Markers for pre-cancer and cancer cells and the method to interfere with cell proliferation therein

Abstract

A novel family of human mitochondrial RNAs, referred to as chimeric RNAs, which are differentially expressed in normal, pre-cancer and cancer cells, are described. Oligonucleotides targeted to the chimeric RNAs are provided. The described oligonucleotides or their analogs can be used for cancer diagnostics and cancer therapy as well as for research. In one embodiment of this invention, these oligonucleotides hybridize with the sense or with the antisense mitochondrial chimeric RNAs, and the result of the hybridization is useful to differentiate between normal proliferating cells, pre-cancer cells and cancer cells. In another embodiment of the invention, the compositions comprise oligonucleotides that hybridize with the human chimeric RNAs resulting in cancer cell and pre-cancer cell death, while there is no effect in normal cells, constituting therefore, a novel approach for cancer therapy.

1 .- 41 . (canceled) 
     
     
         42 . A hybridization method to distinguish between normal, pre-cancer or cancer cells by determining the presence or absence of the antisense mitochondrial chimeric RNA of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and the sense mitochondrial chimeric RNA of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, in mammalian cells, said method comprising
 a) contacting the sample with two or more independently detectable or labeled compounds of 10 to 50 nucleobases in length complimentary to the antisense mitochondrial chimeric RNA of SEQ ID NO 4, SEQ ID NO 5, or SEQ ID NO 6 and/or the sense mitochonchodrial chimeric RNA of SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3 as probes, such that at least one detectable probe hybridizes to the target mitochondrial chimeric RNA sequences   b) detecting, identifying or quantitating the hybridization of the detectable compounds to the target sequences   c) correlating and comparing the results obtained to the pattern of differential expression of the sense and antisense mitochondrial chimeric RNA in normal, pre-cancer and cancer cells.   
     
     
         43 .- 59 . (canceled) 
     
     
         60 . A kit for in situ hybridization useful for distinguishing between normal human proliferating cells and transformed or malignant human cells by the method of  claim 42 , which kit comprises the following:
 a) one or more detectable oligonucleotides targeted to the sense and/or antisense mitochondrial chimeric RNA wherein said oligonucleotides are labeled.   b) conjugated antibody reagent for label detection   c) hybridization and wash buffer   d) one or more slides of fixed human normal proliferating cells and one or more slides of fixed transformed or malignant human cells, as controls.   
     
     
         61 . The kit according to  claim 60 , wherein said label of the oligonucleotides comprises a haptene selected from the group of digoxigenin, biotin, fluorescein. 
     
     
         62 . The kit according to  claim 60 , wherein said detection method comprises antibiotin antibodies, antidigoxigenin antibodies and antifluorescein antibodies. 
     
     
         63 . The kit according to  claim 60  wherein said antibodies are conjugated antibodies with alkaline phosphatase or peroxidase. 
     
     
         64 . The kit according to  claim 60  wherein said detection method comprises alkaline phosphatase or peroxidase conjugated antibodies directed against antibiotin antibodies, antidigoxigenin antibodies or antifluorescein antibodies, and amplification reagents. 
     
     
         65 . The kit according to  claim 60  wherein said detection method comprises streptavidin-alkaline phosphatase conjugate and amplification reagents. 
     
     
         66 . The kit according to  claim 60  wherein said amplification reagents comprise the substrates for alkaline phosphatase nitroblue tetrazolium or the substrate for peroxidase. 
     
     
         67 . (canceled) 
     
     
         68 . A kit for in situ hybridization useful for determining the cell localization of the antisense (SEQ ID NOS 4, 5 and 6) or sense mitochondrial chimeric RNA (SEQ ID NOS 1, 2 and 3) in the cytoplasm, nucleus, and nucleolus of the cells present in a sample, which kit comprises the following:
 a) one or more detectable oligonucleotides targeted to the sense or antisense mitochondrial chimeric RNA wherein said oligonucleotides are labeled.   b) conjugated antibody reagent for label detection   c) hybridization and wash buffer   d) one or more slides of fixed human normal proliferating cells and one or more slides of fixed transformed or malignant human cells, as controls.   
     
     
         69 - 76 . (canceled) 
     
     
         77 . A method of treating a human or animal suspected of having cancer, comprising administering to said human or animal a therapeutically effective amount of one or more compounds or oligonucleotides of 10 to 50 nucleobases in length which is sufficiently complementary to a mitochondrial chimeric RNA molecule comprising:
 (a) SEQ ID NO. 1-3, or   (b) SEQ ID NO. 4-6,   
       such that the compounds or oligonucleotides are able to hybridize with that mitochondrial chimeric RNA molecule to form a stable duplex, to induce at least cancer cell death, wherein the cancer is selected from multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphoma, stomach carcinoma, esophagus carcinoma, pancreas ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, small bowel adenocarcinoma, colorectal carcinoma, cervical carcinoma, and ovarian carcinoma. 
     
     
         78 . The method of  claim 77 , wherein the mitochondrial chimeric RNA comprises SEQ ID NO:4. 
     
     
         79 . The method of  claim 78 , wherein said one or more oligonucleotides comprise at least one moiety selected from the group consisting of a modified internucleoside linkage, a modified sugar moiety and a modified nucleobase. 
     
     
         80 . The method of  claim 77 , wherein said compounds or oligonucleotides are administered in conjunction with a chemotherapeutic drug for the treatment of cancer. 
     
     
         81 . A method as claimed in  claim 80 , wherein said combination of said compounds or oligonucleotides and said chemotherapeutic drug for the treatment of cancer is capable of inducing a more effective cancer cell death than said chemotherapeutic drug for the treatment of cancer, when administered alone. 
     
     
         82 . The method of  claim 77 , wherein the one or more oligonucleotides comprise a nucleotide sequence selected from the group consisting of SEQ ID NOS 9-196. 
     
     
         83 . The method of  claim 82 , wherein said one or more oligonucleotides comprise at least one moiety selected from the group consisting of a modified internucleoside linkage, a modified sugar moiety and a modified nucleobase. 
     
     
         84 . The method of  claim 77 , wherein said administering step does not induce apoptosis in normal cells.