Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-05
2001-05-15
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091210, C536S023100, C536S023500, C536S024310, C536S024330
Reexamination Certificate
active
06232073
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates broadly to a method for identifying cancer or neoplastic tissue in a subject.
The Wilms' Tumor 1 (WT1) gene encodes a transcription factor that is critical to normal urogenital development. However, the role of WT1 in prostatic disease is not known. Decreased expression of WT1 and increased expression IGF-1 receptors have been reported in stromal cells from benign hyperplastic prostate tissue, as compared with stromal cells from normal or malignant prostate. WT1 has been shown to regulate the transcription of several growth factors and growth factor receptors that are implicated in prostatic growth control, specifically, the epidermal growth factor receptor (EGFR), insulin-like growth factor (IGF II), the IGF type I receptor (IGFR I), and the androgen receptor.
The WT1 gene product may activate or repress target gene transcription, depending on which of the four isoforms is produced by the cell. The impact of WT1 on target gene transcription is also influenced by cell type and the presence or absence of other proteins that interact with WT1, such as p53, the prostatic apoptosis response protein PAR-4 known as PAWR and CIOA 1 which have been shown to decrease the transcriptional activation of WT1.
SUMMARY OF THE INVENTION
Earlier studies led to the development of a model of human prostate cancer progression that permits comparison of the cellular properties and molecular properties of increasingly aggressive sublines of SV40 large T-antigen immortalized human prostate epithelial cells within the same lineage. These cell lines display the following phenotypes, assessed by orthotopic and/or subcutaneous injection into athymic nude mice: immortal, nontumorigenic; tumorigenic but nonmetastatic, and consistently tumorigenic and metastatic. The expression of EGFR decreased dramatically in metastatic M12 cells, as compared with the parental line. Additional investigations were conducted to determine whether WT1 was expressed in any of the sublines and whether expression correlated with the alteration in the in vivo phenotype and/or the reduction in EGFR and IGFR. These investigations led to the unexpected finding of a truncated WT1 gene transcript with implications for the role of WT1 oncogenesis.
Accordingly, it is an object of the invention to provide a method for detecting cancer or neoplastic tissue in a subject. Further, it is an object of the present invention to provide a method for identifying cancer or neoplastic tissue in a subject by identifying the presence of a truncated Wilms' Tumor 1 (WT1) gene transcript. Further, the present invention is directed to a truncated WT1 gene transcript and a method of using the truncated WT1 gene transcript as a tumor marker for various forms of cancer that express the WT1 gene transcript.
The present invention provides a method for detecting cancer in a subject. The method provides obtaining a sample from the subject and detecting a truncated WT1 gene transcript in the sample, wherein the presence of the truncated WT1 gene transcript indicates the presence of cancer. Further the invention provides detecting a truncated WT1 gene transcript that includes a portion of intron 5. The invention further provides a truncated WT1 gene transcript that lacks a 101 base pair segment of intron 5 between −101 and −1. Still further, the invention includes a truncated WT1 gene transcript characterized by the sequence shown in FIG.
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Further, the invention provides a method where the detection of the truncated WT1 gene transcript includes performing reverse transcriptase-polymerase chain reaction using a 5 prime primer located in intron 5 and a 3 prime primer located in exon 6. Still further, the invention provides using a primer for reverse transcriptase-polymerase chain reaction where the primer is 5′-GAA CCC TGC ATC TAA AGT GG-3′ (SEQ ID NO: 1).
The invention also provides detecting the truncated WT1 gene transcript by probing products from the reverse transcriptase-polymerase chain reaction with an oligonucleotide sense primer in exon 6 wherein the oligonucleotide sense primer is 5′-CCA CAG CAAC AGG GTA CGA-3′ (SEQ ID NO. 2). Further, the invention provides identifying a 95 base pair fragment representing a product of the truncated WT1 gene transcript.
Still further, the invention provides detecting a truncated gene transcript having a length of about two thousand base pairs.
The invention provides a method where positive detection indicates the presence of prostate cancer. The invention also provides a method where positive detection indicates the presence of breast cancer. The invention further provides a method where positive detection indicates the presence of leukemia.
Still further, the invention provides a method for detecting a truncated WT1 gene transcript that includes obtaining a nucleic acid sample from a subject suitable for performing reverse transcriptase-polymerase chain reaction. The method also provides performing reverse transcriptase-polymerase chain reaction on the nucleic acid sample using a 5 prime primer located in intron 5 and a 3 prime primer located in exon 6. The method further provides probing products from the reverse transcriptase-polymerase chain reaction with an oligonucleotide sense primer in exon 6. The method provides identifying a fragment representing a product of the truncated WT1 gene transcript. The invention provides probing products from the reverse transcriptase-polymerase chain reaction with an oligonucleotide sense primer in exon 6, wherein the oligonucleotide sense primer is 5′-CCA CAG CAC AGG GTA CGA-3′ (SEQ ID NO: 2). The invention also provides for performing reverse transcriptase-polymerase chain reaction with a primer, wherein the primer is 5′ GAA CCC TGC ATC TAA AGT GG-3′ (SEQ ID NO: 1). The invention further provides identifying a 95 base pair fragment.
Further, the invention provides an isolated nucleic acid that includes a truncated WT1 gene transcript characterized by the inclusion of a portion of intron 5. The invention further provides an isolated nucleic acid characterized by an absence of a 101 base pair segment of intron 5 between −101 and −1 and has a length of about two thousand base pairs. Still further, the invention provides an isolated nucleic acid characterized by the sequence shown in FIG.
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Dechsukhum Chavaboon
Garrett Carleton T.
Ware Joy L.
McGuireWoods LLP
Myers Carla J.
Virginia Commonwealth University
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