Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1998-10-16
2001-01-09
Elliott, George C. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S252100, C435S320100, C536S023100, C536S023500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06171857
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to nucleic acids encoding a novel leucine zipper protein, KARP-1, which is involved in DNA repair and polypeptides relating thereto. The present invention also includes fragments and biologically functional variants thereof. Also included are KARP-1 inhibitors which inhibit KARP-1 activity by inhibiting the expression or function of KARP-1. The invention further relates to methods of using such nucleic acids, polypeptides, and inhibitors in the treatment and/or diagnosis of disease, such as in a method for treating cancer in combination with a chemotherapeutic agent by administering a KARP-1 inhibitor to inhibit double strand DNA base repair.
BACKGROUND OF THE INVENTION
Although progress has been made in understanding the biological basis of and in treating cancer, it is believed that one-third of all people in the United States will ultimately develop cancer. The slow progress of understanding cancer and developing treatments for cancer is partly due to the broad variety of cancers as well as the heterogeneity of individual
In spite of the diversity of cancers, many cancers share common features such as the abnormal expression or mutation of various genes, suggesting that a common mechanism may underlie many cancers. Mutations in the tumor suppressor gene p53 have been found in more than 50% of human cancers implying that loss of appropriate p53-dependent gene expression represents a fundamental step in oncogenic progression.
One tool commonly used for treating a wide variety of cancers is chemotherapy. More than 50 chemotherapeutic agents have been developed for the treatment of cancer. Included among chemotherapies for cancer is the use of combinational therapy, in which two or more chemotherapeutic agents having different mechanisms of action are given concurrently. The results typically can be additive. Not all tumors, however, respond to chemotherapeutic agents and others although initially responsive to chemotherapeutic agents may develop resistance to them. As a result, the search for effective anti-cancer drugs and drug combinations has intensified in an effort to find even more effective agents for treating the myriad of cancers.
Cancers are generally treated with either surgery, chemotherapy, including radiation, or a combination of these. Ionizing radiation therapy targets DNA by generating free radicals and reactive oxygen intermediates that damage local cellular substituents including DNA. Cells which undergo rapid proliferation are particularly sensitive to radiation therapy. Hypoxic tissues can be resistant to radiation therapy because the presence of oxygen is important to radiation.
DNA repair enzymes are critical to the normal function of a cell. DNA repair enzymes recognize the damage caused by genotoxic substances or by other causes (such as spontaneous damage or misreplication) and repair such damage. Defects in the ability of a cell to repair errors or breaks in DNA can be lethal to cells and can cause the development of serious diseases in the organism. DNA-dependent protein kinase (DNA-PK) is a serine-threonine protein kinase that requires the presence of double-stranded DNA with free ends for its activity and is composed of a Ku heterodimer consisting of 86 kDa (Ku86) and 70 kDa (Ku7O) subunits (Reeves, 1985; Yaneva et al., 1985; Francoeur et al., 1986; Mimori et al., 1986) and a 465 kDa catalytic subunit (DNA-PK
CS
) (Dvir et al., 1992; Gottlieb and Jackson, 1993; Suwa et al., 1994). Biochemical analyses of the Ku subunit of DNA-PK demonstrated that it bound in a sequence non-specific fashion to virtually all double-stranded DNA ends including 5′- or 3′-protruding ends, blunt ends (Mimori and Hardin, 1986), and duplex DNA ending in stem-loop structures (Falzon et al., 1993), apparently by recognizing transitions from double- to single-stranded DNA. Ku has also been reported to be capable of sequence-specific binding, particularly within the promoter elements of genes (Giffin et al., 1996). Recently, extensive genetic and molecular analyses have identified DNA-PK as an integral component of the DNA double-strand break (DSB) repair pathway (reviewed in Jeggo et al., 1995; Jackson, 1996).
In mammals, defects in DNA DSB repair manifest themselves in two easily recognizable phenotypes: ionizing radiation (IR
S
) hypersensitivity and immunodeficiency. These two seemingly unrelated biological processes are in fact linked by the requirement of DNA DSBs as reaction intermediates. Thus, the exposure of mammalian cells to IR induces lesions in chromosomal DNA such as strand scissions, single-stranded breaks, DSBs and base cross-links (Price, 1993). In particular, DNA DSBs appear to be the predominant cytotoxic lesions as even a single unrepaired DNA DSB can be a lethal event (Klar et al., 1984; Frankenberg-Schwager and Frankenberg, 1990). Mammalian IR-sensitive (IRS) mutants have been isolated and in approximately half of these cell lines, IR sensitivity correlated with a greatly decreased ability to repair DNA DSBs (reviewed in Zdzienicka, 1995). Thus, the DSB repair capacity of a cell appears to be a critical, though not the sole, factor in determining cellular IR-sensitivity. Similarly, the development of the mammalian immune system is dependent upon a site-specific DNA recombination process, termed lymphoid V(D)J recombination, that assembles the non-contiguous genomic segments that encode the Variable (V), Diversity (D), and Joining (J) elements of immunoglobulin and T-cell receptor genes (reviewed in Lewis, 1994). Importantly, analyses of V(D)J recombination products in vivo and in vitro has proven that DNA DSBs are an essential intermediate in the V(D)J reaction mechanism (reviewed in Oettinger, 1996). Thus, the repair of DNA DSBs is an integral feature of IR sensitivity and V(D)J recombination.
Mutations in the subunits of DNA-PK have been shown to affect deleteriously both IR sensitivity and V(D)J recombination. DNA-PK
CS
is now known to be the product of the severe combined immune deficiency (scid) gene (Blunt et al., 1995; Hartley et al., 1995; Kirchgessner et al., 1995; Lees-Miller et al., 1995; Danska et al., 1996) and it has long been recognized that animals homozygously defective at this locus were profoundly immune deficient (Bosma et al., 1983), IR
S
(Fulop and Phillips, 1990) and defective in DNA DSB repair (Biedermann et al., 1991; Hendrickson et al., 1991). Recently, it was shown that cell lines belonging to the fifth X-ray cross-complementation group (XRCC5) (Thompson and Jeggo, 1995; Zdzienicka, 1995), which were known to be IR
S
and V(D)J-defective, were deficient in Ku86 gene expression (Smider et al., 1994; Taccioli et al., 1994; Boubnov et al., 1995; Errami et al., 1996; He et al., 1996). Lastly, knock-out mice for Ku86 have been generated by homologous recombination (Nussenzweig et al., 1996; Zhu et al., 1996) and, as expected, these mice have a profound immune deficiency and are IR
S
. Thus, DNA-PK is an important mammalian DNA repair complex and mutations in either DNA-PK
CS
or the 86 kDa subunit of Ku result in severe IR
S
and V(D)J recombination deficits due to impaired DNA DSB repair.
SUMMARY OF THE INVENTION
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and inhibitors of the foregoing nucleic acids and polypeptides which prevent double strand DNA base repair. The foregoing can be used in the diagnosis or treatment of conditions characterized by the loss of KARP-1 activity and can be used in methods in which it is therapeutically useful to inhibit KARP-1 activity such as a treatment for cancer. Here, we present the identification of KARP-1, which plays a role in double strand DNA base repair.
It was discovered according to the invention that the Ku86 locus encodes a second gene termed (Ku86 Autoantigen Related Protein-1) KARP-1. KARP-1 is a protein which extends at least
Brown University Research Foundatiion
Elliott George C.
Epps Janet
Wolf Greenfield & Sacks P.C.
LandOfFree
Leucine zipper protein, KARP-1 and methods of regulating DNA... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Leucine zipper protein, KARP-1 and methods of regulating DNA..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Leucine zipper protein, KARP-1 and methods of regulating DNA... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2466334