Method for identifying a subpopulation of mammalian cells with distinctive ribosome translation profiles

Abstract

The present invention generally relates to subpopulations of mammalian cells with distinctive ribosome translational profiles, i.e. translational activities. The present invention further relates to methods for identifying and isolating such cells, kits comprising the same, or methods which utilize different translational activities of these subpopulations of mammalian cells.

1 .- 63 . (canceled) 
     
     
         64 . A method for verifying that Class I, Class II, or Class III mammalian cells retain their phenotype, the method comprising:
 treating a subset of Class I, Class II, or Class III mammalian cells with at least one toxin, the mammalian cells being stably transformed with a nucleic acid expression cassette comprising a translation regulated (TR) element encoding a mammalian mRNA molecule that is at least 80% homologous to a human Proteolipid Protein (plp) gene of SEQ ID NO: 4, is selectively translated in stressed and/or dying cells and does not direct translation of a PIRP-L or PIRP-M peptide, and a nucleotide sequence operably linked to the TR element, which encodes a reporter gene and is translated from the TR element;   measuring a level of a reporter protein encoded by the reporter gene in the subset of mammalian cells as compared to a level of the reporter protein expressed by a reference standard, the reference standard being mammalian cells stably transformed with the nucleic expression cassette and not treated with the at least one toxin; and   verifying that the mammalian cells retain their phenotype wherein the class I cells are characterized by the expression of the reporter protein that is up to 500% greater than the expression of the reporter protein in the mammalian cells which have not been treated with the toxins, the class II cells are characterized by the expression of the reporter protein that is more than 500% to 1400% greater than the expression of the reporter protein in the mammalian cells which have not been treated with the toxins, and the class III cells are characterized by the expression of the reporter protein that is more than 1400% to about 75000% greater than the expression of the reporter protein in the mammalian cells which have not been treated with the toxins.   
     
     
         65 . A method for determining the ability of a substance to inhibit or stimulate protein translation in mammalian cells, wherein the method comprises:
 contacting Class I, Class II, or Class III mammalian cells with the substance, the mammalian cells being stably transformed with a nucleic acid expression cassette comprising a translation regulated (TR) element encoding a mammalian mRNA molecule that is at least 80% homologous to a human Proteolipid Protein (plp) gene of SEQ ID NO: 4, is selectively translated in stressed and/or dying cells and does not direct translation of a PIRP-L or PIRP-M peptide, and a nucleotide sequence operably linked to the TR element, which encodes a reporter gene and is translated from the TR element; and   determining the expression of the reporter protein produced by the mammalian cells after contact with the substance as compared to the expression of the reporter protein by the cells which have not been treated with the substance, wherein reduction in the expression of the reporter protein produced by the substance-treated cells as compared to the expression of the reporter protein produced by the cells which have not been treated with the substance indicates that the substance inhibits protein translation, and an increase in the expression of the reporter protein produced by the substance-treated cells as compared to the expression of the reporter protein produced by the cells which have not been treated with the substance indicates that the substance enhances protein translation.   
     
     
         66 . A method for determining the ability of a substance to inhibit or stimulate protein translation in mammalian cells, wherein the method comprises:
 treating a subset of mammalian cells with at least one toxin to form toxin-treated cells, the mammalian cells being stably transformed with a nucleic acid expression cassette comprising a TR element encoding a mammalian mRNA molecule that is at least 80% homologous to a human Proteolipid Protein (plp) gene of SEQ ID NO: 4, is selectively translated in stressed and/or dying cells and does not direct translation of a PIRP-L or PIRP-M peptide, and a nucleotide sequence operably linked to the TR element, which encodes a reporter gene and is translated from the TR element;   measuring a level of a reporter protein encoded by the reporter gene in the toxin-treated cells as compared to a level of the reporter protein expressed by a reference standard to identify whether the subset of mammalian cells exhibit the phenotype of the desired subpopulation of mammalian cells;   isolating at least one cell from the mammalian cells to form a cell culture if the toxin-treated cells of the mammalian cells exhibit the phenotype of the desired subpopulation of the mammalian cells;   growing the cell culture to form a subpopulation of mammalian cells; and   optionally treating the subpopulation of mammalian cells with the at least one toxin and repeating the measuring, isolating, and growing steps until the desired subpopulation of mammalian cells is identified;   contacting the desired subpopulation of the mammalian cells with the substance; and determining the expression of the reporter protein produced by the desired subpopulation of mammalian cells after contact with the substance as compared to the expression of the reporter protein by the cells which have not been treated with the substance, wherein reduction in the expression of the reporter protein produced by the substance-treated cells as compared to the expression of the reporter protein produced by the cells which have not been treated with the substance indicates that the substance inhibits protein translation, and an increase in the expression of the reporter protein produced by the substance-treated cells as compared to the expression of the reporter protein produced by the cells which have not been treated with the substance indicates that the substance enhances protein translation.   
     
     
         67 . The method of  claim 65 , wherein the mammalian cells stably transformed with the nucleic acid expression cassette are used to determine the ability of the substance to inhibit or stimulate cap-independent translation. 
     
     
         68 .- 70 . (canceled) 
     
     
         71 . A method for restoring phenotype of a desired subpopulation of mammalian cells, wherein the method comprises:
 culturing at least a subset of the mammalian cells to form cultured cells, wherein the mammalian cells are stably transformed with a nucleic acid expression cassette comprising a nucleotide sequence for a selection marker and TR element encoding a mammalian mRNA molecule that is at least 80% homologous to a human Proteolipid Protein (plp) gene of SEQ ID NO: 4, is selectively translated in stressed and/or dying cells and does not direct translation of a PIRP-L or PIRP-M peptide, and a nucleotide sequence operably linked to the TR element, which encodes a reporter gene and is translated from the TR element;   treating the cultured cells with a substance to which the selection marker provides resistance, such that the cultured cells which no longer contain the nucleic acid expression cassette die;   growing the treated cells to form a subpopulation of mammalian cells; and   optionally repeating the culturing, treating and growing steps until the phenotype of the desired subpopulation of mammalian cells is restored.   
     
     
         72 . The method of  claim 71 , wherein the selection marker is selected from the group consisting of neomycin, ampicillin, kanamycin, tetracycline, chloramphenicol, hygromycin B, blasticidin and G418. 
     
     
         73 .- 74 . (canceled) 
     
     
         75 . The method of  claim 71 , wherein the subpopulation of the mammalian cells are class I cells characterized by the level of the reporter protein being up to 500% greater than the level of the reporter protein in a reference standard, the reference standard being mammalian cells stably transformed with the nucleic expression cassette and not treated with the at least one toxin. 
     
     
         76 . The method of  claim 71 , wherein the subpopulation of the mammalian cells are class II cells characterized by the level of the reporter protein being more than 500% and not more than 1,400% greater than the level of the reporter protein in a reference standard, the reference standard being mammalian cells stably transformed with the nucleic expression cassette and not treated with the at least one toxin. 
     
     
         77 . The method of  claim 71 , wherein the subpopulation of the mammalian cells are class III cells characterized by the level of the reporter protein being more than 1,400% and not more than 75000% greater than the level of the reporter protein in a reference standard, the reference standard being mammalian cells stably transformed with the nucleic expression cassette and not treated with the at least one toxin. 
     
     
         78 . The method of  claim 71 , wherein the mammalian cells are cancer cells or cancer stem cells. 
     
     
         79 .- 86 . (canceled) 
     
     
         87 . A method for recovering a desired subpopulation of mammalian cells either retaining phenotype of parental cells, or exhibiting increased translational activity as compared to the translational activity of parental cells, wherein the method comprises:
 treating a subset of Class I, Class II, or Class III mammalian cells with at least one toxin, the mammalian cells being derived from a cell culture exhibiting altered translational activity as compared to the translational activity of parental cells, and stably transformed with a nucleic acid expression cassette comprising a translation regulated (TR) element encoding a mammalian mRNA molecule that is at least 80% homologous to a human Proteolipid Protein (plp) gene of SEQ ID NO: 4, is selectively translated in stressed and/or dying cells and does not direct translation of a PIRP-L or PIRP-M peptide, and a nucleotide sequence operably linked to the TR element, which encodes a reporter gene and is translated from the TR element;   measuring a level of a reporter protein encoded by the reporter gene in the subset of mammalian cells as compared to a level of the reporter protein expressed by a reference standard and to a level of the reporter protein expressed by a reference standard of parental cells, the reference standard being mammalian cells stably transformed with the nucleic expression cassette and not treated with the at least one toxin, and the reference standard of parental cells being parental mammalian cells stably transformed with the nucleic expression cassette and not treated with the at least one toxin;   isolating at least one cell from the mammalian cells to form a cell culture if the toxin-treated cells of the mammalian cells exhibit the phenotype of the desired subpopulation of the mammalian cells;   growing the cell culture to form a subpopulation of mammalian cells; and   optionally treating the subpopulation of mammalian cells with the at least one toxin and repeating the measuring, isolating, and growing steps until the desired subpopulation of mammalian cells is identified; and   verifying that the desired subpopulation of mammalian cells retain the phenotype of the parental cells or exhibit increased translational activity as compared to the translational activity of the parental cells wherein the class I cells are characterized by the expression of the reporter protein that is up to 500% greater than the expression of the reporter protein in the mammalian cells which have not been treated with the toxins, the class II cells are characterized by the expression of the reporter protein that is more than 500% to 1400% greater than the expression of the reporter protein in the mammalian cells which have not been treated with the toxins, and the class III cells are characterized by the expression of the reporter protein that is more than 1400% to about 75000% greater than the expression of the reporter protein in the mammalian cells which have not been treated with the toxins.   
     
     
         88 . The method of  claim 66 , wherein the mammalian cells stably transformed with the nucleic acid expression cassette are used to determine the ability of the substance to inhibit or stimulate cap-independent translation. 
     
     
         89 . The method of  claim 87 , wherein the mammalian cells are cancer cells or cancer stem cells.