This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gong, J. Articles by Darzynkiewicz, Z. Search for Related Content PubMed PubMed Citation Articles by Gong, J. Articles by Darzynkiewicz, Z.

Simultaneous Analysis of Cell Cycle Kinetics at Two Different DNA Ploidy Levels Based on DNA Content and Cyclin B Measurements¹

The Cancer Research Institute, New York Medical College, Valhalla, New York 10595

The methods of cell cycle analysis that rely on DNA content measurements cannot discriminate between cells at different phases of the cycle if these cells have similar DNA content. This limitation can be circumvented by measurement of another cell cycle phase-specific cell constituent in addition to DNA content, followed by bivariate analysis of the correlated data. The aim of the present study was to explore the utility of a monoclonal antibody against the G₂- and M phase-specific regulatory protein cyclin B for discrimination of cell populations with overlapping DNA content. This analysis, which was based on correlated DNA/cyclin B content measurements by flow cytometry, was applied to human lymphocytic leukemic MOLT-4 cells. The onset of cyclin B synthesis was observed in the last one third of S phase with its maximum accumulation in G₂ and M phases; cells in G₁ and early- and mid-S phases were negative. Cells arrested in metaphase by vinblastine expressed high levels of this protein, although not as high as in cells arrested in G₂ by the DNA topoisomerase II inhibitor m-AMSA. Disruption of cytokinesis by the protein kinase inhibitor staurosporine led to DNA rereplication, cell progression through the chromatin cycle at higher DNA ploidy, and induction of polyploidy. It was possible, utilizing the cyclin B antibody, to discriminate between G₂ + M cells with a 2C level of DNA and G₁ cells with 4C DNA, as well as to distinguish doublets of G₁ cells with a 2C DNA level. Thus, the rate of cell entrance to G₁ at the 4C DNA level and the rates of progression through the cycle at both the 2C and 4C DNA levels could be simultaneously estimated. The data indicate that, in the presence of 0.1 µM staurosporine, cytokinesis of all MOLT-4 cells is impaired and the cells enter to and progress through the chromosome cycle at 4C DNA at the same rates as at 2C DNA. This approach can be helpful in the analysis of DNA ploidy and the cell cycle of human tumors when there is an overlap in DNA content values between normal stromal or infiltrating cells and aneuploid tumor cell population and may be the method of choice to investigate the activity of antitumor drugs which impair cytokinesis but do not interfere with progression of cells through the chromatin cycle.

¹ This work was supported by the This Close Foundation for Cancer Research and by USPHS Grant RO1 CA 28704, as well as the Carl Inserra Fund.

² To whom requests for reprints should be addressed, at The Cancer Research Institute, New York Medical College, 100 Grasslands Road, Elmsford, NY 10523.

Received 8/17/93. Accepted 9/20/93.

This article has been cited by other articles:

				B. A. Narayanan, N. K. Narayanan, L. Davis, and D. Nargi RNA interference-mediated cyclooxygenase-2 inhibition prevents prostate cancer cell growth and induces differentiation: modulation of neuronal protein synaptophysin, cyclin D1, and androgen receptor Mol. Cancer Ther., May 1, 2006; 5(5): 1117 - 1125. [Abstract] [Full Text] [PDF]

				L. Xu and E. H. Blackburn Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules J. Cell Biol., December 6, 2004; 167(5): 819 - 830. [Abstract] [Full Text] [PDF]

				H. Zhu and N. Gooderham Neoplastic Transformation of Human Lung Fibroblast MRC-5 SV2 Cells Induced by Benzo[a]pyrene and Confluence Culture Cancer Res., August 15, 2002; 62(16): 4605 - 4609. [Abstract] [Full Text] [PDF]

				G. Juan and C. Cordon-Cardo Intranuclear Compartmentalization of Cyclin E during the Cell Cycle: Disruption of the Nucleoplasm-Nucleolar Shuttling of Cyclin E in Bladder Cancer Cancer Res., February 1, 2001; 61(3): 1220 - 1226. [Abstract] [Full Text]

				H. Zhu, A. R. Boobis, and N. J Gooderham The Food-derived Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine Activates S-Phase Checkpoint and Apoptosis, and Induces Gene Mutation in Human Lymphoblastoid TK6 Cells Cancer Res., March 1, 2000; 60(5): 1283 - 1289. [Abstract] [Full Text]

				K. Mashimo, T. Haseba, and Y. Ohno FLOW CYTOMETRIC AND FLUORESCENCE MICROSCOPIC ANALYSIS OF ETHANOL-INDUCED G2+M BLOCK: ETHANOL DOSE-DEPENDENTLY DELAYS THE PROGRESSION OF THE M PHASE Alcohol Alcohol., May 1, 1999; 34(3): 300 - 310. [Abstract] [Full Text] [PDF]

				N. Vitrat, K. Cohen-Solal, C. Pique, J. P. LeCouedic, F. Norol, A. K. Larsen, A. Katz, W. Vainchenker, and N. Debili Endomitosis of Human Megakaryocytes Are Due to Abortive Mitosis Blood, May 15, 1998; 91(10): 3711 - 3723. [Abstract] [Full Text] [PDF]