Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-12-01
2001-07-10
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S007100, C435S069100, C424S009362, C536S023500
Reexamination Certificate
active
06258536
ABSTRACT:
BACKGROUND OF THE INVENTION
Many biological functions are accomplished by altering the expression of various genes through transcriptional (e.g. through control of initiation, provision of RNA precursors, RNA processing, etc.) and/or translational control. For example, fundamental biological processes such as cell cycle, cell differentiation and cell death, are often characterized by variations in the expression levels of groups of genes.
Gene expression is also associated with pathogenesis. For example, the lack of sufficient expression of functional tumor suppressor genes and/or the over expression of oncogene/protooncogenes could lead to tumorigenesis (Marshall, Cell, 64: 313-326 (1991); Weinberg, Science, 254: 1138-1146 (1991), incorporated herein by reference for all purposes). Thus, changes in the expression levels of particular genes (e.g. oncogenes or tumor suppressors) serve as signposts for the presence and progression of various diseases.
BRCA1 encodes a tumor suppressor gene that is mutated in the germline of women with genetic predisposition to breast and ovarian cancer. Germline mutations are found in approximately half of breast-ovarian cancer pedigrees and in ~10% of women with early onset of breast cancer, irrespective of family history. Most mutations result in premature protein truncation and as predicted heterozygous germline mutations show reduction to homozygosity (LOH) within tumor specimens. However, somatic inactivation of BRCA1 is uncommon in sporadic breast cancer, pointing to potentially distinct genetic mechanisms.
Functional properties of BRCA1 have been inferred from its pattern of subnuclear localization. During S phase, endogenous BRCA1 is present within nuclear dots that are colocalized with RAD51, the mammalian homolog of bacterial recA, involved in homologous recombination and the repair of double-strand breaks in DNA following ionizing radiation. BRCA1 and RAD51 are also colocalized during meiosis, a process that involves programmed homologous recombination and in which both BRCA1 and RAD51 proteins are found along unsynapsed chromosomes. BRCA1 is also colocalized with the product of the second breast cancer predisposition gene, BRCA2, whose role in the maintenance of chromosomal integrity is suggested by analysis of mice with a partial loss of function phenotype. Consistent with a potential role in the repair of DNA damage, treatment of cultured cells with ionizing radiation leads to the hyperphosphorylation of BRCA1 and the disappearance of BRCA1 nuclear dots.
In addition to its potential role in homologous recombination, BRCA1 demonstrates properties of a transcription factor or cofactor. Protein purification studies have shown that it coelutes with the RNA polymerase II holoenzyme complex and interacts with RNA helicase, consistent with its involvement in some aspect of transcriptional regulation The C-terminal domain of BRCA1 mediates transcriptional activation when fused to a heterologous DNA binding domain, and a potential target promoter is that of the cyclin-dependent kinase inhibitor p21. In transient transfection assays, BRCA1 mediates transcriptional activation of the p21 promoter, through a site that is distinct from that implicated in its regulation by p53. However, BRCA1 has also been shown to enhance p53-mediated activation of the p21 promoter in transient transfection assays, suggesting that this effect may have both p53 dependent and independent components.
Recently, a defect in transcription-coupled repair of oxidative, but not UV-induced DNA damage has been demonstrated in mouse embryo fibroblasts with attenuated BRCA1 function. BRCA1 has therefore, been implicated in three distinct functional pathways, namely RAD51-dependent homologous recombination, transcriptional activation of p21, and transcription-coupled repair of oxidative DNA damage. The physiological significance of these properties and their implications for the function of BRCA1 as a tumor suppressor gene remain to be defined.
Definitive studies of BRCA1 function have been hampered by the absence of BRCA1-null cell lines and the difficulty in achieving stable expression of a transfected full length cDNA. Cells stably transfected with a truncated BRCA1 construct lacking the large central exon 11 undergo accelerated apoptosis following serum withdrawal, while retroviral infection using a 5′ truncated BRCA1 construct encoding a protein of 190 kD that lacks the N terminal ring domain, inhibits colony formation. Analysis of thymidine incorporation in transiently transfected cells has suggested a block in S phase entry, associated with induction of p21.
Thus there is a need in the art for information on the functional properties of BRCA1 and its effect on endogenous target genes. Because of the important role of BRCA1 in inherited breast and ovarian cancers, such information can be useful diagnostically as well as in developing new generations of therapeutic agents.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a method for detecting a BRCA1 gene functional mutation in target cells.
It is another object of the invention to provide an in-cell functional assay for a BRCA1 sequence alteration.
It is still another object of the invention to provide a computer assisted method for detecting a mutation in a target BRCA1 gene.
It is yet another object of the invention to provide a method of diagnosing neoplasia.
It is another object of the invention to provide a method of identifying potential anti-cancer drugs.
These objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention, a method for detecting a BRCA1 gene functional mutation in target cells is provided. The method comprises detecting expression of a plurality of down-stream genes of BRCA1 in a sample of (a) target cells, and (b) reference cells having a wild-type BRCA1 gene. The reference cells are otherwise substantially similar to the target cells. The down-stream genes are up- or down-regulated by the wild-type BRCA1 gene. The expression of the down-stream genes in the target cells and the reference cells are compared. A difference in the expression between the target cells and reference cells suggests a BRCA1 functional mutation in the target cells.
According to another embodiment of the invention, an in-cell functional assay for a BRCA1 sequence alteration is provided. The assay comprises detecting expression of a plurality of BRCA1 down-stream genes in a target sample from target cells having a BRCA1 sequence alteration and in a reference sample from reference cells having a wild-type BRCA1 gene. The reference cells are otherwise substantially similar to the target cells. The down-stream genes are up- or down-regulated by wild-type BRCA1 gene. The expression in the target sample is compared to the expression in the reference sample A difference in the expression between the two samples suggests that the BRCA1 sequence alteration affects the biological function of BRCA1.
According to still another aspect of the invention, a method is provided for detecting a mutation in a target BRCA1 gene using a computer. Wild-type expression data of a plurality of down-stream genes in a wild-type sample containing a wild-type BRCA1 gene is input into a computer. The down-stream genes are transcriptionally regulated by the wild-type BRCA1 gene. Target expression data of the plurality of down-stream genes in a target sample containing the target BRCA1 gene is also input into the computer. The target and wild-type expresssion data are compared by the computer to determine differences. Differences suggest a mutation in the target BRCA1 gene.
In yet another embodiment of the invention a method of diagnosing neoplasia of a test cell is provided. A transcription indicator of a test cell is hybridized to a set of nucleic acid probes. The transcription indicator is selected from the group consisting of mRNA, cDNA and cRNA. The set of nucleic acid probes comprises a plurality of nucleic acid molecules each of which is a portion of a gene whi
Bean James
Christians Fred
Haber Daniel
Harkin Denis Paul
Miklos David
Banner & Witcoff , Ltd.
Chakrabarti Arun K.
Fredman Jeffrey
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