Reprinted with permission of Cytometry Part A, John Wiley and Sons, Inc. Cytometry of Cyclin Proteins Zbigniew Darzynkiewicz, Jianping Gong, Gloria Juan, Barbara Ardelt, and Frank Traganos The Cancer Research Institute, New York Medical College, Valhalla, New York Received for publication January 22, 1996; accepted March 11, 1996 Cyclins are key components of the cell cycle pro- gests that the partner kinase CDK4 (which upon ac- gression machinery. They activate their partner cy- tivation by D-type cyclins phosphorylates pRB com- clin-dependent kinases (CDKs) and possibly target mitting the cell to enter S) is perpetually active them to respective substrate proteins within the throughout the cell cycle in these tumor lines. Ex- cell. CDK-mediated phosphorylation of specsc sets pression of cyclin D also may serve to discriminate of proteins drives the cell through particular phases Go vs. GI cells and, as an activation marker, to iden- or checkpoints of the cell cycle. During unper- tify the mitogenically stimulated cells entering the turbed growth of normal cells, the timing of expres- cell cycle. Differences in cyclin expression make it sion of several cyclins is discontinuous, occurring possible to discrirmna* te between cells having the at discrete and well-defined periods of the cell cy- same DNA content but residing at different phases cle. Immunocytochemical detection of cyclins in such as in G2vs. M or G,/M of a lower DNA ploidy vs. relation to cell cycle position (DNA content) by GI cells of a higher ploidy. The expression of cyclins multiparameter flow cytometry has provided a new D, E, A and B1 provides new cell cycle landmarks approach to cell cycle studies. This approach, like that can be used to subdivide the cell cycle into no other method, can be used to detect the un- several distinct subcompartments. The point of cell scheduled expression of cyclins, namely, the pre- cycle arrest by many antitumor agents can be esti- sentation of GI cyclins by cells in G# and of G2/M mated with better accuracy in relation to these com- cyclins by G, cells, without the need for cell syn- partments compared to the traditional subdivision chronization. Such unscheduled expression of cy- into four cell cycle phases. The latter applications, clins B1 and A was seen when cell cycle progression however, pertain only to normal cells or to tumor was halted, e.g., after synchronization at the GI/S cells whose phenotype is characterized by sched- boundary by inhibitors of DNA replication. The un- uled expression of cyclins. As sensitive and specilk scheduled expression of cyclins B1 or E, but not of indicators of the cell’s proliferative potential, the A, was also observed in some tumor cell lines even cyclins, in particular D-type cyclins, are expected to when their growth was unperturbed. Likewise, be key prognostic markers in neoplasia. whereas the expression of cyclins D1 or D3 in non- 0 19% Wiley-Liss, hc. tumor cells was restricted to an early section of GI, the presentation of these proteins in many tumor Key terms: Cell cycle, cell proliferation, cell cycle cell lines also was seen during S and G2/M.This sug regulation, cancer Cyclins are the key components of the cell cycle pro- gression machinery. In combination with their respective cyclin-dependent protein kinases ( CDKs), cyclins form the holoenzymes that phosphorylate different sets of pro- teins at consecutive stages of the cell cycle, thereby driv- This work was supported by NCI Grant CA 28704 and the Chemo- ing the cell through the cycle (reviews, see refs. therapy Foundation. J.G., on leave from the Department of Surgery, 8,17,19,33,34,51,53,56-58,65,71).The role of cyclins is Tongji University, Wuhan, China, was supported by a Fellowship from to activate their partner CDKs and possibly to target them the “This Close” for Cancer Research Foundation. GJ. is on leave from the University of Valencia, Valencia, Spain. to specific protein substrates. Nine types of cyclins, de- Address reprint requests to Zbigniew Darzynkiewicz, who is now at noted cyclins A-I, have been identified thus far. Expres- the Cancer Research Institute, N.Y. Medical College, 100 Grasslands sion of cyclins Bl, A, E, or D is cyclic and occurs at Road, Elmsford, N.Y. 10523. E-mail: [email protected]. Reprinted with permission of Cytometry Part A, John Wiley and Sons, Inc. 2 CYTOMETRY OF CYCLIN PROTEINS cyclins is regulated not only at the transcriptional and translational level but also by the altered rate of their degradation via the ubiquitin pathway (22). Thus, e.g., the relatively long half-life of cyclin B1 during G, is short- ened manyfold following the cell division, when the cell resides in G, or is forced to overexpress cyclin E (2). At particular phases of the cycle, therefore, the message level may not always correlate with the amount of the respective cyclin protein. It has recently become possible to detect cyclins im- munocytochemically in individual cells and to relate their S expression to cell cycle position (estimated by simulta- neous measurement of cellular DNA content) by multi- FIG 1. Cellular levels of D type cyclins, cyclin E, cyclin A, and cyclin parameter flow cytometry (5,23-32,43-47,67,72,77). B1 at different phases of the cell cycle. In normal cells and in some This approach allows one to assess the intercellular vari- tumor cell lines during their exponential growth, the expression of D ability in cyclin expression, detect cell subpopulations type cyclins is transient during G, and the cells entering S phase are sharing similar or distinct features, and determine the cyclin D negative (e.g.,see Fig. 2). Also, Go cells are cyclin D negative (see Fig. 3). In many tumor lines, however, the level of D-type cyclins presence of thresholds in expression of these proteins at remains high and invariable thorough the whole cell cycle (see Fig. 6). particular phases of the cycle (30). The relationship be- tween expression of individual cyclins and their actual cell cycle position can be studied in asynchronous cell pop- specific and well-defined points of the cycle (Fig. 1, Table ulations in a manner that does not perturb cell cycle 1). Cyclin B1 activates CDKl (formerly denoted as progression or induce the growth imbalance that almost CDC2) forming Maturation Promoting Factor (MPF), always accompanies attempts to synchronize cells in the whose kinase activity is essential for cell entrance to M cycle (31,77). Multiparameter analysis of the cyclin pro- (51,65). Cyclin B1 begins accumulating at the time of teins also offers entirely new possibilities to study the cell cell exit from S, reaches maximal levels as the cell enters cycle and the cell cycle perturbations caused by intrinsic mitosis, and breaks down at the transition to anaphase or exogenous factors. The aim of this article is to dem- (66). Cyclin A may associate with either CDC2 or CDK2; onstrate these possibilities and to provide a comprehen- the kinase activity of the complex drives the cell through sive overview of this explosively growing area of research. S and G, (21,42,61). Cellular accumulation of cyclin A This article updates our earlier review on the subject of starts in mid-S, is maximal at the end of G, and the protein cyclins (25), focusing primarily on the literature subse- is rapidly degraded in prometaphase. The kinase partner quent to this earlier publication. Emphasis is given to the of cyclin E is CDK2 and this holoenzyme is essential for unique applications of the multiparameter analysis of cy- cell entrance to S phase. Cyclin E starts to accumulate in clins, namely, to those applications that yield information the cell in mid-GI, is maximally expressed at the time of on the biology of the cell cycle and cancer, or on mech- cell entrance to S, followed by its continuous breakdown anisms of antitumor drug action and cannot be obtained as the cell progresses through S (18,40,41). using the tools of molecular biology. Expression of the different members of the D family of cyclins is tissue and cell-type specific, eg, whereas cy- Critical Aspects of the Methodology clin D1 is predominant in fibroblasts, cyclins D2 and D3 Clevenger et al. (9), and Jacobberger et al. (38) pio- prevail in cells of lymphocytic lineage, which are cyclin neered development of the methodology for immunocy- D1 negative (1,3,70,75). D-type cyclins are maximally tochemical detection of the intracellular antigens by flow expressed following mitogenic stimulation of Go cells or cytometry. The critical steps are cell fixation and perme- in response to growth factors; their level appears to de- abilization, which often have to be customized to partic- crease during exponential cell growth (4,48,49,54, ular antigens for their optimal detection. The fixative is 59,68). The only documented role for cyclin D1 is acti- expected to stabilize the antigen in situ and preserve its vation of CDK4: the complex phosphorylates the epitope in a state where it continues to remain reactive retinoblastoma tumor suppressor gene protein RB with the available antibody. The cell has to be permeable (71,73,78). It is likely that cyclins D2 and D3 have a to allow access of the antibody to the epitope. General similar role. Phosphorylation of pRB releases E2F factor, strategies of cell fixation and permeabilization have been which activates transcription of the components of the recently described by Bauer and Jacobberger (7). DNA replication machinery, thereby committing the cell Most studies on cyclins have employed precipitating to S phase (7578). The periodic and timely expression of fixatives such as 70-80% ethanol (14), absolute metha- these cyclins, as with DNA replication and mitosis, rep- nol (43,72), or a 1:l mixture of methanol and acetone resents landmarks of the cell cycle to which timing of (5,4547) cooled to -20-40°C.