EP0573564A1 - Expression de la nestine comme indicateur de tumeurs neuroepitheliales - Google Patents

Expression de la nestine comme indicateur de tumeurs neuroepitheliales

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Publication number
EP0573564A1
EP0573564A1 EP19920907686 EP92907686A EP0573564A1 EP 0573564 A1 EP0573564 A1 EP 0573564A1 EP 19920907686 EP19920907686 EP 19920907686 EP 92907686 A EP92907686 A EP 92907686A EP 0573564 A1 EP0573564 A1 EP 0573564A1
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Prior art keywords
nestin
cells
dna
brain tumor
rat
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German (de)
English (en)
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Ronald D. G. Mckay
Urban Lendahl
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Massachusetts Institute of Technology
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Massachusetts Institute of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

Definitions

  • Brain tumors are a leading cause of cancer deaths in people under the age of 35.
  • An important concept in our understanding of brain tumors is that the neoplastic cells may arise from undifferentiated neuroectodermal cells (Rubinstein, L.J. J. Neurosurg 62 : 795-805 (1985)).
  • neuroectoderm forming the neural plate is derived from ectoderm which has been induced by underlying mesoderm. (Spemann, H. Yale University Press 1938)).
  • the neural plate then folds, in the process called neurulation, to form the neural tube.
  • the cells of the neural tube are the precursors for the major differentiated cell types in the central nervous system: astrocytes, oligodendrocytes, and the various types of neuron (Sauer, F.C. J. Comp. Neurol. 62: 377-405
  • the cells of the neural tube may also be the precursors to neoplastic cells which form brain tumors.
  • Tissue culture systems have provided additional information about the differentiation potential of CNS stem cells.
  • Single-cell microculture of embryonic day 14 rat forebrain has produced clones containing both neurons and glia (Temple, S. Nature 340: 471-473 (1989)).
  • Antibodies have also proven useful for analyzing stem cells. For example, antibodies A2B5 (Raff, M.C.
  • Rat 401 was found to recognize a transient population of embryonic columnar epithelial cells and radial glial cells in many regions of the CNS (Hockfield, S. and McKay J. Neurosci. 5: 3310-3328 (1985)).
  • the number of Rat 401 positive cells, their proliferative rate, and the developmental kinetics of Rat 401 expression relative to neuronal differentiation revealed that the immediate precursors to neurons are also Rat 401 positive (Frederiksen and McKay, J. J. Neurosci 8 : 1144-1151 (1988)).
  • Neither neurons nor glia in the adult brain express the epitope recognized by the Rat 401 antibody.
  • medulloblastoma a common brain tumor of children, is associated predominantly with the cerebellum and brain stem.
  • Medulloblastoma tumors contain cell types with differentiated characteristics of neurons, glia and muscle (Rubinstein, supra 1985; Coakham H.B., et al., J. Clin. Pathol. 38: 165-173 (1985); Velasco, M.E. et al., Surg. Neurol. 23: 177-182 (1985); Hayashi, K. et al., Acta Pathologica Japonica 37 : 85-96 (1987); Cras, P. et al., Acta. Neuropathol 75: 377-384 (1988)).
  • the present invention relates to a gene encoding a protein whose expression distinguishes neural multipotential stem cells and brain tumor cells from the more differentiated neural cell types (e.g. neuronal, glial and muscle cells) of the mammalian brain.
  • the present invention relates to the nestin gene, particularly a nestin gene of mammalian origin, and the encoded nestin protein.
  • nestin gene particularly a nestin gene of mammalian origin
  • two nestin genes of mammalian origin, the rat nestin gene and the human nestin gene have been isolated and sequenced.
  • the coding sequence of the rat nestin gene transcript is 5415 bp , which corresponds to an expected molecular weight of approximately 200 kD for the nestin protein.
  • nestin protein as determined by SDS polyacrylamide gels is approximately 240 kD.
  • the predicted amino acid sequence of the rat nestin gene product shows that nestin defines a distinct sixth class of intermediate filament protein.
  • the present invention also relates to the human nestin gene and the encoded human nestin protein.
  • the coding sequence of the human uestin gene transcript is 4854 bp.
  • the predicted amino acid sequence is 1618 amino acids in length, and shows significant sequence homology with the rat nestin sequence.
  • the present invention further relates to methods of detecting the expression of nestin as a means of
  • DNA present in a sample from the brain of an adult individual is hybridized to a DNA probe which is complementary to all or a portion of the nestin gene.
  • the nestin gene includes the human nestin gene whose sequence is represented herein, the rat nestin gene whose sequence is also represented herein and equivalent genes from other species, such as those which are substantially homologous to the human or the rat gene sequence. Detection of hybridization is an indication of a predisposition to the development of a brain tumor or the presence of a brain tumor.
  • cerebral spinal fluid or a serum sample from the brain of an adult individual can be stained with anti-nestin antibodies. Detection of stained cells is an indication of a predisposition to the development of a brain tumor or the presence of a brain tumor.
  • Figure 1 is the nucleotide and deduced amino acid sequences of the nestin transcript derived from the central nervous system of rat embryos. The three intron positions (912, 1038 and 1111) are indicated. The canonical poly A addition signal (5924-5929) is underlined.
  • Figure 2 is the nucleotide sequence of human nestin.
  • Figure 3 is the deduced amino acid sequence of human nestin.
  • the present invention relates to a gene of mammalian origin encoding a protein whose expression distinguishes neural multipotential stem cells and brain tumor cells from the more differentiated neural cell types (e.g., neuronal, glial and muscle cells of the adult brain).
  • a gene of mammalian origin encoding a protein whose expression distinguishes neural multipotential stem cells and brain tumor cells from the more differentiated neural cell types (e.g., neuronal, glial and muscle cells of the adult brain).
  • the present invention more particularly relates to the nestin gene and the encoded proteins and their use in diagnosing tumors of the brain, such as medulloblastomas, gliablastomas and oligodendroglioma.
  • the human nestin gene and the rat nestin gene have been isolated and sequenced.
  • expression of the nestin gene and its structural features are described.
  • the intracellular distribution of the nestin gene product, both in an embryonic rat brain cell line (ST15A) and human brain tissue, including tumor cells, using immunocytochemical and immunohistochemical techniques is also described.
  • nestin expression in the developing human central nervous system and brain tumor samples is also described herein.
  • a cDNA library in the expression vector lambda gt11 was constructed from poly(A) RNA from CNS of embryonic day 15 (E15) rat embryos as described in detail in
  • Example 1 The library was screened with the monoclonal antibody Rat 401 after induction of protein expression and immunopositive plaques were identified.
  • gt10.401:16, gt10.401:18 and gt10.401:9 in a lambda gt10 library from rat E15 CNS constructed in parallel with the gtll library.
  • a number of hybridizing clones were identified and sequenced and together represent 5333 base pair (bp) of transcribed sequence, including 404 bp of 3' untranslated sequence and a consensus polyA addition site.
  • the remaining portion of the nestin gene was obtained from genomic sequences. Genomic Southern blots, even when hybridized at low stringency, revealed a pattern consistent with a single rat gene. A genomic rat library in the vector Charon 4A was screened with the nestin cDNA clones. Hybridizing clones were organized by restriction mapping and the region upstream of the cDNA clone gt10.401:9 was sequenced.
  • the exact location of the 5' end was determined by a combination of S1 nuclease mapping and primer extension.
  • Figure 1 shows the nucleotide sequence of the rat nestin gene.
  • the total length of the nestin gene transcript is 5945 bp, excluding the poly A tail.
  • the similarity between the nestin gene and the genes of the other five classes of intermediate filaments ranges from 16 to 29 % at the amino acid level in a 307 amino acid long region starting close to the N-terminus of the nestin gene, corresponding to the conserved alpha-helical rod or "core" domain of the intermediate filaments.
  • This region of the predicted nestin amino acid sequence also contains a repeated hydrophobic heptad motif characteristic of intermediate filaments.
  • the degree of amino acid similarity is comparable to that found between different classes of intermediate filament, both in degree and in location, i.e., conserved heptad- containing alpha-helical stretches within the core domain are separated by less well conserved, non-heptad spacers (Steinert, P.M. and D.R. Roop, Ann. Rev. Biochem. 57 :
  • the N-terminal domain is only 11 amino acid residues long, shorter than in other intermediate filaments.
  • the large C-terminal domain loosely resembles those of neurofilaments in that it is highly charged, bears glutamate-rich regions, and features a repeat, in this case the 11 amino acid motif S/P-L-E-E/K-E-X-Q-E-S/L-L-R (underlined residues are strongly conserved).
  • the deduced amino acid sequence of nestin suggests that it is a member of the intermediate filament protein family.
  • its degree of sequence homology to other intermediate filaments in the core domain, its dissimilarity in the head and tail domains, and its different splicing pattern suggest that it defines a new class of intermediate filament protein.
  • the human nestin gene was isolated using low stringency DNA hybridization of a human genomic bacteriophage lambda library with a rat nestin probe.
  • Figure 2 shows the nucleotide sequence of the human nestin gene.
  • the total length of the human nestin gene sequence is 4854 bp.
  • Figure 3 shows the deduced amino acid sequence encoded by the human nestin gene which is 1618 amino acid residues in length.
  • the alignment of the deduced amino acid sequences of human nestin with the deduced amino acid sequence of rat nestin showed greater than 75% sequence similarity between the two sequences, with greater than 60% sequence identity.
  • CNS stem cells differentiate into neurons and glia on a stereotyped schedule
  • the nestin protein was originally identified by the Rat 401 antibody, which transiently stains radial glial cells of the neural tube in cross sections of rat embryos (Fredericksen, K. and R. McKay, J. Neurosci 5: 3310-3328 (1988); Hockfield and McKay, supra (1985)).
  • Rat 401 antibody transiently stains radial glial cells of the neural tube in cross sections of rat embryos
  • RNA from E15 rat CNS or from the Rat 401 positive cell line ST15A were probed with nestin cDNA inserts, a single 6.2 kb mRNA species was
  • nes tin expression was found to decline from a maximum on day E16, such that only extremely low levels of RNA could be detected at day P9 and P12 and none at all in the adult cerebral cortex, even after long exposures.
  • the loss of nestin mRNA precisely parallels the decline of Rat 401 positive stem cells in cerebrum during neurogenesis (Frederiksen and McKay, supra
  • the postnatal cerebellum expresses the nestin gene with a maximum on day P5; no expression can be detected after day P9 . Again, this pattern closely matches the numbers of Rat 401 positive cells in
  • Nestin is not detected by Northern blot analysis in adult tissue. The loss of nestin expression coincides with terminal differentiation of these early multipotential cells. There are two important implications of these results. First, nestin appears to be a general marker for the CNS stem cell from the newly closed Ell neural tube through the postnatally developing
  • nestin expression is a consequence of transcriptional regulation of the gene.
  • the associations of nestin with different components of the cytoskeleton were analyzed by a series of doublelabel immunocytochemistry experiments. These experiments were carried out in the immortalized CNS precursor cell line ST15A which expresses, in addition to components of microtubules and microfilaments, nestin and the inter mediate filament vimentin. ST15A produces a nestin mRNA indistinguishable by Northern blot analysis from that found in E15 CNS.
  • microtubules are more evenly distributed in the cell with no particular aggregation around the nucleus, whereas nestin is found in a fiber-like pattern, with a pronounced perinuclear accumulation.
  • microfilaments and nestin are stained in the same cell: microfilaments form straight and parallel fibers, in contrast to the gently curved fibers of nestin.
  • ST15A was derived from postnatal day 4 rat cerebellum. In the rat, as in other mammals, much of cerebellar development occurs after birth, so that there are a large number of dividing precursor cells present at the time from which this line was derived. Thus, ST15A is derived from the appropriate tissue and stage of development. It has previously been shown that ST15A cells can differentiate along neuronal and glial pathways. The following experiments, which are described in detail in Example 3, show that ST15A cells can also differentiate into muscle. This result suggests that a single neuroectodermal cell can give rise to the different cell types found in brain tumors.
  • the cerebellar cell line ST15A was one of several cell lines obtained by infecting primary rat cerebellar cells with a recombinant retrovirus carrying SV40 Tantigen. A temperature sensitive allele of T-antigen was used to establish these cell lines. At the permissive temperature (i.e., when the T-antigen protein is active) the cells proliferate and express the intermediate filament protein nestin.
  • the T-antigen protein is rapidly degraded and the cells differentiate into either neurons or glial cells depending on the conditions.
  • the differentiation into neurons and glial cells is best accomplished by growing ST15A cells in co-culture with primary cerebellar cells. Partial differentiation has also been observed when ST15A cells are cultured alone; serum-free medium promotes neuronal differentiation and medium containing fetal calf serum promotes glial differentiation.
  • ST15A cells also can differentiate into muscle cells. This fate was first noted when spontaneously contracting cells were observed in long term cultures of ST15A at 39°C, the non-permissive temperature for T-antigen. Horse serum is often used to support the differentiation of primary myoblasts. When ST15A cells are grown in horse serum at 39oC, they reproducibly differentiate into multi-nucleate cells which express muscle specific proteins, regenerative action potentials and spontaneously contract.
  • the monoclonal antibody Rat 401 recognizes the 220 kD intermediate filament protein nestin. ST15A cells express nestin at both 33°C and 39°C.
  • a second monoclonal antibody which recognizes a skeletal and cardiac muscle isotype of troponin T was reacted with the cells (Lin, J. Biol. Chem. 263: 7309 (1988)). Immunofluorescence shows that troponin T expression is induced when ST15A cells are cultured at 39°C in horse serum.
  • Immunoblots of proteins extracted from ST15A cells verify that anti-troponin T antibody recognizes a strong band of 38 Kd, the appropriate molecular weight, in the cells grown at 39°C in horse serum; at 33 C no band is present.
  • the troponin band in ST15A co-migrates with troponin extracted from rat postnatal day 4 muscle.
  • the double band seen in both muscle cells and differentiated ST15A cells may represent the adult and embryonic isoforms of troponin T which differ by 3,000 daltons (Lin, J. Biol. Chem. 263 : 7309 (1988)). These two isoforms are derived from the same primary transcript by differential splicing.
  • a family of DNA binding factors has been shown to play a critical role in the differentiation of muscle.
  • These proteins, myoD1, myogenin, myf5 and herculin/MRF4 contain DNA binding motifs of the helix-loop-helix type in a region of homology to c-myc (Wright, W.E., et al., Cell. 56: 607 (1989); Miner, J.H. and B.Wold, Proc. Nat. Acad. Sci. 87: 1089-1093 (1990); Davis, R.L. et al.,
  • Herculin/MRF4 was found in adult skeletal muscle but was not detected in ST15A.
  • Myf5 gave a very weak signal in ST15A cells compared to the myoblast cell line C2C12.
  • Nestin mRNA accumulates with time at 39°C, as does the protein.
  • ⁇ -actin serves as a control for the amount of RNA at 33°C and in the short time points at 39°C ; the decrease in ⁇ -actin signal at 3 and 7 days at 39°C may be due to the changing composition of the cytoskeleton accompanying differentiation, as levels of myoD1 mRNA appear constant throughout the experiment.
  • ST15A cells become multinucleate as they differentiate.
  • the initial action potentials were generated by a series of hyperpolarizing pulses.
  • the final hyper-polarizing pulse was followed by a train of spontaneous action potentials.
  • Spontaneous action potentials were seen after 12 days at 39°C in 10% horse serum.
  • the resting membrane potential falls and it becomes easier to stimulate trains of action potentials with hyperpolarizing current pulses.
  • ST15A cells also
  • ST15A cells An interesting question raised by the properties of ST15A cells is whether primary cerebellar cells can differentiate into the muscle fate. Dissociated cells from postnatal day 5 rat cerebellum were cultured for 6 days under conditions which promote muscle differentiation of ST15A cells (39°C in the presence of 10% horse serum) and stained with monoclonal antibody against troponin T. These culture conditions promote extensive differentiation of neurons and glia. Troponin T positive cells with an elongated, multinucleate morphology of muscle cells were also seen, but these cells were very rare, occurring less than one per hundred thousand cells.
  • CNS central nervous system
  • Human nestin exhibited biochemical and immunochemical properties similar to those of rat nestin. Further, in the human, nestin was detected immunohistochemically in several different types of immature human CNS cells, i.e. germinal matrix cells, neuroepithelial cells lining the central canal, radial glia and endothelial cells. Nestin appeared in these cells at the earliest gestational age (i.e., 6 weeks) examined here and then it declined in all but the endothelial cells at later embryonic stages. Nestin also was
  • the spinal cord of the 6 weeks GA human fetus was composed of three distinct layers, i.e. a layer of primitive neuroepithelial cells lining the central canal, a mantle layer and an outer marginal layer.
  • Anti-nestin 129 stained most cells in the primitive neuroepithelial layer, i.e. the presumptive miltipotential stem cells that give rise to CNS neurons and glia.
  • thin elongated radial glial fibers extending from the primitive neuro epithelial layer to the subpial region were stained by this antiserum.
  • These neuroepithelial stem cells and radial glial fibers also expressed vimentin but not GFAP. Neuroblasts in the mantle layer (identified by their morphology and NF protein positively) did not express nestin.
  • telencephalic germinal matrix of two cases with GAs of 17 and 20 weeks were examined with anti-nestin 129.
  • many presumptive neuroepithelial stem cells in the germinmal matirx cells were nestin positive. These neuroepithelial precursor cells also were labeled by the anti-vimentin MAb, but no germinal matrix cells were stained by the MAbs to GFAP or NF proteins.
  • GA a well defined germinal matrix was not evident, but clusters of morphologically immature cells (presumably residual, multipotential precursor cells) were seen in the subependymal region. Nestin immunoreactivity was recognized in only a few of these cells.
  • the cerebellar cortex has four distinct layers, i.e. the internal and external granular layers, the Purkinje cell layer and the molecular layer. However, these layers were not yet evident at 17 weeks GA and Purkinje cells were not recognizable by morphological criteria. Nevertheless, immature NF positive cells were observed at 17 weeks GA in the superficial internal granular layer, and these cells probably correspond to nascent Purkinje cells. Nestin immunoreactivity was detected at this time in radial glial cells in the internal granular layer and in radial glial processes that extended to the external granular layer.
  • NF-H are the low, middle and high molecular weight neurofilament subunits, respectively.
  • the following antibodies were used to obtain these data; nestin
  • GFAP (2.2B10), NF-L (NR-4, RMS12), NF-M (RMd020,
  • RM0254 H014)
  • NF-H DP1, TA51, RM024
  • CHOP707m which was derived from a cerebral neuroblastoma (Baker, D.L. et al., Am. Neurol. 28:136 (1990))
  • the other 6 PNET cell lines were obtained from cerebellar medulloblastomas (see Hockfield, S. et al., J. Neurosci 5:3310 (1985)) and citations therein). Daoy does not exhibit any evidence of glial or neuronal differentiation and it was nestin negative, while the other 6 PNET cell lines resembled that of embryonic neuroblasts (He, X. et al., J. Neuropathol. Exp. Neurol. 48:48 (1989)).
  • vimentin positive filament bundles were present throughout these cells.
  • the Daoy line expresses vimentin, but not NF proteins, GFAP or other molecular markers of neurons or glia (He, X. et al., J. Neuropathol. Exp.
  • Daoy is the least differentiated PNET cell line, and it did not express nestin.
  • the foregoing co- localization studies also indicated that the anti-nestin 129 did not cross react with vimentin or NF proteins.
  • the Daoy cell line may resemble a CNS precursor that subsequently develops into a vimentin and nestin positive CNS stem cell like those observed here in the human spinal cord at a GA of 6 weeks.
  • tumor cell lines contain genetic mutations and their phenotype may not fully replicate that of normal cells.
  • the 2 glioma cell lines (U251 MG and U373 MG) resembled immature spinal cord radial glia (i.e. at 11 weeks GA) since these cells co-expressed nestin, GFAP and vimentin in the triple fluorescence studies.
  • ST15A cells express properties expected of the precursor to brain tumor cells as described in Example 3.
  • brain tumor cells were tested to determine whether they express features in common with ST15A, such as expression of nestin. As described in detail in
  • Example 6 tissue from five medulloblastomas was
  • Three of these samples came from recurrent tumors occurring in a 16 year old male, a 26 year old male and a 5 year old female. The remaining tumors were newly diagnosed in an 11 year old male and a 10 year old female. The primary tumor in the 11 year old male was also used as a source of cultured cells.
  • Sections of normal fetal and adult human cerebellum were also stained with polyclonal antiserum against nestin.
  • the fetal tissue showed many positive cells in the proliferative, external granular layer of the
  • cerebellum developing cerebellum and in the outer part of the internal granular layer. In contrast, there was no immunoreactivity in cerebellum from a 2.5 year old.
  • neurofilaments are expressed by neurons, peripherin by a subset of neurons (Leonard, Gorham, et al., (1988)), and glial fibrillary acidic protein by astrocytes.
  • the expression of nestin defines an earlier stage in the pathway of intermediate filament gene expression.
  • Early embryonic cells are nestin negative but express certain cytokeratins
  • neurectodermal cells become nestin positive.
  • CNS cells stop producing nestin and express class III or IV intermediate filaments typical of their differentiated cell type.
  • Nestin positive neurectodermal cells have been shown to be capable of differentiating into neuronal, glial and muscle cells (i.e., the different cell types found in tumors).
  • Nestin expression has also been directly detected in tumor tissue and in a medulloblastoma derived cell line.
  • Nestin positive cells derived from tumors are likely to be renewing stem cells which give rise to the differentiated cells in the tumor.
  • the detection of nestin expression in brain cells obtained from an adult using known methods is indicative of the presence of a brain tumor or of neural precursor cells capable of developing into a brain tumor.
  • an "adult” is an individual whose central nervous system is developed and, therefore, contains differentiated cells.
  • the central nervous system of human neonates is in continual development up until the age of about one year. Therefore, a human adult, for the purposes of the subject invention, is an individual who is older than about one year.
  • Nestin mRNA expression in a brain tissue sample can be detected using a DNA probe; this is indicative of expression of nestin, which is, in turn an indication of a predisposition to the development of a brain tumor or of the presence of a brain tumor in the individual.
  • a sample of brain tissue from of an individual can be hybridized to a DNA probe which is complementary to all or a portion of the nestin gene. Detection of hybridization is an indication of a predisposition to the development of a brain tumor or the presence of a brain tumor.
  • expression of the nestin protein can be detected using polyclonal antibodies, (e.g., antinestin antiserum 129) or monoclonal antibodies (e.g., Rat 401).
  • polyclonal antibodies e.g., antinestin antiserum 129
  • monoclonal antibodies e.g., Rat 401
  • cerebral spinal fluid or a serum sample from the brain of an individual can be stained with anti-nestin antibodies. Detection of stained cells is an indication of a predisposition to the development of a brain tumor or the presence of a brain tumor.
  • nestin marker enables further investigation into the proliferation and differentiation of the stem cells of a brain tumor, so that existing chemotherapies can be implemented at an earlier stage in the tumor development and so that new methods of inhibiting tumor development can be developed.
  • ⁇ gt10 and ⁇ gt11 cDNA libraries were constructed from oligo-dT-primed poly(A)+ RNA from embryonic day 15 dissected rat CNS according to procedures which were slightly modified from those described (Young, R.A. and R.W.Davis, Proc. Natl. Acad. Sci. USA. 80: 1194-1198 (1983)).
  • the second strand cDNA was synthesized using the RNase H modification of the Okayama-Berg method
  • the ⁇ gtll library was screened with the monoclonal antibody Rat 401 (undiluted hybridoma supernatant) and positive clones were visualized with an anti-mouse second antibody conjugated to alkaline phosphatase (Promega Protoblot). Immunopositive clones were selected and the cDNA insert from the clone giving the strongest hybridization signal, ⁇ gt11.401 : 16, was 32 P-labelled (Feinberg, A. P. and B. Vogelstein, Anal. Biochem. 137: 266-267 (1983) and used as probe to screen the ⁇ gt10 library (Benton W.D. and R.W. Davis, Science.
  • cDNA inserts and genomic subclones were sequenced after subcloning into M13mpl8, M13mpl9, or Bluescript KS+ (Stratagene). M13 phage particles and Bluescript were grown and circular DNA prepared according to standard procedures. Both single stranded (M13) and double stranded (Bluescript) sequencing was performed using a modified T7 DNA polymerase (Sequenase, USB), as suggested by the manufacturer. Approximately 90% of the gene, including all exons and cDNA inserts, was sequenced on both strands. Ambiguous regions were further resolved using deoxyinosine in the standard Sequenase protocol. Computer analysis was performed using the University of Wisconsin Genetics Computer Group program package, including the FASTP and TFASTA algorithms (Devereux, Haeberli et al., 1984)). The complete sequence,
  • RNA molecules of known size HMW RNA ladder, BRL
  • RNA or 50 ug of yeast total RNA according to standard procedures, and the S1 resistant hybrids separated on denaturing polyacrylamide gels (Maniatis, T., E.F.
  • cells from the immortalized cell line ST15A were grown for 2 days (to 40% confluence) in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum on sterile, polyornithine coated glass cover slips, as described in U.S. Patent Applications Serial Nos. 07/603,803; 07/201,762 and 180,548. All antibody incubations lasted 60 minutes at room temperature and were followed by three rinses in PBS (0.14M) NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 and 8 mM KH 2 PO 4 pH 7.2).
  • the ST15A cells were briefly rinsed in PBS, presoaked in microtubule stabilizing buffer (MTSB - 2M glycerol, 0.1 M PIPES pH 6.9, 1 mM MgSO 4 , 2 mM EDTA) for 2 minutes and then fixed for 5 minutes in 4% paraformaldehyde, 0.5% NP-40 in MTSB for 5 minutes.
  • MTSB microtubule stabilizing buffer
  • the first antibodies (rabbit-anti-tubulin and mouse-antinestin) were omitted in two separate experiments under otherwise identical conditions. No cross-hybridization or significant spillover was observed.
  • the Stl5A cells were grown as above and treated with colchicine at 25 ug/ml for 24 hours followed by fixation and staining as described here.
  • ST15A cells were grown as above and fixed in 4% paraformaldehyde, 0.1% Triton X-100 in PBS for 5 minutes. After blocking as described above, the cells were incubated with 0.16 uM rhodamine-conjugated phalloidin, as
  • mice received two intraperitoneal immunizations with fixed tissue homogenized in saline and suspended in an equal volume of Freud's complete adjuvant and a final intravenous boost of unfixed tissue with adjuvant.
  • Spleen cells from immunized mice were fused with NS1 myeloma cells and resulting hybrid cell lines were screened immunohistochemically on 100 ⁇ m-thick Vibratome sections of 4% paraformaldehyde-fixed E15 rats. Lines producing antibodies of interest were cloned in soft agar or by limiting dilution and antibody subclass was determined by the Ouchteriony method (Ouchteriony and Nilsson, Immunodiffusion and Immunoelectrophoresis. In Handbook of Experimental Immunology D.M. Weir, ed., Blackwell
  • Tissue for immunohistochemistry was fixed either by intravascular perfusion or by immersion. All postnatal material was fixed by intravascular perfusion (under ether anesthesia) with 4% paraformaldehyde at pH 7.4 for light microscopy or with either 4% paraformaldehyde at pH 10.0 or 4% paraformaldehyde, 0.1% glutaraldehyde at pH 7.4 for electron microscopy.
  • the CNS was dissected out and stored in 0.1 m phosphate buffer (pH 7.4) with 0.1% sodium azide.
  • pregnant females were ether anesthetized, uteri were dissected into cold 0.1 m phosphate buffer and the females were killed by cervical dislocation.
  • Embryos were dissected individually into Sylgard-coated Petri dishes and pinned in place. Intravascular perfusion was performed by opening the skin of the thoracic cavity, nicking the right atrium with a fine forceps and placing a 25 gauge needle into the left ventricle. One to 5 ml of fixative (as above) were injected into the heart. The embryos were placed in a vial containing the same fixative for 4 hr and then stored in phosphate buffer. E12 to term embryos were fixed by perfusion (using a 30 gauge needle for E12 embryos); E10 and E11 embryos were fixed by immersion in fixative. E13 and older animals were sectioned at 50 to 100 ⁇ m on a Vibratome without additional support. E10 to E12 embryos were embedded in 15% gelatin before
  • Immunohistochemical reactions were performed on free-floating sections by sequential incubations in monoclonal antibody as full-strength supernatant (12 to 20 hr); horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody at a dilution of 1:100 in tissue culture medium with 10% serum; and 3.3'-diaminobenzidine (DAB; 0.025%) with H 2 O 2 (0.002%).
  • HRP horseradish peroxidase
  • DAB 3.3'-diaminobenzidine
  • ST15A cells were derived from rat postnatal day 2 cerebellum after infection of a primary culture with a retrovirus transducing the temperature sensitive, tsA58 allele of SV40 T antigen (Frederiksen et al., 1988)). ST15A cells were plated on tissue culture plastic coated with polyornithine and passaged at the permissive temperature, 33oC, in Dulbecco's modified Eagle's medium (DME) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 15 mM HEPES, pH 7.2, containing 10% fetal calf serum (FCS).
  • DME Dulbecco's modified Eagle's medium
  • the medulloblastoma cell line was derived by growing cells from dispersed, fresh medulloblastoma tissue. The tissue was removed from a previously untreated tumor in a 10 year old female. The cell line has not been cloned. Cells were passaged in Ham's F12 supplemented with 20mm L-glutamine and 10% FCS. Immunocytochemistry
  • mice monoclonal Rat 401 Hockfield and McKay, supra (1985), mouse monoclonal antibody against troponin T (Sigma T-6277), and rabbit polyclonal serum raised against the C-terminal region of nestin expressed in bacteria.
  • Fixed cells were incubated in primary antibody for one hour, rinsed 3 times in PBS, incubated for one hour with flourescein-conjugated goat anti-rabbit serum (Organon-Tetnika) and mounted in Immumount (Shandon, Inc.) with 2% DABCO. Medulloblastoma tissue and normal human cerebellum were frozen and subsequently fixed in formalin. 50 urn sections were cut and stained with rabbit polyclonal serum raised against the C-terminal region of nestin.
  • RNA pelleted through a CsCl step gradient (Chirgwin et al., 1979). 20 ug of total RNA was electrophoresed through a 1% agarose/2.2M formaldehyde gel and transferred to nitrocellulose.
  • Probes were prepared for hybridization by random priming on fragments isolated on low melting temperature agarose gels (Feinberg and Vogelstein, supra (1984). The following plasmids were used to generate probes : mouse myoD1 cDNA pEMC11s (Davis, R.L. et al., Cell. 51: 987-1000 (1987); mouse myogenin cDNA p65
  • Proteins were extracted from cultured cells or rat postnatal day 5 limb muscle (Sprague-Dawley; Taconic,
  • anti-nestin 129 a new antiserum, designated anti-nestin 129, was raised to recombinant rat nestin expressed in E. coli since the rat-401 mAb failed to recognize human nestin in immunohistochemical and immunochemical assays. Production of Anti-nestin Antiserum
  • the insert from the clone ⁇ gt10 401:16 (Lendahl, U. et al., Cell 60 : 585 (1990)) was isolated and ligated into the pATH1 vector (Tzagaloff, A. et al., J. Bio l .
  • TrpE-nestin fusion protein migrates at greater than 200 kD in 6% SDS PAGE gels, making it the largest protein in the bacterial lysate.
  • the fusion protein was purified by cutting the top band from the gel.
  • Anti-nestin antiserum 129 was produced by injecting the fusion protein subcutaneously into a female NZW rabbit. The characterization of anti-nestin 129 and its specificity for human nestin is described here.
  • GFAP fibrillary acidic protein
  • NF neurofilament
  • the rabbit anti-nestin antiserum produced as described herein i.e. anit-nestin 129
  • anti-nestin 129 identified a band with an apparent M r of 240 kD. This band was identical to the band labeled by Rat-401.
  • both antibodies labeled similar bands in lysates of the induced bacteria producing the TrpE-nestin fusion protein.
  • anti-nestin 129 labelled radial glial cells very intensively. The processes of these cells radiated from the internal granular layer to the external granular layer. These radial glia also were labeled with Rat-401 and the anti-vimentin MAb. Neither of the anti-nestin antibodies stained immature Purkinje cells or other neurons. In contrast to the P6 rat cerebellum, the anti-nestin 129 and Rat-401 antibodies only stained blood vessel endothelial cells in the adult rat cerebellum, while the anti-vimentin MAb (V9) labelled blood vessels, white matter astrocytes and radial glial fibers.
  • nestin immunoreactivity was demonstrated by both anti-nestin antibodies in immature skeletal muscle of the P6 rat as well as in Schwann cells of adult rats (data not shown).
  • the human nestin gene was isolated using lowstringency DNA hybridization of a human genomic bacteriophage lambda library with a rat nestin probe.
  • a genomic library in the vector EMBL 3 made from partially Sau3A-digested human genomic DNA (Clontech) was screened using a 32 P-labelled probe derived from the rat nestin gene starting at 202 nucleotides upstream of the translation start and ending at nucleotide 483 after the translation start (Lendahl, et al., 1990) under the following conditions: prehybridization in 6X SSC, 5X
  • PNET+N , G PNET with neuronal and glial differentation
  • PNET+N PNET with neuronal differentiation
  • PNET + G PNET with glial differentiation
  • PNET-NOS PNET, not otherwise specified.
  • the results from frozen and ethanol-fixed paraffinembedded samples from the same biopsy are combined in the 10 cases from which these paired samples were available. Notably, the immunohistochemical results obtained from each of these paired samples were identical, although the primary antibodies were used at slightly higher dilutions in the frozen material compared with the paraffin
  • PNETs The anti-nestin antiserum stained tumor cells in 12 of the 15 PNETS examined here. Nestin immunoreactivity was found in the cytoplasm of individual neoplastic cells. Reactivity was also observed in the coarse processes of large stellate cells, as well as in blood vessel endothelial cells. In the PNETs with an insular architecture, nestin reactive cells were found both within and outside the islands. Since PNETs express SYP and other neuroendocrine markers as well as all classes of IF proteins (Gould, V.E. et a l . , Human Pathol. 21:245 (1990)), the PNETs were probed for SYP. These studies demonstrated SYP in 14 of 15 PNETs.
  • This immunohisto-chemical phenotype might represent primitive neuroepithelial cells or immature radial glial cells in the developing CNS.
  • the other PNET-NOS case was negative for nestin and it might correspond to extremely immature CNS precursor cells just like the most undifferentiated PNET cell line (i.e. Daoy).
  • nestin is the dominant IF protein of early CNS precursor cells. However, nestin is rapidly extinguished in neurons, glia and other CNS cell types derived from these precursors. The mechanisms responsible for these rapid changes in IF protein expression are unknown.
  • nestin In contrast to the restricted expression of nestin in normal developing CNS cells, this IF protein was ubiquitously present in a wide variety of CNS neoplasms. Nestin was expressed in a subset of PNETs, but was more abundant in gliomas. The presence of nestin in diverse types of neuroepithelial tumors is similar to vimentin, but distinct from the distribution of GFAP and NF proteins in CNS tumors.

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Abstract

Est décrit un gène encodant une protéine, la nestine, dont l'expression permet de distinguer les cellules souches pluripotentes nerveuses et les cellules tumorales cérébrales des types de cellules nerveuses plus différenciées (par exemple des cellules neuronales, gliales et musculaires). Sont également décrits des procédés pour détecter l'expression de la nestine comme moyen pour diagnostiquer et traiter des tumeurs cérébrales.
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