Nucleic acids comprising regions of the rat PEG-3 promoter that display elevated expression in human cancer cells and uses thereof

Abstract

This invention provides an isolated nucleic acid comprising a PEG-3 promoter comprising the nucleotide sequence of −270 to +194 of FIG. 2. The invention also provides a method for identifying an agent that modulates PEG-3 promoter activity using a cell which comprises a PEG-3 promoter operatively linked to a reporter gene, wherein reduced reporter gene expression in the presence of the agent is indicative of an agent that inhibits PEG-3 promoter activity and wherein increased reporter gene expression in the presence of the agent is indicative of an agent that enhances PEG-3 promoter activity. The invention provides a method for treating cancer in a subject which comprises administering a nucleic acid comprising a PEG-3 promoter operatively linked to a gene-of-interest, wherein the gene-of-interest is selectively expressed in cancerous cells in the subject and such expression results in growth suppression or death of the cancerous cells.

1 - 14  (Cancelled)  
     
     
         15 . A method for identifying an agent which modulates PEG-3 promoter activity in a cell which comprises: 
 (a) contacting the cell with the agent wherein the cell comprises a nucleic acid comprising a PEG-3 promoter operatively linked to a reporter gene;    (b) measuring the level of reporter gene expression in the cell; and    (c) comparing the expression level measured in step (b) with the reporter gene expression level measured in an identical cell in the absence of the agent,    wherein a lower expression level measured in the presence of the agent is indicative of an agent that inhibits PEG-3 promoter activity and wherein a higher expression level measured in the presence of the agent is indicative of an agent that enhances PEG-3 promoter activity, thereby identifying an agent which modulates PEG-3 promoter activity in the cell.    
     
     
         16 . The method of  claim 15 , wherein the cell is a melanoma cell, a neuroblastoma cell, a cervical cancer cell, a breast cancer cell, a lung cancer cell, a prostate cancer cell, a colon cancer cell or a glioblastoma multiforme cell.  
     
     
         17 . The method of  claim 15 , wherein the agent comprises a molecule having a molecular weight of about 7 kilodaltons or less.  
     
     
         18 . The method of  claim 15 , wherein the agent is an antisense nucleic acid comprising a nucleotide sequence complementary to at least a portion of the sequence shown in SEQ ID NO:1 and is at least 15 nucleotides in length.  
     
     
         19 . The method of  claim 15 , wherein the agent is a DNA molecule, a carbohydrate, aglycoprotein, a transcription factor protein or a double-stranded RNA molecule.  
     
     
         20 . The method of  claim 15 , wherein the agent is a synthetic nucleotide sequence, a peptidomimetic, or an organic molecule having a molecular weight from 0. 1 kilodaltons to 10 kilodaltons.  
     
     
         21 . The method of  claim 15 , wherein the reporter gene encodes beta-galactosidase, luciferase, chloramphenicol transferase or alkaline phosphatase.  
     
     
         22 . The method of  claim 15 , wherein expression of PEG-3 promoter activity measured is equal to or greater than a 2.5 to 3.5 fold increase or decrease.  
     
     
         23 . The method of  claim 15 , wherein the PEG-3 promoter is a PEG-3 promoter having promoter activity comprising the nucleotide sequence beginning with the guanosine (G) at position 1507 and ending with the cytosine (C) at position 1970 of SEQ ID NO:1.  
     
     
         24 . A method for treating cancer in a subject which comprises administering a nucleic acid comprising a PEG 3 promoter operatively linked to a gene-of-interest wherein the gene of interest is selectively expressed in cancerous cells in the subject and such expression regulates expression of PEG-3 resulting in growth suppression or death of the cancerous cells, thereby treating cancer in the subject.  
     
     
         25 . The method of  claim 24 , wherein the nucleic acid consists essentially of 
 (i) a PEA3 protein binding sequence consisting of the nucleotide sequence beginning with the thymidine (T) at position 1672 and ending with the thymidine (T) at position 1677 of SEQ ID NO:1,    (ii) a TATA sequence consisting of the nucleotide sequence beginning with the thymidine (T) at position 1748 and ending with the adenosine (A) at position 1753 of SEQ ID NO:1, and    (iii) an AP1 protein binding sequence consisting of the nucleotide sequence beginning with the thymidine (T) at position 1781 and ending with the adenosine (A) at position 1787 of the nucleotide sequence shown in SEQ ID NO:1.    
     
     
         26 . The method of  claim 24 , wherein the nucleic acid has a sequence complementary to at least a portion of SEQ ID NO: 1 of at least 25 nucleotides in length.  
     
     
         27 . The method of  claim 24 , wherein the cancer is melanoma, neuroblastoma, astrocytoma, glioblastoma multiforme, cervical cancer, breast cancer, colon cancer, prostate cancer, osteoscarcoma or chrondosarcoma.  
     
     
         28 . The method of  claim 24 , wherein the administering is carried out via injection, oral administration, topical administration, adenovirus infection, liposome-mediated transfer, topical application to the cells of the subject, or microinjection.  
     
     
         29 . The method of  claim 24 , wherein the subject is a mammal.  
     
     
         30 . The method of  claim 29 , wherein the mammal is a human.  
     
     
         31 . The method of  claim 24 , wherein the gene of interest is a gene whose expression causes apoptosis of a cell.  
     
     
         32 . The method of  claim 24 , wherein the gene comprises an Mda-7 gene or a p53 gene.  
     
     
         33 . The method of  claim 24 , wherein the gene of interest is a tumor suppressor gene.  
     
     
         34 . The method of  claim 33 , wherein the suppressor gene is mda-7.  
     
     
         35 . The method of  claim 24 , wherein the gene of interest is a cytotoxic gene.  
     
     
         36 . The method of  claim 35 , wherein expression of the cytotoxic gene causes cell death.  
     
     
         37 . The method of  claim 36 , wherein the cytotoxic gene is selected from the group consisting of HSV-TK, p21, p27, and p10.