Method for measuring ATR inhibition mediated increases in DNA damage

• US 9,791,456 B2
• Filed: 02/27/2015
• Issued: 10/17/2017
• Est. Priority Date: 10/04/2012
• Status: Active Grant

• Current Assignee: University of Newcastle Upon TYNE, Vertex Pharmaceuticals Incorporated
• Original Assignee: University of Newcastle Upon TYNE, Vertex Pharmaceuticals Incorporated
• Inventors: Falcon, Susanna, Reaper, Philip, Pollard, John, Curtin, Nicola, Middleton, Fiona, Chen, Tao
• Primary Examiner(s): Long, Scott
• Assistant Examiner(s): MARTIN, PAUL C
• Application Number:
  US14/633,394
• Publication Number:
  US 20150247866A1
• Time in Patent Office: 963 Days
• CPC Class Codes: G01N 2333/4703 Regulators; Modulating activity
• CPC Class Codes: G01N 2333/912 transferring phosphorus containing groups, e.g. kinases (2.7)
• CPC Class Codes: G01N 2440/14 phosphorylation
• CPC Class Codes: G01N 33/5047 Cells of the immune system
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

The present relates to methods for detecting DNA damage in subjects treated with an ATR inhibitor. More specifically, this invention relates to a method for measuring changes in levels of γH2AX and/or pChk1^Ser345 in, e.g., surrogate tissue cells, following ex vivo stimulation with a DNA damaging agent.

Claims

1. A method for monitoring DNA damage in a subject by measuring accumulation of γ

H2AX in blood, the method comprising;

a) treating a subject with an ATR inhibitor;

b) taking blood samples from the subject at various intervals;

c) treating the blood samples with a DNA damaging agent;

d) isolating white blood cells from the blood samples; and

e) measuring γ

H2AX levels in the white blood cells by using antibodies specific for γ

H2AX, wherein the ATR inhibitor is;

2. The method of claim 1, wherein the DNA damaging agent is UV light.

3. The method of claim 1, wherein the measurement of γ

H2AX levels is done by immunofluorescence.

4. The method of claim 1, wherein the measurement of γ

H2AX is done by western blotting.

5. The method of claim 1, wherein the DNA damaging agent is 4-nitroquinoline.

6. The method of claim 1, wherein the measurement of γ

H2AX levels is done by flow cytometry.

7. A method for monitoring DNA damage in a subject by measuring changes in γ

H2AX and/or pChk1^Ser345, the method comprising;

administering an ATR inhibitor to a subject;

taking surrogate tissue cell samples from the subject at various intervals;

treating the surrogate tissue cell samples with a DNA damaging agent;

measuring γ

H2AX and/or pChk1^Ser345 levels in the surrogate tissue cell samples using antibodies specific for γ

H2AX and/or pChk1^Ser345.

8. The method of claim 7, wherein the DNA damaging agent is 4-nitroquinoline.

9. The method of claim 7, wherein the surrogate tissue cell samples are peripheral blood mononuclear cells.

10. The method of claim 7, wherein the surrogate tissue cell samples are whole blood.

11. The method of claim 9 or 10, wherein the ATR inhibitor is:

12. The method of claim 7, wherein the DNA damaging agent is UV light.

13. The method of claim 7, wherein the measurement of γ

H2AX and/or pChk1^Ser345 levels is done by western blotting.

14. The method of claim 7, wherein the measurement of γ

H2AX and/or pChk1^Ser345 levels is done by immunofluorescence.

15. The method of claim 7, wherein the measurement of γ

H2AX and/or pChk1^Ser345 levels is done by flow cytometry.

16. The method of claim 7 further comprising isolating white blood cells from the surrogate tissue cell samples after the samples have been treated with a DNA damaging agent.

17. The method of claim 16, wherein the measurement of γ

H2AX levels is performed in CD3+ white blood cells.