Method for detecting radiation exposure

• US 7,563,573 B2
• Filed: 03/06/2006
• Issued: 07/21/2009
• Est. Priority Date: 02/26/1999
• Status: Expired due to Fees

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Trent, Jeffrey M., Amundson, Sally A., Fornace, Albert J. Jr.
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US11/370,079
• Publication Number:
  US 20070166726A1
• Time in Patent Office: 1,233 Days
• Field of Search: None
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

A method is disclosed for detecting exposure of organisms to biologically significant or hazardous amounts of ionizing radiation. The method uses nucleic acid microarray hybridization to evaluate biological effects, such as patterns of expression of genes after radiation exposure. Numerous genes are provided which have been found to be responsive to radiation exposure in a variety of cell lines. These genes are incorporated into probe sets, which are exposed to a labeled nucleic acid composition from a test cell, such as cDNA reverse transcribed from mRNA in the test cell, which specifically hybridizes to members of the probe set when the cell has been exposed to a biologically significant amount of ionizing radiation. Whether the nucleic acid composition hybridizes to the nucleic acid molecules representing genes that are differentially expressed is determined. The invention also includes methods for determining a dose response relationship between radiation exposure and differential expression of one or more genes, for example to determine a probable radiation dose in cells that have actually or potentially been exposed to the ionizing radiation. The invention also includes probe sets and microarrays used in this method.

Claims

1. A method of identifying cells that have been exposed to radiation induced biological stress, comprising:

exposing a probe set comprising nucleic acid molecules representing genes that are differentially expressed in cells that have been exposed to an amount of ionizing radiation sufficient to cause differential expression of genes in the cell to a labeled nucleic acid composition from a test cell which specifically hybridizes to members of the probe set when the test cell has been exposed to an amount of ionizing radiation sufficient to cause differential expression of stress genes in the test cell, wherein at least one nucleic acid molecule of the probe set comprises SEQ ID NO;

14; and

determining whether the probe set hybridizes to the nucleic acid composition.

2. The method of claim 1, wherein the probe set is bound in an array to a surface.

3. The method of claim 2, wherein the probe set comprises oligonucleotides that specifically hybridize to sequences in the nucleic acid composition.

4. The method of claim 3, wherein the nucleic acid composition comprises cDNA reverse transcribed from mRNA in the test cell.

5. The method of claim 1, wherein the labeled nucleic acid is labeled with a fluorophore.

6. The method of claim 1, further comprising exposing the probe set to a labeled nucleic acid composition from a control cell, such that the labeled nucleic acid composition from the control cell specifically hybridizes to members of the probe set.

7. The method of claim 1, further comprising determining a probable time of radiation exposure, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been obtained more than four hours after exposure to the biologically significant amount of ionizing radiation, and wherein hybridization to the probe set indicates that the cells have been exposed to the biologically significant amount of ionizing radiation at least four hours previously.

8. The method of claim 1, further comprising determining a probable time of radiation exposure, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been obtained more than 24 hours after exposure to the biologically significant amount of ionizing radiation, and wherein hybridization to the probe set indicates that the cells have been exposed to the biologically significant amount of ionizing radiation at least 24 hours previously.

9. The method of claim 1, further comprising determining a probable time of radiation exposure, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been obtained more than 48 hours after exposure to the biologically significant amount of ionizing radiation, and wherein hybridization to the probe set indicates that the cells have been exposed to the biologically significant amount of ionizing radiation at least 48 hours previously.

10. The method of claim 1, wherein the probe set comprises probes that specifically hybridize to the labeled nucleic acid composition, when the nucleic acid composition has been exposed to less than about 25 cGy of ionizing radiation.

11. The method of claim 1, wherein the probe set comprises genes that are differentially expressed by at least 1.5 fold following exposure to a biologically significant amount of ionizing radiation.

12. The method of claim 11, wherein the genes are differentially expressed by at least 2-fold following exposure to a biologically significant amount of ionizing radiation.

13. The method of claim 1, wherein the probe set comprises:

(a) at least 10% of the probes identified in Tables 13 wherein the at least 10% of the probes comprises SEQ ID NO;

14;

or

(b) at least 10 of the probes identified in Table 13 wherein the at least one of the 10 probes comprises SEQ ID NO;

14;

or

(b) at least 20 of the probes identified in Table 13 wherein the at least one of the 20 probes comprises SEQ ID NO;

14.

21. The method of claim 1, wherein the probe set comprises nucleic acid molecules identified by Image ID number:

203132, 251591, 489805, 753447, or 1493160.

22. The method of claim 1, wherein the probe set is DNA.

23. The method of claim 22, wherein the probe set is cDNA.

24. The method of claim 23, wherein the labeled nucleic acid composition is cDNA obtained from mRNA expressed by the test cell.

25. The method of claim 1, wherein the probe set nucleic acids are about 10 to about 1,000,000 nucleotides in length.

26. The method of claim 1, further comprising contacting the probe set with a plurality of control nucleic acids obtained from mRNA of a control cell that has not been exposed to a biologically significant amount of ionizing radiation;

and

determining whether the nucleic acids from mRNA of the control cells hybridize differentially to the probe set than the nucleic acid composition from the test cell.

49. The method of claim 1, wherein at least 50% of the nucleic acid sequences of the probe set are differentially expressed following exposure to the ionizing radiation.

50. The method of claim 1, wherein the probe set comprises:

(a) at least 10% of the probes identified in any one of Tables 9, 10 ,11, 12, or 14 wherein the at least 10% of the probes comprises SEQ ID NO;

14;

or

(b) at least 10 of the probes identified in any one of Tables 9, 10 , 11, 12, or 14 wherein the at least one of the 10 probes comprises SEQ ID NO;

14;

or

(c) at least 20 of the probes identified in any one of Tables 9, 10 ,11, 12, or 14 wherein the at least one of the 20 probes comprises SEQ ID NO;

14.

51. The method of claim 1, wherein the probe set comprises nucleic acid molecules identified by Image ID number:

268652, 34396, 35318, 50615, 110589, 247483, 360778, 415817, 469954, 489805, or 884719.

14. A method of identifying cells that have been exposed to radiation induced biological stress, comprising:

providing a plurality of probe elements bound to a surface, each probe element comprising a nucleic acid that is differentially expressed by a cell following ionizing radiation induced biological stress, wherein at least one probe element comprises SEO ID NO;

14;

contacting the plurality of probe elements with a plurality of test nucleic acid sequences obtained from a test cell, under conditions that allow the test nucleic acid sequences to specifically hybridize to one or more of the probe elements, and provide a signal which indicates differential expression of one or more genes if the cell has been exposed to an amount of ionizing radiation sufficient to cause differential expression of stress genes in the cell; and

detecting the presence or absence of the signal.

15. The method of claim 14, wherein the plurality of probe elements is a selected set of nucleic acids from a set of nucleic acids that hybridize to nucleic acids obtained from cells exposed to ionizing radiation.

16. The method of claim 14, wherein the probe elements are nucleic acid sequences that are differentially expressed by a cell more than four hours after exposure to the ionizing radiation.

17. The method of claim 16, wherein the probe elements are nucleic acid sequences that are differentially expressed at a time at least 24 hours after exposure to the ionizing radiation.

18. The method of claim 16, wherein the probe elements are nucleic acid sequences that are differentially expressed more than 48 hours after exposure to the ionizing radiation.

19. The method of claim 16, wherein the probe elements comprise nucleic acid sequences that are differentially expressed by at least 1.5-fold following exposure to a biologically significant amount of ionizing radiation.

20. The method of claim 19, wherein the nucleic acid sequences are differentially expressed by at least 2-fold following exposure to a biologically significant amount of ionizing radiation.

27. The method of claim 14, wherein the test nucleic acid sequences are labeled with a fluorophore that detects hybridization of the test nucleic acid sequences to the probe elements.

28. The method of claim 27, wherein the test nucleic acid sequences are labeled with a first label that detects hybridization of the test nucleic acids to the probe elements, and a plurality of control nucleic acids are labeled with a second label that detects hybridization of the plurality of control nucleic acids to the probe elements, and the first and second labels interact to indicate whether expression of a nucleic acid in the test cell has increased or decreased, relative to expression of the control nucleic acids.

29. The method of claim 28, wherein the first and second labels are fluorophores of different colors.

30. The method of claim 14, wherein the cells are animal cells, microbial cells, or plant cells.

31. The method of claim 30, wherein the animal cells are human cells.

32. The method of claim 30, wherein the animal cells are peripheral blood cells.

33. The method of claim 32, wherein the peripheral blood cells are peripheral blood mononuclear cells.

34. The method of claim 33, wherein the peripheral blood mononuclear cells are lymphocytes.

35. A method of making a microarray for identifying cells that have actually or potentially been exposed to an amount of ionizing radiation sufficient to cause differential expression of stress genes in the cell, comprising:

identifying genes that are differentially expressed by a cell following exposure to the ionizing radiation; and

providing a plurality of probe elements bound to a substrate surface, the probe elements comprising probe nucleic acids from genes that are identified as differentially expressed by a cell following ionizing radiation induced biological stress, wherein at least one probe element comprises SEQ ID NO;

14, and wherein the probe nucleic acid is capable of hybridizing to a nucleic acid differentially expressed by the cell following exposure of the cell to the ionizing radiation.

36. The method of claim 35, wherein identifying genes that are differentially expressed by a cell following exposure to biologically significant amounts of ionizing radiation comprises exposing the cell to a biologically significant amount of ionizing radiation, obtaining mRNA expressed by the cell, reverse transcribing the mRNA into cDNA, labeling the cDNA, and hybridizing the labeled cDNA to a probe set and identifying members of the probe set that hybridize with the labeled cDNA.

37. The method of claim 36, where in the genes comprise p53 regulated genes.

38. The method of claim 35, further comprising determining a dose response relationship between radiation exposure and differential expression of one or more genes.

41. The method of claim 35, wherein identifying genes that are differentially expressed comprises identifying genes that are differentially expressed in a cell type that is to be obtained from a subject for testing.

39. A method of diagnosing ionizing radiation exposure in a subject, comprising:

obtaining a biological sample from the subject;

synthesizing cDNA from mRNA expressed in one or more cells of the biological sample, and labeling the cDNA with a detectable label;

exposing the labeled cDNA to a probe set which represents genes that are differentially expressed in the biological sample following exposure to ionizing radiation, and determining if the labeled cDNA selectively hybridizes to one or more probes of the probe set that are associated with the radiation exposure, wherein the probe set comprises SEQ ID NO;

14.

40. The method of claim 39, wherein the probe set comprises one or more of the probes identified in Table 13.

42. The method of claim 39, further comprising detecting patterns of differential expression associated with biologically significant radiation exposure.

52. The method of claim 39, wherein the probe set comprises one or more of the probes identified in any one of Tables 9, 10 ,11, 12, or 14.

43. A method for measuring a biological response to in a subject comprising:

obtaining a biological sample from the subject;

synthesizing cDNA from mRNA expressed in one or more cells of the biological sample;

labeling the cDNA with a detectable label;

exposing the labeled cDNA to a probe set comprising nucleic acid molecules representing genes that are differentially expressed in cells that have been exposed to an amount of ionizing radiation sufficient to cause differential expression of genes in the cell, wherein at least one nucleic acid molecule of the probe set comprises SEQ ID NO;

14; and

determining if the labeled cDNA selectively hybridizes to one or more probes of the probe set differentially expressed following exposure to ionizing radiation.

44. The method of claim 43 wherein the subject is undergoing radiotherapy for the treatment of cancer and the probe set is used to monitor the subject'"'"'s biological response to the radiotherapy.

45. The method of claim 43 wherein the biological response is a biological response to potential radiation exposure in the subject.

46. The method of claim 45, wherein the biological response is measured in a cell type.

47. The method of claim 46, wherein the cell type is a peripheral blood cell obtained from the subject.

48. The method of claim 43, further comprising determining a relationship between radiation exposure and differential expression of one or more genes to determine a probable radiation dose in cells that have actually or potentially been exposed to the ionizing radiation.