Modulating response to genotoxic stress

• US 20020102248A1
• Filed: 09/05/2001
• Published: 08/01/2002
• Est. Priority Date: 09/06/2000
• Status: Abandoned Application

• Current Assignee: The United States of America As Represented By The Secretary of Agriculture
• Original Assignee: Government of the United States of America
• Inventors: Chung, Jay H.
• Application Number:
  US09/947,258
• Publication Number:
  US 20020102248A1
• Time in Patent Office: Days
• US Class Current: 424/94.5
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Abstract

Methods are disclosed to modify cellular sensitivity to genotoxic stress by introducing into a cell a biological macromolecule that alters phosphorylation of BRCA1 by Cds1. Such biological macromolecules may be used to enhance genotoxin-induced cell death, or alter genotoxin-induced gene expression. Alternatively, such biological macromolecules may be used to enhance cell survival following exposure to genotoxic agents. Methods are also disclosed for determining exposure to genotoxic stress by assessing the phosphorylation status of BRCA1, for example by using antibody molecules disclosed herein.

Claims

1. A method of modifying sensitivity to genotoxic stress in a cell, comprising exposing the cell to a biological macromolecule that alters phosphorylation of BRCA1 by Cds1 at one or more BRCA1 amino acid residues that affect a cellular response to genotoxic stress.

2. The method of claim 1, where the biological macromolecule alters phosphorylation of at least one amino acid residue between amino acid residues homologous to residues 758-1064 of human BRCA1.

3. The method of claim 1, wherein the biological macromolecule alters phosphorylation of a residue homologous to serine 988 (S988) of human BRCA1.

4. The method of claim 1, where the biological macromolecule comprises a nucleic acid operably linked to a promoter, where the nucleic acid expresses a polypeptide in the cell.

5. The method of claim 4, where the polypeptide comprises a Cds1 polypeptide or a functional fragment or variant thereof.

6. The method of claim 5, where the functional fragment or variant relative to unmutated Cds1, has altered ability to phosphorylate BRCA1.

7. The method of claim 6, where the functional fragment or variant of Cds1 has reduced ability to phosphorylate BRCA1.

8. The method of claim 7, where the functional fragment or variant of Cds1 comprises an amino acid substitution at a residue homologous to lysine 249 (K249) of hCds1.

9. The method of claim 8, where the substitution is arginine for K249.

10. The method of claim 4, wherein the polypeptide comprises a BRCA1 polypeptide or functional fragment or variant of BRCA1, which includes a residue homologous to S988 of human BRCA1.

11. The method of claim 10, wherein the BRCA1 polypeptide comprises residues homologous to 758-1064 of human BRCA1.

12. The method of claim 10, wherein the polypeptide comprises a BRCA1 polypeptide or functional fragment or variant thereof, further comprising an amino acid substitution at a residue homologous to S988.

13. The method of claim 12, wherein the amino acid substitution comprises a nonpolar or hydrophobic substitution.

14. The method of claim 12, wherein the amino acid substitution comprises a polar or charged substitution.

15. The method of claim 12, wherein the amino acid substitution is alanine for serine.

16. The method of claim 12, wherein the amino acid substitution is glutamic acid or aspartic acid for serine.

17. The method of claim 10, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 8 residues having at least 70% homology to a human BRCA1 polypeptide.

18. The method of claim 17, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 30 amino acid residues having at least 70% homology to a human BRCA1 polypeptide.

19. The method of claim 18, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 100 residues having at least 70% homology to a human BRCA1 polypeptide.

20. The method of claim 1, wherein the biological macromolecule comprises a polypeptide.

21. The method of claim 20, wherein the polypeptide comprises a Cds1 polypeptide or a functional fragment or variant thereof.

22. The method of claim 21, wherein the polypeptide comprises a functional fragment or variant of Cds1 with altered ability to phosphorylate BRCA1.

23. The method of claim 21, wherein the functional fragment or variant has reduced ability to phosphorylate BRCA1.

24. The method of claim 21, wherein the functional fragment or variant comprises an amino acid substitution at a residue homologous to K249.

25. The method of claim 24, where the amino acid substitution is arginine for lysine.

26. The method of claim 20, wherein the polypeptide comprises a BRCA1 polypeptide or functional fragment or variant of BRCA1 which includes a residue homologous to S988 of human BRCA1.

27. The method of claim 20, wherein the BRCA1 polypeptide comprises residues homologous to 758-1064 of human BRCA1.

28. The method of claim 20, wherein the polypeptide comprises a BRCA1 polypeptide or functional fragment or variant thereof, further comprising an amino acid substitution at a residue homologous to S988.

29. The method of claim 28, wherein the amino acid substitution comprises a nonpolar or hydrophobic substitution.

30. The method of claim 28, wherein the amino acid substitution comprises a polar or charged substitution.

31. The method of claim 28, wherein the amino acid substitution is alanine for serine.

32. The method of claim 28, wherein the amino acid substitution is glutamic acid or aspartic acid for serine.

33. The method of claim 28, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 8 residues having at least 70% homology to a human BRCA1 polypeptide.

34. The method of claim 33, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 30 amino acid residues having at least 70% homology to a human BRCA1 polypeptide.

35. The method of claim 34, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 100 residues having at least 70% homology to a human BRCA1 polypeptide.

36. The method of claim 1, wherein exposing the cell to the biological macromolecule comprises administering a therapeutically effective amount of the biological macromolecule to a subject to affect the subject'"'"'s cellular response to genotoxic stress.

38. The method of claim 37, wherein the nucleic acid comprising a sequence homologous to amino acid residues 758-1064 of human BRCA1.

39. The method of claim 37, further comprising an amino acid substitution at a residue homologous to S988.

40. The method of claim 39, wherein the amino acid substitution comprises a nonpolar or hydrophobic substitution.

41. The method of claim 39, wherein the amino acid substitution comprises a polar or charged substitution.

42. The method of claim 39, wherein the amino acid substitution is alanine for serine.

43. The method of claim 39, wherein the amino acid substitution is glutamic acid or aspartic acid for serine.

44. The method of claim 37, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 8 residues having at least 70% homology to a human BRCA1 polypeptide.

45. The method of claim 44, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 30 amino acid residues having at least 70% homology to a human BRCA1 polypeptide.

46. The method of claim 45, wherein the BRCA1 polypeptide or functional fragment or variant thereof comprises at least 100 residues having at least 70% homology to a human BRCA1 polypeptide.

47. The method of claim 37, where the amino acid substitution modulates expression of p21waf1/cip1.

48. The method of claim 37, wherein the amino acid substitution modulates expression of genes operably linked to one or more estrogen response elements (EREs).

49. The method of claim 37, wherein the amino acid substitution alters binding between BRCA1 and an estrogen receptor (ER).

50. The method of claim 49, wherein the amino acid substitution is at a residue corresponding to S988 of human BRCA1.

51. The method of claim 50, wherein the amino acid substitution is alanine or glutamic acid for S988.

52. The method of claim 37, wherein the amino acid substitution alters binding between a transcriptional repressor and BRCA1.

53. The method of claim 52, wherein the transcriptional repressor is CTIP.

54. The method of claim 37, wherein the amino acid substitution alters ER interaction with nuclear receptor coactivation.

55. The method of claim 54, wherein the nuclear receptor coactivation is PCAF.

56. The method of claim 37, wherein introducing into the cell the nucleic acid encoding BRCA1 or a functional fragment or variant thereof comprising a mutation that encodes an amino acid substitution that alters Cds1 phosphorylation of BRCA1, comprises administering a therapeutically effective amount of the nucleic acid encoding BRCA1 or a functional fragment or variant thereof comprising a mutation that encodes an amino acid substitution that alters Cds1 phosphorylation of BRCA1 to a subject to affect the subject'"'"'s expression of the nucleic acid sequence that is modulated by BRCA1 in a cell.

58. The BRCA1 peptide-specific antibody of claim 57, wherein the antibody is polyclonal.

59. The BRCA1 peptide-specific antibody of claim 57, wherein the antibody is monoclonal.

61. The BRCA1 peptide-specific antibody of claim 58, wherein the amino acid residue other than serine is alanine or glutamic acid.

63. The method of claim 62, wherein the antibody of claim 57 is used to determine whether S988 is phosphorylated.

37. A method of modulating expression from a nucleic acid sequence that is modulated by BRCA1 in a cell, comprising introducing into the cell a nucleic acid encoding BRCA1 or a functional fragment or variant thereof, wherein the nucleic acid comprises a mutation that encodes an amino acid substitution that alters Cds1 phosphorylation of BRCA1.

57. A BRCA1 peptide-specific antibody, wherein the BRCA1 peptide comprises a phosphorylated serine residue at a position corresponding to amino acid residue 988 of a human BRCA1 polypeptide.

60. A BRCA1 peptide-specific antibody, wherein the BRCA1 peptide comprises an amino acid residue other than serine at a position corresponding to 988 of a human BRCA1 polypeptide.

62. A method of determining exposure to genotoxic stress, comprising determining whether S988 of BRCA1 in a cell is phosphorylated.