There’s More than One Way to Skin a Cat: Yet Another Way to Assess Mitotic Index by Cytometry

Mitotic index (MI; or “mitotic rate”) is an important marker to grade malignancy of a variety of tumors (1). It has a stronger prognostic value than other clinical prognosticators (2,3). Many methods to measure the rate of cell cycle progression are based on the estimation of MI (4). The effectiveness of mitotic poisons used as antitumor drugs is also being assessed by the analysis of MI (4). It is not surprising, there-fore, that since the early stages of development of cytometric methods, attempts have been made to identify mitotic cells and rapidly and accurately estimate MI by flow cytometry (5). Initially, the differences in structure between highly condensed mitotic chromosomes versus diffuse chromatin of G₂ cells that affected DNA in situ susceptibility to denaturation and stain-ability with some fluorochromes (5,6), or cell light scattering properties (7), provided the bases to discriminate between G₂ and M cells, otherwise having identical DNA content. The subsequent methods relied on immunocytochemical detection of cellular constituents that were different in mitotic than in interphase cells (8–11). One of them was AF-1 antigen, inaccessible during interphase but reactive with the Ab during mitosis (8). Another Ab, raised against nuclei of mitogen-activated lymphocytes and detecting epitope of a p105 protein, was present only in mitotic cells (9). An increased reactivity with Ki-67 Ab combined with low expression of proliferating cell nuclear antigen (PCNA) (10) or concurrent presence of cyclin B and absence of cyclin A (11) provided further markers to identify mitotic cells. Still another reported marker of mitotic cells was sensitivity of their DNA to single-strand specific S1 or mung bean nucleases, which allowed one to mark the cleaved DNA in the TUNEL reaction (12).

Further approaches relied on Abs reacting with proteins that are phosphorylated during mitosis. Abs reactive with his-tone H3 phosphorylated on Ser-10 (13) or on Ser-28 (1), as well as with a subset of proteins phosphorylated late in G₂ and dephosphorylated at the end of mitosis (MPM-2 Ab) (14), have now become the most widely used immunocytochemical markers of mitotic cells for flow cytometry. On the other hand, the morphometric features that are so distinctly different in mitotic versus interphase cells provide a definite marker for the imaging cytometers such as laser scanning cytometer, with no need for additional markers (15).

A new biomarker to identify mitotic cells by flow cytometry has been presented by Jacobberger et al. (16) in the recent issue of this journal. Unexpectedly, the authors observed that Ab raised to detect a short fragment of human retinoblastoma protein (pRb) phosphorylated on Ser-780 (RPPTLS^780PPIPHIPR) intensely labeled mitotic cells. In elegant and very thorough experiments, they correlated expression of this epitope (“phospho-S780-Rb”) with other established markers of mitotic cells such as phosphorylation of histone H3 on Ser-10 and phosphoproteins detected by MPM-2 Ab. They also did a meticulous correlation of this epitope with the expression of cyclin A, which demonstrated its presence in several cell types of different lineages, and by immonoblotting-characterized reactivity of this Ab with several peptides.

With so many markers of mitotic cells now available, one may ask why we need one more. Indeed, the array of available markers may be initially confusing when making a decision to select the one. However, there are advantages and limitations of each of them and the possibility of choice, therefore, should be appreciated. The limitation of histone H3 phospho-Ser-10 Ab, for example, is its reactivity with monocytes (17), which may contribute to their falsepositive identification as mitotic cells. Its use as mitotic marker is also limited in studies of inhibitors of aurora B, by the fact that H3 phosphorylation on Ser-10 is solely mediated by this kinase. One advantage of the phospho-S780-Rb Ab epitope is its resistance to treatment with acid (16), which makes it compatible with the standard assay for DNA replication that relies on immunochemical detection of BrdU incorporation. In well-designed experiments, the authors measured kinetics of cell entrance to mitosis and from mitosis to G₁ by analyzing progression of a cohort of pulse-labeled BrdU-labeled cells through the phospho-S780-Rb detection-window (16). This Ab, therefore, can be conveniently used for assessment of the duration of individual phases of the cell cycle using the highly accurate “fraction of labeled mitoses” assay (4,18), as well as in other studies combining mitotic count with detection of incorporated BrdU.

Further tests are needed to conclude if phospho-S780-Rb-Ab can detect mitotic cells in sections from routinely prepared paraffin-embedded histopathological blocks. Such an application, as mentioned, is of particular interest to pathologists who are finding that MI is highly prognostic in numerous neoplastic diseases (1–3). Many of them will be looking forward to see the data on comparison of the robustness of this marker with the Abs detecting Ser-10 or Ser-28 phosphorylated histone H3, or with MPM-2 Ab, the reagents already used in histopathology (1,2). It would also be of interest to reveal whether phosphorylation of the epitopes detected by phospho-S780-Rb Ab also occurs during premature chromosome condensation, in conjunction with phosphorylation of histone H3, ATM, and histone H2AX (19).

Availability of diverse markers to detect particular phase of the cell cycle is very helpful in mechanistic studies, particularly in analyzing mechanism of action of drugs targeting individual constituents of the cell cycle machinery. Comparison of “time windows” of expression of particular cell attributes to be detected by different biomarkers, overlap of these windows with each other, and changes in length of the windows induced by the investigated drug, often provide clues on the mode of drug action. Many anti-tumor drugs, either already widely used in the clinic (e.g., paclitaxel, vinca alkaloids) or in different phases of trials, target G₂ to M transition. The availability of different markers of mitotic cells, thus, provide the much needed research tools to assess molecular mechanisms of their interaction with the machinery of the cell cycle progression and value of these drugs, when used alone or in combination with other modalities, as antitumor weapons.