Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-02-07
2002-09-03
Nguyen, Dave T. (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S320100, C435S455000, C536S023100, C536S023500, C536S024310
Reexamination Certificate
active
06444463
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to molecular biology and in particular to genes and proteins involved in vertebrate neural development and to methods for classifying and prognosticating neuroectodermal tumors.
BACKGROUND OF THE INVENTION
Transcription factors of the basic-helix-loop-helix (bHLH) family are implicated in the regulation of differentiation in a wide variety of cell types, including trophoblast cells (Cross et al.,
Development
121:2513-2523, 1995), pigment cells (Steingrimsson et al.,
Nature Gen
. 8:251-255, 1994), B-cells (Shen, C. P. and T. Kadesch.,
Molec
. &
Cell. Biol
. 15:3813-3822, 1995; Zhuang et al.,
Cell
79:875-884, 1994), chondrocytes and osteoblasts (Cserjesi et al.,
Development
121:1099-1110, 1995; Tamura, M. and M. Noda.,
J. Cell Biol
. 126:773-782, 1994), and cardiac muscle (Burgess et al.,
Develop. Biol
. 168:296-306, 1995; Hollenberg et al.,
Molec
. &
Cell. Biol
. 15:3813-3822, 1995). bHLH proteins form homodimeric and heterodimeric complexes that bind with DNA in the 5′ regulatory regions of genes controlling expression.
Perhaps the most extensively studied sub-families of bHLH proteins are those that regulate myogenesis and neurogenesis. The myogenic bHLH factors, (MyoD, myogenin, Myf5, and MRF4), appear to have unique as well as redundant functions during myogenesis (Weintraub, H.,
Cell
75:1241-1244, 1993; Weintraub et al.,
Science
251:761-766, 1991). It is thought that either Myf5 or MyoD is necessary to determine myogenic fate, whereas myogenin is necessary for events involved in terminal differentiation (Hasty et al.,
Nature
364:501-506, 1993; Nabeshima et al.,
Nature
364:532-535, 1993; Rudnicki et al.,
Cell
75:1351-1359, 1993; Venuti et al.,
J. Cell Biol
. 128:563-576, 1995). Moreover, Myf expression has been observed in a number of rhabdomyosarcomas, and has been proposed as a marker for that category of tumor (Clark et al.,
Br. J. Cancer
64:1039-1042, 1991).
Recent work on neurogenic bHLH proteins suggests parallels between the myogenic and neurogenic sub-families of bHLH proteins. Genes of the
Drosophila melanogaster
achaete-scute complex and the atonal gene have been shown to be involved in neural cell fate determination (Anderson, D. J.,
Cur. Biol
. 5:1235-1238, 1995; Campuzano, S. and J. Modolell.,
Trends in Genetics
8:202-208, 1992; Jaman et al.,
Cell
73:1307-1321, 1993), and the mammalian homologs, MASH1 and MATH1, are expressed in the neural tube at the time of neurogenesis (Akazawa et al.,
J. Biol. Chem
. 270:8730-8738, 1995; Lo et al.,
Genes
&
Dev
. 5:1524-1537, 1991). Two related vertebrate bHLH proteins, neuroD1 and NEX-1/MATH-2, are expressed slightly later in CNS development, predominantly in the marginal layer of the neural tube and persisting in the mature nervous system (Bartholoma, A. and K. A. Nave,
Mech. Dev
. 48:217-228, 1994; Lee et al.,
Science
268:836-844, 1995; Shimizu et al.,
Eur. J. Biochem
. 229:239-248, 1995). NeuroD1 was also cloned as a factor that regulates insulin transcription in pancreatic beta cells and named “Beta2” (Naya et al.,
Genes
&
Dev
. 9:1009-1019, 1995). Constitutive expression of neuroD1 in developing Xenopus embryos produces ectopic neurogenesis in the ectodermal cells, indicating that neuroD genes are capable of regulating a neurogenic program. A neuroD1 homolog having 36,873 nucleotides has been identified in
C. elegans
(Lee et al., 1995; Genbank Accession No. 010402), suggesting that this molecular mechanism of regulating neurogenesis may be conserved between vertebrates and invertebrates.
A human achaete-scute homolog has been identified and cloned whose predicted protein of 238 amino acids is 95% homologous to the mouse hash1 gene (Ball et al.,
Proc. Natl. Acad. Sci. USA
90:5648-5652, 1993). Northern blots revealed that transcripts from this bHLH gene were detectable in two types of cancer with neuroendocrine features, namely small cell lung cancer, and the calcitonin-secreting medullary thyroid carcinoma (id.). Thus, hash1 was proposed to provide a marker for cancers with neuroendocrine features.
Primitive neuroectodermal tumors (PNET) are the most common of the malignant central nervous system tumors that occur in children (Biegel et al.,
Genes, Chrom
. &
Cancer
14: 85-96, 1995). Both supratentorial and infratentorial PNETs occur. Medulioblastoma, an infratentorial tumor that expresses neuronal intermediate filaments, synaptic vesicle proteins, growth factor receptors, and adhesion molecules, is the prototypic PNET. The location (posterior fossa) and properties of medulloblastomas suggests that they arise from neuroblasts that escape terminal differentiation (Trojanowski, J. Q., et al.
Mol. Chem. Neuropathol
. 17:121-135, 1992). Abnormalities in chromosome 17 have been observed in PNET biopsies, though the significance of these abnormalities has not been determined (Biegel et al., 1995; Schultz, et al.,
Genes, Chrom
. &
Cancer
16:196-203, 1996). Factors presently used to classify and prognosticate brain tumors include location, histopathology, patient age, and biological behavior of the tumor. However, such bases for tumor identification are not always sufficient for accurate prognostication. Supratentorial and infratentorial PNETs cannot always be distinguished, though they may respond differently to therapy. (Heideman, R. L. et al. “Tumors of the central nervous system.” In: P. A. Pizzo and D. G. Poplack (eds.),
Principles and Practice of Pediatric Oncology
, 2nd. ed., pp. 633-682, 1993; Rorke, L. B., et al.
Cancer
56:1869-1886, 1996; Packer, R. J., et al.
J. Neurosurg
. 81:690-698, 1994; Cohen, B. H. et al.
J. Clin. Oncol
. 13:1687-1696, 1995).
Because different types of brain tumors respond differently to various therapeutic regimens, accurate classification is highly useful to the physician in determining the best course of treatment for a particular patient. Medulloblastomas about half of the time are confined to the bridge that connects the two halves of the cerebellum (vermis). Medulloblastoma often is an aggressive and highly malignant type of tumor, and may invade other portions of the brain and spinal column. Thus, diagnosis based on location is not always reliable. Histologically, medulloblastoma is highly cellular, consisting of undifferentiated small dark round cells. Other PNETs with an identical histologic appearance, occurring predominantly in infants, are found at other locations in the brain. Thus, markers for a more accurate determination of PNET origin would facilitate the assignment of the therapeutic regimen most likely to be effective. Currently, no effective biologic markers exist for facilitating the stratification of treatment groups, assisting in prognosis, or providing targets for therapeutic intervention (Heideman et al., 1993).
SUMMARY OF THE INVENTION
The presently disclosed neuroD proteins represent a new sub-family of bHLH proteins and are implicated in vertebrate neuronal, endocrine and gastrointestinal development. Mammalian and amphibian neuroD proteins have been identified, and polynucleotide molecules encoding neuroD proteins have been isolated and sequenced. NeuroD genes encode proteins that are distinctive members of the bHLH family. In addition, the present invention provides a family of neuroD proteins that share a highly conserved HLH region. Representative polynucleotide molecules encoding members of the neuroD family include neuroD1, neuroD2 and neuroD3.
A representative nucleotide sequence encoding murine neuroD1 is shown in SEQ ID NO:1. The HLH coding domain of murine neuroD1 resides between nucleotides 577 and 696 in SEQ ID NO:1. The deduced amino acid sequence of murine neuroD1 is shown in SEQ ID NO:2. There is a highly conserved region following the helix-2 domain from amino acid 150 through amino acid 199 of SEQ ID NO:2 that is not shared by other bHLH proteins.
A representative nucleotide sequence encoding Xenopus neuroD1 is shown in SEQ ID NO:3. The HLH coding domain of Xenopus neuroD1 resides between nucleotides 376 and 495 in SEQ ID NO:3. The deduced a
Christensen O'Connor Johnson & Kindness PLLC
Fred Hutchinson Cancer Research Center
Nguyen Dave T.
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