Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-11-29
2004-03-16
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S007210
Reexamination Certificate
active
06706491
ABSTRACT:
FIELD OF THE INVENTION
This invention is related to cellular senescence and changes in cellular gene expression that accompany senescence. In particular, the invention is related to the identification of genes the expression of which is modulated by a cellular gene product, p21, induced in cells at the onset of senescence. More specifically, the invention provides markers of cellular senescence that are genes whose expression in induced or repressed by p21. The invention provides methods for identifying compounds that inhibit or potentiate cellular senescence by detecting inhibition of repression or induction of these marker genes. Also provided are reagents that are recombinant mammalian cells containing a recombinant expression construct encoding p21 that is experimentally-inducible, and recombinant mammalian cells containing a recombinant expression construct that expresses a reporter gene under the transcriptional control of a promoter for a gene that is regulated by p21.
SUMMARY OF THE RELATED ART
p21
WAF1/CIP1/SDI1
is an important mediator of growth arrest and senescence in mammalian cells. p21 has been independently identified by several groups as a protein that binds and inhibits cyclin-dependent kinases (CDK) (Harper et al., 1993
, Cell
75: 805-816), as a gene upregulated by wild-type p53 (el-Deiry et al., 1993
, Cancer Res
. 55: 2910-2919), and as a growth-inhibitory gene overexpressed in senescent fibroblasts (Noda et al., 1994
, Exp. Cell. Res
. 211: 90-98). Because of its pivotal role in p53-regulated growth arrest, p21 is usually regarded as a tumor suppressor. Nevertheless, p21 mutations in human cancer are rare (Hall & Peters, 1996
, Adv. Cancer Res
. 68: 67-108), and p21 knockout mice develop normally and do not show an increased rate of tumorigenesis (Deng et al., 1995
, Cell
82: 675-684).
Cellular levels of p21 are increased in response to a variety of stimuli, including DNA-damaging and differentiating agents. Some of these responses are mediated through transcriptional activation of the p21 gene by p53, but p21 is also regulated by a variety of p53-independent factors (reviewed in Gartel & Tyner, 1999
, Exp. Cell Res
. 227: 171-181). Increased p21 expression leads to cell growth arrest (Noda et al., 1994, ibid.), which occurs in both G1 and G2 (Niculescu et al., 1998
, Mol. Cell. Biol
. 18: 629-643) and is accompanied by the development of morphologic and phenotypic markers of senescence (Vogt et al., 1998
, Cell Growth Differ
. 9: 139-146; McConnell et al., 1998
, Curr. Biol
. 8: 351-354; Bates et al., 1998
, Oncogene
17: 1691-1703; Fang et al., 1999, Oncogene 18: 2789-2797).
Transient induction of p21 mediates different forms of damage-induced growth arrest, including transient arrest that allows cell to repair DNA damage, as well as permanent growth arrest (also termed “accelerated senescence”), which is induced in normal fibroblasts (DiLeonardo et al., 1994
, Genes Develop
. 8: 2540-2551; Robles & Adami, 1998
, Oncogene
16: 1113-1123) and tumor cells (Chang et al., 1999
, Cancer Res
. 59: 3761-3767) by DNA damage or introduction of oncogenic RAS (Serrano et al., 1997
, Cell
88: 593-602). A surge of p21 expression also coincides with the onset of terminal growth arrest during replicative senescence of aging fibroblasts (Noda et al., 1994, ibid.; Alcorta et al., 1996, Proc. Natl. Acad. Sci USA 93:13742-13747; Stein et al., 1999
, Mol. Cell. Biol
. 19: 2109-2117) and terminal differentiation of postmitotic cells (El-Deiry et al., 1995, ibid.; Gartel et al., 1996
, Exp. Cell Res
. 246: 280-289). Analysis of cells that cannot express p21 (p21−/−homozygotes) demonstrated the requirement of p21 in transient G1 and G2 arrest (Deng et al., 1995, ibid.; Waldman et al., 1995
, Cancer Res
. 55: 5187-5190; Bunz et al., 1998
, Science
282: 1497-1501), in replicative senescence of normal fibroblasts (Brown et al., 1997
, Science
277: 831-834), and in accelerated senescence of tumor cells (Chang et al., 1999
, Oncogene
18: 4808-4818).
While p21 is not a transcription factor per se, it has indirect effects on gene expression that may play a role in its cellular functions. The best-known biochemical function of p21 is the inhibition of CDK complexes that regulate transitions between different phases of the cell cycle (reviewed in Cartel & Tyner, 1998, “The growth-regulatory role of p21 (WAF1/CIP1),” in
Inhibitors of Cell Growth, Progess in Molecular and Subcellular Biology
, Vol. 20 (A. Macieir-Coelho, ed.), Springer-Verlag: Berlin Heidelberg, pp. 43-71.). One of the consequences of CDK inhibition is dephosphorylation of Rb, which in turn inhibits E2F transcription factors that regulate many genes involved in DNA replication and cell cycle progression (Nevins, 1998
, Cell Growth Differ
. 9: 585-593). A comparison of p21-expressing cells (p21+/+) and p21-nonexpressing cells (p21−/−) has implicated p21 in radiation-induced inhibition of several E2F-regulated cellular genes (de Toledo et al., 1998
, Cell Growth Differ
. 9: 887-896). Another result of CDK inhibition by p21 is stimulation of transcription cofactor p300 that augments NF&kgr;B (Perkins et al., 1988
, Science
275: 523-527). Activation of histone acetyltransferase p300, that enhances many inducible transcription factors, may have a pleiotropic effect on gene expression (Snowden & Perkins, 1988
, Biochem. Pharmacol
. 55: 1947-1954). p21 may also affect gene expression through its interactions with proteins other than CDK. For example, p21 has been found to inhibit the expression of keratinocyte differentiation markers; this effect was dependent on the C-terminal portion of p21, which is not required for CDK inhibition but is known to bind the proliferating cell nuclear antigen (Di Cunto et al., 1998
, Science
280: 1069-1072). p21 was also reported to bind JNK kinases (Shim et al., 1996
, Nature
381: 804-807), apoptosis signal-regulating kinase 1 (Asada et al., 1999
, EMBO J
. 18: 1223-1234), and Gadd45 (Kearsey et al., 1995
, Oncogene
11: 1675-1683); these interactions may affect the expression of genes regulated by the corresponding pathways.
There remains a need in this art to identify genes whose expression is modulated by induction of p21 gene expression. There is also a need in this art to develop targets for assessing the effects of compounds on cellular senescence, carcinogenesis and age-related diseases.
SUMMARY OF THE INVENTION
This invention provides reagents and methods for identifying genes whose expression is modulated by induction of p21 gene expression. The invention also provides reagents and methods for identifying compounds that inhibit or potentiate the effects of p21 on cellular gene expression, as a first step in rational drug design for preventing cellular senescence, carcinogenesis and age-related diseases or for increasing the efficacy of anticancer therapies.
In a first aspect, the invention provides a mammalian cell containing an inducible p21 gene. In preferred embodiments, the mammalian cell is a recombinant mammalian cell comprising a recombinant expression construct encoding an inducible p21 gene. More preferably, the construct comprises a nucleotide sequence encoding p21, most preferably human p21, under the transcriptional control of an inducible promoter. In alternative embodiments, the construct comprises a nucleotide sequence encoding the amino-terminal portion of p21 comprising the CDK binding domain, more preferably comprising amino acids 1 through 78 of the p21 amino acid sequence. In more preferred embodiments, the inducible promoter can be induced by contacting the cells with an inducing agent, most preferably a physiologically-neutral inducing agent, that induces transcription from the inducible promoter or by removing an agent that inhibits transcription from such promoter. In a preferred embodiment, the mammalian cell is a fibrosarcoma cell.
In another embodiment of the first aspect of the invention are provided recombinant mammalian cells comprising a recombinant expression construct in which a reporter gene is under the
Chang Bey-Dih
Roninson Igor B.
Ketter James
Loeb Bronwer M.
McDonnell & Boehnen Hulbert & Berghoff
The Board of Trustees of the University of Illinois
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