Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/82—Translation products from oncogenes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Oncogenes are genes that, when activated or altered, may be involved in transformation of normal cells to a neoplastic phenotype. Numerous oncogenes have been identified in both humans and animals, and the transformation mechanisms associated with each vary widely. Frequently, the oncogene has a corresponding normal cellular gene referred to as a proto-oncogene, and modification of either the expression or the structure of the proto-oncogene may result ' in neoplastic transformation.
• N-myc is a human proto-oncogene which was originally identified because of its similarity to the viral c-myc oncogene. Gene amplification and/or increased expression of N-myc has been found in certain tumor cell lines as well as both primary and metastatic tumors.
• N-myc has been associated with neuroblastomas, retinoblastomas, and small-cell lung cancers (SCLC) . Additionally, expression of N-myc has been found to complement mutant ras genes in tumorigenic conversion of rat fibroblasts. Thus, N-myc appears to be involved in the pathogenesis of certain human malignancies.
• N-myc proto-oncogenes were first identified in human neuroblastoma cell lines which showed a 25 to 700-fold amplification of the gene. Schwab et al.
• the present invention provides methods and compositions for identifying and monitoring human can ⁇ cers, particularly cancers which have neural or neuroendo ⁇ rine properties such as neuroblastomas, retinoblastomas, and small-cell lung cancers.
• the methods rely on detection of N-myc protein in a biological specimen, usually a cell sample such as a tissue sample or sputum sample. Presence of the N-myc protein in the biological specimen may be diagnostic and/or prognostic of the cancer.
• Polypeptides and antibodies thereto are utilized for detecting the N-myc proteins, where the polypeptides are associated with immunogenic sites on the protein.
• the polypeptides may be natural or synthetic and will include at least six contiguous amino acids of the natural protein, usually including at least 9 contiguous amino acids, more usually including at least 12 contiguous amino acids, representing detectable epitope(s) on the N-myc protein.
• Such polypeptides include the N-myc protein in substantially pure form as well as fragments thereof.
• Monoclonal or polyclonal antibodies against the polypeptides are prepared by conventional techniques, and the antibodies and polypeptides are utilized in a variety of conventional immunologic assay and histochemical staining techniques for detection of N-myc protein in the biological specimen.
• the present invention is particularly useful for distinguishing among morphologically similar tumor types. For example, it is frequently difficult to distinguish various round cell tumors of childhood, such as neuroblastomas, neuroepitheliomas, Ewing sarcomas, rahbdomyosarcomas, and lymphomas. Since the treatment required depends greatly on the nature of the tumor, it is important to be able to distinguish the tumor type at an early stage. Use of the present invention will allow early identification of neuroblastomas and other tumors expressing N-myc.
• the present invention will also be useful for identifying the cellular origin of micrometastases in non-primary sites such as lymph nodes and bone marrow, and the amount of the N-myc protein expressed, measured by the intensity of staining, may correlate with the disease prognosis.
• the present invention also promises to provide early detection of small cell lung cancers, particularly by detection of cancer cells in sputum and/or lung lavage samples.
• novel DNA molecules are provided for express ⁇ ing the N-myc polypeptides.
• the DNA molecules include at least a portion of the N-myc gene under the transcriptional control of a heterologous promoter, and usually also include an origin of replication.
• the N-myc gene may be natural or synthetic, and the DNA molecule expressed in vitro to produce the N-myc polypeptides.
• Fig. 1 represents the nucleotide sequence together with the deduced amino acid sequence for the N-myc 1 clone described in the Experimental section hereinafter.
• Fig. 2 illustrates the P414/936 plasmid used in expressing various N-myc 1 fragments, as described in detail in the Experimental section hereinafter.
• DESCRIPTION OF THE SPECIFIC EMBODIMENTS - methods for identifying cancers in human patients rely on detection of the expression product of the human N-myc proto-oncogene in a biological specimen, typically body fluids, tissue, or sputum samples. Conveniently, detection is accomplished by immunological techniques, such as immunohistochemical staining ot a cell sample employing either monoclonal or polyclonal antibodies specific for the N-myc protein. Detection of the N-myc protein is particularly useful in distinguishing specific cancers, such as neuroblastomas, from morphologically similar cancers and for the early detection and differential diagnosis of cancers, such as small-cell lung cancers.
• N-myc is a human proto-oncogene which has been associated with the pathogenesis of a number of human tumors, particularly neural and neuroendocrine associated tumors such as neuroblastomas, retinoblastomas, and small-cell lung cancers.
• the ex ⁇ pression product of the N-myc gene is a doublet phosphoprotein including polypeptides of about 62 kD and 64 kD, respectively, where it is believed that the 62 kD species represents the gene product prior to phosphorylation.
• the protein has a half-life of about 30 to 50 minutes, and is located within the cell nucleus where 40 to 50% is associated with the nuclear matrix.
• neoplastic transformation of certain neural and neuroendocrine associated cells is characterized by detectable expression of the N-myc protein.
• the polypeptides of the present invention will be either haptenic or antigenic, including at least 6 amino acids, usually at least 9 amino acids, and more usually 12 or more amino acids found contiguously within the natural N-myc protein.
• the contiguous amino acids may be located within any region of either of the doublet polypeptides and will correspond to at least one epitopic site which is characteristic of the protein.
• characteristic it is meant that the epitopic site will allow immunologic detection of the N-myc protein in a cell sample with reasonable assurance, in most cases allowing N-myc to be immunologically distinguished from other related proteins, such as c-myc. In other cases, however, the epitopic site(s) will be cross-reactive with other proteins, • such as c-myc.
• the N-myc polypeptides may be natural, i.e., the entire N-myc protein or fragments thereof isolated from a natural source, or may be synthetic.
• the natural polypeptides may be isolated from natural sources, such as neuroblastoma and retinoblastoma cell lines known to produce the N-myc protein, by conventional techniques such as affinity chromatography.
• polyclonal or monoclonal antibodies obtained according to the present invention may be used to prepare a suitable affinity column by well known techniques. Such techniques are taught, for example, in Hudson and Hay, Practical Immunology, Blackwell Scientific Publications, Oxford, United Kingdom, 1980, Chapter 8.
• Synthetic polypeptides which are i munologically cross-reactive with the natural N-myc protein may be produced by either of two general approaches.
• polypeptides having fewer than about 50 amino acids, more usually fewer than about 20 amino acids may be synthesized by the well-known Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain (Merrifield (1963) J. Am. Che . Soc. 85:2149-2156).
• the amino acid sequences of such synthetic polypeptides may be based on the sequence of Fig. 1 described in the Experimental section hereinafter, or on the sequence for the entire N-myc gene reported in Kohl et al. (1986) , supra.
• the second and preferred method for syn ⁇ thesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of the N-myc gene.
• the N-myc gene may itself be natural or synthetic with the natural gene obtainable from cDNA or genomic libraries using available probes, such as pNb-1 (Schwab et al. (1983) Nature 305:245). Alternatively, probes may be synthesized based on the DNA sequences reported in Fig. 1, as described in the Experimental section hereinafter. Suitable cDNA and genomic libraries may be obtained from human cell lines known to contain the N-myc gene, such as the LA-N-5 and IMR-32 human neuroblastoma cell lines.
• polynucleotides may be synthesized by well-known techniques.
• short single-stranded DNA fragments may be prepared by the phosphoramidite method described by Beaucage and Carruthers (1981) Tett. Letters 22:1859-1862.
• a double-stranded fragment may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
• Particular DNA sequences may be based on Fig. 1 herein, or on the sequence reported by Kohl et al. (1986) , supra.
• DNA constructs capable of introduction to and ex ⁇ pression in an in vitro cell culture will be incorporated in DNA constructs capable of introduction to and ex ⁇ pression in an in vitro cell culture.
• the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction and integration within the genome of cultured mammalian or other eukaryotic cell lines.
• DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the N-myc DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the N-myc DNA se ⁇ quence, and transcriptional and translational termina ⁇ tion regulatory sequences joined to the 3'-end of the N-myc sequence.
• the transcriptional regulatory se ⁇ quences will include a heterologous promoter which is recognized by the host.
• available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the N-myc DNA sequence may be employed.
• the polypeptides are obtained in substantially pure form, that is, typically about 50% w/w or more purity, substantially free of interfering proteins and contaminants.
• the N-myc polypeptides are isolated or synthesized in a purity of at least about 80% w/w and, more preferably, in at least about 95% w/w purity.
• homogeneous polypeptides of at least 99% w/w can be obtained.
• the proteins may be purified by use of the antibodies described hereinafter using immunoads ⁇ rbent affinity chromatography.
• Such affinity chromatography is performed by first linking the antibodies to the solid support and then contacting the linked antibodies with the source of the N-myc proteins, e.g., lysates of cells which naturally produce N-myc or which produce N-myc as a result of introduction of a recombinant N-myc DNA molecule.
• the source of the N-myc proteins e.g., lysates of cells which naturally produce N-myc or which produce N-myc as a result of introduction of a recombinant N-myc DNA molecule.
• polyclonal antibodies specific for the N-myc protein may be produced by in vitro or in vivo techniques.
• In vitro techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides, while in vivo techniques require the- injection of the polypeptides into a wide variety of vertebrates. Suitable vertebrates are non-human, including mice, rats, rabbits, sheep, goats, and the like. Polypeptides having more than about 30 amino acids, particularly more than about 50 amino acids, may serve directly as immunogens.
• the polypeptide is smaller than about 10 kD, particularly less than about 6 kD, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response.
• the immunogens are then injected into the animal according to the predetermined schedule, and the animals are bled periodically with successive bleeds having improved titer and specificity.
• the injections may be made intramuscularly, intraperitoneally, subcutaneously, or the like, and usually an adjuvant, such as incomplete Freund's adjuvant, will be employed.
• monoclonal antibodies can be obtained by preparing immortalized cell lines capable of producing antibodies having the desired specificity.
• immortalized cell lines may be produced in a variety of ways. Conveniently, a small vertebrate, such as a mouse, is hyperimmunized with the desired antigen by the method just described. The vertebrate is then killed, usually several days after the final immunization, the spleens removed, and the spleen cells immortalized. The manner of immortalization is not critical. Presently, the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519. Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies.
• the manner of fusion is not critical and various techniques may be employed.
• the spleen cells and myeloma cells are combined in the presence of a nonionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's Medium, for a few minutes.
• a nonionic detergent usually polyethylene glycol
• other additives such as Dulbecco's Modified Eagle's Medium
• the nonionic detergent is rapidly removed by washing the cells.
• the fused cells are promptly dispensed in small culture wells (usually in a microtiter plate) at « 5 relatively low density, ranging from about 1-5x10 per well, in a selective medium chosen to support growth of the hybrid cells while being lethal to the myeloma cells.
• the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT sensitive.
• hybrids After sufficient time, usually from one to two weeks, colonies of hybrids are observed and plates containing hybrid positive wells are identified. The plates and wells having only one colony per well are selected, and supernatants from these wells are tested for binding activity against the N-myc protein or the isolated antigen.
• the cell line can be maintained as viable cultures and/or by lyophilization and frozen storage. Depending on the desired use for the antibodies, further screening of the hybridomas may be desirable. Hybridomas providing high titers are desirable.
• cytotoxic antibodies e.g., IgG2a, IgG2b, IgG3 and IgM, may be selected for use in therapeutic treatment of colorectal cancers.
• monoclonal antibodies may be isolated from the supernatants of the growing colonies.
• the yield of antibodies obtained is usually low.
• the yield may be nehanced by various techniques, such as injection of the hybridoma cell line into the peritoneal cavity of a vertebrate host which accept the cells.
• Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Proteinaceous and other contaminants will usually be removed from the monoclonal antibodies prior to use by conventional technique, e.g., chromatography, gel filtration, precipitation, extraction, or the like.
• polypeptides used as the immunogen antibodies having high specificity and affinity for the N-myc protein can be obtained.
• the polypeptide selected should represent one or more epitopic sites which are unique to the N-myc protein and which can distinguish N-myc from closely related proteins such as c-myc. Such unique epitopes are found on polypeptides II and III shown above.
• the polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antis- bodies will be labelled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature.
• Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
• Antibodies and polypeptides prepared as described above can be used in various immunological techniques for detecting N-myc protein in biological specimens, particularly cell samples such as neurocytes, retina cells, and small lung 'cells (neuroendocrine-derived) and body fluid samples, including blood, plasma, serum, urine, and the like.
• cell samples such as neurocytes, retina cells, and small lung 'cells (neuroendocrine-derived) and body fluid samples, including blood, plasma, serum, urine, and the like.
• liquid phase assays and solid-phase immunohistochemical staining techniques will find use.
• immunohistochemical staining techniques may be used with cell samples including tissue samples, sputum, and lung lavage samples.
• a tissue sample may be fixed in formalin, B-5, or other standard histological preservative, dehydrated and embedded in paraffin as is routine in any hospital pathology laboratory.
• Sections may then be cut from the paraffinized tissue block and mounted on glass slides.
• the N-myc protein if present, may then be detected in the nucleus by exposure with labelled anti-N-myc antibody or exposure to unlabelled anti-N-myc antibody and a labelled secondary antibody.
• Sputum and lavage samples are typically prepared in a similar manner where the sample is first dehydrated by exposure to a dehydrating agent, typically a low molecular weight alcohol.
• Liquid phase immunoassays or Western Blot analysis will also find use in the detection of the N-myc protein particularly in body fluids when the protein is shed into such fluids, e.g., blood or urine.
• Solid tissue and sputum samples may also be assayed in liquid phase systems by lysing the cellular sample in order to release the intracellular protein. Once the protein is released, the sample will be placed in a suitable buffer, the sample buffer subjected to a suitable immunoassay.
• Numerous competitive and non-competitive immunoassays are available and de ⁇ scribed in the scientific and patent literature.
• anti-N-myc antibodies may be coupled to toxins, such as diptheria toxin and the ricin A chain, and administered to patients suffering from neural and neuroendocrine cancers.
• toxins such as diptheria toxin and the ricin A chain
• the use of antibody conjugated toxins in cancer therapy is described generally in U.S. Patent Nos. 4,093,607; 4,340,535; 4,379,145; and 4,450,154.
• Antibodies alone may also find use in treatment, particularly by blocking or interrupting, the activity of the N-myc protein which contributes to the neoplastic phenotype.
• N-myc a cDNA library was constructed from the LA-N-5 human neuroblastoma cell line (insert citation) using the Okayama-Berg vector (Okayama and Berg (1982) Mol. Cell Biol. 2:101). Approximately 80,000 clones were screened with pNb-1 which is a plasmid containing a human N-myc fragment isolated from a neuroblastoma cell line (Schwab et al. (1983), Supra.) . One positive clone, designated N-myc 1, was obtained. Dideoxy sequencing of N-myc 1 was performed on independent Ml3 clones containing either overlapping fragments or complementary DNA strands. The nucleotide sequence together with the deduced amino acid sequence is shown in Fig. 1. The amino acids in Fig. 1 are numbered corresponding to the complete sequencing as reported by Kohl et al. (1986) Supra.
• N-myc 1 The largest open reading frame in N-myc 1 is 1182 nucleotides long and would encode the carboxyl terminal 394 amino acids of the putative N-myc protein. Comparison of the N-myc 1 sequence and that reported by Kohl et al. (1986) Supra, reveals only one notable difference between the large open reading frame from the N-myc 1 clone and the published sequence.
• the third base of the codon for amino acid 226 in the N-myc 1 clone is a cytosine, while the first base of the next codon is a guanine.
• the published sequence shows a reversal of these two residues. This difference results in a change in amino acid 227; i.e., alanine instead of proline.
• a series of vectors for expressing portions of the putative N-myc encoded protein in E . coli were constructed by fusing the 5'-region of a bovine growth hormone gene (bGH) in frame, to six different regions of the N-myc 1 clone as follows:
• bGH/N-myc constructions I, III, IV., V and VI The three sites in the bGH gene used for the fusion to N-myc specific sequences were SstI, PstI, and Hindlll, generating fusion proteins containing 76, 92, and 114 residues of the amino terminus of bGH, respectively.
• Expression vector p414/936 insert citation
• Fig. 2 a modified temperature-sensitive runaway plasmid containing transcription termination and translation termination signals in all three reading frames adja ⁇ cent to a polylinker used for introduction of genes linked to a tryptophan synthetase promoter
• the basic components of the p414/936 vector are: 1) a selectable drug marker. Amp ; 2) transcription termination signal, and 3) a poly linker containing translation termination signals in all three reading frames shown in boxes. All five fusion genes expressed using this vector were promoted by a tryptophan synthetase promoter depicted by the black box.
• the bGH/N-myc genes, including the promoter, were all cloned into the EcoRI site 5' to the promoter and at either the Hpal, BamHI or Sstll sites (underlined) at the 3'-end of the genes. The specific 3'-cloning site is shown in brackets beside each construct.
• each bar construct represents the bGH domain, while the open portion represents the N-myc domain.
• the number of amino acids representing either bGH or N-myc in each construct is shown in brackets within each bar construct.
• the letters opposite the bracketed numbers in the shaded portion refer to the restriction endonuclease sites in bGH where the N-myc gene segments were fused: H (Hindlll) , S (SstI) , and P (PstI) .
• bGH/N-myc I was constructed by cloning the MluI/BamHI fragment (Fig. 1) of N-myc into M13mp20.
• N-myc fragment was then recovered by cleavage with Hindlll and Sstll for cloning into the p414/936 expression vector with the appropriate bGH fragment (EcoRI/HinduI) .
• bGH/N-myc III was generated by cleaving N-myc 1 with TagI and blunt ending the site by filling in the appropriate nucleotides with Klenow.
• N-myc 1 was then cut with SstI and the N-myc fragment ligated to the vector as shown above.
• bGH/N-myc IV was constructed by cloning the small N-myc Mspl fragment into M13ir.pl0 (AccI) and recovering it by cleavage with PstI and BamHI.
• bGH/N-myc V was formed in the same manner as bGH/N-myc IV, using the largest Mspl fragment of N-myc 1.
• bGH/N-myc VI was generated using an Mspl/Hindll fragment of N-myc 1 cloned into M13mp8 (Accl/Hindll) . The fragment was recovered using Hindlll and HindiI for cloning into the vector.
• the dotted portion of the bar construction for bGH/N-myc VI represents the 3'-untranslated sequence present in this construction.
• Fusion gene bGH/N-myc II was expressed using vector pCFM414 previously described for obtaining expression of the human c-myb antigen.
• bGH/N-myc II was constructed by ligating an Sstl/Rsal fragment of N-myc 1 in frame with the SstI site in the bGH gene and allowing the encoded product to terminate at a stop codon provided in the vector.
• the bGH/N-myc fusion proteins obtained as described above were used to produce polyclonal antisera in rabbits by the method described by Slamon et al. (1985) Science 228:1427. 3. Characterization of the N-myc Protein Using the Polyclonal Antisera.
• Antisera generated to the various N-myc protein fragments were tested in a liquid phase immunoprecipitation assay using human neuroblastoma cell lines (LA-N-5 and IMR-32) known to express N-myc transcripts, but not c-myc.
• the human promyelocytic cell line, HL-60 was used as a negative control, since these cells express high levels of c-myc transcripts, but not N-myc.
• two other human cell lines, the HT 29 (colon carcinoma) and U251 (glioma) were used as negative controls since neither express N-myc transcripts. The criteria used to identify a protein as N-myc encoded were two-fold.
• the protein had to be immunoprecipitated from neuroblastoma cell lysates by antisera to at least two different N-myc-encoded fragments representing separate areas of the putative amino acid sequence, thus greatly reducing the possibility that the immunoprecipitation was due to chance sequence homology between the vector-expressed fragment and a cellular protein other than N-myc.
• the protein should not be found in other human tumor cell lines (including the neural derived glioma cells) where the N-myc transcript is not found.
• the c-myc protein is similar in size (64-67 kD) and appearance (doublet) to the N-myc protein (Hann and Eisenman (1984) Mol. Cell Biol. 4:2486). Evidence that this is not the c-myc protein, however, is provid ⁇ ed by two sets of data. First, the protein identified using the anti-bGH/N-myc fusion antibodies is found in cells (LA-N-5) which lack detectable c-myc transcripts. Second, five of the six antisera fail to identify any specific protein when used in immunoprecipitation assay with lysate from the HL-60 cell line which is rich in c-myc transcripts, indicating that they do not recog ⁇ nize the c-myc protein.
• the antisera directed against • N-myc fragment V did recognize a 64-67 kD doublet in lysate from HL-60 cells on long exposure (96 hours) of the immunoprecipitation radioautograph. Comparison of the deduced amino acid sequence from this fragment of N-myc and the analogous area in the c-myc protein shows significant sequence homology which would account for the ability of this antiseru to recognize the c-myc protein. None of the anti-bGH/N-myc antisera precip ⁇ itated a 62-64 kD protein in the U251 or HT-29 control cell lines. Thus, the data demonstrate that the 62-64 kD protein is the N-myc-encoded protein.
• 32 P-orthophosphate (ICN Biochemicals, Inc., Irvine, CA) by the method of Osterman, in: Methods of Protein and Nucleic Acids Research, Springer-Verlag, New York, New York, 1984, pp. 144-150.
• the lysate was then subjected to immunoprecipitation using antisera to the bGH/N-myc II fusion protein and analyzed on SDS-PAGE gel. A band at 62-64 kD was found to be labelled with 32P-orthophosphate.
• the N-myc protein is a phosphoprotein.
• a known amount of radioisotope-labelled whole cell lysate was subjected to immunoprecipitation using a combination of N-myc-specific antisera (anti-bGH/N-myc II and IV) .
• the resulting precipitated proteins were then subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) analysis, and the doublet band at 62-64 kD was eluted from the gel, counted and compared to the total trichloroacetic acid-preciptable counts in the whole cell lysate as described by Slamon et al. (1985) , supra. Approximately 0.076% of the total SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) analysis, and the doublet band at 62-64 kD was eluted from the gel, counted and compared to the total trichloroacetic acid-preciptable counts in the whole cell lysate as described by Slamon et al. (1985) , supra. Approximately 0.076% of the total SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) analysis, and the doublet
• LA-N-5 human neuroblastoma cells were labelled with 35S-methionine for sixty minutes and chased with excess unlabelled methionine (Slamon et al.
• each of anti-N-myc antisera were used in immunocytochemical analyses of LA-N-5 cells.
• N-myc II and III did not stain HL-60 cells, indicating specificity for the N-myc protein.
• the antisera directed against N-myc fragment V stained both LA-N-5 and HL-60 cells.
• the ability of this antiserum to immunocytochemically recognize both the N-myc and c-myc proteins is likely due to the high degree of homology between the two proteins in this region.
• the nuclear localization of the 62-64 kD protein was confirmed by biochemical fractionation of LA-N-5 cells into nuclear, cytoplasmic and membrane fractions as described by Slamon et al. (1985) supra.
• the data placed the majority of the N-myc protein within the nucleus of neuroblastoma cells, and thus were consistent with those obtained by immunocyto- chemical analysis.
• Experiments to evaluate the sub- nuclear localization of the N-myc protein were done by obtaining intact nuclei from LA-N-5 cells and subject ⁇ ing them to differential fractionation. The nuclei were fractionated into three components; nucleoplasm, chromatin and nuclear matrix as described by Boyle et al. (1985) Mol. Cell Biol. 5:3017.
• N-my_c-specific antisera were used; i.e., anti-N-myc fragment II.
• the antiserum was used at dilutions of 1:2000 to 1:4000.
• Tissue from two separate primary tumors were examined; one from a patient with stage II disease, and one with stage IV disease. In both instances, there was intense and specific staining of malignant neuroblasts, with virtually no staining of the adjacent vascular or stromal tissue.
• the tumor from the patient with stage II disease was stroma rich, with nodular.
• the tumor from the patient with stage IV disease consisted of predominantly undifferentiated neuroblasts with surrounding vascular and stromal elements. It was shown to contain 200 copies of the N-myc oncogene (Id.)

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