Claims
- 1. A method for identifying a compound that modulates the cell cycle, the method comprising the steps of:
(i) a cell comprising an E1 polypeptide from FIG. 1, column 1; an E2 polypeptide from FIG. 2, column 1; or an E3 polypeptide from FIG. 3, column 1, or a fragment thereof, with the compound, the polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid encoding an E1, E2, or E3 polypeptide having an amino acid sequence as identified in FIG. 1, column 4, FIG. 2, column 4, or FIG. 3, column 3; and (ii) determining the effect of the compound upon the cell comprising the polypeptide or fragment thereof, thereby identifying a compound that modulates the cell cycle.
- 2. The method of claim 1, wherein modulation of the cell cycle is associated with cancer.
- 3. The method of claim 1, wherein modulation of the cell cycle causes cellular proliferation.
- 4. The method of claim 1, wherein modulation of the cell cycle causes apoptosis.
- 5. The method of claim 1, wherein modulation of the cell cycle causes cell cycle arrest.
- 6. The method of claim 1, wherein the effect of the compound on the cell is determined by measuring cell cycle arrest, lymphocyte proliferation, cancer cell proliferation, or apoptosis.
- 7. The method of claim 1, wherein the effect of the compound on the cell is determined by cell tracker dye assay and FACS analysis.
- 8. The method of claim 1, wherein the effect of the compound on the cell is determined by BRDU staining assay and FACS analysis.
- 9. The method of claim 6, wherein the cell cycle modulation is measured by assaying DNA synthesis, DNA content, or fluorescent marker level.
- 10. The method of claim 9, wherein DNA synthesis or content is measured by 3H thymidine incorporation, BrdU incorporation, or Hoescht staining.
- 11. The method of claim 9, wherein the fluorescent marker is selected from the group consisting of a cell tracker dye or green fluorescent protein.
- 12. The method of claim 1, wherein modulation is activation of cell cycle arrest.
- 13. The method of claim 1, wherein modulation is activation of cancer cell cycle arrest.
- 14. The method of claim 1, wherein the host cell is a lymphocyte.
- 15. The method of claim 1, wherein the host cell is a cancer cell.
- 16. The method of claim 15, wherein the cancer cell is a breast, prostate, colon, or lung cancer cell.
- 17. The method of claim 15, wherein the cancer cell is a transformed cell line.
- 18. The method of claim 17, wherein the transformed cell line is PC3, H1299, MDA-MB-231, MCF7, A549, or HeLa.
- 19. The method of claim 15, wherein the cancer cell is p53 null or mutant.
- 20. The method of claim 15, wherein the cancer cell is p53 wild-type.
- 21. The method of claim 1, wherein the polypeptide is recombinant.
- 22. The method of claim 1, wherein the polypeptide is encoded by a nucleic acid comprising a sequence selected from FIG. 1, column 3, FIG. 2, column 3, or FIG. 3, column 3.
- 23. The method of claim 1, wherein the compound is an RNAi molecule.
- 24. The method of claim 1, wherein the compound is an antibody.
- 25. The method of claim 1, wherein the compound is an antisense molecule.
- 26. The method of claim 1, wherein the compound is a small organic molecule.
- 27. The method of claim 1, wherein the compound is a peptide.
- 28. The method of claim 27, wherein the peptide is circular.
- 29. A method for identifying a compound that modulates the cell cycle, the method comprising the steps of:
(i) contacting an E1 polypeptide from FIG. 1, column 1; an E2 polypeptide from FIG. 2, column 1; or an E3 polypeptide from FIG. 3, column 1, or a fragment thereof, with the compound, the polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid encoding an E1, E2, or E3 polypeptide having an amino acid sequence as identified in FIG. 1, column 4, FIG. 2, column 4, or FIG. 3, column 3; (ii) determining the effect of the compound upon the polypeptide or fragment thereof; and (iii) contacting a cell comprising the polypeptide with the compound; and (iv) determining the effect of the compound upon a cell comprising the polypeptide or fragment thereof, thereby identifying a compound that modulates the cell cycle.
- 30. A method for identifying a compound that modulates the cell cycle, the method comprising the steps of:
(i) contacting an E1 polypeptide from FIG. 1, column 1; an E2 polypeptide from FIG. 2, column 1; or an E3 polypeptide from FIG. 3, column 1, or a fragment thereof, with the compound, the polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid encoding an E1, E2, or E3 polypeptide having an amino acid sequence as identified in FIG. 1, column 4, FIG. 2, column 4, or FIG. 3, column 3; and (ii) determining the effect of the compound upon the polypeptide or fragment thereof in vitro using a ubiquitin ligase assay, thereby identifying a compound that modulates the cell cycle.
- 31. A method of identifying a cell cycle modulating compound, comprising:
(i) combining (a) a ubiquitin activating agent (E1) selected from the E1s listed in FIG. 1, column 1, (b) a ubiquitin moiety which binds with said E1 in the process of ubiquitin activation and (c) a compound, under conditions in which said E1 normally forms a bond with said ubiquitin moiety and; (ii) determining the binding of said E1 and said ubiquitin moiety, wherein an alteration in binding between said E1 and said ubiquitin moiety as compared with binding in the absence of said compound indicates that said compound modulates the cell cycle.
- 32. The method of claim 31, wherein the E1 is SAE1 or UBE1L.
- 33. A method of identifying a cell cycle modulating compound, comprising:
(i) combining (a) a ubiquitin conjugating agent (E2) selected from the E2s listed in FIG. 2, column 1, (b) a ubiquitin activating agent (E1) capable of transferring an activated ubiquitin moiety to said E2, (c) a ubiquitin moiety or capable of being activated by said E1 and subsequently binding said E2 and (d) a compound, under conditions in which said E1 normally transfers an activated ubiquitin moiety to said E2 and; (ii) determining the binding of said E2 and said ubiquitin moiety, wherein an alteration in binding between said E2 and said ubiquitin moiety as compared with binding in the absence of said compound indicates that said compound modulates the cell cycle.
- 34. The method of claim 33, wherein the E1 is SAE1 or UBE1L and the E2 is XM—054332, UbcH9, or Ubc12.
- 35. A method of identifying a cell cycle modulating compound, comprising:
(i) combining (a) a ubiquitin ligating agent (E3) selected from the E3s listed in FIG. 3, column 1, (b) a ubiquitin conjugating agent (E2) capable of transferring an activated ubiquitin moiety to said E3, (c) a ubiquitin activating agent (E1) capable of transferring an activated ubiquitin moiety to said E2, (d) a ubiquitin moiety capable of being activated by said E1 and subsequently transferred, via said E2, and binding said E3 and (e) a compound, under conditions in which said E2 normally transfers an activated ubiquitin moiety to said E3 and; (ii) determining the binding of said E3 and said ubiquitin moiety, wherein an alteration in binding between said E3 and said ubiquitin moiety as compared with binding in the absence of said compound indicates that said compound modulates the cell cycle.
- 36. The method of claim 35, wherein the E1 is SAE1 or UBE1L and the E2 is XM—054332, UbcH9, or Ubc12.
- 37. A method for identifying a compound that modulates the cell cycle, the method comprising the steps of:
(i) contacting a cell comprising an E1 polypeptide from FIG. 1, column 1; an E2 polypeptide from FIG. 2, column 1; or an E3 polypeptide from FIG. 3, column 1, or a fragment thereof, with the compound, the polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid encoding an E1, E2, or E3 polypeptide having an amino acid sequence as identified in FIG. 1, column 4, FIG. 2, column 4, or FIG. 3, column 3; and (ii) determining the effect of the compound upon the cell comprising the polypeptide or fragment thereof using an assay comprising application of a dye selected from the group consisting of a cell tracker dye and BRDU, and FACS analysis of the cells, thereby identifying a compound that modulates the cell cycle.
- 38. A method of modulating the cell cycle of a cell comprising,
(i) providing a cell comprising a polypeptide, or naturally occurring variant thereof, selected from: a peptide of FIG. 1, column 1, a peptide of FIG. 2, column 2; and a peptide of FIG. 3, column 3; and (ii) contacting said cell with an inhibitor of said polypeptide, whereby the cell cycle of said cell is modulated.
- 39. A method of modulating the cell cycle in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a compound identified using the method of claim 1.
- 40. The method of claim 39,wherein the modulation of the cell cycle is associated with cancer.
- 41. The method of claim 39, wherein the subject is a human.
- 42. The method of claim 41, wherein the subject has cancer.
- 43. The method of claim 39, wherein the compound is an RNAi molecule.
- 44. The method of claim 39, wherein the compound is an antibody.
- 45. The method of claim 39, wherein the compound is an antisense molecule.
- 46. The method of claim 39, wherein the compound is a small organic molecule.
- 47. The method of claim 39, wherein the compound is a peptide.
- 48. The method of claim 47, wherein the peptide is circular.
- 49. The method of claim 39, wherein the compound inhibits cancer cell proliferation.
- 50. A method of modulating the cell cycle in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of an E1 polypeptide from FIG. 1, column 1; an E2 polypeptide from FIG. 2, column 1; or an E3 polypeptide from FIG. 3, column 1, or a fragment thereof, the polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid encoding an E1, E2, or E3 polypeptide having an amino acid sequence as identified in FIG. 1, column 4, FIG. 2, column 4, or FIG. 3, column 3.
- 51. A method of modulating the cell cycle in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a nucleic acid encoding an E1 polypeptide from FIG. 1, column 1; an E2 polypeptide from FIG. 2, column 1; or an E3 polypeptide from FIG. 3, column 1, or a fragment thereof, THe polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a nucleic acid encoding an E1, E2, or E3 polypeptide having an amino acid sequence as identified in FIG. 1, column 4, FIG. 2, column 4, or FIG. 3, column 3.
- 52. A method for identifying a compound capable of interfering with binding of a component of the ubiquitin ligation pathway or fragment thereof, the method comprising the steps of:
(i) combining a first polypeptide of FIG. 1, column 1, and a second polypeptide of FIG. 2, column 1, or fragment thereof, and (ii) determining the binding of the first polypeptide or fragment thereof to the second polypeptide or fragment thereof.
- 53. The method of claim 52, wherein the first polypeptide or fragment thereof and the second polypeptide are combined first.
- 54. The method of claim 52, wherein the first polypeptide or fragment thereof and the second polypeptide are expressed in a cell.
- 55. The method of claim 54, wherein the cell is a yeast cell or a mammalian cell.
- 56. The method of claim 55, wherein the first polypeptide or fragment thereof is fused to a heterologous polypeptide.
- 57. The method of claim 52, wherein the binding of the first polypeptide or fragment thereof to the second polypeptide is determined by measuring reporter gene expression.
- 58. A method for identifying a compound capable of interfering with binding of a component of the ubiquitin ligation pathway or fragment thereof, the method comprising the steps of:
(i) combining a first polypeptide of FIG. 1, column 1, and a second polypeptide of FIG. 4, column 1, or fragment thereof; and (ii) determining the binding of the first polypeptide or fragment thereof to the second polypeptide.
- 59. A method of inducing cell cycle arrest of a cell, the method comprising the step of administering to the cell a vector expressing an siRNA molecule, wherein the siRNA molecule is from 21 to 30 nucleotide base pairs in length and wherein the siRNA molecule is specific for a nucleic acid encoding a polypeptide having an amino acid sequence as identified in FIG. 1, column 4, FIG. 2, column 4, or FIG. 3, column 3.
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.SNo. 60/407,487, filed Aug. 30, 2002, and U.S.SNo. 60/435,427, filed Dec. 20, 2002, each herein incorporated by reference in their entirety.
Provisional Applications (2)
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Number |
Date |
Country |
|
60407487 |
Aug 2002 |
US |
|
60435427 |
Dec 2002 |
US |