Human Central Nervous System Primitive Neuroectodermd Tumor Expressing Nerve Growth Factor Receptors: CHP707m David L. Baker, MD," Usha Rani Reddy, PhD," Samuel Pleasure," Mattie Hardy," Marge Williams," Margaret Tartaglione,"Jaclyn A. Biegel, PhD," Beverly S. Emanuel, PhD," Patrizia Lo Presti, MD," Barbara Kreider, PhD," John Q. Trojanowski, MD, PhD," Audrey Evans, MD," Amit R. Roy, PhD,t Gita Venkatakrishnan, PhD,t Jie Chen, MD,T Alonzo H. Ross, PhD,? and David Pleasure, MD"

A primitive neuroectodermal tumor (PNET) presented as a cerebral hemispheric mass in a 33-year-old man. Bone marrow metastases were discovered 11 months later. A cell line (CHP707m) was derived from these metastases. In culture, the cells showed features of neuronal differentiation, forming short neurites and synthesizing low-molecularweight neurofilament protein. Northern blotting showed the tumor cells express nerve growth factor (NGF) receptor messenger RNA, and fluorescence-activated cell-sorting demonstrated NGF receptors on the cell surface. Western blotting showed CHP707m NGF receptors are truncated. The receptors are functional; they bind iodine 125-labeled M, and short-term treatment with NGF induces expression by the tumor mouse NGF with an affinity of 1.6 X cells of the proto-oncogene, c-fos. Although CHP707m is the first central nervous system PNET cell line proven to express NGF receptors, immunohistologicd survey of tissue sections prepared from human central nervous system PNETs showed that 13 of 35 contained NGF receptor-positive tumor cells. Thus, more than one-third of such tumors might be responsive to the effects of NGF. Baker DL, Reddy UR, Pleasure S, Hardy M, Williams M, Tartaglione M, Biegel JA, Emanuel BS, Lo Presti P, Kreider B, TrojanowskiJQ, Evans A, Roy AR, Venkatakrishnan G, Chen J, Ross AH, Pleasure D. Human central nervous system primitive neuroectodermal tumor expressing nerve growth factor receptors: CHP707m. Ann Neurol 1990;28:136-145

Primitive neuroectodermal tumors (PNETs) arise at any age and anywhere in the central nervous system (CNS) but are most frequent in childhood and in the cerebellum [1-3}. In the latter site, they are referred to as medulloblastomas, but cerebral variants are often designated cerebral neuroblastomas. The tumors are composed primarily of small- to medium-sized neuroepithelial cells that appear undifferentiated by light microscopy 11-31 but often contain cells that express cytoskeletal proteins characteristic of differentiated neurons or astroglia 14, 51. Cytogenetic analysis of fresh PNETs or short-term PNET cultures has revealed extra copies of chromosomes, chromosomal deletions or translocations, double minutes, or in some instances, apparently normal karyotypes 16, 71. The most frequent finding in CNS PNETs is an isochromosome 17q 171. More detailed analyses of the extent of differentiation of PNET cells and of abnormalities in PNET genotype that contribute to pathogenesis would be

facilitated by the availability of permanent cell lines derived from the tumors. Such permanent lines have already proved valuable in the analysis of PNETs arising outside the CNS. Cytogenetic and molecular studies have defined three biologically distinct neural crest-derived PNET syndromes (neuroblastoma, neuroblastoma with N-myc amplification, and neuroepithelioma), and strongly suggested a neuroepithelial origin for yet a fourth childhood malignancy, Ewing's sarcoma IS- 181. It is, therefore, unfortunate that only five permanent human CNS PNET cell lines, DAOY 1191, D283 MED [20}, D341 MED [Zl}, ONS-76, and ONS-81 1221, are thus far available for study. Even this small sample has been profitably used. Four of the lines, D283 MED, D341 MED, ONS-76, and ONS-81, express neurofilament proteins 123-251. One of these neuronally differentiated lines, D34 1 MED, demonstrates genomic amplification of the proto-oncogene cmyc 1211, and it is reasonable to speculate that this

From the "Children's Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, PA; and the Shrewsbury, Foundation for MA.

Received Nov 21, 1989, and in revised form Jan 22, 1990. Accepted for publication Jan 25, 1990. Address correspondence to Dr David pleasure, Neurology Research, Children's Hospital of Philadelphia, Philadelphia, PA 19104.

136 Copyright 0 1990 by the American Neurological Association

A Fig 1. Radiological and histological appearance ofthe cerebral tumor. (A)An injected computed tomographic scan shows an irregular frontal mass with an enhancing border. (8)The tumor was composed of small round and oval cells with darkly staining nuclei. The celhfirmed occasional Homer Wright rosettes (arrow). Connective tissue stronla was scant. Paraffinsection, stained with H 6 E . overexpression of c-myc contributed to progression of this tumor. Establishment of more CNS PNET lines to search for other genetic mechanisms contributing to the etiology and progression of C N S PNETs is clearly in order. T h e present study describes a sixth permanent human C N S P N E T line, C H P 7 0 7 m , which has the following interesting features: it expresses low-molecularweight neurofilament (NF-L) protein but not amplified c-myc; like neuroepithelioma and Ewing’s sarcoma { 11, 13, 15, 171, it has a stable derivative 22 from a t( 11;22) translocation and synthesizes functional nerve growth factor (NFG) receptors.

B

A second operation was required 2 days later to drain an epidural hematoma. The patient was treated with 5,000 rads to the tumor bed and 3,000 rads to other regions of the neuraxis. Computed tomography 4 months later showed no evidence of residual tumor. The patient was entirely well until 11 months after surgery, at which time he developed pain in the limbs and was found to have metastases to the bone marrow. H e received VP-16 and cis-platinum chemotherapy, and was referred to the Children’s Hospital of Philadelphia (CHP) for consideration for further chemotherapy and bone marrow transplantation. Reexamination of the bone marrow at CHP revealed progressive disease, and it is from this marrow aspirate that CHP707m originated. The tumor became widespread, and the patient died 21 months after the initial diagnosis.

Case Report A previously well 33-year-old man presented with symptoms of increased intracranial pressure of 1 week‘s duration. Computed tomography showed a 5 x 7-cm mass in the right frontal lobe with enhancing margins (Fig 1A). Craniotomy was performed, with subtotal resection of the tumor, which consisted of apparently undifferentiated neuroepithelial cells occasionally arranged in Homer Wright rosettes (Fig 1B). The location and histological appearance of the tumor were consistent with a cerebral PNET El], also known as a cerebral neuroblastoma [ 3 , 261.

Methods Cell Cultwe CHP707m cells were cultured in RPMI 1640 medium containing the following additions: 4 mM L-glutamine, 1.1 mM cis-oxaloacetic acid, 0.45 mM pyruvic acid, 200 U L insulin, 100 Uiml penicillin, 100 pdml streptomycin, and 10% (voli vol) fetal calf serum. The cultured cells adhered to tissue culture plastic surfaces. The immunochemical, NGF binding, and messenger RNA (mRNA) studies were done with cells in the 7th to 17th passage, but CHP707m has now been

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passaged 40 times without change in growth characteristics or immunochemical properties. Immunohistological analysis of the proportion of CHP707m cells incorporating the thymidine analogue, bromodeoxyuridine (BrdU) 1271, demonstrated a 15% nuclear labeling index with a 6-hour labeling period during the log-growth phase. Other lines used in this study included the melanoma A875 [28), the human neuroepithelioma line TC32 {14}, the rat pheochromocytoma line PC12 1291, the human rhabdomyosarcoma line RDTE671 [30-32}, and the human PNET lines DAOY [19}, D283 MED [20), and D341 MED 121). These were cultured in RPMI 1640 medium with the following additions: 2 mM L-glutamine, 100 pdml gentamicin, and 10% (voYvo1) fetal calf serum. Fresh medium was added to each of the lines twice weekly. Periodic testing for Mycoplasma was negative. When confluent, adherent cells were released from the substratum using 0.1% ethylendiaminetetraacetate (EDTA) with 0.25% trypsin.

Cytogenetics Colcemid (final concentration 0.01 l d m l ) was added to the medium of a T-25 flask of exponentially growing CHP707m cells in the 4th passage, and the flask was incubated for 1 hour at 37°C. Cells were treated with 1% sodium citrate for 20 minutes, fixed in methanol: acetic acid (3:1, vollvol), and dropped onto slides that were then air-dried. Chromosomebanding was achieved by trypsin and Wright's staining. Twenty metaphases were examined. Karyotypes of each of the other CNS PNET lines used in this study were consistent with those previously described {19, 21, 261.

Immunocbemicul and Moleczlhr Biological Methods Fluorescence-activated cell sorting, Western blotting, and Northern blotting were as previously described 1181. The cytoskeletal preparation used for characterization of neurofilament triplet peptides expressed by CHP707m was prepared by incubating a whole cell pellet in 0.1 Mmorpholinoethanesulfonic acid, 1 mM magnesium chloride, 1 mM dithiothreitol, 1 mM EGTA, and 1% (volivol) Triton x-100 for 30 minutes at 5°C. After centrifugation at 100,000 g for 30 minutes at 5"C, the pellet was dissolved in Laemmli buffer prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter and overlaid, using primary antibodies to the high- (NF-H),middle- (NF-M), and low-molecular-weight neurofilament (NFL) proteins [23, 241. Total RNA was extracted from the cell cultures [33], resolved by electrophoresis on 1% agaroseformaldehyde gels, and then transferred to Gene Screen Plus membranes (NEN Research Products). The membranes were hybridized with phosphorus 32-labeled 2'-deoxycytidine 5 '-triphosphate nick translation-labeled probes for NF-L [34}, N-myc [9}, c-myc 1353, NGF receptor {36], glial fibrillary acidic protein (GFAP) [37], glutamine synthetase [3S}, myelin basic protein [391, chromogranin 1401, and phosphoglycerate kinase 1411. Paraffin or frozen sections were prepared from biopsy or autopsy specimens from 35 patients with CNS PNETs. Thirty-three of these tumors were cerebellar, and the other 2 were cerebral. Immunoreactive NGF receptor in the sections was demonstrated by an immunoperoxidase procedure as previously described 242, 431.

138 Annals of Neurology Vol 2 8 N o 2 August 1990

NG F Studirs Iodine 125 (1251)-labeled NGF was prepared [44}, and its binding and internalization by CHP707m were estimated as previously described 245,461. Effects of NGF on CHP707m phenotype were examined by adding 50 to 1,000 ng/ml mouse 2.5 S NGF for up to 10 days (three changes of medium). An effect of NGF on the level of CHP707m c-fos mRNA was sought by treating the cells with 50 ngiml NGF for 15 minutes to 1 hour in the presence of 50 &ml cycloheximide; RNA was prepared from the NGF-treated cultures, and from cultures exposed to cycloheximide but not NGF, and c-fos mRNA was detected by Northern blotting 247, 483.

Results A karyotype representative of CHP707m is shown in Figure 2. There were 52 chromosomes, extra copies of a Y, 8, 13, and 17, and missing copies of 14 and 20. Several clonal structural abnormalities were noted, including 2 copies of an isochromosome of lq, a marker containing part of a chromosome 3, a deletion of 5q, two different deletions of 7, a t(9;18), and a t(11;22) with subsequent rearrangement of the derivative 11 chromosome. This rearrangement appears to be an insertion of unidentified chromosomal material into llq22. In addition, there were two marker chromosomes that could not be characterized. Two of the PNET lines, D283 MED and D 3 4 1 MED, expressed much higher levels of mRNAs coding for c-myc than did CHP707m, DAOY,the rhabdomyosarcoma line RDTE67 1, o r the neuroepithelioma line T C 3 2 (Fig 3). Northern blot comparison of the steady-state levels of N-myc mRNA expressed by CHP707m, D281 MED, D 3 4 1 MED, and DAOY with those of N-myc amplified and nonamplified neuroblastomas [183 indicated that N-myc overexpression was not present in either CHP707m or the other three CNS PNET lines (not shown). Several lines of evidence indicated that CHP707m was neuronally differentiated. The cultured tumor cells expressed considerably higher steady-state levels of NF-L mRNA than did the other human CNS PNET lines examined (see Fig 3). The presence of NF-L protein in CHP707m was demonstrated by Western blot (Fig 4). Further Western blots, using monoclonal antibodies recognizing phosphorylated and nonphosphorylated epitopes in NF-M and NF-H [24],failed to detect these peptides in CHP707m (not shown). Indirect immunofluorescence microscopy with a polyclonal NF-L antiserum confimed that all CHP707m cells in the cultures are NF-L + , and was helpful in visualizing the short neurites formed by most CHP707m cells (Fig 5). Light microscopic immunohistological examination of a cell pellet prepared from CHP707m indicated that the cells do not express synaptophysin, a protein characteristic of neuroendocrine cells and neuronally differentiated tumors 149, SO]. The cell pellet

Fig 2. Karyotype of CHP707m. The numerical and structural abnormalities in a representative G-banded kayotype from CHP707m are indicated by arrows. The stemline karyotype is 5 2 , X Y , +Y, -1, -3, +8, - 1 1 , +13, -14, +17, -20, -22, i(Iq), + i f l q ) , +der(3), de1(5)(q31),de117)(q2IqS?), &1(7)(q11.2), t(9;18)(ql3;qlI .2J, f &r(l ])ins (der(1l)t(11;22)(q23-24;qi 1.2- 12j;?j (q22;?)), +&r(22)t(I 1;22)(q23-24;ql1.2-q12),+2 markers.

+

+

was faintly positive for another panneuroendocrine marker protein, chromogranin {5l, 521, but chromogranin mRNA was not detected by Northern blotting of CHP707m total RNA (data not shown). Northern blotting of portions of total RNA from the PNET lines indicated that neither CHP707m nor the other lines express detectable levels of mRNA coding for GFAP or myelin basic protein (data not shown). As previously demonstrated at the protein level [25], three of the four PNET lines (CHP707m, D283 MED, and D341 MED) do express mRNA coding for another protein characteristic of neuroglia, glutamine synthetase. The steady-state level of this mRNA is much lower in CHP707m than in the MED lines, and we were unable to detect glutamine synthetase mRNA in DAOY (data not shown).

CHP707m synthesized and expressed NGF receptor mRNA, whereas D283 MED, D341 MED, and DAOY did not. The size of the CHP707m NGF receptor mRNA was similar to that expressed by the human melanoma A875 (Fig 6). Translation of NGF receptor mRNA by CHP707m was demonstrated by fluorescence-activatedcell sorting (Fig 7) and by Western blotting (Fig 8); immunoreactive NGF receptor was not detected in the three other PNET lines by these methods. Trace amounts of immunoreactive NGF receptor were also detected in the human rhabdomyosarcoma line “RDTE67 1,” previously classified as a CNS PNET (“TE671”) {30-321. Figure 8 also demonstrates that the electrophoretic mobility of CHP707m immunoreactive NGF receptor under denaturing conditions is slightly greater than that of A875, suggesting that CHP707m NGF receptor protein is smaller than A875 melanoma NGF receptor protein. A series of functional studies of the CHP707m NGF receptor were performed. Scatchard analysis [46} demonstrated only a single class of NGF receptors, which bound the mouse {1251)NGF with an dfinity of 1.6 x l o p 9 M (Fig 9). This is intermediate Baker et

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Fig 3. Northern blot show that CHP707m expresses lowmolecular-weight neurofilament (NF-Lj messenger R N A (mRNA) but does not exhibit ampliJZcationof c-myc mRNA. Fifeen-microgram portions of total R N A from the sources indicated at the top of the figure were applied to the lanes. The “8-wk-brain”sample was from total brain of 8-week postnatal rats. The blot was hybridizedfirst with a complementary D N A (cDNAj probe codingfor NF-L (lower panel). Aftw radioautography and stripping, the same blot was rehybridized with a cDNA probe coding for c-myc (upper panel). (Lower panel) CHP707m was strongly positivefor NF-L mRNA. NFL mRNA was also detected in the primitive neuroectodermal tumors (FNETs),D283 MED and DAOY, was barely a’etectable in the PNET 0 3 4 1 MED, and was not detected in the rhabdomyosarcomaRDTE671 or the neuroepithelioma, TC32. (Upperpanel) 0 2 8 3 MED and D341 MED overexpressed cmyc mRNA, whereas CHP707m, DAOY, and RDTE671 did not. TC32 expressed an intermediate level of c-myc mRNA.

between the high affinity (9.6 x 10-l’ M) and low affinity (1.1 X lo-* M) NGF-binding sites expressed by TC32 neuroepithelioma but is comparable to the single class of binding sites (binding affinity, 3.3 x l o T 9 M)present on A875 melanoma cells. The data also indicated that CHP707m expresses 7.1 X lo4 NGF-binding sites per cell, in comparison with the 3.9 X lo5 sites per cell of A875 and the 3.2 X lo4 high affinity and 1.2 X lo5 low afhnity sites per cell of TC32. CHP707m cells internalized [1251)NGF to a greater extent than did A875 melanoma but to a much less extent than the rat NGF-responsive chromaffin cell line, PC12 (Fig 10) [47]. Brief exposure of CHP707m to NGF increased expression by the cells of the proto-oncogene C ~ O J (Fig 11) 147, 48, 531. Treatment of CHP707m cultures with 50 to 1,000 ng/ 140 Annals of Neurology Vol 28 No 2 August 1990

Fig 4. CHP707m expresses lw-molecular-weight neurofilament (NF-L)protein. Equal amounts of cytoskehtal protein extracts from an adult human nerve root (lane 1) and from CHP707m (lane2) were applied to a sodium dodecyl sulfate-polyacvylamide gel. After electrophoresis and transfer t o nitrocellulose, the blot was overlaid with a rabbit antiserum recognizing the carboxyterminal region of NF-L protein. Visualization of bound immunoghbulin was achieved by an auidin-biotin-peroxidaseprocedure.

A

B

Fig 5 . CHP707mf o m short low-molecular-weightneurofilament protein (NF-L) +- neurites. Representative fields of a CHP707m coverslip culture, visualized by indirect immunofluorescence microscopy with a rabbit antiserum recognizing the carboxy-terminal region of NF-L protein. The tumw cells express short neurites that contain NF-L protein, better seen when the cells are not in groups (A).

CHP707m

,*,.--Control MoAb -Anti-NGF-R MoAb

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I 1 I I

I

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Fig 6. CHP707m expresses nerve growth factor (NGFJ receptor messenger R N A (mRNA). Fifteen-microgram portions of total R N A from the soums indicated above the lanes were anahzed by Northern Blotting with a complementary D N A (cDNA)-probe coding for humdn NGF receptor (A) and then, after stripping, with a cDNA probe coding for the “housekeepinggene” phosphoglycerate kinase (B). CHP707m expressed NGF receptor mNRA of similar size to that expressed by the human melanoma line A875. The rhabdomyosarcomd line RDTE671 had trace quantities of NGF receptor mRNA, whereas the primitive neumectodemal tumor lines D341 MED, 0 2 8 3 MED, and DAOY did not.

ml NGF for up to 10 days, however, failed to diminish the rate of proliferation of the cells, to increase neurite length, or, as judged by Western blotting, to qualitatively or quantitively alter expression by the tumor cells of neurofilament protein (54, 551 (data not shown). To determine the frequency with which CNS PNETs express NGF receptors by CNS PNETs, we immunohistologically surveyed sections prepared from tumor specimens from 35 patients with CNS PNETs (33 cerebellar medulloblastomas and 2 cerebral PNETs). Thirteen (3795) contained NGF receprorpositive turnor cells. Discussion Cytogenetic analysis of CNS PNETs has revealed that most have structural or numerical chromosomal abnor-

Log fluorescence Fig 7. CHP707m has immunoreactive cell sudace nerve growth factor (NGF) receptors. CHP707m and DAOY were harvested with ethylenediaminetetraacetate,and the intact cells were incubdted with either a control monoclonal antibody (P3X63Ag8) or a monoclonalanti-human NGF receptor antibody (ME20.4), both I g G I isotype, then washed, and incubated with ajuorescein-conjugated goat anti-mouse immunoglobulin.Fluorescenceactivated cell sorting was with an Ortho Cytojluorograf 50 HH. Results with CHP707m are shown in the upper panel, and with DAOY in the lower panel. CHP707m cells dmonstrated strong su$ace binding of the NGF receptor antibody, whmus DAOY did not. Similar experiments with 0283 MED and 0 3 4 1 MED (not shown)produced results like those with DAOY.

malities, or both. The most commonly involved chromosomes have been 5, 6, 11, 16, 17, and a sex chromosome, and the most frequent structural abnormalities are deletions and nonreciprocal translocations [6, 71. The most consistent cytogenetic alteration is an i(17q), present in approximately one-thrd of tumors, sometimes in the absence of other structural abnormalities [7}.Though CHP707m lacks an i( 17q), it does manifest multiple structural and numerical chromosomal abnormalities, most notably a t( 11;22) with subsequent rearrangement of the derivative 11 chromosome. This is of interest in that a t( 11;22) translocation Baker et al: CNS Tumor Line

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NGF Bound (fmoles) Fig 8. CHP707m expresses immunoreactive newe growth factor (NGF) receptor protein. Fify-microgram portions of total pmtein from the sources indicated above the lanes were analyzed by Western blotting {l8). CHP707m is intensely positive for immunoreactive NGF receptor protein, which has an electrophoreticmobility slight&faster than that expressed by the A875 melanoma cell line. The three other primitive neuroectodermaltumor lines, and the rhabdomyosarcoma, RDTEG71, are NGF receptor negative.

is a constant finding in two neuroepithelial malignancies arising outside the CNS, neuroepithelioma and Ewing's sarcoma 111, 13, 157. It must be emphasized, however, that the karyotype of CHP707m was obtained by examination of cells derived from a metastasis after radiotherapy and chemotherapy. Some or all of the chromosomal abnormalities in CHP707m, including the t( 11;22) translocation, may have been induced by these treatments. CHP707m does not demonstrate high levels of cmyc, a proto-oncogene overexpressed by two other neuronally differentiated CNS PNET lines, D283 MED and D341 MED 1211. Further, it does not overexpress N-myc, a developmentally regulated neuronal proto-oncogene 1561 implicated in the progression of neuroblastomas 116, 57-59} and reported to be amplified in DNA prepared from some neuronally differentiated CNS PNETs 160, 611. CHP707m has a neuron-like phenotype in culture, forming neurites and expressing neurofilaments. Unlike D283 MED, D341 MED, ONS-76, and ONS-81, which contain NF-M and NF-H 122-251, only NF-L was detected in CHP707m. NF-H appears after NF-L during normal development of CNS neurons, and largely after the neurons have become postmitotic 162-641. If the order of induction of NF proteins in PNETs recapitulates that in normal neurons, as suggested by Molenaar et al 151, then the neuronal cyto142 Annals of Neurology

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Fig 9. Scatchard plot of the binding of iodine 125-labeled newe growth factor ({i2sI}-NGF)by CHP707m. {"'I)-NGF binding to CHP707m cells was measured as previous& described {46}. The data points on the Scatchard plot are wellfit by a single straight line, indicating the presence of 7.1 x 104 NGF-binding sites per cell, with a binding afinity of 1.6 x 10 M. ~

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Internalization of NGF

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Fig 10. CHP707m internalizes iodine 125-labeled nerve growth factor (NGF) to a lesser extent than PC12 cells. NGF internalization by CHP707m, A875 melanoma, and PC12 pheochromocytomacells was measured afer a 30-minute incubation at 3 7°C as previousb described {45), and is expressed as the ratio of counts intwnalized (I) t o counts on the snrface (Ej. Resnlts are the mean of 4 determinations, with the bars indicating standard deviations.

Fig 11. Nerve gmwth factor (NGE;)induces CHP707m c-foJ messenger RNA. CHP707m cells were incubated with 50 pglml cycloheximide with (lane 1)or without (lane 2 ) 50 nplml NGF for 30 minutes. Fifty-microgram portions of total R N A were studied by Northern blotting with a complementary D N A (cDNA)probe coding for v-fos (upper panel) and then, after stripping, with a cDNA probe coding for the “housekeeping gene” phosphoglyceratekznase (lower panel). The upper panel shows that expression by CHP707m of c-fos was induced by exposure to NGF. The lower panel shows that RNA equivalent in amount and quality were loaa’ed on lanes 1 and 2.

skeletal differentiation of CHP707m terminates at an earlier stage than that of D283 MED or D341 MED. Unlike the three other CNS PNET lines we studied, but like many neurons in the developing cerebellum and cerebrum [65, 661 and cholinergic neurons in the adult forebrain 167, 681, CHP707m expresses NGF receptors. Survey of histological sections prepared from a group of human CNS PNETs (chiefly cerebellar medulloblastomas) showed that tumor cells in 13 (379%) of 35 cases expressed immunoreactive NGF receptors. Thus, a significant proportion of human CNS PNETs might be responsive to NGF. Earlier studies provided evidence that NGFresponsive cells have nonlinear Scatchard curves suggesting two classes of NGF-binding sites, whereas cells with linear Scatchard curves fail to respond to NGF {53, 691. The present study indicates, however, that this generalization does not always hold; CHP707m yielded a linear Scatchard plot, yet the CHP707m NGF receptors are functional, in that they are capable of mediating the induction by NGF of the protooncogene c-fm {17, 531. The CHP707m NGF receptors bind NGF with an affinity intermediate between the high-affinity and low-affinity sites expressed by

NGF-responsive human neuroblastoma lines [70, 7 11. Both high-affinity and low-affinity NGF receptors contain the same NGF-binding protein 1531. The region of this protein missing in the truncated CHP707m receptor has not as yet been established. It seems unlikely that the missing segment is the same as that lost in the truncated form of Schwann cell NGF receptors [72, 731, since these Schwann cell receptors are released into biologic fluids and, hence, may not retain the normal transmembrane hydrophobic domain, whereas the CHP707m NGF receptor is plasma membrane bound. In a prior study of peripheral nervous system PNETs [18), we observed that two of five neuroepithelioma lines examined, SKNMC and, to a less degree, TC32, expressed three bands of immunoreactive NGF receptor protein, one migrating at the same rate as that of A875 melanoma and the other two more rapidly, like the NGF receptor band of CHP707m. Further studies will be required to determine whether these apparently truncated versions of NGF receptor expressed by CHP707m and the neuroepithelioma lines arise by a posttranslational mechanism or, instead, because of alternate patterns of RNA splicing [39, 72-74]. This work was supported by National Institutes of Health grants NS25044, NS21716, CA47983, and CA36245, and by the Juvenile Diabetes Foundation. We are grateful to Drs H. Friedman and D. signer (Duke University) for providing us with the D283 MED and D341 MED cell lines, to Dr V. M.-Y. Lee for access to her library of anti-NF protein antibodies, to Dr Richard Miller (Case Western Reserve University) for the rat glutamine synthetase cDNA clone, to Dr John Kamholz (University of Pennsylvania) for the human myelin basic protein cDNA clone, to Dr Pamela Cohen (National Institutes of Health) for the chromogranin cDNA clone, and to Dr Moses Chao (Cornell University School of Medicine) for the human NGF receptor cDNA clone. The cDNA probes for N-myc and c-nzyc were obtained from the Cell Center, University of Pennsylvania, and the cDNA probe f0rf.r from the American Type Culture Collection, Rockville, MD.

1. Rorke, LB. The cerebellar meddoblastoma and its relationship to primitive neuroectodermal tumors. J Neuropathol Exp Neurol 1983;42:1-15 2. Dehner Lp. Peripheral and central primitive neuroectodermal tumors. Arch Pathol Lab Med 1986;110:997-1005 3. Russell DS, Rubinstein LJ. Pathology of tumors of the nervow system. 5th ed. Baltimore: Williams & Wilkins, 1989 4. Dehner LP, Abenoza P, Sibley RK. Primary cerebral neuroectodermal tumors: neuroblastoma, differentiated neuroblastoma, and composite neuroectodermal tumor. Ultrastruct Pathol 1988; 12:479-494 5. Molenaar WM, Jansson D. Gould VE, et al. Molecular markers of primitive neuroectodermal tumors and other pediatric central nervous system tumors. Monoclonal antibodies to neuronal and gllal antigens distinguish subsets of primitive neuroectodermal tumors. Lab Invest 1989;61:635-643

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