Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-08-06
2004-08-24
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S377000, C536S023100, C536S024100, C536S024500, C514S04400A
Reexamination Certificate
active
06780642
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to the fields of molecular biology, genomics, bioinformatics, pathology, and medicine. More particularly, the invention relates to a new utility of a gene whose expression is modulated in select cancers.
BACKGROUND
Recent efforts to sequence the entire human genome have resulted in the identification of tens of thousands of genes. See, e.g., Venter et al., Science, 291:1304-51, 2001. Despite this achievement, many of these identified genes have yet to be functionally characterized. As the function of these genes are elucidated they should prove to be useful for identifying new diagnostic and therapeutic targets for a variety of different diseases.
SUMMARY
The invention relates to the discovery of specific polynucleotide sequences that are expressed at higher levels in select cancer cells than in non-diseased cells. The polynucleotide sequences were identified using a modified datamining tool referred to herein as DDDM (for Digital Differential Display tool, Modified) to analyze the Cancer Gene Anatomy Project (CGAP) database of the National Cancer Institute. In particular, DDDM was used to identify several expressed sequence tags (ESTs) more prevalent in cancer tissue libraries than in corresponding non-cancerous tissue libraries. The identified ESTs were than used to identify specific UniGenes associated with cancer. Based on the identified polynucleotide sequences, a gene termed SIM2 (for Single Minded homolog 2), whose expression is selectively upregulated in colon, prostate and pancreas tumors was identified.
The native human SIM2 gene has previously been cloned and sequenced. Chrast et al., Genome Res. 7: 615-624, 1997. Northern blot analyses indicated that several different species of mRNA are expressed from the SIM2 gene, including those of 2.7, 3, 4.4, and 6 kb. The multiple mRNAs are believed to be due to alternative splicing, overlapping transcription, or different utilization of 5′ or 3′ untranslated sequences. At least two different forms of the SIM2 gene have been characterized. The long form (GenBank ACC# U80456; SEQ ID NO: 1) is 3901 bp and codes for a protein of 667 amino acid with an apparent molecular weight of 74 kD. The short-form (GenBank ACC# U80457; SEQ ID NO: 2) is 2859 bp and codes for a protein of 570 amino acid with an apparent molecular weight of 64 kD. The N-termini of both the forms of SIM2 protein show extensive sequence identity to each other as well as to another member of the family, SIM1. The N-terminus of all of these proteins contains four recognized domains, namely, bHLH, PAS1, PAS2 and HST. These domains are often seen in transcription factors. The C-terminal ends of the proteins show some similarity, but also contain unique sequences.
SIM2 has previously been associated with Down's Syndrome, but not cancer.
Accordingly, the invention features a method for detecting a cancer in a tissue sample. This method includes the steps of: (a) providing the tissue sample; and (b) analyzing the tissue sample for the presence of a SIM2 marker. The presence of the SIM2 marker in the tissue sample indicates that the tissue sample contains a cancer. In this method, the tissue sample can be a colon tissue sample, a prostate tissue sample, or a pancreas tissue sample.
SIM2 markers utilized within the invention can be, e.g., a SIM2 nucleic acid such as a SIM2 mRNA or a native SIM2 nucleic acid. The native SIM2 nucleic acid can have a nucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 2. The SIM2 marker can also be a SIM2 protein such as a native SIM2 protein, e.g., one having an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
In the foregoing method, the step of providing a tissue sample can include obtaining the tissue sample from a human subject; and the step of analyzing the tissue sample can include isolating RNA from the tissue sample, generating cDNAs from the isolated RNA, amplifying the cDNAs by PCR to generate a PCR product, and electrophoretically separating the PCR product to yield an electrophoretic pattern. The step of amplifying the cDNAs by PCR can be performed using an oligonucleotide primer, e.g., one that includes a nucleotide sequence of SEQ ID NOs: 7, 8, 15, and 16. Also in this method, the step of amplifying the cDNAs by PCR can be performed using a first oligonucleotide primer and a second oligonucleotide primer. The first oligonucleotide primer can include the nucleotide sequence of SEQ ID NOs: 7 or 15. The second oligonucleotide primer can include the nucleotide sequence of SEQ ID NOs: 8 or 16. In a particular embodiment of this method, the presence of a 472 base pair nucleic acid in the electrophoretic pattern indicates that the tissue sample contains a cancer.
Also in the foregoing method, the step of analyzing the tissue sample for the SIM2 nucleic acid can include contacting the tissue sample with an oligonucleotide probe that hybridizes under stringent hybridization conditions to a polynucleotide having a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, the complement of SEQ ID NO: 1, or the complement of SEQ ID NO: 2. For example, the oligonucleotide probe can include the nucleic acid of SEQ ID NO: 9. The oligonucleotide probe of this method can also include a detectable label.
In a variation of the foregoing method, the SIM2 marker is a SIM2 protein such as a native SIM2 protein (e.g., one having an amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4). In this variation, the step of providing a tissue sample can include obtaining the tissue sample from a human subject, and the step of analyzing the tissue sample can include contacting at least a portion of the tissue sample with a probe that specifically binds to the SIM2 protein. The probe can include a detectable label and/or an antibody (e.g., an antibody that specifically binds to the peptide of SEQ ID NO: 14). In another variation of the method, the tissue sample includes a cell isolated from feces, urine, or peripheral blood.
In another aspect, the invention features a method of modulating SIM2 gene expression. This method includes the steps of: (a) providing a cell that expresses a SIM2 gene; and (b) introducing into the cell an agent that modulates the expression the SIM2 gene in the cell. The agent can be an oligonucleotide such as an antisense oligonucleotide. For example, an antisense oligonucleotide that hybridizes under stringent hybridization conditions to a polynucleotide that encodes a SIM2 protein can be used, as can an antisense oligonucleotide that is at least 18 nucleotides in length and includes a sequence that is a complement of a nucleic acid that encodes the SIM2 protein. For instance, the antisense oligonucleotide can include a nucleic acid sequence of SEQ ID NOs: 11 or 12.
Also within the invention is a method of identifying a test compound that modulates expression of a SIM2 gene in a cell. This method includes the steps of: (a) providing a cell expressing a SIM2 gene; (b) contacting the cell with the test compound; and (c) detecting a modulation in the expression of the SIM2 gene. Detecting the modulation indicates that the test compound modulates expression of the SIM2 gene. In this method, the cell can be derived from a colon tissue sample, a prostate tissue sample, or a pancreas tissue sample. Also in this method, the step of detecting the modulation in the expression of the SIM2 gene can include analyzing the cell for a change in the intracellular concentration of a SIM2 marker.
The invention additionally features a method for reducing the growth rate of a cancer includes a cell expressing a SIM2 protein. This method includes the step of: contacting the cell with an agent that inhibits the expression of the SIM2 protein in the cell.
The agent can an oligonucleotide such as an antisense oligonucleotide. For example, an antisense oligonucleotide that hybridizes under stringent hybridization conditions to a polynucleotide that encodes a SIM2 protein can be used, as can an antisense oligonucleotide that is at least 18 nucleotides in length and includes a sequence t
Edwards & Angell LLP
Florida Atlantic University
McGarry Sean
McLaren, Esq. Margaret J.
LandOfFree
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