Flow and image cytometry contribute significantly to cell cycle and cell kinetic studies, clinical evaluation of tumor cells, and investigations into the effects of pharmacologic agents. Relatively simple analyses such as DNA content measurements provide an estimate of the fraction of cycling cells (S phase) [1] and apoptotic indices (sub-G1 events) [2]. For mixed populations of tumor and normal cells, the mean fluorescence intensity (MFI) of the tumor G1 phase divided by the MFI of the normal G1 cells provides an estimate of the tumor DNA content, or DNA index [3].

More complex analyses involve coupling measurements of DNA, RNA, and/or protein to provide additional (more precise) information about the fraction of cycling cells. With these global measurements, the cycling and non-cycling cells in a population can be fractionated into important cell cycle compartments [4]. Labeling cells with bromo-deoxyuridine (BrdUr) and subsequently measuring DNA content and BrdUr-substituted DNA either by dye quenching [5, 6] or immunofluorescence [7] provides a means to determine cell cycle times and phase durations in addition to cycling population frequencies [8, 9] (Figure 1, Figure 2).

DNA content coupled with multi-color immunofluorescence provides the ability to measure the levels of molecules that identify differentiated sub-populations (e.g., cytokeratin staining of epithelial cells [10]), molecules that regulate the cell cycle (e.g., viral proteins [11], Figure 3, or the cyclins [12-14], Figure 4, and molecules that are related through gene epression dependencies (e.g., the p53 pathway [15]).

Fluorescence detection of DNA strand breaks induced at BrdUr-substituted sites by ultra violet irradiation (SBIP) facilitates multi-color assays of cell cycle kinetics and expression of antigens [16].

These brief paragraphs provide a sampling of ideas and references to a very small fraction of the literature and methods of cytometry applied to cell cycle research. A search of Medline through PubMed on "cell cycle" [All Fields] AND "cytometry" [All Fields] produces 10143 references (Search Date: April 11, 2001). To begin, an interested reader can browse Cytometry or obtain copies of the books listed below. Figure 5 shows that early on the fraction of papers that mentioned "cytometry AND cell cycle" constituted between 30 - 55% of the papers published that mentioned "cytometry". The contribution of "cytometry AND cell cycle" papers to the cell cycle literature has averaged 7½ % for the last decade. Numbers that reflect (more or less) contributions to cell cycle literature are presented in Table 1.

Submitted by: Zbigniew Darzynkiewicz, James W. Jacobberger, Peter Rabinovitch, T. Vincent Shankey, Nicholas Terry, and Frank Traganos

References

1. Gray, J.W. and P. Coffino, Cell cycle analysis by flow cytometry. Methods Enzymol, 1979. 58: p. 233-48.

2. Darzynkiewicz, Z., et al., Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry, 1997. 27(1): p. 1-20.

3. Shankey, T.V., et al., Guidelines for implementation of clinical DNA cytometry. International Society for Analytical Cytology [published erratum appears in Cytometry 1993 Oct;14(7):842]. Cytometry, 1993. 14(5): p. 472-7.

4. Darzynkiewicz, Z., F. Traganos, and M.R. Melamed, New cell cycle compartments identified by multiparameter flow cytometry. Cytometry, 1980. 1(2): p. 98-108.

5. Rabinovitch, P.S., et al., BrdU-Hoechst flow cytometry: a unique tool for quantitative cell cycle analysis. Exp Cell Res, 1988. 174(2): p. 309-18.

6. Poot, M., et al., Continuous bromodeoxyuridine labeling and bivariate ethidium bromide/Hoechst flow cytometry in cell kinetics [published erratum appears in Cytometry 1989 Sep;10(5):670]. Cytometry, 1989. 10(2): p. 222-6.

7. Dolbeare, F., et al., Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci U S A, 1983. 80(18): p. 5573-7.

8. Bergstrom, C., et al., Labelling indices in human tumours: to apply corrections or not--that is the question. Br J Cancer, 1999. 80(10): p. 1635-43.

9. Terry, N.H. and R.A. White, Cell cycle kinetics estimated by analysis of bromodeoxyuridine incorporation [In Process Citation]. Methods Cell Biol, 2001. 63: p. 355-74.

10. Glogovac, J.K., et al., Cytokeratin labeling of breast cancer cells extracted from paraffin- embedded tissue for bivariate flow cytometric analysis. Cytometry, 1996. 24(3): p. 260-7.

11. Sladek, T.L., and Jacobberger, J.W., Dependence of SV40 large T-antigen cell cycle regulation on T-antigen expression levels. Oncogene, 1992. 7: p. 1305-1313.

12. Juan, G. and Z. Darzynkiewicz, Detection of cyclins in individual cells by flow and laser scanning cytometry. Methods Mol Biol, 1998. 91: p. 67-75.

13. Juan, G., S. Gruenwald, and Z. Darzynkiewicz, Phosphorylation of retinoblastoma susceptibility gene protein assayed in individual lymphocytes during their mitogenic stimulation. Exp Cell Res, 1998. 239(1): p. 104-10.

14. Darzynkiewicz, Z., et al., Cytometry of cyclin proteins. Cytometry, 1996. 25(1): p. 1-13.

15. Jacobberger, J.W., et al., Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy. Cytometry, 1999. 38(5): p. 201-13.

16. Juan, G., X. Li, and Z. Darzynkiewicz, Correlation between DNA replication and expression of cyclins A and B1 in individual MOLT-4 cells. Cancer Res, 1997. 57(5): p. 803-7.