CYTOMETRY OF CYCLIN PROTEINS

Cyclins are key components of the cell cycle progression machinery. Th ey activate their partner cyclin-dependent kinases (CDKs) and possibly target them to respective substrate proteins within the cell. CDK-med iated phosphorylation of specific sets of proteins drives the cell thr ough particular phases or checkpoints of the cell cycle. During unpert urbed growth of normal cells, the timing of expression of several cycl ins is discontinuous, occurring at discrete and well-defined periods o f the cell cycle. lmmunocytochemical detection of cyclins in relation to cell cycle position (DNA content) by multiparameter how cytometry h as provided a new approach to cell cycle studies. This approach, like no other method, can be used to detect the unscheduled expression of c yclins, namely, the presentation of G(1) cyclins by cells in G(2)/M an d of G(2)/M cyclins by G(1) cells, without the need for cell synchroni zation. Such unscheduled expression of cyclins B1 and A was seen when cell cycle progression was halted, e.g., after synchronization at the G(1)/S boundary by inhibitors of DNA replication. The unscheduled expr ession of cyclins hi or E, but not of A, was also observed in some tum or cell lines even when their growth was unperturbed. Likewise, wherea s the expression of cyclins D1 or D3 in nontumor cells was restricted to an early section of G(1), the presentation of these proteins in man y tumor cell lines also was seen during S and G(2)/M. This suggests th at the partner kinase CDK4 (which upon activation by D-type cyclins ph osphorylates pRB committing the cell to enter S) is perpetually active throughout the cell cycle in these tumor lines. Expression of cyclin D also may serve to discriminate G(0) vs. G(1) cells and, as an activa tion marker, to identify the mitogenically stimulated cells entering t he cell cycle. Differences in cyclin expression make it possible to di scriminate between cells having the same DNA content but residing at d ifferent phases such as in G(2) vs. M or G(2)/M of a lower DNA ploidy vs. G(1) cells of a higher ploidy. The expression of cyclins D, E, A a nd B1 provides new cell cycle landmarks that can be used to subdivide the cell cycle into several distinct subcompartments. The point of cel l cycle arrest by many antitumor agents can be estimated with better a ccuracy in relation to these compartments compared to the traditional subdivision into four cell cycle phases. The latter applications, howe ver, pertain only to normal cells or to tumor cells whose phenotype is characterized by scheduled expression of cyclins. As sensitive and sp ecific indicators of the cell's proliferative potential, the cyclins, in particular D-type cyclins, are expected to be key prognostic marker s In neoplasia. (C) 1996 Wiley-Liss, Inc.