Proc. Natl. Acad. Sci. USAVol. 87, pp. 8496-8500, November 1990Neurobiology

Introduction of nerve growth factor (NGF) receptors into amedulloblastoma cell line results in expression of high- andlow-affinity NGF receptors but not NGF-mediated differentiation

(primitive neuroectodermal tumors/endocytosis/neuronal cell line/signal transduction)

SAMUEL J. PLEASURE*t, USHA RANI REDDYf, GITA VENKATAKRISHNAN§, AMIT K. ROy§, JIE CHEN§,ALONZO H. ROSs§, JOHN Q. TROJANOWSKI*, DAVID E. PLEASURE4, AND VIRGINIA M.-Y. LEE*¶

*Department of Pathology and Laboratory Medicine, Medical Pathology Section, tDavid Mahoney Institute of Neurological Sciences, and *Department ofNeurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and §Worcester Foundation for Experimental Biology,Shrewsbury, MA 01545

Communicated by Eliot Stellar, August 13, 1990 (received for review April 30, 1990)

ABSTRACT Expression of the cloned human nerve growthfactor receptor (NGFR) cDNA in cell lines can generate bothhigh- and low-affinity binding sites. Since the inability torespond appropriately to differentiation factors such as NGFmay contribute to determing the malignant phenotype ofneuroblastomas, we sought to determine whether the same istrue of medulloblastomas. To generate a human central ner-vous system neuronal cell line that would respond to NGF, weinfected the medulloblastoma cell line D283 MED with adefective retrovirus carrying the cDNA coding for the humanNGFR. The resultant cells (MED-NGFR) expressed abundantlow- and high-affinity NGFRs, and NGF treatment induced arapid transient increase of c-fos mRNA in the NGFR-expressing cells but not in the parent line or in cells infectedwith virus lacking the cDNA insert. However, the MED-NGFRcells did not internalize the NGFR at high efficiency, nor didthey differentiate in response to NGF. Three important con-clusions emerge from this study: (i) internalization of NGFRsis not necessary for some early rapid transcriptional effects ofNGF; (ii) an unknown factor(s) that cooperates with the clonedNGFR in allowing high-affmity NGF binding is found in aprimitive central nervous system cell line; and (iii) NGFRsintroduced into and expressed by D283 MED (i.e., MED-NGFR) cells are partially functional but are unable to inducedifferentiation in these primitive neuron-like tumor cells, im-plying that high-efficiency receptor-mediated endocytosis ofNGF and its receptor may be a necessary step in the cascade ofevents leading to NGF-mediated differentiation.

Medulloblastomas, a subtype of primitive neuroectodermaltumors of the central nervous system (CNS), are poorlydifferentiated cerebellar neoplasms that are among the mostcommon intracranial malignancies in childhood (1). Thesetumors are believed to be derived from primitive neuroecto-dermal precursor cells in the cerebellum and to be related toprimitive neuroectodermal tumors elsewhere in the neu-roaxis, including retinoblastomas, pineoblastomas, and so-called cerebral neuroblastomas (1). Medulloblastomas havebeen observed in situ to exhibit neuronal and/or glial differ-entiation, thus emphasizing the multipotential and primitivenature of this type of neoplasm (2). These characteristics ofmedulloblastomas make it vitally important to study thepossible pathways of differentiation as mediated by growthand differentiation factors in these cells. Such studies willlead to a better appreciation ofthe similarities and differencesbetween the neoplastic medulloblastoma cell and its normalcounterpart in the developing human nervous system. The

major tools available for the study of medulloblastomas areseveral cell lines of primarily neuronal phenotype, amongwhich D283 MED is perhaps the most differentiated (3, 4).Nerve growth factor (NGF) is known to play an important

role in the development and maintenance of sympathetic andsensory neurons ofthe peripheral nervous system (5-7), but itsrole in CNS function is less clear. NGF is expressed and hassome effects on gene expression in the CNS (8-10), and NGFreceptors (NGFRs) are found in numerous immature cellpopulations in the CNS of rats, felines, and primates, includingthe granule cells of the cerebellum, which may be the cell oforigin for medulloblastomas (11-14). In the cerebellum thesereceptors have been shown to include both the high- andlow-affinity NGFR (15). In all cases, except the cholinergicbasal forebrain neurons, the expression ofNGFRs is transientduring development (12, 14). Studies of NGF action in theperipheral nervous system have been facilitated by severalvaluable cell lines, including PC12 rat pheochromocytomacells and various neuroblastoma cell lines (16,17), which allowthe study of homogeneous populations of cells synchronizedwith respect to their response to NGF. Previously no medul-loblastoma cell line with NGF-mediated responses has beendescribed to allow the study of NGF effects in this highlymalignant and common childhood brain tumor.

Since the putative cell of origin of medulloblastomas ex-presses NGFRs during development, and since medulloblas-tomas in situ frequently express NGFRs (see Discussion), wedecided to use NGFR cDNA (18) to engineer a medulloblas-toma cell line so that it might be a model for NGF-mediatedresponses in medulloblastomas. Defects in responses to NGFhave been proposed to be important in the pathogenesis ofneuroblastomas and we wished to determine whether suchdefects also exist in medulloblastomas.

MATERIALS AND METHODSTissue Culture, Retroviral Infection, Indirect Immunofluo-

rescence, and Northern Blotting. Uninfected D283 MED cellsand D283 MED cells infected with retroviruses either contain-ing or lacking NGFR cDNA were maintained in suspensionculture with RPMI 1640 medium containing 10% fetal bovineserum and 2 mM glutamine. The retroviral construct used isshown in Fig. la; however, a full description of the construc-tion and packaging of the retroviral vector containing thehuman NGFR cDNA will be described elsewhere. Infections

Abbreviations: NGF, nerve growth factor; NGFR, NGF receptor;CNS, central nervous system.$To whom reprint requests should be addressed at: Department ofPathology and Laboratory Medicine, University of PennsylvaniaSchool of Medicine, Hospital of the University of Pennsylvania,Maloney Basement, Room A009, Philadelphia, PA 19104.

8496

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. NatL. Acad. Sci. USA 87 (1990) 8497

a

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FIG. 1. (a) Schematic representation of the retroviral construct used to infect D283 MED cells. The NGFR cDNA is located downstreamfrom a neomycin-resistance gene (NeoR) and a simian virus 40 (SV40) regulatory sequence. Flanking retroviral long terminal repeats (LTR) arealso shown. kb, Kilobase. (b) Localization of the NGFR to the cell surface of infected MED A009.3 [Upper (two cells shown)] and MED-MTV[Lower (seven cells shown)] by indirect immunofluorescence. (Bar = 20 ,m). (c) RNA blot analysis of NGFR expression in A875 (lane 1),MED-MTV (lane 2), and MED-NGFR (lane 3). The heterogeneity of hybridizing bands and slightly larger size of the NGFR mRNA in theMED-NGFR RNA are due to alternative sites of transcription initiation and termination that are included in the retroviral construct.

were performed and the cells were selected with G418(GIBCO/BRL) at 0.6 pg/ml, as described (19). D283 MEDcells that expressed NGFR were subcloned by limiting dilu-tion. Indirect immunofluorescence techniques were con-ducted on unfixed, unpermeabilized cells (20). For Northernblot analysis, totalRNA was prepared (20), electrophoresed in1% agarose gels containing 2.2M formaldehyde, transferred tonylon membrane, and baked in vacuo at 800C for 2 hr. Theimmobilized RNAs were probed and washed as described (20).

Binding, Internaflation, and Cr kingofNGF. NGF waspurchased from Bioproducts for Research (Indianapolis, IN)and was iodinated by the lactoperoxidase technique of Sutteret al. (21). Binding of 125I-labeled NGF to intact cells wasassayed according to Vale and Shooter (22). 12-I-NGF wascrosslinked to its receptor by a 2-min treatment with 30 mMethyldimethylisopropylaminocarbodiimide and then extractedwith solubilizing buffer and analyzed by SDS/PAGE (23). Thiscrosslinking was completely blocked by addition of unlabeledNGF (100 nM; data not shown). Internalization of '2-I-NGF

was measured as described by Bernd and Greene (24). In brief,5 x 101 cells were plated on poly(L-lysine)-coated plastic35-mm dishes and cultured for 48 hr prior to use. 12-I-NGF,and in some cases nonradioactive NGF, in complete mediumwas incubated with the cells at 37°C. External NGF wasstripped from the cells with 0.2M acetic acid and 0.5M NaCI.Internalized NGF was solubilized with 0.2 M NaOH for y

counting. Although widely varying values for the numbers andaffinities of PC12 cell NGFRs have been reported, the PC12cells used here resemble those used by Buxser et al. (25), whoreported that PC12 cells have 57,000 low-affinity [dissociationconstant (Kd) = 5.2 nM) and 15,000 high-affinity (Kd = 0.3 nM)NGFRs per cell. The rate of increase ofthe ratio of internal toexternal counts was calculated by taking the derivative of thestraight-line functions for the internalization ofNGFRs duringa 5- to 30-min period.

RESULTSD283 MED cells were infected either with a defective murineretrovirus (Fig. la) containing the NGFR cDNA (full virus)

LTR

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8498 Neurobiology: Pleasure et al.

or with the same virus lacking the insert (empty virus). Theinfected cells were selected with G418, and a population ofresistant cells was derived for each of the two viruses. Thefull virus-infected cells were cloned by limiting dilution andscreened for NGFR expression by indirect immunofluores-cence with the monoclonal antibody ME20.4, which is spe-cific for an extracellular epitope on the human NGFR (26).NGFR-positive cells (MED-NGFR) were identified, and foursubclones (MED A009.1-4) were derived that expressed highlevels ofNGFR. Although selective data are presented here,all experiments described in this report have been performedon a mixed population ofNGFR-positive cells (MED-NGFR)and one subclone of this population (MED A009.3). Indirectimmunofluorescence detected NGFR on the surface of theMED A009.3 subclone, whereas the empty virus-infectedcells (MED-MTV) showed no NGFR immunoreactivity (Fig.lb). MED-MTV, MED-NGFR, and A875 (a human mela-noma line with abundant low-affinity NGFRs) were exam-ined for the presence of NGFR mRNA by Northern blotting(Fig. ic). MED-NGFR and A875 cells expressed abundantNGFR mRNA, but no NGFR mRNA was detected in MED-MTV cells.

125I-NGF binding studies revealed that MED-NGFR andthe MED A009.3 subclone expressed 92,000 (Kd = 20 nM)and 124,000 (Kd = 5 nM) low-affinity receptors per cell and10,000 (Kd = 0.31 nM) and 37,000 (Kd = 0.53 nM) high-affinityreceptors per cell, respectively (Fig. 2a). These values aresimilar to those reported for PC12 cells (25). In contrast,MED-MTV cells showed no specific NGF binding activity(data not shown). Treatment ofMED A009.3 with '2'I-NGFand crosslinking agents followed by SDS/PAGE resulted ina major band at 100 kDa representing complexes of NGFR

a

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FIG. 2. (a) 125I-NGF binding of MED-NGFR. Abscissa, NGFbound (fmol); ordinate, bound/free (B/F). (b) Chemical crosslinkingof 125I-NOF to its binding sites. Molecular size markers (kDa) are atright, and a star marks the major 100-kDa band; these and other bandsrepresent complexes of NGFR and NGF. Lane 1, no crosslinkingagent added (0.2 MM NGF); lanes 2-4, increasing amounts of 125I-NGF present in the crosslinking reaction (0.2, 0.5, and 3.0 AM,respectively). (c) Internalization of 1251I-NGF by A875 cells, PC12cells, and MED A009.3 cells. I/E, internal/external.

and NGF (Fig. 2b), as observed previously for PC12 andA875 cells (23, 26). Assays for receptor-mediated endocyto-sis revealed that MED A009.3 internalized '25I-NGF at alevelsimilar to that of the A875 cells, which express only low-affinity receptors (25). This value is much lower than thatobserved for PC12 cells (Fig. 2c) but is similar to values forvariants of PC12 cells that express low-affinity but nothigh-affinity receptors (27). Furthermore, the rates of in-crease for the ratio of internal to external counts were 0.0064,0.0076, and 0.056 min- for A875, MED A009.3, and PC12cells, respectively. This nearly flat plot for A875 cells hasbeen reported previously and was interpreted as reflecting anon-receptor-mediated process (25); we believe this to be thecase with MED A009 cells as well. Previous studies haveshown that the high-affinity NGFR is mainly responsible forreceptor-mediated endocytosis (24, 27, 28), but our resultsprove that high-affinity binding sites alone are not sufficientto confer high-efficiency internalization. Further, receptor-mediated endocytosis may not be linked to the conversion oflow-affinity sites to high-affinity sites (29).

Since one ofthe earliest cellular effects ofNGF is the rapidinduction of immediate early response genes such as c-fos(30-32), we examined this response in MED-NGFR cells.NGF stimulation of MED-NGFR cells resulted in a largeincrease in c-fos mRNA at 15 min and a further increase by30 min (Fig. 3 Upper). In repeated experiments with the sametreatment, MED-MTV cells showed no change in c-fosmRNA (data not shown). This is convincing evidence that atleast some of the early transcriptional effects mediated byNGF are not dependent on high-efficiency receptor-mediatedinternalization ofNGF. Attempts to determine whether othertypes of NGF responses observed in PC12 cells also arefound in MED-NGFR cells all yielded negative results. (i)NGF caused no short- or long-term change in morphology orrate of cell division ofMED-NGFR cells grown in suspensionor adherent to poly(L-lysine) (16). (ii) There was no short- orlong-term change in phosphorylation of neurofilament pro-teins (33, 34). (iii) There was no long-term induction of otherneuronal cytoskeletal proteins, i.e., X and microtubule-associated protein 2 (35). (iv) There were no changes in theprofile of total phosphoproteins (36). (v) NGF had no effecton phosphotyrosine-containing proteins as measured by an-tibodies to phosphotyrosine (37, 38). (vi) NGF treatmentcaused no increase in ornithine decarboxylase mRNA (39).

DISCUSSIONOur study has important implications regarding the mecha-nism of action of NGF, and it resolves some questionsconcerning the relationship of low- and high-affinity NGFRs.First, experiments with MED-NGFR cells show that earlytranscriptional responses to NGF (i.e., c-fos induction) canoccur without high-efficiency receptor-mediated endocyto-sis, but other short- and long-term responses (e.g., increasedexpression and phosphorylation of neuronal cytoskeletalproteins) require more than occupancy of high-affinity NGFbinding sites. We conclude from our data that internalizationof NGFR and/or NGF is probably a necessary step in thecascade of many, but not all, NGF-mediated effects. Second,our system conclusively shows that low- and high-affinityNGFRs are not, as previously suggested (29), distinguishablesolely by their differential ability to undergo receptor-mediated endocytosis. MED-NGFR cells and subclonesthereof may provide valuable tools for characterizing factorsthat allow the high-efficiency internalization of NGFRs.Third, the existence of high-affinity NGFRs in aCNS neuron-like cell line implies that factors necessary for the conversionof low-affinity binding sites to high-affinity ones are notlimited to neural crest-derived tissues and cell lines (40).Recent data from experiments using PC12 cells and purine

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Proc. Nad. Acad Sci. USA 87 (1990)

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FIG. 3. (Upper) Induction of c-fos by NGF treatment in MED-NGFR cells. Lane 1, unstimulated MED-NGFR; lanes 2 and 3,MED-NGFR cells stimulated with NGF (50 ng/ml) for 15 and 30 min,respectively; lane 4, unstimulated NIH 3T3 cells (mouse fibroblastline); lane 5, NIH 3T3 cells following 15 min of treatment with 1 ,uMphorbol 12-myristate 13-acetate. Positions of 28S and 18S rRNA areindicated. (Lower) First three lanes of the same blot probed with ahuman phosphoglycerate kinase (PGK) probe to show uniformity ofRNA loading. One hundred micrograms of total RNA from MED-NGFR cells or 15 gg from NIH 3T3 cells was used per lane.

analogues support the dissociation of early transcriptionaleffects from other effects ofNGF (41) that we have observedin NGFR-expressing D283 MED cells.Our data also are highly significant for understanding the

biology of medulloblastomas because we show that a medul-loblastoma cell line with a well defined neuronal phenotypeis able to express high- and low-affinity NGFRs and respondto NGF by inducing the expression of the protooncogenec-fos but is unable to differentiate further. This is significantfor the in vivo behavior ofmedulloblastomas because 37% (13of 35 biopsy specimens) of these tumors express NGFRs insitu (20). Whether or not any ofthese tumors respond to NGFremains to be determined, but the present data suggest thatthese NGFRs may be incapable of promoting terminal dif-ferentiation in these cells. A new cerebral neuroblastoma-derived cell line, CHP707m, lends further support to thishypothesis since it is aCNS primitive neuroectodermal tumorcell line that expresses endogenous NGFR (20). CHP707mexpresses truncated NGFRs of intermediate affinity (Kd = 2nM) that are able to induce c-fos in response to NGF, butthese cells also fail to undergo further differentiation withNGF treatment (20). Previous work on human peripheralneuroblastoma cell lines has led to the hypothesis that themalignant phenotype of these tumors is related in part toaberrant or blocked responses to NGF (42). Our report isnoteworthy in the context of what is now known about thestate of differentiation and expression of NGFRs in medul-loblastomas (2, 20) and the expression of NGFRs in normaldevelopment. In development, NGFRs are expressed tran-siently in a number of sites (12-14) including the premigratorycerebellar granule cells, (12, 13); these cells may be thosefrom which medulloblastomas are derived. Hence our datasuggest that tumor progression in primitive neuroectodermalneoplasms of the CNS may share with peripheral neuroblas-tomas a defect in their ability to differentiate in response toNGF despite the presence ofabundant NGFRs on the plasmamembranes of some of these tumor cells.

We thank Dr. Darrell Bigner for the opportunity to study the D283MED cell line, C. Page for unfailing technical support, Dr. JamesKeen for his critical reading of the manuscript, and Dr. J. Wang for

anti-phosphotyrosine antibodies. This work was supported by grantsfrom the National Institute ofNeurological Disorders and Stroke andthe National Cancer Institute. S.J.P. was supported by a MedicalScientist Training Program predoctoral fellowship.

1. Zeltzer, P. M. & Pochedly, C., eds. (1986) in Medulloblasto-mas in Children: New Concepts in Tumor Biology, Diagnosisand Treatment (Praeger, New York).

2. Molenaar, W. M., Jansson, D. S., Gould, V. E., Rorke, L. B.,Franke, W. W., Lee, V. M.-Y., Packer, R. J. & Trojanowski,J. Q. (1989) Lab. Invest. 61, 635-643.

3. Trojanowski, J. Q., Friedman, H. S., Burger, P. C. & Bigner,D. D. (1987) Am. J. Pathol. 126, 358-363.

4. He, X., Skapek, S. X., Wikstrand, C. J., Friedman, H. S.,Trojanowski, J. Q., Kemshead, J. T., Coakham, H. B., Big-ner, S. H. & Bigner, D. D. (1989) J. Neuropathol. Exp. Neurol.48, 48-68.

5. Levi-Montalcini, R. & Booker, B. (1960) Proc. Nat!. Acad. Sci.USA 46, 384-391.

6. Gorin, P. & Johnson, E. M., Jr. (1979) Proc. Natl. Acad. Sci.USA 76, 5382-5386.

7. Thoenen, H. & Barde, Y.-A. (1980) Physiol. Rev. 60, 1285-1335.

8. Large, T. H., Bodary, S. C., Clegg, D. O., Weskamp, G.,Otten, U. & Reichardt, L. F. (1986) Science 234, 352-355.

9. Shelton, D. L. & Reichardt, L. F. (1986) Proc. Nat!. Acad. Sci.USA 83, 2714-2718.

10. Mobley, W. C., Neve, R. L., Prusiner, S. B. & McKinley,M. P. (1988) Proc. Natl. Acad. Sci. USA 85, 9811-9815.

11. Taniuchi, M., Schweitzer, J. B. & Johnson, E. M., Jr. (1986)Proc. Natl. Acad. Sci. USA 83, 1950-1954.

12. Schatteman, G. C., Gibbs, L., Lanahan, A. A., Claude, P. &Bothwell, M. (1988) J. Neurosci. 8, 860-873.

13. Yan, Q. & Johnson, E. M., Jr. (1988) J. Neurosci. 8, 3481-3498.14. Allendoerfer, K. L., Shelton, D. L., Shooter, E. M. & Shatz,

C. J. (1990) Proc. Nat!. Acad. Sci. USA 87, 187-190.15. Cohen-Cory, S., Dreyfus, C. F. & Black, I. B. (1989) Exp.

Neurol. 105, 104-109.16. Greene, L. A. & Tischler, A. S. (1976) Proc. Nat!. Acad. Sci.

USA 73, 2424-2428.17. Sonnenfeld, K. H. & Ishii, D. N. (1982) J. Neurosci. Res. 8,

375-391.18. Johnson, D., Lanahan, A., Buck, C. R., Sehgal, A., Morgan,

C., Mercer, E., Bothwell, M. & Chao, M. (1986) Cell 47,545-554.

19. Keller, G., Paige, C., Gilboa, E. & Wagner, E. F. (1985) Nature(London) 318, 149-153.

20. Baker, D. L., Reddy, U. R., Pleasure, S. J., Hardy, M., Wil-liams, M., Tartaglione, M., Biegel, J. A., Emanuel, B. S.,LoPresti, P., Kreider, B., Trojanowski, J. Q., Evans, A., Roy,A. R., Venkatakrishnan, G., Chen, J., Ross, A. H. & Pleasure,D. E. (1990) Ann. Neurol. 28, 136-145.

21. Sutter, A., Riopelle, R. T., Harris-Warrick, R. M. & Shooter,E. M. (1979) J. Biol. Chem. 254, 4972-4982.

22. Vale, R. D. & Shooter, E. M. (1985) Methods Enzymol. 109,21-39.

23. Green, S. H. & Greene, L. A. (1986) J. Biol. Chem. 261,15316-15326.

24. Bernd, P. & Greene, L. A. (1984) J. Biol. Chem. 259, 15509-15516.

25. Buxser, S. E., Watson, L. & Johnson, G. L. (1983) J. Cell.Biochem. 22, 219-233.

26. Ross, A. H., Grob, P., Bothwell, M., Elder, D. E., Ernst,C. S., Marano, N., Ghrist, B. F. D., Slemp, C. C., Herlyn, M.,Atkinson, B. & Koprowski, H. (1984) Proc. Nat!. Acad. Sci.USA 81, 6681-6685.

27. Green, S. H., Rydel, R. E., Connolly, J. L. & Greene, L. A.(1986) J. Cell Biol. 102, 830-843.

28. Hosang, M. & Shooter, E. M. (1987) EMBO J. 6, 1197-1202.29. Eveleth, D. D. & Bradshaw, R. A. (1988) Neuron 1, 927-936.30. Greenberg, M. E., Greene, L. A. & Ziff, E. B. (1985) J. Biol.

Chem. 260, 14101-14110.31. Milbrandt, J. (1986) Proc. Nat!. Acad. Sci. USA 8R3, 4789-4793.32. Visvader, J., Sassone-Corsi, P. & Verma, I. M. (1988) Proc.

Natl. Acad. Sci. USA 85, 9474-9478.33. Lee, V. M.-Y. & Page, C. D. (1984) J. Neurosci. 4, 1705-1714.

Neurobiology: Pleasure et al.

.If

Dow

nloa

ded

by g

uest

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July

21,

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8500 Neurobiology: Pleasure et al.

34. Lindenbaum, M. H., Carbonetto, S. & Mushynski, W. E.(1987) J. Biol. Chem. 262, 605-610.

35. Black, M. M., Aletta, J. M. & Greene, L. A. (1986) J. CellBiol.103, 545-557.

36. Halegoua, S. & Patrick, J. (1980) Cell 22, 571-581.37. Wang, J. Y. J. (1985) Mol. Cell. Biol. 5, 3640-3643.38. Maher, P. A. (1988) Proc. Natl. Acad. Sci. USA 85, 6788-6791.

Proc. Nail. Acad. Sci. USA 87 (1990)

39. Feinstein, S. C., Dana, S. L., McConlogue, L., Shooter, E. M.& Coffino, P. (1985) Proc. Natl. Acad. Sci. USA 82, 5761-5765.

40. Hempstead, B. L., Schleifer, L. S. & Chao, M. V. (1989)Science 243, 373-375.

41. Volont6, C., Rukenstein, A., Loeb, D. M. & Greene, L. A.(1989) J. Cell Biol. 109, 2395-2403.

42. Weston, J. A. (1986) Curr. Top. Dev. Biol. 20, 195-210.

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