Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1998-05-12
2003-06-24
Falk, Anne-Marie (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S013000, C800S014000, C800S018000, C435S325000, C435S352000, C435S354000, C435S355000, C424S009100, C424S009200
Reexamination Certificate
active
06583333
ABSTRACT:
BACKGROUND OF THE INVENTION
Tumor suppressors are generally identified as genes in which loss of function causes tumor formation, either as seen by transformation of cells in culture, or by association of mutations with tumors in animals. The usual normal function of these genes is to impose some constraint on the cell cycle or cell growth. In certain cancers, patients develop tumors which have mutations in both alleles of the tumor suppressor gene. p53, Rb and p16 are among the best characterized of the tumor suppressor genes.
In vitro analysis of human tumor cell lines from some tumor types show a correlation between p53 mutations and resistance to treatment. Burkitt's lymphoma cell lines with mutant p53 are more resistant to a variety of treatments when compared to those with wild type p53 (Fan S., et al., Cancer Res. 54,5824-30, 1994). Consistent with in vitro studies, p53 status is linked to drug resistance in several tumor types. Perhaps the most striking examples occur in lymphoid malignancies, including non-Hodgkin's lymphoma, acute myeloid leukemia, myelodysplastic syndrome, and chronic lymphocytic leukemia (Wattel, E., et al.,
Blood
84, 3148-57 1994; Wilson, W. H., et al.,
Blood
89, 601-9 1997). In these tumor types, p53 mutations are rare but generally associated with disease progression and poor prognosis. When patients are classified by p53 status, tumor response (i.e. remission vs. nonresponsive), and survival, patients with p53 mutations are remarkably resistant to therapy and display very short survival times. In this regard, a particularly informative tumor type is acute lymphoblastic leukemia. Here, p53 mutations in primary tumors are exceedingly rare, and most patients typically respond to therapy. However, a subfraction of patients relapse, and approximately 30% of relapsed tumors harbor mutant p53. Moreover, patients with p53 mutant tumors are less likely to enter a second remission compared to patients with relapsed tumors harboring wild-type p53 (Diccianni, M. B., et al.,
Blood
84, 3105-12 1994; Hsiao, M. H., et al.,
Blood
83, 2922-30 1994).
It is now known that most anticancer agents induce apoptosis, a genetically-regulated form of cell death (reviewed in Kerr, J. F. R. et al.,
Cancer
73:2013-2026, 1994). Since drugs with distinct primary targets can induce apoptosis through similar mechanisms, mutations in apoptotic programs can produce multiple drug resistance (Dive, C., and Hickman, J. A.,
Br. J. Cancer
64:192-196, 1991). These observations raise the possibility that the chemosensitivity of human tumors is determined, in part, by the combined effects of oncogenic mutations on apoptosis (Lowe, S. W. et al.,
Cell
74:957-967, 1993).
Considerable progress has been made in identifying components of apoptotic programs, although the interaction between these components and their precise modes of action remain elusive. A schematic diagram that illustrates a loose hierarchy of an apoptotic “pathway' is shown in FIG.
1
.
Resistance to cytotoxic agents used in cancer therapy remains a major obstacle in the treatment of human malignancies, including leukemia and lymphoma. Since most anticancer agents were discovered through empirical screens, efforts to overcome resistance are hindered by our limited understanding of why these agents are ever effective. The role of apoptosis in malignancy provides a new explanation for drug sensitivity and resistance. This view suggests that responsive tumors must readily undergo apoptosis in response to cytotoxic agents and that resistant tumors may have acquired mutations that suppress apoptosis. Also, the fact that apoptosis is controlled by genes raises the prospect that the problem of drug sensitivity and resistance will be amenable to molecular biology.
One of the difficulties in identifying determinants of drug cytotoxicity in vivo is the limited availability of appropriate material. Human tumor lines grown as xenographs are unphysiological, and the wide variation between human individuals, not to mention treatment protocols, makes clinical studies difficult. Consequently, oncologists are forced to perform correlative studies with a limited number of highly dissimilar samples, often leading to confusing results.
SUMMARY OF THE INVENTION
The invention encompasses a mouse which expresses the oncogene myc in its B cells, and is thereby susceptible to lymphoma. The expression of myc in B cells can result from a number of genetic events, either natural or created by man. Preferred embodiments include a mouse of genotype E&mgr;-myc/mutated tumor suppressor gene, such as E&mgr;-myc/p53
+/−
, E&mgr;-myc/Rb
+/−
, and E&mgr;-myc/p16
+/−
.
The invention also encompasses cell lines that can be cultured from lymphoma cells arising in a mouse of the invention, preferably a mouse of genotype E&mgr;-myc/p53
+/−
, E&mgr;-myc/Rb
+/−
, or E&mgr;-myc/p16
+/−
. The cells of these cultured cell lines can have genetic changes beyond those in non-lymphoma cells of the mouse from which they are derived.
The invention also includes methods for testing a lymphoma, either a primary tumor or one that has recurred after one or more remissions, for sensitivity to an anti-tumor treatment, by giving the treatment to a mouse of the invention, such as an E&mgr;-myc/mutated tumor suppressor gene mouse, and monitoring the mouse for a response to the treatment, such as remission from the lymphoma. A treatment can be tested for its effectiveness against lymphoma by the use of similar steps.
Further methods employ the cell lines of the invention. For example, E&mgr;-myc/mutated tumor suppressor gene lymphoma cells can be cultured in vitro, given a treatment, and monitored for slowed or stopped growth relative to an untreated or sham-treated population of the same cell type. A further test of a treatment involves transplanting lymphoma cells from an E&mgr;-myc/mutated tumor suppressor gene mouse into a mouse of a type which normally does not develop lymphoma (preferably an immunocompetent mouse), administering the treatment to the recipient mouse after it develops lymphoma, and monitoring the treated recipient mouse for remission. A transplanted, recipient mouse which does not receive the treatment can serve as a control for comparison.
The invention further encompasses a method to assess an effect of a gene on the response of a lymphoma to a treatment, using a cell line derived from the lymphoma. A gene can be introduced into a population of cells of the cell line to make “test” cells, and another population of cells of the same cell line can be used as controls (e.g., left unaltered, transfected with control virus not bearing gene, transformed with control vector not bearing gene). Test or control cells can be introduced into one or more mice, and the two groups of mice can be given an identical treatment and monitored for remission.
REFERENCES:
patent: 5087571 (1992-02-01), Leder et al.
patent: 5175383 (1992-12-01), Leder et al.
patent: 5709844 (1998-01-01), Arbeit et al.
LA Donehower et al., Molecular Carcinogenesis, “Effects of Genetic Background on Tumorigenesis in p53-Deficient Mice,” (1995), 14:16-22.*
D-H Cho et al., Journal of Biomedical Science,“IL-6 undergoes transition from in vitro autocine growth factor to in vivo growth inhibitor of B lymphoma cells,” 1997,4/5, pp. 201-207.*
Jacks, T. et al., “Effects of an Rb mutation in the mouse,”Nature, vol. 359, pp. 295-300, Sep. 24, 1992.
Strasser, A. et al., “Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bc1-2,”Nature, vol. 348, pp. 331-333, Nov. 22, 1990.
Serrano, M. et al., “Role of the INK4a Locus in Tumor Suppression and Cell Mortality,”Cell, vol. 85, pp. 27-37, Apr. 5, 1996.
Harris, A. W. et al., “The E&mgr;-myc Transgenic Mouse A Model for High-incidence Spontaneous Lymphoma and Leukemia of Early B Cells,”J. Exp. Med., vol. 167, pp. 353-371, Feb. 1988.
Hsu, B. et al., “Evidence that c-myc mediated apoptosis does not require wild-type p53 during lymphomagenes
Lowe Scott W.
Wallace-Brodeur Rachel R.
Cold Spring Harbor Laboratory
Falk Anne-Marie
Hamilton Brook Smith & Reynolds P.C.
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