Methods of determining cellular chemosensitivity

• US 8,221,966 B2
• Filed: 04/02/2007
• Issued: 07/17/2012
• Est. Priority Date: 03/31/2006
• Status: Active Grant

• Current Assignee: Dana-Farber Cancer Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Letai, Anthony
• Primary Examiner(s): AEDER, SEAN E
• Application Number:
  US11/695,321
• Publication Number:
  US 20080199890A1
• Time in Patent Office: 1,933 Days
• Field of Search: None
• US Class Current: 435/4
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12Q 1/025 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/521 Single-layer analytical elements
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/5748 involving oncogenic proteins

Abstract

The present invention provides methods of determining cell sensitivity to a therapeutic agent.

Claims

1. A whole cell method for determining chemosensitivity of a patient'"'"'s cancer cells to apoptosis-inducing chemotherapeutic agents, comprising:

permeabilizing the patient'"'"'s cancer cells, and

determining change in mitochondrial membrane potential upon contacting the permeabilized cells with one or more BH3 domain peptides so as to probe the extant cellular apoptotic machinery, wherein the BH3 domain peptide is selected from a PUMA BH3 domain, a BAD BH3 domain, a BIK BH3 domain, a NOXA BH3 domain, a HRK BH3 domain, and a BMF BH3 domain; and

correlating a loss of mitochondrial membrane potential with chemosensitivity of the cells to apoptosis-inducing chemotherapeutic agents.

2. The method of claim 1, wherein the cancer cells are a lymphoma.

3. The method of claim 1, wherein the cancer cells are small cell lung cancer.

4. The method of claim 1, wherein the BH3 domain peptide is a PUMA BH3 domain or BMF BH3 domain.

5. The method of claim 1, wherein the BH3 domain peptide has an amino acid sequence consisting essentially of SEQ ID NO:

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

10, or SEQ ID NO;

11.

6. The method of claim 5, wherein the BH3 domain peptide has an amino acid sequence consisting essentially of SEQ ID NO:

10 or SEQ ID NO;

11.

7. The method of claim 1, wherein the 131-13 domain peptides are chemically synthesized.

8. The method of claim 1, further comprising, separately contacting the permeabilized cells with a BH3 domain peptide selected from a BID BH3 domain or BIM BH3 domain, and correlating loss of mitochondrial membrane potential with chemosensitivity of the cells.

9. The method of claim 1, wherein loss of mitochondrial membrane potential is determined by contacting the permeabilized cells with a potentiometric dye, and detecting a change in emission of said potentiometric dye.

10. The method of claim 9, where said potentiometric dye is JC-1 or dihydrorhodamine 123.

11. The method of claim 1, wherein the cells are permeabilized with digitonin.

12. The method of claim 6, wherein the BH3 domain peptide has the amino acid sequence of SEQ ID NO:

10.

13. The method of claim 1, wherein the cancer cells are determined to be sensitive to apoptosis-inducing chemotherapeutic agents, and an apoptosis-inducing chemotherapeutic agent is selected for treatment.

14. The method of claim 13, wherein the apoptosis-inducing chemotherapeutic agent does not directly target an anti-apoptotic Bcl-2 family protein.

15. The method of claim 14, wherein the chemotherapeutic agent is vincristine.

16. The method of claim 1, wherein the cells are contacted with BH3 domain peptides to prepare a BH3 profile for the cancer cells.

17. The method of claim 16, wherein an apoptosis-inducing agent that targets a Bel-2 family protein is selected for treatment on the basis of the BH3 profile.

18. The method of claim 17, wherein the apoptosis-inducing agent is ABT-737.