Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-01-14
2001-10-02
Huff, Sheela (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024100, C435S325000
Reexamination Certificate
active
06297366
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to genetic and cellular factors associated with cellular senescence and immortalization, apoptosis, neoplastic transformation and sensitivity to chemical and physical environmental insult. More particularly, the invention also provides methods and reagents for activating a particular cellular factor, the cellular tumor suppressor known as p53, and specifically provides reagents for activating p53-dependent transcriptional activity. The reagents provided by the invention comprise recombinant expression constructs encoding all or a portion of a particular cellular gene, termed ING1, and the protein produced by expression of this gene, known as p33
ING1
. The invention provides methods for using such recombinant expression constructs for activation of p53-related transcription involved in expression of apoptosis and other cellular expression pathways. The invention also provides reagents and methods for preparing genetic suppressor elements (GSEs) from ING1-encoding nucleic acid species, and methods for using such GSE for inhibiting apoptosis, delaying cellular aging, facilitating anchorage-independent growth, protecting the cell from the effects of certain cytotoxic drugs, and inhibiting the activity of p53. This invention also provides methods for characterizing mammalian cells on the basis of whether such cells express the ING1 gene, and embodiments of such methods directed at malignant or pre-malignant tissues in an animal for assaying the risk of developing malignant disease by the animal.
2. Summary of the Related Art
Cancer remains one of the leading causes of death in the United States. Clinically, a broad variety of medical approaches, including surgery, radiation therapy and chemotherapeutic drug therapy are currently being used in the treatment of human cancer (see the textbook
CANCER: Principles
&
Practice of Oncology
, 2d Edition, De Vita et al., eds., J. B. Lippincott Company, Philadelphia, Pa., 1985). However, it is recognized that such approaches continue to be limited by a fundamental lack of a clear understanding of the precise cellular bases of malignant transformation and neoplastic growth.
The beginnings of such an understanding of the cellular basis of malignant transformation and neoplastic growth have been elucidated over the last ten years. Growth of normal cells is now understood to be regulated by a balance of growth-promoting and growth-inhibiting genes, known as proto-oncogenes and tumor suppressor genes, respectively. Proto-oncogenes are turned into oncogenes by regulatory or structural mutations that increase their ability to stimulate uncontrolled cell growth (see Varmus, 1989, “A historical overview of oncogenes”, in
Oncogenes and the Molecular Origin of Cancer
, Weinberg, ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 3-44).
It is likely, however, that there are at least as many cancer-associated genes that are involved in suppression rather than induction of abnormal cell growth. This class of genes, known as anti-oncogenes or tumor suppressors, has been defined as comprising “genetic elements whose loss or inactivation allows a cell to display one or another phenotype of neoplastic growth deregulation” by Weinberg (1991
, Science
254: 1138-1146). Changes in a tumor suppressor gene that result in the loss of its function or expression are recessive, because they have no phenotypic consequences in the presence of the normal allele of the same gene. The recessive nature of mutations associated with tumor suppressors makes such genes very difficult to analyze or identify by gene transfer techniques and explains why oncogene research is far more advanced than studies of tumor suppressors.
In normal cells, tumor suppressor genes may participate in growth inhibition at different levels, from the recognition of a growth inhibiting signal and its transmission to the nucleus, to the induction (or inhibition) of secondary response genes that finally determine the cellular response to the signal. The known tumor suppressor genes have indeed been associated with different steps of the regulatory pathway. Thus, the DCC and ErbA genes encode receptors of two different classes (Fearon et al., 1990
, Science
247: 49-56; Sap et al., 1986
, Nature
324: 635-640; Weinberger et al., 1986
, Nature
324: 641-646). The gene NF-1 encodes a polypeptide that resembles ras-interacting proteins, that are members of the signaling pathway (Xu et al., 1990
, Cell
62: 599-608; Ballester et al., 1990
, Cell
62: 851-859; Buchberg et al., 1990
, Nature
347: 291-294; Barbacid, 1987
, Ann. Rev. Biochem
. 56: 779-827). p53, RB and WT genes encode nuclear regulatory proteins (Fields et al., 1990
, Science
249: 1046-1049; Raycroft et al., 1990
, Science
249: 1049-1051; Kern et al., 1991
, Oncogene
6: 131-136; O'Rourke et al., 1990
, Oncogene
5: 1829-1832; Kern et al., 1991
, Science
252: 1708-1711; Lee et al., 1987
, Nature
329: 642-645; Friend et al., 1987
, Proc. Natl. Acad. Sci. USA
84: 9059-9063; Call et al., 1990
, Cell
60: 509-520; Gessler et al., 1990
, Nature
343: 774-778).
Two approaches have been previously used for cloning tumor suppressor genes. The first approach is based on isolating the regions associated with nonrandom genetic deletions or rearrangements observed in certain types of tumors. This approach requires the use of extremely laborious linkage analyses and does not give any direct information concerning the function of the putative suppressor gene (Friend et al., 1991
, Science
251: 1366-1370; Viskochil et al., 1990
, Cell
62: 187-192; Vogelstein et al., 1988
, N. Engl. J. Med
. 319: 525-532). In fact, among numerous observations of loss of heterozygosity in certain tumors (Solomon et al., 1991
, Science
254: 1153-1160; LaForgia et al., 1991
, Proc. Natl. Acad. Sci. USA
88: 5036-5040; Trent et al., 1989
, Cancer Res
. 49: 420-423), there are only a few examples where the function of the affected gene is understood. In two of these rare cases the gene function was identified using another method, analysis of dominant negative mutant proteins (Herskowitz, 1987
, Nature
329: 219-222).
Specifically, the tumor suppressor gene p53 was first discovered as an altered forms which encoded a mutant protein (Sap et al., 1986
. ibid
.; Weinberger et al., 1986
, ibid
.; Raycroft et al., 1990
, ibid
.; Milner et al., 1991
, Molec. Cell. Biol
. 11: 12-19). p53 is a tumor suppressor gene that mediates cell response to various types of stress leading the cell growth arrest, apoptosis or other responses (i.e. differentiation) through modulation of expression of p53-responsive genes. p53 pathway is inactivated in the majority of human cancers making its restoration a major goal of gene therapy. At the same time, p53 pathway determines sensitivity of several tissues to DNA damaging treatments, including chemotherapy and gamma radiation. Therefore, stimulation or restoration of the p53 pathway could be critically important for the efficacy of anti-cancer therapy, while suppression of p53 pathway could be used to defend sensitive tissues from genotoxic stress and for the generation of immortal cell lines also requiring p53 functional inactivation. Oncogenic mutant p53 protein forms functionally inactive complexes with the wild-type protein; such complexes fail to provide the normal negative regulatory function of the p53 protein (Herskowitz, 1986
, ibid
.; Milner et al., 1991
, ibid
.; Montenarh & Quaiser, 1989
, Oncogene
4: 379-382; Finlay et al., 1988
, Molec. Cell. Biol
. 8: 531-539).
The discovery and analysis of new recessive genes involved in neoplastic transformation has been greatly accelerated through the use of genetic suppressor elements (GSEs), derived from such genes and capable of selectively suppressing their function. GSEs are dominant negative factors that confer the recessive-type phenotype for the gene to which the particular GSE corresponds. Recently, some developments have been made in the difficult area of isolating recessive genes using GSE te
Garkavstev Igor
Gudkov Andrei
Riabowol Karl
Board of Trustees of the University of Illinois
Helms Larry R.
Huff Sheela
McDonnell & Boehnen Hulbert & Berghoff
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