Method for identifying metastatic sequences

Abstract

The invention relates to methods for the identification of metastatic sequences. Cells from a cell line or an animal tissue are treated to form a cell line predisposed to metastasis. Treated cells are implanted in an animal of at a primary site and incubated for a period of time sufficient for the cells to proliferate and develop metastases at secondary sites. Expressed sequences from cells at the primary and secondary sites are amplified by differential display polymerase chain reaction and compared. Differentially expressed sequences are identical identified and can be cloned and sequenced. These sequences can be used as probes in the diagnosis of metastatic disorders, as probes to isolate metastatic sequences and as a therapeutic agent.

I claim:  
     
       1. A method for identifying a sequence expressed in a metastasis comprising the steps of: 
       a) transfecting an oncogenic sequence into a mammalian cell to form a population of transfected cells;  
       b) administering tranfected cells to a primary site of a host mammal to form a primary tumor;  
       c) maintaining said mammal for a period of time sufficient to develop a metastasis at a secondary site;  
       d) amplifying expressed RNA sequences of the transfected cells and expressed RNA sequences of the metastasis by differential-display PCR; and  
       e) comparing the amplified expressed RNA sequences of the transfected cells with the amplified expressed RNA sequences of the metastasis and identifying the sequence expressed at a higher level in the metastasis as compared to the expressed RNA sequences of the transfected cells.  
     
     
       2. The method of  claim 1  wherein the mammalian cell is transfected by calcium phosphate transfection, viral transduction, lipofection, dextran sulfate transfection or electroporation. 
     
     
       3. The method of  claim 1  wherein the oncogenic sequence is a sequence of the gene that erodes the oncoproteins p21, p34, p53, myc, ras or src. 
     
     
       4. The method of  claim 1  wherein the oncogenic sequence is a sequence that enhances metastatic potential. 
     
     
       5. The method of  claim 4  wherein the oncogenic sequence is a sequence of the gene that encodes cyclin D1, caveolin or TGF-β1. 
     
     
       6. The method of  claim 1  wherein the mammalian cell is treated with an agent that alters gene expression prior to the administration of said cell to said host mammal. 
     
     
       7. The method of  claim 6  wherein the agent is benzanthracene (BA), dimethyl benzanthracene (DMBA) or 5-azacytidine. 
     
     
       8. The method of  claim 1  wherein the mammalian cell is a primary cell or an established cell line. 
     
     
       9. The method of  claim 1  wherein the mammalian cell is isolate from urogenital sinus tissue. 
     
     
       10. The method of  claim 1  wherein the mammalian cell is a fetal cell. 
     
     
       11. The method of  claim 1  wherein the mammalian cell contains a gene selected from the group consisting of TGF-β1, cyclin D1, p21, p34, p53, ras, and myc. 
     
     
       12. The method of  claim 1  wherein the mammalian cell is isolated from the same species as the host mammal. 
     
     
       13. The method of  claim 1  wherein the mammalian cell and the host mammal are histocompatible. 
     
     
       14. The method of  claim 1  wherein the mammalian cell and the host mammal are syngeneic. 
     
     
       15. The method of  claim 1  wherein the transfected cell is isolated and maintained in vivo or in vitro for a period of time prior to introduction of said cell to the host mammal. 
     
     
       16. The method of  claim 1  wherein the expressed sequences of the transfected cells are obtained from a cell line of immortalized transfected cells. 
     
     
       17. The method of  claim 1  wherein the transfected cells are administered to the primary site by subcutaneous implantation. 
     
     
       18. The method of  claim 1  wherein the host mammal is a mouse, a rabbit or a primate. 
     
     
       19. The method of  claim 1  wherein the host mammal is a syngeneic, xenogeneic, immunocompromised or transgenic host mammal. 
     
     
       20. The method of  claim 1  further comprising suppressing expression of TGF-α in the host mammal prior to the introduction of transfected cells into said host mammal. 
     
     
       21. The method of  claim 1  wherein the primary site is the renal capsule, the prostate or the testis. 
     
     
       22. The method of  claim 1  wherein the secondary site is selected from the group of sites consisting of lung, kidney, liver, lymph nodes, brain, bone, testis, spleen, ovaries and mammary. 
     
     
       23. The method of  claim 1  wherein differential display PCR is performed with an anchor primer and a variable primer. 
     
     
       24. The method of  claim 22  wherein the anchor primer comprises a polythymidine sequence and a dinucleotide sequence connected to a 3′-terminus. 
     
     
       25. The method of  claim 24  wherein the polythymidine sequence comprises between about 5 to about 30 thymidines. 
     
     
       26. The method of  claim 24  wherein the dinucleotide sequence is selected from the group of sequences consisting of AA, AG, AC, AT, GA, GG, GC, GT, CA, CG, CC and CT. 
     
     
       27. The method of  claim 23  wherein the anchor primer or the variable primer comprise a detectable moiety selected from the group consisting of radioactive moieties, phosphorescent moieties, magnetic moieties, luminescent moieties and conjugatable moieties. 
     
     
       28. The method of  claim 23  wherein the anchor primer and the variable primer have a common sequence. 
     
     
       29. The method of  claim 7  wherein the agent is a retinoid. 
     
     
       30. A method for identifying a sequence expressed in metastasis comprising the steps of: 
       a) pretreating a mammalian cell with an agent that enhances metastatic potential to form a population of cells predisposed to metastasis;  
       b) introducing the pretreated cells to a primary site of a host mammal;  
       c) maintaining said mammal for a period of time sufficient to develop a metastasis at a secondary site;  
       d) amplifying expressed RNA sequences of pretreated cells and expressed RNA sequences of the metastasis by differential-display PCR; and  
       e) identifyg the sequence expressed at a higher level in the metastasis as compared to expressed RNA sequences of the pretreated cells.  
     
     
       31. The method of  claim 30  further comprising the step of treating cells of the primary or secondary sites with a genotoxic agent prior to amplification. 
     
     
       32. The method of  claim 31  wherein the genotoxic agent is benzanthracene (BA), dimethyl benzanthracene (DMBA) or 5-azacytidine. 
     
     
       33. The method of  claim 30  further comprising the step of comparing the expressed sequences amplified from the metastasis with expressed sequences amplified from mammalian cells before pretreatment to identify the sequence selectively expressed in the metastasis. 
     
     
       34. The method of  claim 30  wherein the chemical compound is a benzanthracene, dimethyl benzanthracene, or 5-azacytidine. 
     
     
       35. The method of  claim 30  wherein the mammalian cell is transfected, prior to the administration of said cell to the host mammal, with an oncogenic sequence before or after treatment of said cell with the agent that enhances metastatic potential. 
     
     
       36. The method of  claim 30  wherein the mammalian cell is a cell line. 
     
     
       37. The method of  claim 30  wherein the mammalian cell is isolated from lymphatic tissue, hematopoietic cells, reproductive tissues or urogenital sinus tissue. 
     
     
       38. The method of  claim 30  wherein the mammalian cell is a fetal cell. 
     
     
       39. The method of  claim 30  wherein the mammalian cell is isolated from a transgenic animal. 
     
     
       40. The method of  claim 30  wherein the primary site is the renal capsule, the prostate or the testis. 
     
     
       41. The method of  claim 30  wherein the secondary site is selected from the group of sites consisting of lung, kidney, liver, lymph nodes, brain, bone, testis, spleen, ovaries and mammary. 
     
     
       42. The method of  claim 30  wherein differential display PCR is performed using an anchor primer and a variable primer. 
     
     
       43. A method of screening a biological tissue for the presence of a metastasis comprising contacting the tissue with a nucleic acid probe, wherein the probe detects the presence of a nucleic acid molecule comprising SEQ ID NO: 89  or its complement, and wherein an increased level of a nucleic acid molecule comprising SEQ ID NO: 89  or its complement in the biological tissue relative to the level of a nucleic acid molecule comprising SEQ ID NO: 89  or its complement in a primary tumor is indicative of a metastasis.  
     
     
       44. The method of  claim 1 , wherein the tissue is lung, kidney, liver, lymph node, brain, testis, bone, spleen, ovary, or mammary tissue.  
     
     
       45. The method of  claim 1 , wherein the tissue is renal capsule, testis, prostate, or ovary tissue.  
     
     
       46. The method of  claim 1 , wherein the method comprises in situ hybridization of the probe with the tissue.  
     
     
       47. The method of  claim 1 , wherein nucleic acids are extracted from the tissue prior to contact with the probe.  
     
     
       48. The method of  claim 5 , wherein the nucleic acids are amplified prior to contact with the probe.  
     
     
       49. The method of  claim 6 , wherein the method comprises different display polymerase chain reaction.  
     
     
       50. The method of  claim 1 , wherein the primary tumor in a prostate tumor.