Methods for screening cellular proliferation using isotope labels

• US 6,808,875 B2
• Filed: 05/24/2002
• Issued: 10/26/2004
• Est. Priority Date: 05/15/1997
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Hellerstein, Marc K.
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US10/155,536
• Publication Number:
  US 20030068634A1
• Time in Patent Office: 886 Days
• Field of Search: 435/4, 435/6, 435/29, 435/77, 435/91.1, 435/91.2
• US Class Current: 435/4
• CPC Class Codes: A61K 31/665 having oxygen as a ring hetero atom, e.g. fosfomycin
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/5088 of vertebrates
• CPC Class Codes: G01N 33/60 involving radioactive labelled substances

Abstract

The present invention relates to methods for measuring the proliferation and destruction rates of cells by measuring deoxyribonucleic acid (DNA) synthesis and/or destruction. In particular, the methods utilize non-radioactive stable isotope labels to endogenously label DNA synthesized through the de novo nucleotide synthesis pathway in a cell. The amount of label incorporated in the DNA is measured as an indication of cellular proliferation. The decay of labeled DNA over time is measured as an indication of cellular destruction. Such methods do not involve radioactivity or potentially toxic metabolites, and are suitable for use both in vitro and in vivo. Therefore, the invention is useful for measuring cellular proliferation or cellular destruction rates in humans for the diagnosis, prevention, or management of a variety of disease conditions in which cellular proliferation or cellular destruction is involved. The invention also provides methods for measuring proliferation or destruction of T cells in a subject infected with human immunodeficiency virus (HIV) and methods of screening an agent for a capacity to induce or inhibit cellular proliferation or destruction. In addition, the invention provides methods for measuring cellular proliferation in a proliferating population which utilize both radioactive isotope labels and stable isotopes to endogenously label DNA through the de novo nucleotide synthesis pathway.

Claims

1. A double labeling method for measuring cellular proliferation in a proliferating population of cells, said method comprising:

(a) contacting the proliferating population of cells with a detectable amount of a first label, wherein the first label comprises a stable isotope label which is incorporated into DNA via the de nova nucleotide synthesis pathway;

(b) detecting the first label incorporated into the DNA to measure cellular proliferation in the proliferating population of cells;

(c) contacting the proliferating population of cells with a detectable amount of a second label, wherein the second label comprises a radioactive isotope label which is incorporated into DNA via the de nova nucleotide synthesis pathway; and

(d) detecting the second label incorporated into the DNA to measure cellular proliferation in the proliferating population of cells.

2. The method of claim 1, wherein steps (a) and (b) are performed before steps (c) and (d).

3. The method of claim 1, wherein steps (c) and (d) are performed before steps (a) and (b).

4. The method of claim 1, herein steps (a) and (c) are performed simultaneously and steps (b) and (d) are performed simultaneously.

5. The method of claim 1, wherein the stable isotope label and the radioactive isotope label are each attached to a precursor of deoxyribose in the de nova nucleotide synthesis pathway, each said precursor being incorporated into deoxyribose.

6. The method of claim 5, wherein the stable isotope and the radioactive isotope label each comprise a labeled glucose.

7. The method of claim 1, comprising hydrolyzing the DNA incorporating the stable isotope label to deoxyribonucleosides prior to detecting the label in the DNA.

8. The method of claim 1, comprising hydrolyzing the DNA incorporating the radioactive isotope label to deoxyribonucleosides prior to detecting the label in the DNA.

9. The method of claim 1, comprising measuring the stable isotope label in intact DNA polymers.

10. The method of claim 1, comprising measuring the radioactive isotope label in intact DNA polymers.

11. The method of claim 1, wherein the radioactive isotope label is detected by accelerator mass spectrometry, liquid scintillation counting, or gamma counting.

12. The method of claim 1, wherein the stable isotope label is detected by mass spectrometry.

13. The method of claim 1, wherein the proliferating population of cells comprises cancer cells.

14. The method of claim 1, wherein the proliferating population of cells comprises lymphocytes.

15. The method of claim 14, wherein the lymphocytes comprise CD4⁺cells.

16. The method of claim 14, wherein the lymphocytes comprise CD8⁺cells.