Modulation of line-1 reverse transcriptase

Abstract

A reverse transcriptase encoded by L-1 (LINE-1) has been identified as a target molecule for treating or preventing cancers induced or mediated by this molecule. Method of treating or preventing such cancers in patients involves administration of a therapeutically effective amount of a composition having an inhibitor or antagonist of the reverse transcriptase in cells of the patients. The inhibitor or antagonist blocks lengthening of telomeres in telomerase negative cells. Methods and kits for detecting pathologically proliferating cells expressing L1RT are also disclosed.

What is claimed is: 
     
         1 . A method of selecting a compound capable of shortening telomeres in telomerase negative cancer cells, the method comprising:
 administering a test compound to said cells; evaluating anti-L-1 (LINE-1) retrotransposon reverse transcriptase (L1RT) activity of the test compound or evaluating whether the compound down-regulates expression of L1RT in said cells; and   selecting the compound that exhibits anti-L-1 retrotransposon activity or down-regulates the L1RT expression,   wherein said cells are either in vitro cultured cells or in a non-human animal model.   
     
     
         2 . The method of  claim 1 , wherein the step of evaluating comprises testing for telomere shortening or G2 arrest in said cells or apoptosis of said cells. 
     
     
         3 . The method of  claim 2 , wherein said cells are in vitro cultured cells. 
     
     
         4 . The method of  claim 1 , wherein the animal model is selected from the group consisting of a mouse, a rat, a rabbit, a pig, a cow, a monkey and a guinea pig. 
     
     
         5 . A method of detecting the presence of cancerous cells in a cell sample that is telomerase negative, the method comprising:
 contacting said sample with an inhibitor or antagonist of L-1 (LINE-1) retrotransposon encoded reverse transcriptase (L1RT); and   testing for cells exhibiting telomere shortening or G2 arrest in said cells or apoptosis of said cells.   
     
     
         6 . A method of detecting cells capable of pathologically proliferating in a sample of cells obtained from a tissue of a mammal, comprising contacting the sample of cells with a nucleic acid probe that is substantially complementary or fully complementary to a subsequence of an L1RT mRNA, or with an antibody specific to L1RT reverse transcriptase; and detecting L1RT expression in said cells. 
     
     
         7 . The method of  claim 6 , wherein the nucleic acid probe or antisense sequence comprises a sequence selected from the group consisting of: 5′-CCAGAGATTCTGGTATGTGGTGTC TTT GTT-3′ (SEQ ID NO: 2), 5′-CTT TCT CTT GTA GTA GGC ATT TAG TGC TAT AAA-3′ (SEQ ID NO: 3), 5′-CTC TTG CTT TTC TAG TTC TTT TAA TTG TGA-3′ (SEQ ID NO: 4), 5′-CTT CAG TTC TGC TCT GAT TTT AGT TAT TTC-3′(SEQ ID NO: 5) and 5′-TCC TGC TTT CTC TTG TAG GCA-3′(SEQ ID NO 6). 
     
     
         8 . The method of  claim 7 , wherein said nucleoside analog is 3′-azido-2′,3′dideoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′3′-didehydro-3′deoxythymidine (d4T), ganciclovir or valganciclovir, or a combination thereof. 
     
     
         9 . The method of  claim 6 , wherein the nucleic acid probe comprises a detectable moiety. 
     
     
         10 . The method of  claim 9 , wherein the detectable moiety is a radioisotope, a fluorescent molecule, biotin or digioxigenin. 
     
     
         11 . An in vitro method of interfering with lengthening of telomeres in telomerase negative tumor cells showing alternative lengthening of telomeres induced or mediated by L-1 (LINE-1) retrotransposon encoded reverse transcriptase (L1RT), the method comprising administering to the cells an effective amount of an antisense sequence wherein the antisense sequence hybridizes with a nucleic acid sequence encoding the reverse transcriptase and blocks lengthening of telomeres in said cells. 
     
     
         12 . The method of  claim 11 , wherein the L1 nucleic acid sequence is a DNA sequence, an RNA transcribed from the DNA or a cDNA reverse transcribed from the RNA. 
     
     
         13 . The method of  claim 11 , wherein the antisense sequence comprises a chimeric RNA-DNA oligonucleotide. 
     
     
         14 . The method of  claim 11 , wherein the antisense sequence comprises a sequence selected from the group consisting of: 5′-CCAGAGATTCTGGTATGTGGTGTC TTT GTT-3′ (SEQ ID NO: 2), 5′-CTT TCT CTT GTA GGC ATT TAG TGC TAT AAA-3′ (SEQ ID NO: 3), 5′-CTC TTG CTT TTC TAG TTC TTT TAA TTG TGA-3′ (SEQ ID NO: 4), 5′-CTT CAG TTC TGC TCT GAT TTT AGT TAT TTC-3′ (SEQ ID NO: 5) and 5′-TCC TGC TTT CTC TTG TAG GCA-3′ (SEQ ID NO 6). 
     
     
         15 . An in vitro method of inhibiting the growth of a telomerase negative cell showing alternative lengthening of telomeres induced or mediated by L-1 (LINE-1) retrotransposon encoded reverse transcriptase (L1RT) in said cell, the method comprising: transfecting the cell with a construct capable of expressing an antisense sequence that is fully complementarily to L1RT nucleic acid sequence or to a subsequence of the L1RT nucleic acid sequence in said cell, wherein said antisense sequence is expressed and the growth of the telomerase negative cell is inhibited. 
     
     
         16 . The method of  claim 15 , wherein the nucleic acid is a human L1RT open reading frame. 
     
     
         17 . The method of  claim 15 , wherein the nucleic acid is an L1RT mRNA. 
     
     
         18 . The method of  claim 15 , wherein the antisense sequence is one resulting from the construct expressing the sequence in SEQ ID NO: 1 in reverse orientation. 
     
     
         19 . The method of  claim 15 , wherein the subsequence of the L1RT nucleic acid is fully complementary to 5′-CCAGAGATTCTGGTATGTGGTGTC TTT GTT-3′ (SEQ ID NO: 2), 5′-CTT TCT CTT GTA GGC ATT TAG TGC TAT AAA-3′ (SEQ ID NO:3), 5′-CTC TTG CTT TTC TAG TTC TTT TAA TTG TGA-3′ (SEQ ID NO: 4), 5′-CTT CAG TTC TGC TCT GAT TTT AGT TAT TTC-3′ (SEQ ID NO: 5) or 5′-TCC TGC TTT CTC TTG TAG GCA-3′ (SEQ ID NO 6). 
     
     
         20 . The method of  claim 15 , wherein the antisense sequence is a DNA oligonucleotide, a 2′-O methyl oligonucleotide, a peptide nucleic acid oligonucleotide or a phosphorothioate oligonucleotide. 
     
     
         21 . The method of  claim 15 , wherein the antisense L1RT nucleic acid has the nucleotide sequence comprising SEQ ID NO: 1. 
     
     
         22 . The method of  claim 15 , wherein the antisense sequence is about 8 to about 50 nucleotides in length. 
     
     
         23 . The method of  claim 15 , wherein the antisense sequence is about 15 to about 25 nucleotides in length. 
     
     
         24 . The method of  claim 17 , wherein the cell is contacted with two or more antisense sequences fully complementary to different subsequences of the nucleic acid. 
     
     
         25 . A composition comprising a polynucleotide capable of encoding a nucleic acid segment capable of interfering with L-1 (LINE-1) retrotransposon reverse transcriptase (L1RT) activity in cells. 
     
     
         26 . The composition of  claim 25 , wherein the nucleic acid segment comprises SEQ ID NO: 1. 
     
     
         27 . An isolated host cell comprising the composition of a polynucleotide capable of encoding a nucleic acid segment capable of interfering with L-1 (LINE-1) retrotransposon reverse transcriptase (L1RT) activity in cells. 
     
     
         28 . The isolated cell of  claim 27 , wherein the cell is a human cell. 
     
     
         29 . The isolated cell of  claim 28 , wherein the cell is a cancer cell.