Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Nucleoproteins – e.g. – chromatin – chromosomal proteins,...
Reexamination Certificate
1999-02-12
2003-09-02
Bugaisky, Gabrielle (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Nucleoproteins, e.g., chromatin, chromosomal proteins,...
C530S350000, C530S300000, C530S324000, C530S326000, C530S327000, C514S002600, C514S012200, C514S015800, C514S013800, C424S185100, C424S277100
Reexamination Certificate
active
06613883
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to methods of screening for compounds capable of inducing apoptosis in certain tumor-cells. The invention also relates to compounds identified by such methods. In addition, the invention relates to methods for the in vitro diagnosis of
Xeroderma pigmentosum
and compounds useful in these methods.
Certain tumors, benign, premalignant, and malignant, are known to have genetic components etiologically. The gene for the nuclear phosphoprotein, p53, is the most commonly mutated gene identified in human cancers. Missense mutations occur in tumors of the colon, lung, breast, ovary, bladder, and several other organs. When mutant forms of the p53 gene are introduced into primary fibroblasts, these cells are immortalized. The wild type p53 gene can suppress the growth of transformed human cells, but oncogenic forms lose this suppressor function. Thus, the p53 gene has been termed a “tumor suppressor” gene.
If the p53 gene of a tumor cell is of the wild type, its p53 gene product may nevertheless be interfered with functionally. For example, a transforming viral infection of the cell can interfere with the p53 protein product. For instance, certain strains of human papillomavirus (HPV) are transforming and are known to interfere with the p53 protein function because the virus produces a protein, E6, which promotes degradation of the p53 protein.
There is also pharmaceutical interest in p53 because p53 protein is capable of inducing certain tumor cells to undergo apoptosis. In apoptosis, or “programmed cell death”, a series of lethal events for the cell appear to be generated directly as a result of transcription of cellular DNA. Thus, apoptosis is a physiologic means for cell death. For example, lymphocytes exposed to glucocorticoids die by apoptosis. Involution of hormone sensitive tissue such as breast and prostate that occurs when the trophic hormone is removed occurs via apoptosis.
In particular, recent studies have indicated that the introduction of wild type (non-mutated) p53 into transformed cell lines that carry a mutant form of p53 induces the cells to undergo apoptosis with disintegration of nuclear DNA. It is believed that p53 may suppress tumor development by inducing apoptosis, thus modulating cell growth.
In addition to p53, there are numerous other genes involved with cell growth. One group of such genes is designated XP because their derangement can result in the disease
Xeroderma pigmentosum. Xeroderma pigmentosum
is a rare disorder characterized by disfigurement, deranged pigmentation of the skin, scarring and heightened incidence of skin cancers, especially on exposure to sunlight. The disease is inherited as an autosomal recessive trait. Eight genetic forms of the disease are known. Phenotypically these forms vary in their symptoms, signs and severity. Two of the more grave forms are associated with mental deficiencies. These two forms are characterized by mutations in the XPB and XPD genes.
Selection of drugs for potential therapeutic use against tumors is an area of medical research which remains fraught with complications and which often present an array of suboptimal treatment choices. There are currently a multitude of potential compounds available to evaluate. Screening procedures are valuable to limit the bewildering array of drug choices for further testing. Improvements in screening methods or reagents are highly desirable. In addition, there is a need for better diagnosis of XP subtypes. These and other needs are addressed by the present invention.
SUMMARY OF THE INVENTION
The invention provides a method for screening a compound for an ability to induce apoptosis. The method includes providing a first cell containing either a normal or mutant p53 gene. The first cell is responsive to p53. For instance, the first cell is typically capable of undergoing apoptosis after microinjection of a DNA construct expressing wild type p53. The method further includes providing a second cell containing at least one mutant
Xeroderma pigmentosum
gene such as a mutant XPB gene, or a mutant XPD gene, or both. The second cell is not usually capable of undergoing apoptosis after microinjection of a DNA construct expressing wild type p53. According to a method of the invention, both the first and second cells are contacted with a compound of interest. An example of a compound of interest is any compound one desires to screen for possible use as a chemotherapeutic agent or drug. The method includes detecting whether or not apoptosis of either the first or second cell, or both, occurs after contact of the compound to the cells. A comparison of the observations for apoptosis is made, thereby determining whether the compound can induce apoptosis.
The first and the second cell can be selected from any of a number of cell types including benign, premalignant, and malignant. The first and the is second cell can be uninfected or infected. If the latter, the infection can be viral, such as from a papilloma virus. The first and second cell can be selected from any histological or anatomical classification. Typically, the cells are selected from the group consisting of fibroblastic, epithelial, and hematopoietic cells. The cells can be derived from a variety of tissues, including tissue of colon, lung, breast, ovary, cervix, liver, kidney, nervous system, and hematopoietic system. Preferably, the cells are fibroblastic or lymphoblastic cells.
The invention also provides a method of screening for a compound capable of inhibiting the binding of p53 protein to a
Xeroderma pigmentosum
protein, such as either XPB or XPD proteins or both. This method includes providing a reagent having at least one
Xeroderma pigmentosum
protein, preferably XPB or XPD, or both. The reagent is contacted with the compound, permitting the compound to compete with wild type p53 protein for a binding site on any or all of the
Xeroderma pigmentosum
protein(s). Subsequently, any binding of the compound to the protein(s) is detected.
Additionally, this method can include contacting the reagent with wild type p53 protein and detecting a binding of the wild type p53 to at least one of the
Xeroderma pigmentosum
proteins such as an XPB and\or XPD protein(s). The method can further comprise attaching a label to at least one of the
Xeroderma pigmentosum
protein(s) and the p53 protein. The label can be any of a number of detectable labels known in the art. Some examples are an antibody, a radioisotope, and a fluorescent molecule. Conveniently, the reagent has a TFIIH complex containing both XPB and XPD proteins.
The invention also provides a method of screening for a compound capable of inhibiting at least one
Xeroderma pigmentosum
helicase activity, such as XPB and\or XPD helicase activity. This method includes providing a reagent having at least one
Xeroderma pigmentosum
protein, contacting the reagent with the compound which permits thee compound to bind to the
Xeroderma pigmentosum
helicase, and determining the helicase activity. Typically, the reagent has a TFIIH complex containing both XPB and XPD proteins.
The invention also provides compositions. In particular this invention describes compounds consisting essentially of an amino acid sequence selected from a group of subsequences from wild type p53 having the 393 amino acids depicted in SEQ ID NO:1 wherein the subsequences are in their native order and selected from the group consisting of: (a) amino acids 319 to 393 (SEQ ID NO:7); (b) amino acids 361 to 393 (SEQ ID NO:8); (c) amino acids 367 to 387 (SEQ ID NO:2); (d) amino acids 350 to 380 (SEQ ID NO:9); (e) amino acids 355 to 375 (SEQ ID NO:10); (f) amino acids 360 to 370 (SEQ ID NO:11); and, (g) a subsequence comprising a, b, c, d, e or f where the subsequence further consists of amino acids of SEQ ID NO:1 which flank subsequence a, b, c, d, e, or f within 10 amino acids or less at either or both of the amino or carboxy terminus and are in their native order with the proviso that the peptide have less than 100 amino acids, and wherein s
Harris Curtis C.
Hoeijmakers Jan H. J.
Wang Xin Wei
Bugaisky Gabrielle
The United States of America as represented by the Secretary of
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