Gong J, Traganos F, Darzynkiewicz Z
Cancer Research Institute, New York Medical College, Valhalla 10595.
Cancer Res. 1993 Nov 1;53(21):5096-9.
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 microM 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.
依赖DNA含量测量的细胞周期分析方法，如果处于细胞周期不同阶段的细胞具有相似的DNA含量，则无法区分这些细胞。通过除测量DNA含量外，再测量另一种细胞周期阶段特异性细胞成分，然后对相关数据进行双变量分析，可规避这一局限性。本研究的目的是探索一种针对G2期和M期特异性调节蛋白细胞周期蛋白B的单克隆抗体，用于区分DNA含量重叠的细胞群体的效用。这种基于流式细胞术对DNA/细胞周期蛋白B含量进行相关测量的分析方法，应用于人类淋巴细胞白血病MOLT-4细胞。细胞周期蛋白B的合成起始于S期的最后三分之一阶段，在G2期和M期积累至最大值；G1期以及S期早期和中期的细胞呈阴性。长春花碱使细胞停滞在中期，这些细胞表达高水平的这种蛋白，尽管不如DNA拓扑异构酶II抑制剂m-AMSA使细胞停滞在G2期时表达的水平高。蛋白激酶抑制剂星形孢菌素破坏胞质分裂，导致DNA再复制，细胞以更高的DNA倍性通过染色质周期，并诱导多倍体形成。利用细胞周期蛋白B抗体，能够区分DNA水平为2C的G2 + M期细胞和DNA为4C的G1期细胞，以及区分DNA水平为2C的G1期细胞双联体。因此，可以同时估计细胞在DNA水平为4C时进入G1期的速率以及在DNA水平为2C和4C时通过细胞周期的速率。数据表明，在存在0.1微摩尔星形孢菌素的情况下，所有MOLT-4细胞的胞质分裂均受损，细胞以与DNA水平为2C时相同的速率进入并通过DNA水平为4C的染色体周期。当正常基质细胞或浸润细胞与非整倍体肿瘤细胞群体的DNA含量值存在重叠时，这种方法有助于分析人类肿瘤的DNA倍性和细胞周期，并且可能是研究损害胞质分裂但不干扰细胞通过染色质周期进程的抗肿瘤药物活性的首选方法。
