Cell Cycle Tools: A Portfolio of Kits and Products for Cell Cycle Research

Cancer, the scourge of the 20th century, is in its most basic form the uncontrolled proliferation of invasive cells as a consequence of perturbations in the genetic and biochemical processes of a normal cell cycle. Deciphering the molecular, genetic, biochemical, and physiological aspects of this process in normal and abnormal cells has received much impetus these past two decades, as researchers seek to understand the nature of cancer, apoptosis, cell differentiation, and tissue regeneration. All these processes are inherently associated with the cell cycle. In addition, explicitly defining the mechanisms of these diseases and physiological phenomena may provide novel avenues to develop pharmaceuticals and therapies to aid in the treatment of these and other medical maladies such as neurodegenerative and autoimmune diseases. Thus, some of the most examined aspects of cells have been the events, molecules, and signals controlling the cell cycle. Conceptually, the regulators of this basic activity are eventually affected by nearly every cellular response, whether initiated internally or externally to the cell. Early work on cell cycle processes was generally confined to histological studies with Allium and Haemanthus cells. The advent of chromatographic media in the 1960s spurred a new era in cell cycle research as researchers were provided with tools to examine the biochemical aspects of the cell cycle. The first major inroad was made in 1971 by Masui and Markert (Y. Masui and C.L. Markert, J. Exp. Zool., 177:129-146, 1971), who partially purified an extract from maturing Xenopus oocytes that could induce maturation (G2 to M phase transition) in immature oocytes. They termed the active element in their extract "Maturation Promoting Factor," or MPF. Subsequently, mutant strains of the budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeasts were utilized by Hartwell and Nurse, respectively, to probe the genetics of the cell cycle (L.H. Hartwell, Bacteriol. Rev 38:164, 1974; P. Russell and P. Nurse, Cell 45:145, 1986). They identified a series of genes that controlled key events in the cell cycle. CDC28 and cdc2 (homologues from S. pombe and S. cervisiae, respectively) were the most notorious becuase they appeared to control the G2 to M transition. The products of these genes remained a mystery until the development of another critical tool in cell cycle research; cell-free extracts. In a series of experiements using cell-free extracts in the assay from MPF activity, Maller, in the late 1980s (M.J. Lokha et al, Proc. Nat. Acad. Sci USA 85:3009-3013, 1988), demonstrated that MPDF consisted of two proteins, later designated p34cdc2 (the product of the CDC28/cdc2+genes) and cyclin. This discovery revitalized interest in cell cycle research. Winthin a few short years, a plethora of resaerch articles was published describing not only the components and mechanisms modulating MPF activity, but also the other factors and proteins that effected or modulated the cell cycle. The cdc2 kinase (p34cdc2 protein) is relatively stable throughout the entire cell cycle; its phosphorylation state, however, uniquely changes relative to the cell cycle. The assembly of the MFP complex and establishment of catalytic activity is controlled by a number of cellular events: synthesis of cyclin, protein-protein complex formation, and phosphorylation and dephosphorylation by other protein kinases and phosphatases. The functional (yet inactive) complex is assembled from monomeric, unphosphorylated cdc2 kinase and cyclin. The binding of cyclin also induces phosphorylation of several key cdc2 kinase amino acid residues, Thr14, Tyr15, and Thr161 by wee1 and CAT protein kinases, respectively. Phosphorylation of Thr161 is required for activity, whereas the phosphorylation of Thr14 and Tyr15 serves as a "regulatory (inhibitory) switch." Activation of the cdc2 complex is achieved by the cdc25 phosphatase-mediated dephosphorylation of Thr14 and Tyr15. The subsequent activation of MFP complex is the critical regulatory step in the progression of the cell cycle from G2 to mitosis. More detailed studies on cdc2 kinase and various isoforms of cdk have been achieved using new kinase inhibitors (i.e., staurosporine, flavopyridol, and olomoucine). These compounds have provided researchers with new means to examine the involvement of specific cdk's in cell cycle events and related processes. These inhibitory compounds range from the non-specific and mildly selective to the very specific and highly selective. Some of the non-specific and non-selective, inhibiting both cdk's and other kinases (i.e., cAMP-dependent protein kinases), include staurosporine and suramin. Others are cdk-specific (i.e., staurosporine, butyrolactone), whereas still others are not only specific to cdk's, but also extremely selective for particular members within a given cdk family (i.e., roscovitine inhibits cdk5 but not sub-family member cdc2 or cdk2). Some of the nuclear targets of the active cyclin-cdc kinase complex are regulatory factors (i.e., the retinoblastoma tumor suppressor protein, pRb) involved in the arrest of the cell cycle in the G2 phase. Un- or hypophosphoryalted pRb is an active inhibitory regulator of the E2F transcription factor protein. The G2 to M phase transition is mediated in part by the cyclin-cdc kinase hyperphosphorylation of pRB, which suppresses its E2F inhibitory properties. In a normal cell cycle, the hyperphosphorylated pRB is eventually dephosphorylated, returning it to its original function. In the presence of a newly discovered class of proteins called oncogenes, some of which bind specifically to the pRB protein, pRB function is constituitively suppressed. This causes a loss of cell cycle control and results in uncontrolled cell proliferation and the subsequent development of a cell mass (tumor). This medical condition occurs most often in retinas of children, thus its name juvenile retinoblastoma. Understanding the molecular aspects of this disease, long after it was originally described in medical annals, has only recently possible using new molecular tools for cell cycle research. Chief among these research tools have been antibodies and the various technologies (i.e., immunoprecipitation, immunoblotting, and immunoaffinity chromatography) that utilize these molecular probes. The development of phosphoprotein-, cyclin-, and pRb- specific antibodies has provided new means to identify the nature of a protein phosphorylation event, determine the intra- and extracellular locations of particular proteins, purify proteins via immunoaffinity media, selectively inhibit protein function (in vivo and in vitro), and identify protein-protein and protein-ligand associations. The use of antibodies and new immunotechniques has been instrumental in opening new windows into the cell cycle. The result has been the generation of a tremendous body of information leading to a better understanding of not only the cell cycle but also kinases in general. In addition to their role in M phase induction, both cyclin and active MPF also function in downstream activities. Cyclin has been shown to be associated with centromeres, where it has been implicated in the site-specific recruitment of cdc2. This correlates well with the finding that MPF can phosphorylate histone H1. Collectively, these findings suggest that both cyclin and MPF play an important role in a cascade of events leading to mitotic chromosomal condensation. MPF has also been shown to media te the phosphorylation of lamin filaments, resulting in their cytosolic disassembly. This results in the subsequent breakdown of the parent cell nuclear lamina, which is required for mitotic division. Analogous to the importance of elevated cyclin levels in M phase induction, the subsequent reduction in cyclin levels is critical to progression out of M phase. Exit out of mitosis is mediated by the inactivation of the MPF complex, the result of ubiquitin-dependent degradation of cyclin. However, this process isn't as straightforward as it seems because the cyclin family is composed of many members (isoforms) that are differentially synthesized and degraded at different points throughout the cell cycle. The consistent cyclic variation in the concentration of this protein during the cell cycle was so prominent that it is the basis of the name cyclin. Although this phenomenon may complicate cell cycle research in one arena, in another arena it provides an invaluable new avenue to explicitly differentiate between various stages of the cell cycle. Using antibodies developed against specific cyclin isotypes, one can identify cells in specific cycle phases via in vivo immunofluorescence techniques. These antibodies have also been invaluable in assessing the temporal and spatial nature of cyclins, as well as examining the involvement of cyclin with other molecules. These studies also extend into the genetic arena. The transcriptional activity of cyclin genes may now be qualitatively and quantitatively assessed using various PCR techniques and cyclin isoform-specific RNA probes in Northern and cytolog ical investigations. Cycle phase-specific studies have also been driven by the availability of new compounds that selectively inhibit or alter various phases of the cell cycle or a specific substage within a phase. For example, nocodazole inhibits exit from M phase by way of stabilizing cyclin-B (in the MPF complex), preventing its ubiquination and subsequent degradation. Delving into the finer details of cell cycle regulation, researchers are unraveling what appears to be a complex web of intra- and extracellular regulatory elements. These elements are involved both in direct modulation of cdk, cyclin, and MPF and in many more cases indirectly via some signaling cascade. Among the growing cast of "regulatory" characters are proteins such as p21 (WAF1), p27, p16, and p53. Deregulation of any of these proteins usually results in uncontrolled cell proliferation or neoplasia. The protein p21 is known to be regulated by the tumor suppressor p53; regulation by the p53 product occurs at the transcriptional level of the p21 gene. The protein p53 has long been associated with at least two major cellular processes: one is a checkpoint in the cell cycle pathway in which cells respond to DNA damage by extending the G1 phase to make appropriate repairs. If repairs are insufficient, the cycle aborts via another pathway leading to apoptosis. This field of study has been hastened by extensions of techniques utilized in other disciplines and within other areas of cell cycle research. Although molecular cloning and expression technologies have been prominent providers of advancement in this area, antibody-based and inhibitor studies have played major roles as well. In many cases, symbioses of these technologies have provided past major findings and will likely provide future ones as well. The stage for examining critical events in cell cycle regulation has expanded dramatically over the last decade. Advances in this field have been the result not only of good science, keen observations, and persistance but also the availability of a multitude of research tools and techniques. When used appropriately, these tools provided the means to analyze the activity of enzymes, identify post-translationally modified proteins, quantitate protein and non-protein macromolecules, and dissect the biochemical events of the many phases of the cell cycle. The available research tools can range from the very specific to broad in application and from the very simple to complex in practice. A good starting point is a recent edition of the Methods in Enzymology series from Academic Press (New York, N.Y.) Cell Cycle Control (volume 283, William G. Dunphy, ed., 1997). It is clearly beyond the scope of this article to list and identify every antibody, probe, or inhibitor that is available. Our goal in this short synopsis was to provide an overview of how a few tools and techniques have been utilized to enhance and expand cell cycle research. These tools and many more are available from a variety of biological research suppliers. In addition to an excellent selection of purified proteins, substrates, phosphoprotein- and kinase-specific antibodies, and inhibitors of both the cell cycle and kinase activities, many also provide comprehensive (printed and web-accessible) indices containing kinetic, physico-chemical, and pharmacolog ical data for many of their cell cycle research products. The following is a list of some of the companies providing tools for cell cycle research: Cell Cycle Chart Christopher Smith is an Associate Staff Scientist at the San Diego SuperComputing Center and can be reached at csmith@sdsu.edu. Thomas Unger can be reached attfunger@aol.com.

Cell Cycle Tools: A Portfolio of Kits and Products for Cell Cycle Research

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