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Title:  Chromosome 3p21.3 genes are tumor suppressors
United States Patent: 
7,902,441
Issued: 
March 8, 2011

Inventors:
 Ji; Lin (Sugar Land, TX), Minna; John Dorrance (Dallas, TX), Roth; Jack (Houston, TX), Lerman; Michael (Rockville, MD)
Assignee:
  Board of Regents, The University of Texas (Austin, TX)
The United States of America as represented by the Department of Health and Human Services (Washington, DC)

Appl. No.:
 10/445,718
Filed:
 May 27, 2003


 

Woodbury College's Master of Science in Law


Abstract

Tumor suppressor genes play a major role in the pathogenesis of human lung cancer and other cancers. Cytogenetic and allelotyping studies of fresh tumor and tumor-derived cell lines showed that cytogenetic changes and allele loss on the short arm of chromosome 3 (3p) are most frequently involved in about 90% of small cell lung cancers and greater than 50% of non-small cell lung cancers. A group of recessive oncogenes, Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), Luca 1 (HYAL1), Luca 2 (HYAL2), PL6, 123F2 (RaSSFI), SEM A3 and Beta* (BLU), as defined by homozygous deletions in lung cancers, have been located and isolated at 3p21.3.

Description of the Invention

SUMMARY OF THE INVENTION

The tumor suppressor genes at 3p21.3 are now disclosed: Gene 26 (CACNA2D2).sup.340, PL6, Beta* (BLU), LUCA-1 (HYAL1), LUCA-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), and SEM A3. The function of the individual 3p genes in suppression of tumor growth and tumor progression, induction of apoptosis, alteration of cell cycle kinetics, and repression of telomerase activity has been characterized by the liposome- and recombinant adenoviral vector-mediated transfer of 3p genes in vitro and in vivo. This also is the initial disclosure of the Beta* gene.

Therefore, it is an objective of the present invention to provide methods of using tumor suppressors having a chromosomal location of 3p21.3. It is also an objective to provide a tumor suppressor, Beta*. Further, it is an objective to provide methods of constructing recombinant adenovirus in which these tumor suppressors may be inserted.

An embodiment of the present invention is an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence of SEQ ID NO:2. There is also provided a nucleic acid with the sequence of SEQ ID NO:1. Further provided is an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2. Another embodiment is a nucleic acid of 15 to about 100 base pairs comprising from 15 contiguous base pairs of SEQ ID NO:1, or the complement thereof. A further embodiment includes from about 20, 25, 30, 40, 50 or 100 contiguous base pairs of SEQ ID NO:1, or the complement thereof.

Another embodiment of the invention is an isolated peptide having between 10 and about 50 consecutive residues of SEQ ID NO:2. Further, the peptide may comprise 15, 20, 25, or 30 consecutive residues of SEQ ID NO:2. In this application, "about" is defined as within + or -2 amino acids.

Yet another embodiment is an expression cassette comprising a polynucleotide encoding a polypeptide having the sequence of SEQ ID NO:2, wherein said polynucleotide is under the control of a promoter operable in eukaryotic cells. In another embodiment, the promoter of this expression cassette is heterologous to the coding sequence. The promoter may be a tissue specific and inducible promoter. In another embodiment, the expression cassette may be contained in a viral vector. The viral vector may be a retroviral vector, an adenoviral vector, and adeno-associated viral vector, a vaccinia viral vector, or a herpesviral vector. In a further embodiment the expression cassette may comprise a polyadenylation signal.

Another embodiment is a cell comprising an expression cassette comprising a polynucleotide encoding a polypeptide having the sequence of SEQ ID NO:2, wherein said polynucleotide is under the control of a promoter operable in eukaryotic cells, said promoter being heterologous to said polynucleotide.

Yet another embodiment of the invention is a monoclonal antibody that binds immunologically to a polypeptide comprising SEQ ID NO:2, or an immunologic fragment thereof. Also provided is a monoclonal antibody with a detectable label. The label may be a fluorescent label, a chemiluminescent label, a radiolabel and an enzyme. Another embodiment of the invention is a hybridoma cell that produces a monoclonal antibody that binds immunologically to a polypeptide comprising SEQ ID NO:2, or an immunologic fragment thereof. A further embodiment is a polyclonal antisera, antibodies of which bind immunologically to a polypeptide comprising SEQ ID NO:2, or an immunologic fragment thereof.

Yet another embodiment is a isolated and purified nucleic acid that hybridizes, under high stringency conditions, to a DNA segment comprising SEQ ID NO:1, or the complement thereof. In a further embodiment the nucleic acid is about 15, 17, 20 or 25 bases in length.

Another embodiment of the invention is a method for constructing a recombinant adenovirus comprising: (a) providing a shuttle vector, said shuttle vector comprising an adenoviral inverted terminal repeat (ITR) sequence, an expression cassette comprising a promoter and a poly-A sequence, a transgene under the control of said promoter, and unique restriction sites at the 5'- and 3'-ends of the ITR-promoter-transgene-poly-A segment; (b) cutting at said restriction enzyme sites; (c) ligating the released segment into an adenoviral vector lacking the entire E1 and E3 regions and transforming the resulting vector a bacterial host cell; (d) obtaining vector from said bacterial host cell and digesting the vector to release the E1/E3-deleted adenovirus genome; and (e) transfecting the adenovirus genome into E1-expressing host cells. In a further embodiment, the transgene is Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3. In another embodiment, the promoter may be a cytomegalovirus (CMV) promoter and said poly A sequence is bovine growth hormone (BGH) poly A sequence.

Yet another embodiment of the invention is a method for constructing a recombinant adenovirus comprising: (a) providing a shuttle vector comprising an adenoviral inverted terminal repeat (ITR) sequence, an expression cassette comprising a promoter and poly-A signal sequence, a transgene under the control of said promoter, a tetracycline resistance-off responsive element, and unique restriction sites at the 5' and 3' ends of the IRT-promoter-transgene-poly-A segment; (b) cutting at said restriction enzyme sites; (c) ligating the released segment into an adenoviral vector comprising a tetracyclin resistant-off transactivator gene and lacking the entire E1 and E3 regions, and transforming the resulting vector a bacterial host cell; (d) obtaining vector from said bacterial host cell and digesting the vector to release the E1/E3-deleted adenovirus genome; and (e) transfecting the adenovirus genome into E1-expressing host cells. In a further embodiment, the transgene is Gene 26, PL6, Beta*, LUCA-1, LUCA-2, 123F2, Fus1, 101F6, Gene 21 or SEM A3. In another embodiment, the promoter may be a cytomegalovirus (CMV) promoter and said poly A sequence is bovine growth hormone (BGH) poly A sequence.

In yet another embodiment, also provided is a shuttle vector comprising an adenoviral inverted terminal repeat (ITR) sequence, an expression cassette comprising a promoter and poly-A sequence, a TetR-Off responsive element, and unique restriction sites at the 5'- and 3'-ends of the ITR-promoter-poly-A segment. In another embodiment of the invention the promoter is a cytomegalovirus (CMV) promoter and said poly A sequence is bovine growth hormone (BGH) poly A sequence. Also provided is a multipurpose cloning site in said segment, positioned between said promoter and said poly-A sequence.

Yet another embodiment is an adenoviral vector comprising a tetracycline resistant-off transactivator gene and lacking the entire E1 and E3-regions.

Another embodiment of the invention is a method of diagnosing cancer in a subject comprising the steps of: (i) obtaining a biological sample from said subject; and (ii) assessing the expression of a functional Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 product in sample. In a further embodiment the sample is a tissue sample. The tissue sample may be brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cells, pancreas, colon, stomach, cervix, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, oral tissue, bone marrow or blood tissue. In another embodiment, the assessing comprises detecting a nucleic acid encoding Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3. Detecting may comprise amplification said nucleic acid, nucleic acid hybridization, or sequencing. In another embodiment, assessing comprises detecting a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide. The detecting of a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide may comprise ELISA or immunohistochemistry. In yet another embodiment, the assessing may comprise wild-type or mutant oligonucleotide hybridization, with said oligonucleotide configured in an array on a chip or wafer. In another embodiment of the invention, the expression of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 is compared with the expression of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 in normal samples. In another embodiment, the comparison involves evaluating the level of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3 expression.

Another embodiment is a non-human transgenic animal lacking one or both functional alleles of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3. Also provided is a non-human transgenic animal that overexpresses Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 as compared to a similar non-transgenic animal. In a further embodiment is a non-human transgenic animal, the genome of which comprises an expression cassette comprising a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 under the control of an inducible promoter.

An embodiment of the invention is a method for suppressing growth of a tumor cell comprising contacting said cell with an expression cassette comprising: (a) a nucleic acid encoding Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3; and (b) a promoter active in said tumor cell, under conditions permitting the uptake of said nucleic acid by said tumor cell. In another embodiment, the tumor cell is derived from a brain tumor, lung tumor, liver tumor, spleen tumor, kidney tumor, lymph node tumor, small intestine tumor, blood cell tumor, pancreatic tumor, colon tumor, stomach tumor, cervix tumor, breast tumor, endometrial tumor, prostate tumor, testicle tumor, ovarian tumor, skin tumor, head and neck tumor, esophageal tumor, oral tissue tumor, or bone marrow tumor. In a further embodiment, the nucleic acid is contained in a viral vector. The viral vector may be a retroviral vector, an adenoviral vector, and adeno-associated viral vector, a vaccinia viral vector, and a herpesviral vector. In yet another embodiment, the nucleic acid is contained in a liposome.

Another embodiment of the invention is a method of altering the phenotype of a tumor cell comprising contacting said cell with an expression cassette comprising: (a) a nucleic acid encoding Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3; and (b) a promoter active in said tumor cell, under conditions permitting the uptake of said nucleic acid by said tumor cell. In another embodiment, the phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth, cell cycling, invasiveness, tumorigenesis, and metastatic potential. In yet another embodiment, the promoter is a cytomegalovirus (CMV) promoter.

Another embodiment is a method of inhibiting cancer in a subject suffering therefrom comprising administering to said subject an expression cassette comprising: (a) a nucleic acid encoding Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide; and (b) a promoter active in tumor cells of said subject, whereby expression of said polypeptide inhibits said cancer. In a further embodiment, the subject is a human. In other embodiments, the nucleic acid encodes Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3. In another embodiment, the cancer is a selected from the group consisting of brain cancer, lung cancer, liver cancer, spleen cancer, kidney cancer, lymph node cancer, small intestine cancer, blood cell cancer, pancreatic cancer, colon cancer, stomach cancer, cervix cancer, breast cancer, endometrial cancer, prostate cancer, testicle cancer, ovarian cancer, skin cancer, head and neck cancer, esophageal cancer, oral tissue cancer, and bone marrow cancer. In yet another embodiment, the expression cassette is contained in a viral vector. The viral vector may be a retroviral vector, an adenoviral vector, and adeno-associated viral vector, a vaccinia viral vector, and a herpesviral vector. In another embodiment, the expression cassette is contained in a liposome. In another embodiment, the expression cassette further comprises a poly-A sequence. The poly-A sequence may be a bovine growth hormone (BGH) poly-A sequence. In a further embodiment, the expression cassette is administered intratumorally, in the tumor vasculature, local to the tumor, regional to the tumor, or systemically.

Also provided in the method of inhibiting cancer is the administering of a chemotherapeutic agent to said subject. In another embodiment, the chemotherapeutic comprises cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate. Also provided is the administering radiation to said subject. In another embodiment, the radiation is delivered local to a cancer site or is whole body radiation. The radiation may comprise .gamma.-rays, X-rays, accelerated protons, microwave radiation, UV radiation or the directed delivery of radioisotopes to tumor cells. In yet another embodiment, a second anticancer gene may be administered to said subject. The second anticancer gene may be a tumor suppressor. The second anticancer gene may be an inhibitor of apoptosis. In another embodiment, the second anticancer gene is an oncogene antisense construct.

An embodiment of the invention is a method of treating a subject with cancer, comprising the step of administering to said subject a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3 polypeptide. In another embodiment, the cancer is a selected from the group consisting of brain cancer, lung cancer, liver cancer, spleen cancer, kidney cancer, lymph node cancer, small intestine cancer, blood cell cancer, pancreatic cancer, colon cancer, stomach cancer, cervix cancer, breast cancer, endometrial cancer, prostate cancer, testicle cancer, ovarian cancer, skin cancer, head and neck cancer, esophageal cancer, oral tissue cancer, and bone marrow cancer. In a further embodiment, the polypeptide is contained within a liposome. the liposome may be comprised of N-(1-[2,3-Dioleoyloxy]propyl)-N,N,N-trimethylammonium (DOTAP) and cholesterol. In another embodiment, the subject is human.

Another embodiment of the invention is a method of screening a candidate substance for anti-tumor activity comprising the steps of: (i) providing a cell lacking a functional Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide; (ii) contacting said cell with said candidate substance; and (iii) determining the effect of said candidate substance on said cell. In another embodiment, the cell is a tumor cell. In another embodiment, the determining may comprises comparing one or more characteristics of the cell in the presence of said candidate substance with the same one or more characteristics of a similar cell in the absence of said candidate substance. In a further embodiment, the characteristic is selected from the group consisting of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), SEM A3 expression, phosphatase activity, proliferation, metastasis, contact inhibition, soft agar growth, cell cycle regulation, tumor formation, tumor progression, metastasis and tissue invasion. In another embodiment, the candidate substance is a chemotherapeutic or radiotherapeutic agent. Also provided is a candidate substance selected from a small molecule library. In further embodiments, the cell is contacted in vitro or in vivo.

An embodiment of the invention is a method of screening a candidate substance for anti-tumor activity comprising the steps of: (i) providing a cell; (ii) contacting said cell with said candidate substance; and (iii) determining the effect of said candidate substance on expression of a Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3 polypeptide.

Another embodiment is a method of producing a Beta* polypeptide in a host cell comprising: (a) providing an expression cassette comprising a nucleic acid encoding Beta* operably linked to an promoter active in said host cell; (b) transferring said expression cassette into said host cell; and (c) culturing said host cell under conditions permitting expression of said Beta* polypeptide.

Yet another embodiment of the invention is a method of diagnosing cancer in a subject comprising the steps of: (i) obtaining a biological sample from said subject; and (ii) detecting hypermethylation of the promoter region of Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYAL1), Luca-2 (HYAL2), 123F2 (RASSF1), Fus1, 101F6, Gene 21 (NPRL2), or SEM A3.
 

Claim 1 of 35 Claims

1. A method for suppressing growth of a non-small cell lung cancer tumor cell comprising contacting said cell with an expression cassette comprising: (a) an isolated nucleic acid encoding FUS1 encoded by SEQ ID NO:3; and (b) a promoter heterologous to said isolated nucleic acid that is active in said tumor cell and is operatively associated with said isolated nucleic acid, under conditions permitting the uptake of said nucleic acid by said tumor cell.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.
 

 

     
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