Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-12
2001-03-06
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S325000
Reexamination Certificate
active
06197521
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to genes and genetic suppressor elements associated with the control of neoplastic transformation of mammalian cells. More particularly, the invention relates to methods for identifying such genes and genetic suppressor elements as well as to uses for such genes and genetic suppressor elements. The invention specifically provides genetic suppressor elements derived from genes associated with the transformed phenotype of mammalian cells, and therapeutic and diagnostic uses related thereto. The invention also provides genes associated with the control of neoplastic transformation of mammalian cells.
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 now understood to be regulated by a balance of growth-promoting and growth-inhibiting genes, known as proto-oncogens and tumor suppressor genes, respectively. Proto-oncogene are turned into oncogenes by regulatory or structural mutations that increase their ability to stimulate uncontrolled cell growth. These mutations are therefore manifested as dominant (e.g. mutant RAS genes) or co-dominant (as in the case of amplification of oncogenes such as N-MYC or HER2/NEU) (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).
Dominant and co-dominant genes can be effectively identified and studied using many different techniques based on gene transfer or on selective isolation of amplified or overexpressed DNA sequences (Kinzler et al., 1987,
Science
236: 70-73; Schwab et al., 1989,
Oncogene
4: 139-144; Nakatani et al.,
Jpn. J. Cancer Res.
8: 707-710). Expression selection has been successfully used to clone a number of cellular oncogenes. The dominant nature of the oncogenes has facilitated the analysis of their function both in vitro, in cell culture, and in vivo, in transgenic animals. Close to fifty cellular oncogenes have been identified so far (Hunter, 1991,
Cell
64: 249-270).
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 at., 1990,
Science
242: 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 genes erbA and p53 were first discovered as altered forms which encoded mutant proteins (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). These altered genes were initially classified as oncogenes, since they induced cell transformation when transfected alone or in combination with other oncogenes (ras in the case of p53 and erbB in the case of erbA; see Eliyahu et al., 1984,
Nature
312: 646-649; Parada et al., 1984,
Nature
312: 649-651; Graf & Beug, 1983,
Cell
34: 7-9; Damm et al., 1989,
Nature
339: 593-597). Later, however, it was recognized that both of these “oncogenes” acted by interfering with the normal function of the corresponding wild-type genes. Thus, the 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 oncogene erbA, found in chicken erythroblastosis virus, is a mutant version of the chicken gene for thyroid hormone receptor, the transcriptional regulatory protein which participates in the induction of erythroid differentiation (Damm et al., 1989, ibid.; Damm et al., 1987,
EMBO J.
6: 375-382). The mutant ErbA protein blocks the function of the wild-type receptor by occupying its specific binding sites in the DNA (Sap et al., 1989,
Nature
340: 242-244).
Thus, naturally arising dominant negative mutants not only allowed the identification of the corresponding tumor suppressor genes but also served as tools for their functional analysis. Such natural tools for recessive gene identification seem to be rare, however, limiting the utility of this approach for the discovery of new tumor suppressor
Gudkov Andrei
Kazarov Alexander
Mazo Ilya
Roninson Igor B.
Brusca John S.
McDonnelll Boehnen Hulbert & Berghoff
University of Illinois
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