WO1999011291A1 - A method of inducing apoptosis in human brain tumor cells using nerve growth factor - Google Patents

Abstract

A method of inducing apoptosis in human brain tumor cells by transfecting human brain tumor cells with a viral vector containing a gene encoding a receptor for nerve growth factor is provided. Viral vectors containing a gene encoding a receptor for nerve growth factor which are used in the method are also provided.

Description

A METHOD OF INDUCING APOPTOSIS IN HUMAN BRAIN TUMOR CELLS

USING NERVE GROWTH FACTOR

Introduction

This invention was made in the course of research sponsored by the National Institutes of Health. The U.S. Government may have certain rights in this invention.

Background of the Invention

Nerve growth factor (NGF) is the most well studied representative of a family of trophic factors (neurotrophins) and has pleiotrophic effects including the ability to induce differentiation, support cell survival, and prevent apoptosis in neuronal progenitor cells and immature neurons of the central (CNS) and peripheral (PNS) nervous system (Levi-Montalcini, R. and P. Angeletti 1968 Physiol . Rev. 48:534-569; Greene, L.A. and A. Tischler 1976 Proc . Na tl . Acad. Sci . 73:2424-2428; ϋnsicker, K. et al. 1978 Proc . Na tl . Acad. Sci . 75:3498-3502; Doupe, A.J. et al. 1985 J. Neurosci . 5:2119-2142; Li, Y. et al . 1995 J. Neurosci . 15:2888-2905). NGF also acts as a mitogen for neuronal precursors or endocrine cells (Lilian, L. and P. Claude 1985 Nature 317:632- 634; Tischler, A.S. et al . 1993 J. Neurosci . 13:1533-1542). Two different receptors for NGF have been identified including the p75LNGFR or p75 receptor (Chao, M.V. et al . 1986 Science 232:518-521; Chao, M.V. and B.L. Hempstead 1995 Trends Neurosci . 7:321-326), a transmembrane protein with no identifiable cytoplasmic catalytic domain (Johnson, D. et al . 1986 Cell 47:545-554; Rade e, M.J. et al. 1987 Nature 325:593- 597), and the Trk receptor (also known as TrkA) , a transmembrane protein with a cytoplasmic tyrosine kinase domain (Kaplan, D.R. et al . 1991a Science 252:544-548; Klein, R. et al. 1991a Cell 65:189-197). Recent studies have shown that TrkA is essential for NGF mediated signal transduction (Cordon-Cardo, C. et al. 1991 Cell 66:173-183; Hempstead, B.L. et al. 1992 Neuron 9:883-896; Kaplan, D.R. et al. 1991a Science 252:544-548; Kaplan, D.R. et al. 1991b Na ture 350:158- 160; Klein, R. et al . 1991a Cell 65:189-197). TrkA is a member of a family of high affinity tyrosine kinase receptor proteins that mediate the biological effects of neurotrophins. Other members of this family include TrkB and TrkC (Klein, R. et al. 1991b Cell 66:395-403; Lamballe, F. et al. 1991 Cell 66:967-969; Barbacid, M. 1995 Curr. Opin . Cell Biol . 7:148- 155) .

Although the role of the different neurotrophin receptors in transducing signals induced by neurotrophins is beginning to be elucidated, the possible roles of neurotrophins in the induction and progression of tumors remain unproven. Analysis of two common childhood tumors of the PNS and CNS, i.e., neuroblastomas and medulloblastomas, respectively, demonstrate that these tumors express one or more neurotrophins and neurotrophin receptors (Nakagawara, A. et al. 1992 Cancer Res . 52:1364-1368; Segal, R.A. et al . 1994 Proc . Na tl . Acad. Sci . 91:12867-12871; Washiyama, K. et al . 1996 Am . J. Pa thol . 148:929-940). Further, the levels of trkA mRNA in neuroblastomas (Nakagawara, A. et al. 1993 N. Engl . J. Med. 328:847-854), and the levels of trkC mRΝA in medulloblastomas (Segal, R.A. et al. 1994 Proc . Na tl . Acad. Sci . 91:12867-12871) correlate with improved survival. However, the mechanism whereby different neurotrophins influence the biology of medulloblastomas has not been elucidated. Medulloblastomas are prototypical primitive neuroectodermal tumors (PΝETs) that arise in cerebellum, and they are among the most common childhood brain neoplasms (Hart, M.Ν. and K.M. Earle 1973 Cancer 32:890-897; Becker, L.E. and D. Hinton 1983 Hum . Pa thol . 14:538-550; Rorke, L.B. 1983 J. Neuropa thol . Exp. Neurol . 42:1-15; Peringa, J. et al. 1995 Curr. Opin . Neurol . 8:437-440). Medulloblastomas resemble CNS neuroepithelial progenitor cells at the morphological level although subsets of medulloblastomas express neuronal and /or glial markers (Tremblay, G.F. et al. 1985 Acta . Neuropa thol . 68:239-244; Molenaar, W.N. et al . 1989 Lab . Invest . 61:635-643; Gould, V.E. et al . 1990 Lab . Invest . 62:498-509). Medulloblastoma derived cell lines that express markers of the neuronal lineage (e.g., D283 MED) are thought to be more differentiated, while medulloblastoma cell lines that do not express markers of neuronal or glial lineage (e.g., DAOY) are thought to be more embryonal (Trojanowski, J.Q. et al. 1994 Am . J. Pa thol . 135:747-758; Peringa, J. et al. 1995 Curr. Opin . Neurol . 8:437-440). Since no medulloblastoma cell lines express p75, one of these lines

(i.e., D283 MED or D283) was engineered to express human p75.

This cell line (known as A009 or D283p75) did not differentiate or cease dividing in response to treatment with exogenous NGF (Pleasure, S.J. et al . 1990 Proc . Na tl . Acad. Sci . 87:8496-8500).

It has now been found that human medulloblastoma cell lines transfected with a viral vector to express human TrkA, a receptor for NGF, undergo massive apoptosis in response to exogenous NGF.

Summary of the Invention

An object of the present invention is to provide a method of inducing apoptosis in human brain tumor cells which comprises transfecting human brain tumor cells with a viral vector containing a gene encoding a receptor for nerve growth factor wherein expression of the receptor results in apoptosis of the brain tumor cells.

Another object of the present invention is to provide a vector for inducing apoptosis in human brain tumor cells which comprises an endogenous gene encoding a nerve growth factor receptor wherein expression of said receptor results in apoptosis of brain tumor cells.

Detailed Description of the Invention Nerve growth factor (NGF) has a number of well documented but diverse biological effects. For example, NGF enhances the survival and differentiation of a variety of PNS and CNS neurons including sympathetic, sensory, and cholinergic neurons (Levi-Montacini, R. and P. Angeletti 1968 Physiol . Rev. 48:534-569; Mobley, W.C. et al. 1989 Neuron 3:655-664; Lindsay, R.M. et al . 1994 Trends Neurosci . 17:182- 190) . NGF also promotes the differentiation and proliferation of neural progenitor cells such as chromaffin precursor cells and neuroepithelial stem cells (ϋnsicker, K. et al . 1978 Proc. Na tl . Acad. Sci . 75:3498-3502; Doupe, A.J. et al. 1985 J. Neurosci . 5:2119-2142; Lillien, L. and P. Claude 1985 Na ture 317:632-634; Cattaneo, E. and R. McKay 1990 na ture 347:762- 765). However, a recent study shows that during early development, endogenous NGF can cause the death of retinal neurons that express p75NGFR but not TrkA (Frade, J.M. et al. 1996 Na ture 383:166-168). These studies provide evidence that NGF can enhance normal developmentally regulated programmed cell death in vivo via interactions with p75NGFR.

Histochemical evidence of apoptosis has been observed previously in biopsy samples of human medulloblastomas

(Schiffer, D. et al. 1994 Acta . Neuropa thol . 87:362-370),.

However, while NGF and other neurotrophins (i.e., BDNF, NT3,

NT4/5, NT6) are known to have pleiotrophic effects on different cell types, these factors are primarily known for their ability to promote the proliferation, survival and maturation of target cells. In contrast, cell death is generally thought to be a consequence of neurotrophin withdrawal rather than the exposure of cells to one of these factors (Deckwerth, T.L. and E.M. Johnson 1993 J. Cell Biol . 123:1207-1222; Freeman, R.S. et al. 1994 Neuron 12:343-355; Pittman, R.N. et al. 1993 J. Neurosci . 13:3669-3680; Barbacid, M. 1995 Curr. Opin . Cell Biol . 7:148-155; Mills, J.C. et al. 1995 Methods Cell Biol . 46:217-242) . Further, although each of the major neurotrophin receptors (including p75) as well as several neurotrophic factors have been detected in biopsy samples of human medulloblastomas (Baker, D.L. et al. 1991 Am . J. Pa thol . 139:115-122; Segal, R.A. et al. 1994 Proc . Na tl . Acad. Sci . 91:12867-12871; Washiyama, K. et al . 1996 Am . J. Pa thol . 148:929-940), and preliminary studies of medulloblastoma biopsies suggest that the levels of trkC mRNA correlate with a better response to therapy (Segal, R.A. et al. 1994 Proc. Na tl . Acad. Sci . 91:12867-12871), the effects of NGF and other neurotrophins on the biology of these or other human brain tumors were unknown. It has now been found that NGF, acting through the TrkA receptor, induces apoptotic cell death in human brain tumor-derived cell lines. This is the first direct demonstration that NGF specifically induces apoptosis in two human medulloblastoma-derived cell lines by activating a cognate high affinity receptor, TrkA. Accordingly, the present invention provides a method of inducing apoptosis in human brain tumor cells by transfecting human brain tumor cells with a viral vector containing a gene encoding a receptor for nerve growth factor wherein expression of the receptor results in apoptosis of the brain tumor cells. Also provided in the present invention are viral vectors which induce apoptosis in human brain tumor cells through expression of an endogenous gene encoding a nerve growth factor receptor. In a preferred embodiment, the receptor for nerve growth factor encoded by the viral vector comprises TrkA. Examples of viral vectors which are useful in the present invention include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses and herpesviruses . It is believed that the method of the present invention and vectors used in this method will provide a means for treatment of brain tumors, and in particular medulloblastomas and neuroblastomas. The method of the present invention will also be useful studying the normal development of brain tissue as well as tumor progression. For example, the induction of apoptosis through activation of the TrkA receptor in TrkA expressing medulloblastoma cells is indicative of a novel signaling mechanism playing a role in normal developmentally regulated programmed cell death because medulloblastomas resemble embryonic neuroectodermal stem cells or their immature neuronal and glial progeny (Molenaar, W.N. et al. 1989 Lab . Invest . 61:635-643; Gould, V.E. et al . 1990 62 : 498-509; Trojanowski, J.Q. et al. 1994 Am . J. Pa thol . 135:747-758). The role of NGF and TrkA in the pathobiology of medulloblastomas was determined in two human medulloblastoma derived cell lines, D283trk and DAOYtrk. Wild-type D283 cells were first confirmed to be cells which do not contain detectable p75 and do not express any endogenous TrkA. A Northern blot analysis was performed on the wild-type D283 cells, as well as other genetically modified forms of D283 cells, using a full-length cDNA probe for human trkA. The analysis showed that wild-type D283 cells, as well as D283 cells infected with an empty vector and D283p75 cells, do not express detectable trkA mRNA. Experiments using primers corresponding to trkA also failed to detect trkA mRNA by RT- PCR.

D283 cells were then infected with retroviral constructs designed for selection with either neomycin (D283Ntrk) or L- histidinol (D283Htrk) . Using these cells, two distinct mRNA bands were demonstrated by Northern blot analysis; both bands were present at higher levels than endogenous trkA transcripts in rodent PC12 cells. The larger size of the trkA bands in D283Ntrk and D283HDtrk cells resulted from inclusion of the 5' and 3' long terminal repeats, the packaging signal, the drug resistant gene, and the internal cytomegalovirus (CMV) promoter with the human trkA coding sequences. There was also a difference in the molecular weight of the two upper bands in the D283Ntrk versus the D283Htrk cells that is attributed to the length of the L-histidinol-resistant gene in the pLHDCX vector, a gene that is approximately 500 bp larger than the neomycin-resistance gene in the pLNCX vector (Miller, A.D. and G.J. Rosman 1989 Biotechniques 7:980-982). Further, the expression level of the trkA transcripts in the D283Ntrk cells was slightly higher than in the 3T3trk cells, which were transfected with a construct harboring rat trkA (Mahadeo, D. et al. 1994 J. Biol . Chem . 269:6884-6891). In parallel experiments, it was demonstrated that the unmodified DAOY cell line also did not contain any endogenous trkA, trkB, or trkC transcripts, whereas trkA mRNA was detected in the DAOYtrk cells .

The expression of functional TrkA receptors on D283trk and DAOYtrk cells was then demonstrated. The expression of TrkA receptor protein was monitored by Western blot analysis using E7, a monoclonal antibody (mAb) that recognizes an epitope in the extracellular domain of human TrkA. Both the D283trk and DAOYtrk cell lines exhibited two distinct TrkA immunoreactive bands of approximately 125 and 110 kDa . To determine whether TrkA was expressed on the plasma membrane of the D283trk cells, indirect immunofluorescence was performed on live D283trk cells using E13, a mAb that binds to an epitope in the extracellular domain of TrkA (Muragaki, Y. et al. 1995 J. Comp. Neurol . 356:387-397). The results of these studies demonstrated that TrkA receptor proteins were expressed on the surface plasma membrane of the D283trk cells, whereas no immunoreactive TrkA was detected on the surface of control D283vec cells (cells with the empty vector) . Similarly, TrkA receptor proteins were expressed on the cell surface of the DAOYtrk cells, but not on the wild-type DAOY cells .

Functionality of the TrkA receptor on the cells was also demonstrated by determining whether TrkA became activated after treatment of the D283trk cells with NGF. In these experiments, the ability of TrkA receptor proteins to undergo NGF-induced autophosphorylation on tyrosine residues was monitored. D283trk cells were treated for 5 minutes with NGF followed by immunoblotting from cell lysates with an antiphosphotyrosine antibody. In the absence of NGF, there was little or no autophosphorylation of the TrkA receptors that were expressed in the D283trk cells. Following brief exposure of these cells to NGF, however, autophosphorylation of tyrosine residues in TrkA was induced. To confirm that the immunoband detected by the anti-phosphotyrosine antibody was TrkA, the nitrocellulose replicas were stripped and reprobed using the E7 mAb. The same TrkA immunoband was detected in cell lysates prepared from NGF-treated or untreated cells.

Once the specificity of the cell system was established, experiments were carried out to determine the nature of the biological response of the Trk-expressing medulloblastoma cells to NGF. Aliquots of D283trk and DAOYtrk cell lines were treated with NGF. After a 3 day incubation with 100 ng/ml of NGF in complete culture medium, the wells plated with D283trk cells contained fewer cells than wells containing the D283vec and D283p75 cells (controls for the experiment) . Similarly, the DAOYtrk cells contained fewer cells after treatment with NGF. Furthermore, a large number of the D283trk and DAOYtrk cells were rounded up and floating, suggesting they had undergone cell death. This effect of NGF was observed regardless of whether the D283trk cells were grown in suspension or in monolayer culture on poly-D-lysine-coated dishes. Additionally, a similar response to NGF was seen after stable infection of D283 cells with either the pLNCtrk or the pLHDCtrk vector as well as in subclones of D283trk and DAOYtrk cells that expressed different levels of TrkA receptor protein. To confirm that the reduction in number of D283trk and DAOYtrk cells was specific to NGF treatment, several sets of control experiments were conducted. First, PC12^nmr5 (a PC12 mutant that does not express detectable TrkA, and therefore does not respond to NGF) was infected with the same trkA retroviral construct (pLNCtrk) . Results showed that these cells expressed functional TrkA receptors by extending long processes in response to treatment with NGF. Second, because the NGF used had been prepared from extracts of salivary glands harvested from male mice (as described by Mobley, W.C. et al. 1976 Biochemistry 15:5543-5551), commercially available 2.5S mouse NGF and commercially available recombinant NGF were also used and it was demonstrated that NGF from both commercial sources had similar effects on the D283trk and DAOYtrk cells. The third control experiment performed involved adding an anti-NGF antibody to the culture medium, blocking the effects of NGF treatment on the D283trk cells in a dose-dependent manner. For example, at an antibody dilution of less than 1:500 of a polyclonal antisera, anti-NGF antibody completely blocked the effects of NGF on the D283trk cells. Similar effects also were observed using a mAb to NGF. A fourth control experiment involved pretreatment of D283trk and DAOYtrk cells with K-252a, a specific blocker of TrkA tyrosine phosphorylation (Berg, M.M. et al. 1992 J. Biol . Chem . 267:13- 16) . The induction of cell death by NGF was completely blocked. The fifth control experiment involved use of NT3, the neurotrophin that is the cognate ligand for the TrkC receptor, but that also binds weakly to the TrkA receptor (Cordon-Cardo, C. et al . 1991 Cell 66:173-183; Ip, N.Y. et al. 1993 Neuron 10:137-149; Clary, D.O. and L.F. Reichardt 1994 Proc . Na tl . Acad. Sci . 91:11133-11137). These experiments showed that treatment of the D283trk and DAOYtrk cells with 100 ng/ml NT3 only had a small effect on the number of D283trk and DAOYtrk cells. However, at 1 μg/ml and higher concentrations, NT3 also induced cell death in TrkA expressing D283 and DAOY cells. Finally, D283 and DAOY cells that had been engineered to stably express the TrkC receptor were treated with NT3; this did not result in any augmentation or enhancement of cell death in these cell lines.

To quantify the reduction in the number of NGF-treated D283trk cells, the viability of D283trk cells was monitored using the MTS (3- (4 , 5-dimethylthiazol-2-yl ) -5- (3- carboxymethoxyphenyl) -2- (4-sufophenyl) -2H-terazolium, inner salt) assay on cells treated with different concentrations of NGF. Standard curves for the MTS assay were established to document the linear relationship between the number of D283trk cells and the absorbance values of MTS at 450 nm. After treatment of the D283trk cells with NGF for 4 days in a complete medium, the MTS assay confirmed that there was a dramatic reduction in the number of viable D283trk cells. Moreover, an inverse does-dependent relationship was seen between the concentration of NGF and the number of remaining D283trk cells. Because 50% of the D283trk cells were eliminated by treatment of these cells with 1-5 ng/ml NGF, it is likely that the effect of NGF is mediated by high-affinity TrkA receptors. Parallel studies of the D283vec and D283p75 cell lines support this conclusion since neither of these cell lines showed any significant reduction in cell viability (as monitored by the MTS assay) after treatment with NGF. To determine whether the response to NGF treatment was independent of factors present in serum, the MTS assay was also performed on cells cultured in serum-free medium. In these experiments, NGF caused a 51% reduction in the viability of the D283trk cells at a dose of 100 ng/ml, when compared to D283trk cells that were not treated with NGF. Considered together, these experiments with the MTS assay demonstrate that NGF selectively compromises the viability of D283trk cells and that the effect is independent of the presence or absence of serum factors in the culture medium. Having proved that the effects of NGF on brain tumor cells is both specific to the TrkA receptor and results in cell death, experiments were performed to characterize the type of cell death induced by NGF in the D283trk cells. Three different complementary assays of cell number and viability were used: Hoechst 33342 staining of apoptotic bodies, gel electrophoresis to detect DNA fragmentation induced by apoptosis, and time-lapse video microscopy. In a parallel experiment, NGF-treated DAOYtrk cells were stained with Hoechst 33342 dye and examined by fluorescence microscopy to determine whether these cells also showed evidence of apoptosis .

Immunofluorescence studies performed on cells stained with the Hoechst 33342 dye revealed condensation and fragmentation of the nuclear chromatin in numerous D283trk cells treated with NGF for 4 days, while similar nuclear changes were extremely rare or absent in untreated D283trk cells, as well as in the NGF treated D283vec cells. Similarly, when DAOYtrk cells were treated with NGF for 2 days and then stained with the Hoechst dye, they also revealed condensation and fragmentation of their nuclear chromatin. These findings are consistent with an apoptotic form of cell death in both of these cell lines.

DNA gel electrophoresis supported the results of the fluorescence studies since treatment of the D283trk cells with NGF induced a DNA "ladder" characterized by DNA fragments of multiples of 180 bp. To further analyze and quantify the extent of DNA fragmentation in the NGF treated D283trk cells, we utilized a 3 ' end labeling method to tag DNA fragments with 32P-ddATP at different times following treatment of the D283trk cells with NGF. These studies revealed a DNA "ladder" indicative of apoptosis as early as 24 hours after treatment of the D283trk cells with NGF, and the intensity of the DNA "ladder" increased even further after 48 and 72 hours of treatment. Since video-microscopy enables prolonged in vi tro observations of individual cells after experimental manipulation, D283trk cells were examined by this method following NGF treatment. These video microscopy studies showed that many of the NGF treated D283trk cells became spherical at 12 hrs, followed by membrane ruffling and blebbing during the next 24-72 hrs. This was followed by a loss of cell volume and disintegration of the cell. Since these morphological features are associated with active apoptosis (Deckwerth, T.L. and E.M. Johnson 1993 J. Cell Biol . 123:1207-1222; Pittman, R.N. et al . 1993 J. Neurosci . 13:3669- 3680; Schiffer, D. et al. 1994 Acta . Neuropa thol . 87:362-370; Mills, J.C. et al. 1995 Methods Cell Biol . 46:217-242), the video-microscopy observations provide additional evidence that NGF induces an apoptotic form of cell death in the D283trk cells. Most of the D283trk cells monitored by video microscopy died after treatment with NGF for 4 days, and many of these cells exhibited the morphological features of apoptosis (described above) . Finally, flow cytometric analysis was performed on NGF treated D283trk cells to determine if NGF induced apoptosis in a cell cycle dependent manner. NGF treatment resulted in about a 70% decrease in the percentage of D283trk cells in the S phase of the cell cycle at 24 hrs post-treatment, with a more modest decrease in the number of these cells in the G0/G1 and G2/M phases of the cell cycle. Although the percentage of D283trk cells in G0/G1 and G2/M gradually decreased further with time, these studies demonstrate an early vulnerability of the S phase D283trk cells to apoptosis following treatment with NGF. The quantitative analysis of the absolute number of NGF treated D283trk cells in each phase of the cell cycle demonstrated a similar preferential loss of S phase D283trk cells. For example, the number of D283trk cells in S phase promptly decreased by 70% within the first 24 hrs of NGF treatment, while cells in the G2/M phase decreased much more gradually, i.e., by 46% after 96 hrs of treatment with NGF. This finding that NGF selectively reduces the percentage of D283trk cells in S phase agrees with previous reports which suggest a tight association between DNA synthesis and apoptosis (Qin, X.Q. et al. 1994 Proc . Na tl . Acad. Sci . 91:10918-10922; Shan, B. and W.H. Lee 1994 Mol . Cell Biol . 14:8166-8173) .

In addition, immunohistochemistry of 51 pediatric brain tumors including 20 biopsy specimens of primitive neuroectodermal tumors and 31 other pediatric brain tumors using antibodies to NGF, brain derived neurotrophic factor and NT-3, their cognate high affinity receptors as well as neuronal and glial markers showed no co-distribution of TrkA and NGF in the 20 biopsy specimens of primitive neuroectodermal tumors. Co-distribution of TrkA and NGF was observed in samples from other tumor types. Thus, these results are compatible with evidence of NGF inducing apoptosis in medulloblastoma cell lines transfected to express TrkA and are indicative of the ability of NGF to induce apoptosis in TrkA-expressing medulloblastoma cells in vivo .

The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES

Example 1 : Tissue Culture , Retroviral Infection , And NGF Treatment

Uninfected D283MED (D283) and DAOY cells and cells infected with retrovirus were maintained with RPMI 1640 medium containing 10% fetal bovine serum and 2 mM glutamine in accordance with procedures described by Friedman, H.S. et al . 1985 J. Neuropa thol . Exp. Neurol . 44:592-605 and Trojanowski, J.Q. et al. 1989 Am . J. Pa thol . 135:747-758. DAOY cells normally grow in a monolayer, while the D283 cells grow in suspension. Thus, for the MTS assays, fluorescence and video microscopy, as well as for the immunoblotting and immunostaining, monolayer cultures of the D283 cells were established by plating the cells onto poly-D-lysine (1 μg/ml) coated tissue culture dishes or cover slips as previously described (Pleasure, S.J. et al . 1990 Proc . Na tl . Acad. Sci . 87:8496-8500). For studies involving the use of serum-free medium, fetal bovine serum was omitted from the culture medium.

Retroviruses bearing a full length human trk cDNA ( trkAI isoform; as described by Barker, P.A. et al. 1993 J. Biol .

Chem . 268:15150-15157) cloned into the retroviral vectors

(Miller, A.D. and G.J. Rosman 1989 Biotechniques 7:980-982) pLNCX (designated as pLNCtrk; Stephens, R.M. et al. 1994

Neuron 12:691-705) and pLHDCX (designated as pLHDCtrk; Verdi, J.M. et al. 1994 Neuron 12:733-745) were packaged by electroporation in the GPenvampl2 packaging cell line (Markowitz, D. et al. 1988 Virology 167:400-406). Supernatants containing the retroviruses were harvested and used directly to infect the medulloblastoma cell lines. Following 16 hours of infection, the viral supernatants were removed and the cells were incubated with media alone for an additional 24 hours before selection with either G418 (GIBCO/BRL) at 0.8 mg/ml for the pLNCtrk construct or with L-histidinol (Sigma) at 8 mM for the pLHDCtrk construct for an additional 3 weeks. The drug resistant D283Ntrk and D283Htrk cells were used either as a mass culture or they were subcloned by limited dilution to obtain clonal lines that express high levels of trkA . Similar responses to NGF were obtained with the mass culture and the subclones of the D283Ntrk and the D283Htrk cells. As controls, D283 cells were also infected with a retrovirus bearing the pLNCX empty retroviral vector (D283vec cells) or infected with a retrovirus containing human p75 (designated D283p75 cells: Pleasure, S.J. et al. 1990 Proc. Na tl . Acad. Sci . 87:8496- 8500) or with the pLNCtrkC construct. The pLNCtrkA construct was also used to infect DAOY, PC12^nnr5 and NIH3T3 cells to generate stable population of cells expressing the TrkA receptor. The pLNCtrkC construct also was used to infect DAOY and D283 cells to generate stable populations of these cells that expressed the human TrkC receptor. Identical infection and selection procedures were used for all cell lines. Subclones of DAOYtrk cells and mass cultures of D283trk cells were used in the experiments. NIH3T3 cells transfected with a rat r^AI cDNA (designated 3T3trk cells; Mahadeo, D. et al . 1994 J. Bio . Chem . 269:6884-6891) and PC12 cells over-expressing TrkA (PC12-615 designated here as PC12trk, Hempstead, B.L. et al . 1992 Neuron 9:883-896) were used as positive controls for monitoring the expression of TrkA in the retrovirally infected medulloblastoma cells. Wild type and transfected NIH3T3 cells were maintained in DMEM supplemented with 10% calf serum, and Penicillin/Streptomycin. G418 (0.2 mg/ml) was added to the medium for continuous selection of 3T3trk cells. Wild type PC12 and PC12trk cells were maintained as described by Hempstead, B.L. et al. 1992 Neuron 9:883-896.

NGF was extracted from mouse salivary glands in accordance with procedures by Mobley, W.C. et al . 1976 Neuron 3:655-664. Two other commercial sources of NGF were also used: 2.5S mouse NGF was purchased from Collaborative Biomedical Products (Bedford, MA) and human recombinant NGF-β was obtained from Sigma Chemical Co. (St Louis, MO) . All NGF preparations were used to examine the biological effects of NGF on the wild type and the genetically modified medulloblastoma cell lines (i.e., D283 and DAOY). The specificity of these effects was monitored by using a rabbit antibody to 2.5S mouse NGF (Sigma) as well as a monoclonal antibody to recombinant NGF (Sigma) in blocking experiments. Example 2 : Northern Blots , Immunoblots , and Indirect Immunofluorescence

Total RNA was extracted with Trizol (World Precision Instruments) , and 25 μg of RNA were then electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde, followed by transfer of the separated RNAs to a nylon membrane. After drying the membranes, the immobilized RNAs were probed with a full length human trkA cDNA probe labeled with ^t32P] -dCTP for 3 hours, and washed twice with 2 X SSC (0.15 M sodium chloride/ 0.15 M sodium citrate pH 7.0) + 0.1% SDS at 42°C, twice with 0.2 X SSC + 0.1% SDS at 42°C, and once with 0.1 X SSC + 0.1% SDS at 55°C. The labeled RNA species in these blots were visualized using a Phosphoimager and analyzed with ImageQuant software (Molecular Dynamics, Sunnyvale, CA) . The conditions for cell lysis, and immunoblot analysis were exactly as described in earlier experiments with cells genetically engineered to express each of the major neurotrophin receptor proteins (Muragaki, Y. et al. 1995 J. Comp . Neurol . 356:387-397). For immunoblot analysis, cells were grown in poly-D-lysine coated 10 cm dishes, harvested with Laemmli sample buffer and the proteins denatured by boiling for 15 minutes. One hundred micrograms of protein were separated by SDS-PAGE (7.5% polyacrylamide) gels and transferred onto nitrocellulose paper. The nitrocellulose replica was then divided into two portions using the Mr marker, bovine serum albumin (i.e., 66 kd) as a guide. The top part of the nitrocellulose replica was probed with E7, a previously described monoclonal antibody (MAb) to the extracellular domain of TrkA (Muragaki, Y. et al . 1995 J. Comp. Neurol . 356:387-397) and the bottom part was probed with a MAb to -tubulin (Sigma) . The relative amount of TrkA and -tubulin in each sample was revealed by enhanced chemiluminescence (ECL) . To detect the activated or autophosphorylated form of the TrkA receptor, D283trk cells were either untreated or treated with 100 ng/ml of NGF for 5 minutes. The cells were then lysed with cell lysis buffer (Tris-buffered saline containing 1% SDS, 0.5 mM EDTA, 1 mM EGTA, and a cocktail of protease inhibitors including 1 mM PMSF, 10 μg/ml aprotonin, 1 μg/ml each of leupeptin, TLCK TPCK and soybean trypsin inhibitors and 0.5 mM sodium orthovanadate) , sonicated on ice and spun at 16,000 x g for 30 minutes. One hundred micrograms of total protein from each cell lysate were then separated on 10% SDS-PAGE gels, transferred onto a nitrocellulose replica and probed with a mouse anti-phosphotyrosine MAb (4G10; Upstate Biotechnology, Lake Placid, NY) . Following development of the nitrocellulose replica to reveal tyrosine phosphorylated proteins, the nitrocellulose replica was stripped with 1% SDS; reprobed with the E7 MAb and then redeveloped with ECL to identify the TrkA receptor in the same replica.

For indirect immunofluorescence experiments to detect cell surface expressed Trk receptor protein, live (unfixed) wild type and genetically modified medulloblastoma cells were incubated with a pan-trk mouse MAb (E13) specific for an epitope in the extracellular domain of Trk receptors (Muragaki, Y. et al. 1995 J. Comp. Neurol . 356:387-397) at 4°C for 90 minutes. After washing to remove unbound antibodies, the cells were fixed with 70% ethanol containing 150 mM NaCl, pH 7.0. A Texas red conjugated rabbit anti-mouse antibody was applied to these cells at room temperature for 60 minutes to reveal bound antibody. To demonstrate that the NGF-induced cell death in D283trk and DAOYtrk cells requires tyrosine phosphorylation, the cells were pretreated with 100 nM of K-252a (Kamiya Biochemical, Thousand Oaks, CA) for 1 hour before the addition of NGF (Berg, M.M. et al. 1992 J. Biol . Chem . 267:13-16). Example 3 : Measurement Of Cell Viability

Cell viability was quantitated using the MTS (3- (4, 5-dimethythiazol-2-yl) -5- (3-carboxymethoxyphenyl ) -2- ( 4-sufophenyl) -2H-tetrazolium, inner salt) assay according to procedures recommended by the vendor (Promega, Madison, WI). To do this, 5 X 10³ cells in serum-containing medium or 1 X 10" cells in serum-free medium were used to quantitate MTS in triplicate wells of 96 well-plates after incubating wild type and genetically modified cells for 4 days in NGF as described in Example 1. The absorbance values at 450 nm were quantitated and compared with the values obtained from wells containing wild type or genetically modified D283 medulloblastoma cells that were not treated with NGF. Each MTS assay was repeated at least 3 times and each experiment was done in triplicate. Furthermore, the results of the MTS assays correlated well with cell counts obtained using trypan blue exclusion assay.

Example 4 : Measurements of Apoptotic Cell Death

Three complementary methods were used to monitor apoptotic cell death in NGF treated TrkA expressing medulloblastoma cells.

Hoechst 33342 staining of apoptotic bodies :

In this assay, cells were grown in suspension, incubated with 100 ng/ml of NGF for 4 days, and stained with 5 μg/ml of the Hoechst 33342 dye for 5 minutes at room temperature. Normal nuclei and nuclear apoptotic bodies stained by the Hoechst 33342 dye were then visualized and monitored in each set of experiments by fluorescence microscopy.

Detection of DNA fragmentation induced by apoptosis : To detect DNA "laddering" in cells undergoing apoptosis, wild type and genetically modified D283 cells were incubated with 100 ng/ml of NGF for 3 days and DNA from approximately 8 X 10⁶ cells was extracted as described by Tilly, J.L. and A.J. Hsueh 1993 J. Cell Physiol . 154:519-526. The DNA was electrophoresed in 2% agarose gels, stained by 2 μg/ml ethidium bromide for 30 minutes at room temperature, and destained using distilled water at 4°C overnight. Alternatively, DNA "laddering" was detected in gels of similar DNA extracts by labeling the DNA fragments with α- [³²P] -ddATP (30 μCi) and TdT (25 U) at 37°C for one hour prior to electrophoresis. In this procedure, unincorporated nucleotides were separated from the labeled DNA by precipitating the DNA twice with ethanol. These DNA samples were electrophoresed in 2% agarose gel, and the gel was denatured with 1.5 M NaCl/0.5 M NaOH for 30 minutes followed by an extensive wash with 1.5 M NaCl/1.0 M Tris (pH 7.0) for 30 minutes and transfer to a nylon membrane. These membranes were then analyzed with a Phosphoimager .

Visualization of apoptosis by time-lapse video microscopy:

Living wild type and genetically modified D283 cells treated with NGF were monitored for evidence of apoptosis

(e.g., membrane ruffling and blebbing, formation of apoptotic bodies) by video microscopy for up to 4 days during treatment with NGF as described by Pittman, R.N. et al. 1993 J.

Neurosci . 13:3669-3680 and Mills, J.C. et al . 1995 Methods Cell biol . 46:217-242).

Example 5 : Flow Cytometric Analysis of Apoptosis During Different Phases of the Cell Cycle

To monitor the occurrence of apoptosis during different phases of the cell cycle by flow cytometry, approximately 2.5 X 10⁶ of the D283trk cells were incubated with NGF for different lengths of time, stained with 50 μg/ml propidium iodide in hypotonic buffer containing 0.1% Triton-X and 0.1% sodium citrate at 4°C overnight (Nicoletti, I. et al. 1991 J.

Immunol . Methods 139:271-279), and analyzed with a FACScan

(Becton-Dickinson Immunocytometry Systems, Mountain View, CA) .

The stained cells traversed the beam of light (488 nm wave length) emitted by an argon laser. A 560 nm dichroic mirror and a 585 ± 21 nm band pass filter were used for data collection. The forward scatter (FSC) and side scatter (SSC) of the red fluorescent particles (due to propidium iodide stained DNA) were measured simultaneously with a threshold set up with FL2H 52. All of the data generated in these experiments were recorded with a Macintosh computer using CELL

Quest research software (BDIS) , and ModFit software (Verity

Software House, Inc) was used to analyze the number of cells in each phase of the cell cycle.

Example 6: Immunohistochemistry

Fifty-one children with CNS tumors were included in this study. Samples included 20 classic posterior fossa medulloblastomas in addition to 19 glial tumors (13 astrocytomas, 2 anaplastic gliomas, 2 glioblastoma multiformes, 1 oligodendroglioma, and 1 mixed oligoglioma) , 6 ependymal tumors (3 ependymomas, 1 ependymoblastoma, and 2 anaplastic ependymomas), 3 gangliomas and 3 teratoid tumors (rhabdoid teratoid tumor, teratoma, and teratocarcinoma) . Fresh surgical biopsy samples were fixed with 70% ethanol containing 150 mmol/1 NaCl for 24 to 36 hours and then embedded in paraffin. In addition, normal post mortem human CNS samples of cerebellum, spinal cord, dorsal root ganglion and hippocampus without evidence of neurological disease were used as positive controls and processed in similar fashion. The diagnosis of each tumor was determined primarily by data from immunohistochemical studies performed with antibodies to GFAP, NFL and synaptophysin.

Serial sections of each tumor were cut at a nominal thickness of 6 μM. The immunohistochemistry was performed using the peroxidase-antiperoxidase (PAP) and the avidin- biotin complex method. Several different previously characterized polyclonal antibodies to neurotrophins and their receptors were used including antibodies specific for TrkA (antibody 256-3), TrkB (antibody Bin), TrkC (antibody Cout), NGF (antibody H-20), BDNF (antibody N-20) and NT-3 (antibody anti-NT-3) .

To assess the co-localization or co-distribution of the antigens detected herein, overlap of immunopositive tumor cells within a defined circle (0.5 mm in diameter) in adjacent serial sections of each tumor biopsy sample was determined. The co-distribution of the neurotrophins with their cognate Trk receptors as well as with neuronal and/or glial markers was defined in this manner since these neurotrophins diffuse more than 0.5 mm through the brain following intracerebral injection.

Claims

What is claimed is :

1. A method of inducing apoptosis in human brain tumor cells comprising transfecting human brain tumor cells with a viral vector containing a gene encoding a receptor for nerve growth factor wherein expression of said receptor results in apoptosis of the brain tumor cells.

2. The method of claim 1 wherein the receptor for nerve growth factor is the TrkA receptor.

3. The method of claim 1 wherein the brain tumor cells are medulloblastoma cells.

4. The method of claim 1 wherein the viral vector is a retrovirus.

5. A vector for inducing apoptosis in human brain tumor cells comprising an endogenous gene encoding a nerve growth factor receptor wherein expression of said receptor results in apoptosis of brain tumor cells.

6. The method of claim 5 wherein the receptor for nerve growth factor is the TrkA receptor.