported by a fellowship and a grant from the National Neurofibromatosis Foundation (to N.R.), grant BRSG 2SO7RRO5408-26 (to N.R.), N I H P5O-HD-20748 (to M.A.L.), and from the Texas Neurofibromatosis Foundation, Dallas, Texas, through its funding of the Baylor College of Medicine Neurofibromatosis Program. We are grateful to Drs Richard Bunge and Luis Glaser, in whose laboratories this work was begun, and to Dr Robert Brackenbury for helpful discussions. We acknowledge Kandace Proffitt for technical assistance, Susan Eder for preparation of the manuscript, and Linda D. Langley for facilitating tumor collection.
References 1. Carey JC, Baty BJ, Johnson JP, et al. The genetic aspects of neurofibromatosis. Ann N Y Acad Sci 1986;486:45-56 2. Crowe FW,Schull WJ, Nee1 JV. A clinical, pathological and genetic study of multiple neurofibromatosis. Springfield, I L Thomas, 1956 3. Riccardi VM. Neurofibromatosis: clinical heterogeneity. Current Probl Cancer 1982;7:1-34
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4. Rubensrein AE. Neurofibromatosis: A review of the clinical problem. Ann N Y Acad Sci 1986;486:1-13 5. Barker D, Wright E, Nguyen K, et al. The gene for von Recklinghausen neurofibromatosis is in the pericentromeric region of chromosome 17. Science 1987;236:1100-1102 6. Collins FS, Ponder BAJ, Seizinger BR, Epstein CJ. The von Recklinghausen neurofibromatosis region on chromosome 17genetic and physical maps come into focus. Am J Hum Genet 1988;44:1-5 7. Seizinger BR, Rouleau GA, Ozelius LJ, et al. Genetic linkage of von Recklinghausen neurofibromatosis to the nerve growth factor receptor gene. Cell 1987;49:589-594 8. Peltonen J, Jaakkola S, Lebwohl M, et al. Cellular differentiation and expression of matrix genes in type I neurofibromatosis. Lab Invest 1988;59:760-771 9. Krone W, Jirikowski G, Muhleck 0, et al. Cell culture studies on neurofibromatosis (von Recklinghausen). 11. Occurrence of glial cells in primary cultures of peripheral neurofibromas. Hum Genet 1983;63:247-251 10. Krone W, Mao R, Muhleck OS, et al. Cell culture studies on neurofibromatosis (von Recklinghausen). Characterization of cells growing from neurofibromas. Ann NY Acad Sci 1986; 486:354-370 11. Pleasure D, Kreider B, Sobue G, et al. Schwann-like cells cultured from human dermal neurofibromas: immunohistological identification and response to Schwann cell mitogens. Ann NY Acad Sci 1986;486:227-240 12. Sobue G, Sonnenfeld K, Rubenstein AE, Pleasure D. Tissue culture studies of neurofibromatosis: effects of axolemmal fragments and cyclic adenosine 3’,5’-monophosphate analogues on proliferation of Schwann-like and fibroblast-like neurofibroma cells. Ann Neurol 1985;18:68-73 13. Salzer JL, Williams AK, Glaser L, Bunge RP. Studies of Schwann cell proliferation. 11. Characterization of the stimulation and specificity of the response to a neurite membrane fraction. J Cell Biol 1980;84:753-766 14. SalzerJL, Bunge RP, Glaser L. Studies of Schwann cell proliferation. 111. Evidence for the surface localization of the neurite mitogen. J Cell Biol 1980;84:767-778 15. Sobue G, Brown MJ, Kim SU, Pleasure D. Axolemma is a mitogen for human Schwann cells. Ann Neurol 1984;15:449452 16. Wood PM, Bunge RP. Evidence that sensory axons are mitogenic for Schwann cells. Nature 1975;256:662-664 17. Janet T, Grothe C, Pettman B, et al. Immunocytochemical demonstration of fibroblast growth factor in cultured chick and rat neurons. J Neurosci Res 1988;19:195-201 18. Riccardi VM. Growth promoting factors in neurofibroma crude extracts. Ann N Y Acad Sci 1986;486:206-226 19. Hansson HA, Lauritzen C, Lossing C. Somatomedin C as a tentative pathogenic factor in neurofibromatosis. Scand J Plast Reconstr Surg 1988;22:7-13 20. Ratner N, Hong D, Lieberman MA, et al. The neuronal cell surface molecule mitogenic for Schwann cells is a heparinbinding protein. Proc Natl Acad Sci USA 1988;85:6992-6996 21. Riss TL, Sirbasku DA. Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using Western immunoblotting and enzyme-linked immunosorbent assays. J Cell Physiol 1988;138:405-414 22. Raines EW, Ross R. Purification of human platelet-derived growth factor. Meth Enzymol 1985;109:749-773 23. Lacey M, Alpert S , Hanahan D. Bovine papillomavirus genome elicits skin tumors in transgenic mice. Nature 1986;322:609612 24. Riccardi VM. Mast-cell stabilization to decrease neurofibroma growth. Arch Dermatol 1987;123:1011-1016 25. Ansari AH, Facoog F, Nagamani M. Pregnancy and neuro-
26. 27.
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fibromatosis (von Recklinghausen’s disease). Obstet Gynecol 1976;47:255-295 Swapp GH, Main RA. Neurofibromatosis in pregnancy. Br J Dermatol 1973;88:431-435 Martuza RL, MacLaughlin DT, Ojemann RG. Specific estradiol binding in schwannomas, meningiomas, and neurofibromas. Neurosurgery 1981;9:665-671 Miller M, Hall J. Possible maternal effect on severity of neurofibromatosis. Lancet 1978;2:1071-1073 Nilsson J. Growth factors and the pathogenesis of atherosclerosis. Atherosclerosis 1986;62:185-199 Wilcox JN, Smith KM, Williams LT, et al. Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in srtu hybridization. J Clin Invest 1988;82:11341143 Huson S, Ponder B. The Runnymede Conference: 1st European Symposium on Neurofibromatosis. Neurofibromatosis 1988;1:55 Ratner N, Bunge RP, Glaser L. A neuronal cell surface heparan sulfate proteoglycan is required for dorsal root ganglion neuron stimulation of Schwann cell proliferation. J Cell Biol 1985; 101~744-754 Lemke GE, Brockes JP. Identification and purification of dial growth factor. J Neurosci 1984;4:75-83 Brockes JP, Breakefield XO, Martuza RL..Glial growth factor-
35. 36.
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like activity in Schwann cell tumors. Ann Neurol 1986;20:317322 Raff MC, Abney E, Brockes JP, Hornby-Smith A. Schwann cell growth factors. Cell 1978;15:813-822 DeCoster MA, DeVries GH. Evidence that the axolemmal mitogen for cultured Schwann cells is a positively charged, heparan sulfate proteoglycan bound, heparin-displaceable molecule. J Neurosci Res 1989;22:283-288 Libermann TA, Friesel R, Jaye M, et al. An angiogenic growth factor is expressed in human glloma cells. EMBO J 1987;6: 1627-1632 Schweigerer L, Neufeld G, Hergia A, et al. Basic fibroblast growth factor in human rhabdomyosarcoma cells: implications for the proliferation and neovascularization of myoblast-derived tumors.Proc Natl Acad Sci USA 1987;84:842-846 Klagsbrun M,Sasse J, Sullivan R, Smith JA. Human tumor cells synthesize an endothelid cell growth factor that is structurally related to basic fibroblast growth factor. Proc Natl Acad Sci USA 1986;83:2448-2452 F o h a n J, Klagsbrun M. Angiogenic factors. Science 1987; 235:442-447 Oshini A, Nada 0. Ultrastructure of the onion bulb-like lamellated structure observed in the sural nerve in a case of von Recklinghausen’s disease. Acta Neuropathol (Berl) 1972;20: 258-263
Ratner et al: Mitogen Accumulation in NF1 303
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Materials and Methods Tumor samples, including both dermal and plexiform neurofibromas, were obtained incidental to therapeutic surgery or at autopsy from patients who met the criteria for NF1 [I]. Tumors were frozen in pieces no larger than 2 inches in diameter, either on dry ice or in liquid nitrogen, and were stored at - 80°C for up to 2 years before analysis. No differences were obtained in data derived from dermal and plexiform neurofibromas. Normal peripheral nerves were obtained from biopsies of non-NF1 individuals and stored at
- 80°C.
~~
From the *Department of Anatomy and Cell Biology and ?Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati Medical Center, Cincinnati, OH, and SNeurofibromatosisProgram, Baylor College of Medicine, Houton, Tx.
to serum mitogens, becoming contact inhibited at high density 19, 101. One possible explanation for increased cellular proliferation in neurofibromas is that aberrantly produced growth factors stimulate proliferation of Schwann cells and fibroblasts. Neurons are candidate growth factor producers, since they are known to stimulate Schwann cell proliferation 113-161 and contain the fibroblast mitogen (and competence factor) basic fibroblast growth factor (bFGF) 1171. In addition, crude extracts of neurofibromas have been reported to stimulate the proliferation of Schwann cells from these tumors 1181. Insulin-like growth factor I, a fibroblast progression factor, has been demonstrated in neurofibromas using immunocytochemistry 1191. The present series of investigations was designed to define mitogens present in neurofibromas that may drive proliferation of tumor cells.
~~
~~
Received May 12, 1989, and in revised form Aug 24. Accepted for publication Aug 25, 1989.
Address correspondence to Dr Department of Anatomy and Cell Biology, University of Cincinnati Medical Center, Cincinnati. OH 45267-0521.
298 Copyright 0 1990 by the American Neurological Association
Primary Schwann cell cultures were prepared from neonatal rat sciatic nerves, as described previously 1201. Swiss 3T3-NR6 cells were grown as described [20). Measurement of the stimulation of DNA synthesis in both neonatal rat Schwann cells [20) by autoradiography and NR6 cells 120) by trichloroacetic acid precipitation has been described previously. Frozen tumor specimens (5 gm; n = 18), stored at -8O"C, were minced into small (1-2 mm) pieces and thawed rapidly in 35 ml ice-cold buffer (0.16 M sucrose, 1 mM MgC12, 10 mM Tris HC1, p H = 7.4, containing 1 mM phenylmethylsulfonylfluoride and 60 unitdm1 aprotinin). Tumors were disrupted by polytron homogenization (2 bursts of 30 seconds each). Two methods were used to generate samples for analysis. In method I both soluble (S1) and particulate (Pl) fractions were generated for further study. The initial homogenate was clarified by centrifugation (10 minutes at 2,000 rpm), and the resultant supernatant was further clarified by a high-speed centrifugation (1 hour at 100,000 g ) , and designated S1. The insoluble material from the first spin (PI, tumor particulate) was separately extracted in the same buffer containing 0.5 M NaCI, 1 mM EGTA (35 ml) for 1 hour on ice and clarified by centrifugation for 1 hour at 100,000 g . Heparin purification of these samples was achieved by the addition of heparin-Sepharose (Pharmacia) (1 mVgm weight tumor) to extracts, followed by 1 hour of mixing at 4°C. Resin was washed with at least 10-column volumes 0.5 M NaCl in the same buffer, then eluted with 2column volumes of buffer containing 0.8 M NaC1. Alternatively (method 11) tumor homogenates were brought to 0.5 M NaCl by the addition of 5 M NaCI, maintained for 1 hour at 4"C, and clarified by centrifugation (1 hour at 100,000 g). In this procedure particulate-associated mitogens would be expected to be released during the high salt incubation, so that both soluble and particulateassociated mitogens should be present in the supernatant. The supernatant was diluted 4 times with water before being loaded onto a heparin-Sepharose column (1 mVgm weight tumor). After loading, the column was washed with 20 mM Tris HC1, p H = 7.4, 1 mM EDTA, 0.1 mM dithiothreitol until the Azso reached baseline and eluted with a 0.7 to 2.0 M NaCl gradient in the same buffer. All samples were dialyzed exhaustively against phosphate-buffered saline before addition to neonatal rat Schwann cells; the removal of salt was not necessary before addition to 3T3-NR6 cells. Western blotting was performed as previously described [20). The IgG fraction of an antiserum was generated against residues 1-24 of bovine pituitary bFGF [21).
Results In order to determine if mitogens are present in neurofibromas, extracts from 14 neurofibromas were tested for their ability to stimulate proliferation of Schwann cells and fibroblasts. Extracts of particulate fractions obtained by method I from all the neurofibromas stimulated incorporation of L3H1thymidine into quiescent primary Schwann cells cultured in the absence of serum (Fig 1). Extracts of neurofibromas were mitogenic for Schwann cells over the same concentrations of protein as are extracts of membranes from neonatal rat
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I
1000
Fig 1. Extraction of mitogenic activity from particulate fractions of neurofibromas.Neurofibromas were disrupted by polytron homogenization and extracts of particulate fractions were prepared by method I (see Materials and Methods) and dialyzed against phosphate-buflered saline; 3 t o 100 pl was then added t o Schwann cells in vitro in a final volume of 250 pl. Prolijeration was assayed by autoradiography;300 to 1,000 cells were counted per sample. A representative sampling of tumor extracts with d i f f i n g speci$c activity is shown; all 18 samples tested using this paradigm were mitogenicfor Schwann cells. As noted in the text, soluble fractions were also mitogenicfor Schwann cells. For comparison with mitogen extracted under identical conditions from neonatal rat membranes, a representative preparation of this mitogen is included in the figure.
brain membranes, a source of highly enriched neuronal membranes, which contain a neuronal cell surface mitogen that stimulates Schwann cell proliferation El 3, 14, 16, 201. To test whether neurofibromas contain mitogens that can stimulate fibroblast proliferation, neurofibroma extracts were assayed on NR6-3T3 fibroblasts, which proliferate in response to a variety of mesodermal cell mitogens. Crude extracts obtained by method I1 from 2 neurofibromas exhibited 4,500 and 4,800 units of mitogenic activity per gram tumor, respectively (not shown). To characterize this activity, the crude extract was fractionated on a heparin-Sepharose column. All activity bound to the column, and in 5 of 7 tumors tested, 2 peaks of mitogenic activity eluted from heparin (Fig 2); the other 2 tumors contained only detectable amounts of the lower-affinity mitogen(s). The 2 peaks of mitogenic activity for NR6-3T3 cells purified by heparin-Sepharose chromatography were tested separately for their ability to stimulate Schwann cell proliferation. Only the low-affinity peak stimulated the incorporation of { 3H]thymidine into Schwann cells. We have previously demonstrated that a neuronal cell surface molecule mitogenic for Schwann cells binds to heparin-Sepharose and elutes at low (0.6-0.8 M) salt concentrations {20} and can stimulate proliferation of NR6-3T3 cells (R. Malhotra and N. Ratner, unpublished observations), suggesting simiRatner et al: Mitogen Accumulation in NF1 2%
0.0
A,
A
2.4 -/
I/ 1
n
b 0.7
I
3
5
7
9 1 1 13 15 FRACTION NUMBER
Fig 2. Heparin-Sepharose chromatography of neurof broma tumor extract (solubleand particulate fractions). Neurofbromas were disrupted by polytron homogenizations and 0.5 M NaCl was added directly to homogenates. After clarification by centrifugation (method II, see Materials and Methods), samples were loaded onto heparin-sepharose columns and eluted using a salt gradient. One-$ aliquots of 0.7-mifractions were assayed for their ability to stimulate {3H}thymidineincorporation on NRG-3T3 cells as described {17}. L & ordinate, Units of mitogenic activity, where 2 units of stimulation are defined as counts per minute (cpm) incorporated in the presence of 10% calf serum. Two peaks of heparin-bindingNR6-3T3 cell mitogen were obtained (n = 5). The two peaks were pooled separately and dialyzed against phosphate-buflered saline, and 3 to 100 $ was added to Schwann cell proliferation assays. Only the h e r affinitypeak contained Schwann cell mitogen. The higheraffinitypeak eluted at 1.3 to 1.5 M NaCl, suggesting identity with basic fibroblast grozuth factor (bFGF).Inset, Western blots of the pooled sample of the 1.3- to 1.5-M NaClpeak. Samples of the 1.3- to 1.5-M NaClpeak were concentrated by centrifugation in Amicon Y M 10 centricons (Amicon Co, Danvers, M A ) , and resolved on sodium dodeqlsulfate gels. ( I = neurofbroma, 1.3- to 1.5-M peak; 2 = 25 ng bFGF (CollaborativeResearch, Bedford, MA); 3 = 25 ng acidic FGF (aFGF; Collaborative Research)).Lanes 1 and 2 were probed with anti-bFGF antiserum; lane 3 was probed with anti-FGF antiserum. (a = migration of aFGF; b = migration of bFGF).
larity of this mitogen with that present in neurofibromas. Several other heparin-binding mitogens were not present in this fraction. Protein from this fraction did not cross-react with either anti-acidic or basic FGF antibodies (not shown). Platelet-derived growth factor (PDGF) also binds with low affinity to heparin 122). We tested the ability of antibodies that block PDGFstimulated cell proliferation to block the activity of neurofibroma-derived mitogen; no inhibition of NR6 cell proliferation was observed (not shown). Mitogenic activity eluted from heparin-Sepharose by 1.2 to 1.5 M NaCl contained material that cross-
300 Annals of Neurology Vol 27 No 3 March 1990
17
19
21
23
reacted with a monospecific polyclonal antiserum raised against amino acids 1-24 of bFGF 121) in solid-phase enzyme-linked immunosorbent assay (not shown) and co-migrated with bFGF in Western blots (Fig 2, inset). N o acidic FGF was detected in Western blots. These data indicate that neurofibromas contain at least 2 fibroblast mitogens, bFGF and molecule(s) that elute from heparin-Sepharose at 0.7 M NaC1. Mitogenic activity is present in both the particulate (Pl) and the soluble S1 fraction of neurofibromas. While all neurofibromas contain mitogenic activity for Schwann cells, the proportion of activity in the soluble fraction varies among tumors from a low of 0% to a high of 93% of the total recovered activity (Table). S i x of 7 tumors contained more than 24% of the total activity in the soluble fraction. Since saturation of Schwann cell proliferation was rarely achieved in the Schwann cell bioassay, estimates of soluble mitogenic activity were corroborated in 3 cases by adding heparin-purified mitogen from soluble and particulate fractions in parallel to NR6-3T3 cells. Using this more sensitive assay, saturation was achieved for both normal nerve and tumor samples. The results corroborated those obtained in Schwann cell proliferation assays; in 3 tumor samples, the percentage of activity in the soluble fraction was 0, 43.3, and 82.5, while no detectable activity was obtained in the soluble fraction from normal human nerve. These results are consistent with earlier observations showing that essentially all of the mitogen associated with normal neurons is associated with membrane fractions C13); our data suggest that, in contrast, a variable amount of mitogenic activity in neurofibromas is soluble.
Discussion Although NF1 is inherited as an autosomal dominant mutation, the development of neurofibromas must be
Solubility of Schwann Cell Mitogen in Neurofibromasa
Tumor N o . 1
2
3
4
5
6
7
Normal H u m a n Nerve
24.0
52.7
39.3
63.0
93.2
55.5
0
0
~~~
% soluble mitogedtotal mitogen
“Heparin-binding mitogen from soluble and particulate fractions of neurofibromas or normal human nerve were purified separately using method I (see Materials and Methods). Samples were dialyzed against phosphate-buffered saline and 3 to 100 pl was added to Schwann cell proliferation assays. The halfmaximal percent labeled nuclei were calculated for soluble and particulate fractions from each sample. The data are expressed as the percentage of labeled nuclei (LI) in the soluble sample using the formula LI (soluble)/LI (soluble + particulate) x 100.
a multistep process since not all patients with a defective NF1 gene form tumors. Mutations in the NF1 gene therefore confer only susceptibility to tumor formation. Since we have detected substantial levels of Schwann cell and fibroblast mitogen in neurofibromas, it is possible that mitogens cause cell proliferation in neurofibromas, and that mitogen accumulation in tumors reflects an obligate step in the multistep process of neurofibroma formation. Mitogen accumulation in neurofibromas might occur as a direct consequence of a first “hit,” a defect in the NF1 gene. In this case, all peripheral nerves from NF1 patients would be expected to contain abnormally high levels of mitogen. Me have been unable to obtain the NF1 peripheral nerves necessary to investigate this possibility. Therefore, we cannot rule out constitutive overproduction of mitogen, but since neurofibromas do not occur on all peripheral nerves in all patients with a defect in the NF1 gene, the simplest hypothesis to explain the data is that mitogen accumulation in tumors is a consequence of a second hit at nascent tumor sites. The proximate cause of neurofibroma formation may be genetic second hits in tumor cells. An argument against a genetic second hit comes from analysis of chromosomal structure in neurofibromas. At least a portion of genetic second hits should result in detectable chromosomal deletions or translocations, but such rearrangements have not been detected in DNA from 30 neurofibromas examined (B. Seizinger, personal communication, 1989). Epigenetic changes at tumor sites are also possible second hits. Several environmental factors have been proposed to modulate neurofibroma formation. Injury is a suggested tumor promoter in NF1, and mice with bovine papilloma virus integrated into their genome develop tumors preferentially at wound sites 123). Mast cell degranulation products have been postulated to facilitate neurofibroma formation 1241. Changes in sex steroid levels, as brought about by puberty or pregnancy, may also augment neurofibroma development; several reports of dramatic tumor enlargement associated with puberty and pregnancy have appeared 125, 261, estrogen binding sites are present in neuro-
fibromas 1271, and it is possible that maternal inheritance of the NF1 gene can affect the severity of the disease 1281. If epigenetic changes cause tumor formation, proliferation of neurofibroma cells might occur only because of excessive mitogen accumulation, and not because of intrinsic abnormality in Schwann cells or fibroblasts. Specific amplification of normal cells by abnormal mitogen production is known to occur in atherosclerotic plaques, in which smooth-muscle cells are driven to proliferate in response to PDGF secreted by endothelial cell, fibroblasts, and mast cells 129, 30). This hypothesis is consistent with analysis of the response of Schwann cells and fibroblasts placed into tissue culture to appropriate mitogens 19- 12). However, some possible differences have been reported between neurofibroma-derived cells and their normal counterparts 112, 311, and no complete studies using matched human control subjects have been carried out, so it remains possible that Schwann cells are abnormal in NF1, either as a function of the NF1 mutation or a second hit. Schwann cells have previously been shown to respond to only one mitogen under the serum-free culture conditions used here. This mitogen is a neuronal cell surface protein 113, 14) associated with a heparan sulfate proteoglycan in the neuronal membrane 120, 32). The data shown in Figures 1 and 2 suggest that neurofibromas contain a molecule similar to the neuronal mitogen, based on ability to stimulate Schwann cell proliferation in the absence of serum and on affinity for heparin-Sepharose. The only other mitogen described to date that will vigorously stimulate Schwann cells is glial growth factor 133); significant amounts of gllal growth factor-like activity were not present in 4 of 4 neurofibromas assayed 134). Schwann cells are refractory to a variety of other mitogens and extracts of normal and tumor tissues do not contain Schwann cell mitogen 113, 34, 35). If the neurofibroma Schwann cell mitogen is similar to the mitogen found in neuronal membranes, the few neurons in neurofibrornas cannot account for the large amount of mitogen present in these tumors. We have shown that neurofibroma mitogen is found both in
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the soluble and particulate fractions prepared from tumors. Since soluble fractions from normal neurons contain little, if any, mitogenic activity, it is hypothesized that during neurofibroma formation neuronal mitogen dissociates from the plasma membrane and stimulates the proliferation of Schwann cells (and fibroblasts) distant from z o n a l processes. Heparin has been shown to dissociate adult axolemmal mitogen from neuronal membranes 1361. Mast cell degranulation, causing release of heparin, might play a role in dissociation of mitogen from neurons. The finding of a bFGF-like factor in neurofibromas is consistent with the finding of bFGF-like molecules in a variety of tumors, such as gliomas 1371, rhabdomyosarcomas 1381, and hepatomas C391. It is not clear if the primary role of FGF in these tumors is as an angiogenic factor 1401, or to promote the proliferation of fibroblasts and endothelial cells. The cells responsible for bFGF production in neurofibromas are not known, but neurons represent a potential source of this mitogen since immunocytochemical localization demonstrated bFGF in neurons C171. Since not all neurofibromas contained detectable levels of bFGF, accumulation of this mitogen may be secondary to development of neurofibromas. This is consistent with the finding that some neurofibromas take the form of “onion bulbs” C411 in which concentric rings of Schwann cells accumulate around neurons, and fibroblast overproduction is not observed. The demonstration of mitogens in neurofibromas predicts that the regulation of mitogen production is important in tumor formation and that, if mitogen production can be blocked, tumor formation will be reduced. Such working models will be useful for the development of treatments for NF1.
This work was supported by a fellowship and a grant from the National Neurofibromatosis Foundation (to N.R.), grant BRSG 2SO7RRO5408-26 (to N.R.), N I H P5O-HD-20748 (to M.A.L.), and from the Texas Neurofibromatosis Foundation, Dallas, Texas, through its funding of the Baylor College of Medicine Neurofibromatosis Program. We are grateful to Drs Richard Bunge and Luis Glaser, in whose laboratories this work was begun, and to Dr Robert Brackenbury for helpful discussions. We acknowledge Kandace Proffitt for technical assistance, Susan Eder for preparation of the manuscript, and Linda D. Langley for facilitating tumor collection.
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