Vet Pathol 35:461-478 (1998) REVIEW ARTICLE The Cell Cych le: A Review K. A. ScHAFER ‘Wyeth-Ayerst Research, Chazy, NY Abstract. The cell cycle is a complex process that cell through a specific sequence of events culminating Central to this process are the cyclin-dependent kinases proteins regulate the cell’s progression through the stages proteins, including p53, p21, pl6, and ede25. Downste involves numerous regulatory proteins that direct the in mitosis and the production of two daughter cells, (caks), which complex with the cyclin proteins, These of the cell cycle and are in turn regulated by numerous sam targets of eyclin-cadk complexes include pRD and EQE The cell cycle can be altered to the advantage of many viral agents, most notably polyomaviruses, pap- illemaviruses, and adenoviruses. The cell eyele often is ‘dysregulated in neoplasia duc to alterations either in ‘oncogenes that indirectly affect the cell cycle or in tumor suppressor genes or oncogenes that directly impact cell cycle regulation, such ax pRh, p53, p16, eyelin DI, or mdm.2. The cell cycle has hecome:an intense suject ‘of research in recent years. This research has led to the development of techniques useful for the determination Of the effects of drugs and toxins on the cell cycle. Any drug or toxin with DNA damaging ability would be expected to alter cell cycle progression, and therefore, the cell cycle should be considered in the design of studies using such chemicals. With the appropriate techniques, cell eycle alterations may also be detected in tnssue sections. Because of the ubiquitous nature of the interpretation of studies in a wide variety of disciplines. Key words: Cancer; cell cyele; eyelin-dependent kin ‘cology: virology. ‘The cell cycle is a ubiquitous, complex process in- volved in the growth and proliferation of cells, organ- ismal development, regulation of DNA damage repair, tissue hyperplasia as a response to injury, and diseases such as cancer. The cell cycle involves numerous reg- ulatory proteins that direct the cell through a specific sequence ot events culmmating in mitosis and the pro- duction of two daughter cells. Central to this process are the cyclin-dependent kinases (edks) and the cyclin proteins that regulate the cell’s progression through the stages of the cell cycle referred 10 as G,, S, Gs, and M phases (Fig. 1). The cell cycle can be morpholog- ically subdivided into interphase and stages of M (ani- totic) phase, which include prophase, metaphase, ana- phase, and telophase.’ Interphase encompasses Gy, S, and G, phases of the eyele represented the “gaps” in the cell cycle that occur between the two obvious landmarks, DNA synthesis and mitosis. In the first gap, G, phase, the cell is preparing for DNA syn- thesis. S phase cells are eynthesizing DNA and there- fore have aneuploid DNA content between 2N and 4N. ‘The G, phase is the second gap in the cell eyele during, which the cell prepares for mitosis or M phase. Gy cells are not actively eycling G, was originally nsed 461 cell eycle, it deserves consideration in the design and ases; cyclins; p53 protein; retinoblastoma protein; (ox- to indicate cells not in the cycle but with the potential for division, such as hepatocytes. G, has since been loosely, and probably incorrectly, used to also include terminally differentiated cells, such as those of the out- et layers of the epidermis and adult neurons, Coll Cycle Regulation ‘The cell cycle is catalyzed primarily by complexes containing cUks and cyclins, Cdks are serine/threonine protein kinases that are activated at specific points in the cell eyele. There are at Teast seven udhs int matu- ‘malian cells.’ The odks are critical for progression through the cell cycle because their inactivation pre- vents mitosis.®'"“" Much of the cell cycle regulation is determined by phosphorylation state. Cde2, also known as cdkl, is the prototype cdk. Cde2 originally ‘was identified in fission yeast, with the ede designation used to name cell division cycle genes and proteins.*! Cdk is now the preferred name, ‘Cais are regulated by several methods, one of which is phosphorylation on threonine and tyrosine residues. Ck has phosphorylation sites that are both inhibitory and stimulatory. Phosphorylation of cdk on threonine 161 by cdk-activating kinase is necessary for cdk ki- nase activity ® Phosphorylation af ed at tyrosine 15462 ‘Terminal Diferentation estrction Poin Synthesis Fig. 1. Schematic drawing of normal cell eycle progres- sion. After exiting from mitosis, a cell can terminally dif feremliate, enter a quiescent state, or reenter the cell cycle. Progression through the cell cycle is regulated by various cedk-cyclin complexes. (¥15) and threonine 14 (T14), which are located within the active site of the kinase, prevents kinase activity. This phosphorylation is performed by the weel, mikl, and mytl protein kinases.”"5""5425!8 The wel kinase phosphorylates cdl upon entry of the cdk1— 6 Fig. 2. Schematic drawing of proposed weel kinase and cede25 phosphatase interactions with cdkl-cyclin B com- plexes. Weel phosphorylates and inactivates edkI-tyclit B Cde25 dephosphorylates and activates edkl-cyclin B. Pro- posed in vivo substrates of edk-l-cyclin B inclide weel and edc25, which are inactivated and activated, respectively, by phosphorylation. Dashed arrows indicate reactions catalyzed by the respective enzymes, and threonine (T), which target them for degradation by ubiquitination at specific times."”*"""'S Thus, a cycling cell enters and exits cell cycle phases in as- sociation with the synthesis and degradation of specific cyclins (Fig. 3). In general, before a cell can enter the next cell eyele phase, the appropriate cyclin of the pre- vious phase is degraded, and the cyclin of the next phase is synthesized. The cdks phosphorylate a variety of substrates, Fig. 3. Schematic drawing of cell eycle-dependdent levels of cyclins. Thickness of hashed bars indicates relative intra- cellular eyelin concentration.©® C1 (ap) Tanseroton 4. Schematic drawing of activation of E2F by eak— n complexes. Cuk4 or edk6 complexed to cyclin D phosphorylates pRb, which scleases E27 to transcribe genes necessary for cell cycle progression thereby catalyzing the proces of cell division. In G, and M, edk substrates include nuclear lamins and mi- ferotubules that form the nuclear cytoskeleton.%'l¢ Cak1 can phosphorylate its own regulators wel and ede2SC in vito and probably in vivo, although this ability is unproven (Fig. 2).” In G, an important target of the edks is the retic noblastoma protein (pRb), and the G, cyclin-cdk com- plexes phosphorylate pRh on multiple residues.” Hy- pophosphorylated pRb binds the E2F transcription fac- tor. making it unavailable for transcription. Once the cyclin-cdk phosphorylates pRb, pRb releases E2R freeing it to participate in transcription of proteins nec- essary for the progression of the cell cycle (Fig. 4)."! pRb remains hyperphosphorylated through the remain- der of the cell cycle, Cyclin A- and eyclin B-dependent kinases (cdk2 and cd!) probably maintain pRb in its hyperphosphorylated form because pRb does not re- vert to the hypophosphorylated form until the end of mitosis." Negative regulators of the cell cycle differ among stages. Inhibition of the cell cycle in G, is thought to occur largely by phosphorylation of cdk1 by weel Cdc25C counteracts wee! by dephosphorylating Y1S and T14 of cdk, and failure of activation of ede25C may also result in failure of activation (dephosphory- lation) of edkt (Fig. 2)." In the G, and S phases of the cell cycle, the most extensively investigated inhibitors, and what appear to be the most important inhibitors, are the p16 and p21 families. The p16 family includes p16, p15, p18, and plY, which mactivate only the G, cdks, cdk+ and cdK6.*1"4 These proteins form stable complexes with the edks before they are bound to eyclins, and ‘The Call Cycle 463 excess expression of cyclin will not dissociate them from the dk. By binding cak4 and cdk6, the p16 family members prevent the phosphorylation and in- activation of pb. The p21 family includes wafl (also known as p21, ipl, and pict), p27 (kipl), and p57 (kip2)."™* The p2l family proteins are of primary importance in G, regulation.” These proteins can inhibit most cdks, pos- sibly excepting edk1."™ The p2 family proteins bind cyclins, thereby preventing the edk froin pluyphoryl- ating pRb and inducing its dissociation from E2E*"* Tu contast p16, cell cycle inhibition by p21 can be prevented by increasing cellular concentrations of cy- lins. p21, but not p27 or p57, also binds and inhibits proliferating cell nuclear antigen (PCNA), a subunit of DNA polymerase 8, which has roles in DNA replica- tion and repair." The p53 protein transcriptionally in duces p21 expression. This is one method by which p53 induces cell cycle arrest."* ‘The Restriction Point and Checkpoints Growth factors primarily act on cells in G, and G ‘The point in G, after which cells no longer respond to withdrawal of growth factors has been termed the re- striction point (Fig. 1)."* Growth factors stimulate the entry of cells into the cell cycle from G,, Early in G,, the removal of growth factors will result in cells ming to Gy, However, in the later parts of G, when the cells have passed the restriction point, despite the removal of the growth factor. the cells will continue to progress into S phase. Therefore, the restriction point is the time after which the cell is committed to entering the cell cycle. The restriction point is thought to be regulated largely by pRb."® The complete path- way from growth factors to pRb is not known. Cell cycle checkpoints have been defined as “bio- chemical pathways that ensure dependence of one pro- cess upon another process that is biochemically unre~ lated.”*” As an example, the completion of cellular DNA replication is a checkpoint that must be passed before the biochemically unrelated event of chromo- some separation in mitosis can begin. Although the term checkpoint, and the above example, suggest a discrete point or time in the cell cycle, checkpoints are often more nebulous. Cell cycle arrest, also referred to as delay, is pro- duced by a variety of factors that may be intrinsic or extrinsic and may affect several different checkpoints. An example of an intrinsic factor is cell size. Cell size is important in determining cell cycle progression in yeast cells, but 1t plays a less important role in mam- malian cells. An example of an extrinsic factor is cell nutrition, which is important in all cells and determines whether and the rate at which a cell progresses through the cell cycle." Cultured cells often become quies-464 cent under conditions of starvation, exit the cell cycle, and cuter Gy. On the whole-animal scale, starvation will induce certain organs and tissues to atrophy due to cell shrinkage and loss. These cells enter back into the cell cycle only alter nutrition is reestablished. ”'** DNA damaging agents trigger checkpoints that pro- duce arrest in G, and G, stages of the cell cycle. Cells can also arrest in S, which amounts to a prolonged phase with slowed DNA synthesis. Arrest in G, allows repair before DNA replication, whereas arrest in G, allows repair before chromosome separation in mito- sis.!0” The G, arrest is p53 dependent because cell cul- tures derived from p53-null transgenic mice do not arrest in G, in response to DNA damage such as ion- izing radiation." In other cell lines, disruption of PSI leads to increased radiow and chemosensitivie ty." However, these p53-null cells preserve the ability to arrest in Ci following exposure to these same agents. The p53 protein has multiple functions related to the G, stage arrest. p53 recognizes and binds several types. of DNA damage including single-stranded DNA. in- sertion/deletion mismatches, and free DNA ends. In addition to these functions, p53 has sequence-spe- cific DNA binding and transcriptional activity.!*!850"5 Following DNA damage, p53 stimulates the transcrip- tion of p21." The p21 protein then inhibits G, cdks. The p53 protein also has an autoregulatory feedback loop in which p53 stimulates the transcription of the mdm-2 oncogene, whose protein product inhibits p53. Another function of p53, beyond the scope of this review, is the p53-dependent stimulation of apoptosis following severe DNA damage. All of these p53 functions indicate roles in DNA damage checkpoints The role of p53 in G, arrest is controversial. pS3- null cells arrest in G, but do not arrest in G,.’? which suggests that p53 does not have a role in G, arrest; several studies support this conclusion. How- ever, other studies indicate a role for p53 in G, and/or M phases."!6'619% Evidence supporting this role also includes studies in p53-deficient mice.:' Whether Or not p53 has a role in G, is yet to be decided, The Cell Cycle and Pathogens A variety of pathogens, primarily viruses, affect the cell cycle. Of the viruses, DNA viruses have received the most attention. Because the small DNA viruses reyuire the host DNA replication machinery w repli- cate their own genome, progression in the cell cycle is beneficial for viral replication, For example, the 12- diomimetic behavior of the parvoviruses is due to their requirement for the host ccll to progress through $ phase for lytic viral replication.""*"* Many parvovi ruses are oncotropic in that they replicate well in ne~ Schafer oplasms.'* A few large DNA viruses and RNA viruses, can also affect the segulation of the cell eycle. The cell cycle aberrations induced by viruses are largely related to the induction of the cell cycle, but in a few cases the aberrations induced are related to cell cycle qui- scence or arrest and viral latency. The immortalizing and transforming properties of some of these viruses in cell culture is one consequence of their propensity for induction of the cell cycle. ‘These viruses override many cell checkpoints, of which the restriction point and DNA damage checkpoints are most notable, and may inchide the abrogation of apoptotic pathways, with which some cell cycle regulators such as p53 are intimately involved. This aspect of these viruses is be- yond the scope of this review. Viruses that induce cell proliferation largely share similar targets for cell cycle activation, ic., p53, pRb, and related proteins. Ry binding p53, vieal proteins can sequester it from its cell cycle checkpoint func- tions.’°8" Binding of pRb by viral proteins overrides the restriction point."®\”" Less common mechanisms for altering the cell cycle include insertional mutagen- esis of cyclin genes, expression of viral cyclinlike pro teins, and expression of viral homologs of cellular growth factors.""!22.0%"" These mechanisms all tend to stimulate cell cycle progression through G, or prevent cell cycle arrest in G,. Several known bio- chemical cell cycle pathways are used to the advan- tages of viruses and other pathogens (Table 1). ‘The pRb pathway One of the cell cycle regulators targeted most often by viruses is pRb. Simian virus 40 (SV40) large T (tumor) antigen, mouse polyomavirus large T antigen, adenovirus early gene 1A (EIA) protein, and papillo- ‘mavirus early gene 7 (E7) protein all bind pRb with similar results: sequestration of pRb from its cell cycle functions.°°*2!2 When these viral proteins bind pRb, EF is dissociated from pRb and E2F is freed to tran- seribe genes necessary for the continuation of the cell cycle (Hg. 1)! ‘The ultimate result 1s the override of the pRb-regulated restriction point, Although some of these viral proteins also bind p107 and p30 and e the release of their E2F partners, neither p107 nor p130 has been shown to have any ceil cycle check- point functions Some viruses promote the cell cycle by acting up- stream of pRb. SV40 small T antigen activates the pathways leading 1 the promotion of cyclin DI2"** SV40 small T antigen also maintains this pathway as continuously active." By incteasing eyclin D1 levels, cedk4 and edk6 can be activated to phosphorylate pRb, which again releases E20 (Fig. 1). The related gamma herpesviruses Herpes saimiri and Kaposi’s sarcoma herpesvirus (KSHV) both have genes that encode a‘Table 1. ‘The Cell Cycle 465 Vial cell eycle effects.* Mechanism of Acton Virus ‘Active Vial Protein ‘sva0 LTAg STAg Mouse polyomavirus LTAg MTAg Human adenovirus EIA EIB Human papillomavirus ES Ey Bovine papillomavirus EI 52 Es Homan hepauis B virus HBx Bovine herpesvirus 1 LR protein Herpes sinplen LR protein Cytomegalovirus Unknown Hospes saimici Kaposi’s sarcoma herpesvirus Epstein-Barr virus Rabbit myxoma virus Malignant rabbit fibroma virus Vaccinia virus Orf virus Lemtiviruses (HIY, SIV) Viral cyclin D homolog Viral cyclin D homolog Zta gene product Myxoma growth factor Vaccinia growth factor VEGF homolog Vpr gene proauct Shope fibroma growth factor Binds and sequesters pRb, binds and inactivates ps3 Stimulates increased cyclin D1 expression Binds and sequesters pRb Binds pp60o Binds and sequesters pko, prevents G, inhib tion by binding p27" Inhibits p$3 transcriptional activation Binds and inactivates pS3, targets p33 for degra- dation Binds and sequesters pRb Persistent edk activation Stabilizes p53, which allows p21 transcription Activates PDGFR and EGER pathways ‘Transcriptional activation, binds p53 Binds cyclin A. Binds cyclin A Binds p53 Initiates the cell cycle Initiates the cell cycle ‘Arrests cells and switches to lytic infection EGF homolog EGF homolog EGF homolog VEGF homolog Arrests cells in G, * Abbreviations: LTAg = large T antigen; STAg ~ small T antigen; MTAg = middle T antigen, TGP = pltcletaerived pum factor recep; EGFR ‘vascular endothelial growth factor; SIV 8 Cdk ~ cyelindependeut Kine, PDGF latency related; EGF = epidermal growah factor; VEGI cyclin D homolog”***"* that substitutes for cellular ¢ clin D and can act in the phosphorylation of pRb and the release of E2E" The Herpes saimiri cyclin D ho- molog preterentially binds edk6 over edk4; the signi icance of this preference is unknown." In experimental cell systems, KSHV can abrogate pRb-mediated blocks in cell proliferation. ‘Another method of abrogating the pRb checkpoint is the binding of viral proteins to cdk inhibitors. Ad- enovirus ELA, in addition to binding pRb, can also bind p27." This binding is thought to prevent the p27 inhibition of cdks and cdk6, upstream cell cycle reg- ulators of pRb. The p53 pathway ‘The p53 protein is also an important and frequent target of viruses. SV40 large T antigen, adenovirus EIA and carly gene 1B (EIB) proteins, and papillo- mavirus early gene 6 (E6) protein bind pS3.!"2"”2 By doing ¢o they can override many of the functions of p53. These proteins are thought to function largely by disrupting pS3-DNA complexes, thereby inhibiting 53-mediated transcription of p2l, an upstream regu- lator of pRb. Human papillomavirus E6 protein can ‘wansforming growih factor epidermal grow facie recepoe: LR simian immunodeficieney virus also target p53 for ubiquitination and degrada- tion." This disruption of p53 function has been shown to correlate with complete loss of G, DNA damage-induced arrest." Various viruses have p53 effects, but it is not clear how all ot them function. Human papillomavirus E7 protein can bypass the p53-induced G, arrest, but not by directly binding to p93." ‘The Epstein-Barr vi- rus Zta gene, a transcriptional activator responsible for switching to lytic infection, arrests cells in G, or G, with increased expression of p53, which in turn in- duces expression of p21 and p27. Experiments with human cytomegalovirus (HCMV), which binds p53 in vivo, have shown conflicting results pertaining to p: Infection of endothelial cells has demonstrated in- taeayed p53 in G, amd 8, but the location is almost exclusively cytoplasmic.” This cytoplasmic sequestra- tion suggests that IICMY prevents p53 from perform- ing its normal nuclear functions. In contrast, in studies of HCMV in coronary restenosis the location of the increased levels of p53 protein in HCMV-positive smooth muscle cells is almost entirely nucleas.'*? One possible explanation for this discrepancy is cell type- specific differences in behavior of HCMV. Finally, bo-466 vine papillomavirus early gene (E2) protein has been studied as an inhibitor of the cell eyele. It can inhibit proliferation of HeLa cells that contain integrated hu- ‘man papillomavirus. E2 transfection into these cells, stabilizes p53, in tum producing p21 expression and inhibition of edks. These celle then accumulate the hy- pophosphorylated (inhibitory) form of pRb and arrest in G0 Other pathways: Many pathogens use other pathways either indepen- dent of or in aeldition te p53 and pRh pathways, in- cluding mitogenic signaling pathways, cyclins, and Gy cell eycle regulators. Of the viruses that use mitogenic signaling pathways, the poxviruses are most remark- able. Rabbit myxomavirus, malignant rabbit fibroma- virus, and vaccinia virus encode polypeptides that are homologs of epidermal growth factor (EGF).'%! ‘The EGF homolog of rabbit myxomavirus has been named myxoma growth factor (MGF), The Shope fi broma virus also encodes a growth factor termed Shope fibroma growth factor (SFGF).""" Transforming {growth factor a, vaccinia growth factor, and SFGF can all substitute for MGF with the resulting diseases in rabbits being indistinguishable, indicating that all four growth factors have similar biological functions." ‘The virus of contagious ecthyma, instead of containing an EGF homolog, contains a vascular endothelial growth factor (VEGF) homolog. Histologically, con- tagious ecthyma has dermal vascular proliferation, which is thought to be due to the VEGF homolog ex- pression because the VEGF homolog is mitogenic for endothelial cells in vitro." By expressing these growth factor homologs, poxviruses indirectly dysre- ulate the cell cycle by inducing mitogenesis. Mito- genesis contributes to the virulence of these viruses, but the metabolic purpose is uncertain because cell cy- cle progression is not necessary for poxviral replica tion. However, cell cycle progression may upregulate cellular metabolism, making viral replication more ef- ficient Other organisms not of the poxvirus group that di turb the host cell cycle through mitogenic signaling are bovine papillomavirus, human hepatitis B virus, mouse polyomavitus, Theileria parva, andl 7: anmudla- 1a, Bovine papillomavirus early gene 5 (E5) protein is thought to induce cell proliferation by activating the platelet-derived growth factor receptor and EGF re- ceptor pathways The human hepatitis B virus HBx protein activates the Ras-Raf-Mapk mitogenic signal transduction cascade by uncertain mechanisms.!*9® Mouse polyomavirus middle T antigen can bind p60" and produce the aberrant pattern of phosphor ylation on p60" that is also observed with the ¥-ste Protein, resulting in activation of pp6O in all stages Schaler of the cell cycle." Theileria parva, the cause of East Coast fever, a leukemia like disease of bovids, pro duces proliferation in subsets of T cells." 7. annulara produces proliferation of B cells. These cells can pro- duce tumors in nude mice and can be maintained in culture indefinitely." Intralymphocytic Theileria or- ganisms divide synchronously with host lymphocytes, providing an inereasing pool of host cells as the par asites increase in number, T. parva encodes a casein Kinase TT (CKTD homolog that is thenght to be the primary transforming protein produced by the para- site 85 CKI ig. a serine/threonine protein kinase im= portant in the regulation of multiple metabolic events, receptor signaling pathways, and cell proliferation and transformation. Substrates that CKII is thought to phosphorylate and therefore regulate include Myc. Max, Fos, Myb, ErbA, and p53." Mitogenic stim- ulation of T and B cells results in increased expression and activation of CK; mitogenesis can be blocked by anti-CKII antibodies." ‘Human hepatitis B virus, bovine herpesvirus 1 (BHY-1), and herpes simplex have cell cycle effects that involve cyclin A. Human hepatitis B virus has been shown on one occasion to be integrated into the cyclin A gene in a hepatocellular carcinoma, resulting in marked increases in cyclin A protein expression. In addition, this cyclin A was truncated with loss of the cyclin degradation signal." This finding suggested that expression of a nondegradable cyclin A may con- tribute to the deregulation of the cell cycle and neo- plasia BHY-1 and herpes simplex have a viral gene ex- pressed in latency known as the latency-related (LR) gene.*'*! When expressed in osteosarcoma cells, LR inhibits their prolileration. LK. binds cyclin A, sug- gesting that it sequesters cyclin A from its normal ac- tivity and prevents cell cycle progression. Lentiviruses, such as HIV and the related simian immunodeficiency virus, encode proteins that produce G, cell cycle arrest in cultured cells. The vpr gene of these viruses is conserved among primate lentiviruses and produces cell cycle arrest in G, in cells from all primate species investigated so fai.°"!” The ypr-in- duced arrest is cytostatic and not cytotoxic and can aust cells fiom a vatiety of linicages, including epi- thelial and lymphoid cells."”"" This arrest is accom- panied by decreased activity of cyclin B-edkl com- plexes with cdkI in the hyperphosphorylated inactive form." In addition, the ede2SC phosphatase is un- phosphorylated and therefore unable to dephosphory- late edkl.' The purpose of this activity of vpr may be explained in several ways. Vpr, by inhibiting edkl, may delay or prevent apoptosis, thereby optimizing the number of virions produced per cell.” Alternatively, pr may impair CD4+ lymphocyte cell eycle iva.‘The Cell Cycle 46 Table 2, Cell cycle regulators in neoplasia. Oncoprotein Tumor Suppressor Normal Cell Cyele Function Oncogenic Akeration p53 Promotes G, arrest after DNA damage Mutation Rb Restriction point regulator Deletion Mam-2 ‘Overrides p53 transcriptional activity Gene amplification or en- hanced mRNA translation pls Iuactivates G, eyclin-edk compere Deletion Cyclin DL Initiates cell eycle Gene amplification Cde25A, ode258 Activates Gy/S edks Overexpression tion and therefore immune surveillance." A third pos- sibility is that the vprinduced G, arrest may allow for optimal viral replication.'” Organisms with potential cell eyele effects Various organisms have demonstrated an ability to produce cellular proliferation, but the mechanisms have not been investigated. Of the viruses, herpes ate- les, Lucke’s frog carcinoma virus, and Marek’s disease virus all may affect the cell cycle because they all can produce tumors or tumorlike growths. In addition, the various pox and papillomaviruses that produce prolif- erative lesions would also be expected to affect the cell cycle. However, in those cases, the cell prolifera- tion may be induced by mechanisms already described for their relatives Viruses with antiproliferative effects include the de- pendoviruses or adeno-associated viruses, defective parvoviruses that require a “helper” virus such as an adenovirus for replication, These viruses are also on- cotropic, but infection of human malignant tumor cells by these viruses results in growth arrest and cell death.” he mechanisms are unknown. Cells intected with a dependovirus and an adenovirus show attenu: ation of the immortalizing and transforming ability of the adenovirus."” No studies of how bacteria may alter the cell cycle in animal cells have been done. However, at least two bacteria may affect the cell cycle: Lawsonia intracel- lularis and Citrobacter freundii. Lawsonia intracellu- laris, the cause of proliferative ileitis in pigs, and C. freundii, the cause of transmissible mutine colonic hy- pesplasia, both produce hyperplasia uf intestinal Tining cells.*:'" The proliferative response of the enterocytes in proliferative ileitis may be produced by damage to the mucosa. However, the epithelial cells that prolif- crate are also those infected by L. intracellularis.'" In the intestinal adenomatosis form, there is little histo- logic evidence of mucosal'damage, yet enterocyte pro- liferation is marked, suggesting ceil cycle dysregula- tion by the bacterium itself. Similarly, transmissible ‘murine colonic hyperplasia is associated with C. freun- dii and produces marked proliferation of colonic glands." Citrobacter does not enter but instead attach- es ta the colonic epithelium, suggesting a cell surface receptor-mediated proliferation. However, ultrastruc- inrally the cells proliferating in the erypts are less ma- ture and lack the adherent bacteria, which suggests that the proliferation replaces colonic epithelinm with at tached bacteria and is not due to direct bacterial ef fects.** This same response is not seen in animals with Escherichia coli or Enterococcus durans adherent to the intestinal mucosa. calling into question how simple bacterial adherence stimlulates protiferation. How or- ganisms such as Lawsonia and Citrobacter might alter cell cycle regulation is a question ripe for investiga tion, ‘The Cell Cycle and Carcinogenesis Oncogenes and tumor suppressor genes, by defini- tion, must in some way be linked to the cell cycle, because a neoplasm is defined as “an abnormal tissue that grows by cellular proliferation more rapidly than normal and continues to grow after the stimuli that initiated the new growth cease.’ Oncogenes and tu- mor suppressor genes must, therefore, be involved with some aspect of mitogenic signal transduction or checkpoint control. Mitogeme signal transduction in- volves numerous recognized oncoproteins, including the extracellularly secreted growth factors, growth fac- tor receptors, cytoplasmic’ signal transduction cas- cades, and transcription factors. These oncoproteins, in the normal cell, carry a signal from the cell surface to the nucleus with the end result being transcription and initiation of the cell cycle. In transformed cells, these signal transduction pathways are always “turned on" or their inhibitory mechanisms are “turned off.” Be- cause of the sheet volume of information knows about these numerous oncoproteins and the fact that many are not the immediate regulators of the cell cycle, this review will focus on those cell cycle regulators that hhave been either firmly established or putatively iden- tified as being encoded by oncogenes or tumor sup- pressor genes. These regulators largely include pro teins located in two important G, checkpoint path- ‘ways, that of p53 and pRb. Table 2 cummarizes the68 roles of the cell cycle regulators in neoplasia. Also recently recognized but not a part of the definition giv~ cn here is the important role that apoptosis plays in acoplasia, which is beyond the scope of this review. ps3 p53 mutations have been identified in as much as 50% of malignancies.“ Single amino acid changes throughout large regions of the p53 molecule can have ‘marked influences on its function. Mutations frequent ly result in conformational changes in the p53 mole- cule, which can be distinguished by antibodies that recognize either wild-type or mutated conforma- tions. Another consequence of p53 mutation is in creased intracellular concentrations of the protein due to increased stability and decreased degradation of the mutated protein.!"” 53 plays an important role in the DNA damage checkpoint. Following DNA damage, p53 is stabilized and initiates the transcription of p21." This results in G, cell cycle arrest. In p53-null cells or cells with a ‘mutated p53. this DNA damage checkpoint is abro- gated. Following DNA damage, cells with mutat- ed p53 do not arrest and p21 transcription is not stim- ulated. Cells Tacking normal p53 function therefore continue through the cell cycle despite DNA damage. Thus, the ability of the cell to repair DNA damage before mitosis is curtailed, and after mitosis the dam- age is fixed in the genome. ‘These results are compatible with findings in vivo. ‘Transgenic mice have been developed that are p53-null homozygotes (p53~'-), p53-deficient heterozygotes (p33), p53-mutant heterozygotes (p53°"™), and p53-mutant-null heterozygotes (p53%-).t967°2000:02 p53" mice develop normally but are predisposed to a variety of spontaneous tumors, predominately lym- Phosarcomas, osteosarcomas, and pulmonary adeno- carcinomas. Single doses of ionizing radiation markedly shorten the latency of tumor development in transgenic mice. p53 wild-type mice (p53“"*) have very low tumor incidences, whereas p53" have a me- dian tumor latency of 70 weeks. A single dose of ion- izing radiation shortens this median latency to 40 weeks. p53~ mice have an even shorter tumor latency of 21 weeks, with radiation further shortening it to a median of 14 weeks”? In a second study, where p53" mice were exposed 10 ultraviolet radiation, the tran genic mice had no decrease in tumor latency but did have increased tumor incidence and increased inci- dence of multiple tumors."® In other studies, the p53" genotype also was associated with increased tumvri- genicity, indicating that the mutated p53 allele had a dominant negative activity over the wild-type allele. ‘At the same time, p53" and p53-'~ genotypes were associated with similar behaviors, indicating no gain Schater of function of the mutated p53 allele.“ However, other rniutations in p53 mice may in fact demonstrate gain of function.” Although the tansgenie animal studies uf p53 do not demonstrate a direct cell cycle effect, the effect may not be seen in this complex model. In cell eultuse, 53-null or -mutated cells lack DNA damage-induced G, arrest” Actively dividing cells in the transgenic mice probably also lack this G, arrest in vivo. If so, they will have a curtailed opportunity for DNA dam- age repair, with damage becoming fixed in the genome with subsequent mitoses. Mam-2 ‘The mdn-2 oncogene is overexpressed as much as 100-fold in as many as one-third of human sarcomas because of amplification or enhanced mRNA transla- ion"? The mdm-2 protein binds wild-type p53, sta- bilizes it, and inhibits its transcriptional activity.*%65""” Thiy inhibition probably prevents the p53 induction of cell cycle inhibitors, such as p21, thus abrogating the DNA damage checkpoint function of p53. p33 in tur activates the transcription of mdi-2, providing an au- toregulatory feedback Iovp."** Because p53 aud midair 2 could be considered to be in the same pathway, itis not surprising that mdm-2 overexpression is associated, with wild-type p53 and not mutated p53.%""" Mdm-2 also binds the same region of pRb required for stable pRb-E2F binding and interferes with E2F binding." Mdm-2 is an interesting protein in that like SV40 large T antigen and adenovirus EIA it can override the growth-suppressing propertics of both pS3 and pRb. pat Although the role of p21 in oncogenesis is unclear, its role as an inhibitor of the cell cycle suggests that it might act as a tumor suppressor. A variety of tissues and types of human neoplasia have been examined for p21 expression. In the normal tissue adjacent to the tumors, p21 is expressed exclusively in terminally dif- ferentiated cells.” This p21 expression is com teutly mutually exclusive with expression of Ki-67, which is a marker of proliferation.”*" In the tumors themselves, p21 is frequently expressed, occasionally with the highest expression in the more malignant sions." Yet other tumors had decreased p21 expres- sion correlated with invasion and metastasis." In some of these studies, p21 expression was inversely related to p53 levels.™"!"" In others, there were no correlations between p21 and p53 expression. Ex- pression of p21 in neoplastic tissues suggests that p21 is altered in some way to be nonfunctional. Deletions of portions of the p2/ gene and mutations of the p27 ‘gene have been detected, but their functional signifi-cance is uncertain." Whether or not p21 truly is a tumor suppressor is yet to be determined. pRb The retinoblastoma protein pRb, like p53, is a tumor suppressor. Alterations in the retinoblastoma gene ‘most commonly associated with human neoplasia are deletions and missense mutations that result in a trun- cated, nonfunctional pRb or complete lack of detect- able pb.” Because of the prominent role ot pXb in regulating the cell cycle, itis easy to speculate on how its cell cycle regulatory role may lead to neoplasia pRb is suspected to be the key inhibitory regulator of the restriction point in cells." After inactivation of Rb by phosphorylation, E2F is released, and the cell cycle is initlated.'=" If pRb function is thus abrogat- ed, pRb does not bind E2F and E2F initiates transerip- tion. The cells have then lost one cell cycle inhibitory mechanism, namely the restriction point, which may explain how loss of pRb cell cycle function produces neoplasia. p16 Family proteins The p16 family proteins are G, cell cycle inhibitors that inactivate cyclin-cdk complexes." Once the G, oyclin-edk complescy ate inactivated, they can uv Lou ‘ger maintain pRb in its hyperphosphorylated, inactive state. Therefore, p16 proteins are indiseet activators of pRb, and p16 and p15 could be considered tumor sup- pressors.®*'"8" p16 is frequently deleted in human tu- mors and tumor cell fines.'*"® p/5, which is located close to p16 on the same chromosome, is often si- multaneously deleted." However, in some instances only p15 is deleted.” In tumors, p16 deletions are associated with a normal pRb, whereas pRb mutations are associated with intact p16.'"""! Like pRb, p16 is associated with familial neoplasia in humans, most no- tably melanoma.'"®® Deletions of p16 and/or p15 result in the loss of the pRb-regulated restriction point. With- ut the normal cell eycle inhibitory funetion of the p16 family proteins, cdk-cyclin complexes phosphorylate and inactivate pRb.*9! Cyclin DI Cyclin D1 is another cell cycle restriction point reg- ulator that is oncogenic. Cyclin DI forme complexes, with cdk4 and cdk6. These complexes phosphorylate and inactivate pRb, and these functions may be im- portant for the oncogenic activity of cyclin D1, The ‘oncogene PRAD-J, which was fist identified as being located adjacent to the parathyroid hormone gene, was later identified as cyclin D1." Gene amplification of cyclin D1 has since been identified in a variety of hu- man neoplasms, including breast cancer and squamous cell carcinoma of the head and neck." Expression ‘The Cell Cyele 469 of cyclin DI has also been observed in leukemias." Cyclin D2 and cyclin D3, but not cyclin Di, are nor- mally expressed in Iymphocytes.'* In experiments of mouse skin tumorigenesis, amplification of the cyclin DI gene was an early change." Also consistent with the role of cyclin DI as an oncoprotein, transgenic mice with the cyclin D1 gene linked to the mouse mammary tumor virus promoter overexpress cyclin D1 in the mammary gland and develop mammary hyper- plasia and adenocaremomas.""" cacas The cdc25A and ede25B genes of the cde25 family have been proposed 10 be oncogenes." Both the cde25A and cdc25B proteins, but not cdc25C, coop- erate with mutated. H-ras and pRb loss In transfor mation assays. In addition, cdc2S5B is overexpressed in 32% of primary breast cancers. Others have found similar results with high expression of cde25B in mul- tiple cancer cell lines, including some transformed by SV40 large T antigen and papillomavirus genes £6 and 7 Functionally, cde25 proteins dephosphorylate cdks at aminy avid residues in dhe edk catalytic sites.” By overexpressing or activating the cde25 phospha- tases, the cdks may be maintained in a constitutively activated state. There is evidence that some activators of cde25s include other oncogene products. The Myc/ Max heterodimer has binding and transcriptional ac- tivation affinity for the cdc25A gene. The Raf protein kinase, a member of the Ras-Raf-Mapk cascade, binds ede2S proteins and activates them in vitro.‘* At least two members of the 14-3-3 protein family that are reg- ulators of Mapk cascade kinases bind both cde25A and ede25B in vivo. These 14-3-3 proteins do not alter the phosphatase activities of the ede25s but arc thought instead to regulate or facilitate the binding of Raf to ede25. These data suggest one means of activating the cell cycle, possibly to the point of being oncogenic, is by mitogenic signal transduction cascades. The Cell Cycle in Toxicology Understanding the basics of the cell cycle is impor- tant in toxicology. Many toxins affect the cell cycle, often at specific points, as best illustrated by the nu- merous cancer chemotherapeutics. Because these agents act at a vatiety of often discrete points in the cell cycle, understanding the cell eycle greatly assists in understanding their mechanisms of action. The cell cycle, then, can be used as a tool to explore the mech- anisms of various toxins and drugs. For example, if a toxin induces arrest at the beginning of $ phase, it t that the DNA synthesis machinery is A variety of markers have been used to determine470 Schafer Table 3. Cell cycle markers.* Marker Technique Information Gained Advaniages Disadvantages Beat FOM,THC Cell cycle kinetics, § Very sensitive when de Cannot be used in ret- phase fractions tected with MAb rospective studies H-thymidine AR Cell eycle kineties, S Very sensitive Radioactive, cannot be ‘phase fractions used in retrospective studies Ploidy FM Coll eyele kaneties, S Very precise and accu» Cannot distinguish , pphase fractions, cell rate, highly repeat from M, overlap in cycle arrest able, easily combined cell eyele phases with detection of oth- fer markers KioT FCM, IHC Proliferative index Expressed in all cycling Very labile antigen, un- cells known function PCNA, FCM, IHC Proliferative index, cell Staining patterns allows Induced by DNA-dam- cycle distribution distinetion of cell ey- aging agents, long (ic) cle phases, stable an- half-life may overes tigen timate proliferative fractions Cyclin FCM, IHC, Proliferative index Can detect cells in spe- Can be altered in neo- WB IHC), potential prog, cific phases depend plastic or transformed nostic indicator (cy- lin D) © Abbreviations: AR = auioradiographys BrdU ‘monoclonal antibody; WB = westem blot cell cycle status, including DNA ploidy, bromodeoxy- uridine (BrdU) incorporation, tritiated thymidine CH- thymidine) incorporation, Ki-67 expression, PCNA ex- pression, cyclin expression, cdk expression, and cdk phosphorylation status. The use of these markers can range trom the simplest measurements, such as the rel- ative proportion of cells in a population in the cell cycle, to the most complex, such as the pinpointing of the location of a given cell in the cycle or the deter- mination of cell cycle Kinetics. Table 3 outlines the markers and techniques that are used to measure cell cycle alterations, DNA ploidy is one of the simpler measurements and is most easily measured by flow cytometry. By apply- ing fluorescent stains with affinity for nucleic acids and measuring relative Muvresceuce intensity on x celle by-cell basis, cell cycle distributions of populations of cells can be determined. With comparisons to normally cycling cells, shifts in cell cycle distributions can be detected following treatment of the cells with a toxin, ‘Measurement of DNA ploidy can then identify arrest in various stages of the cell eyele, including G,, $, and GJM. Simple measurement of ploidy on its own does not distinguish between G, and M phase colls.'* ‘The addition of other parameters can further refine cell cycle measurements. For example, BrdU, a thy- Twomedonyoriding; FCM ing on cyclin select- ed. may subdivide cell eyele phases when used with other ‘markers (FCM) cells, requires syn- chronization of cells cw) Tlow eytometry; IHC midine analog, is incorporated into the DNA during S phase."*"" With the application of anti-BrdU antibod- ies, relative incorporation can be measured. BrdU has replaced ‘H-thymidine incorporation in many studies because of its ease of use and nonradioactive nature. With the appropriate protocols, S-phase fractions can then be identified more accurately, transit times of the various cell cycle stages can be measured, and time to traverse the entire cell cycle can be determined." ‘Some workers have proposed the measurement of the scheduled expression of cyclins as a means of fur- ther subclassifying normal ceils in the cell cycle.”* For example, the presence of cyclin D1 in cells with 2N chromatin would distinguish Gy cells from Gy (quies= cent) cells. Also, cyclin A expression may distinguish G, fiom M phase cells biochemically. G and M arc easily distinguished morphologically, but not in the mixed cell populations frequently examined with flow cytometry. Whereas many drugs or toxins arrest cells in particular phases of the cell eyele, the simultancous measurement of cyclins may help localize more pre- cisely at what point within a given phase the drug or toxin acts (Fig. 3). For example, if the arrest can be identified as coming before cyclin BI expression but after cyclin E expression, then the arrest can be placed in the first half of § phase. Once the location of thecell cycle arrest is more precisely determined, corre- lations with ccll cycle biochemical events can be made, possibly identifying mechanisms of action of a given toxin. Altemative methods for assessing stage of arrest in- clude the determination of levels of eyelin expression and edk phosphorylation state by western blotting or immunoprecipitation.'2! These techniques are amena- ble largely to cell culture systems where cell cycles ean be synchronized. With unsynchronized popula- tions, the wide variability of location within the cell cycle of individual cells in the population masks any cell cycle-dependent alterations that may be induced hy a particular experimental agent At a more basic level, a wide range of markers have heen used to measure cellular proliferation. Cellular proliferation indices or growth fractions provide a nu- merical value for the proportion of a population's cells, progressing through the cell cycle. These indices can be determined by PCNA. Ki-67. or cyclin immuno- reactivity or incorporation of *H-thymidine or BrdU. PCNA is a subunit of the mammalian DNA poly- merase 3 and is synthesized primarily during the S phase of the cell cycle.%* Levels of PCNA expression are therefore highest during S phase, with little to no expression during G, and intermediate levels in G, and M phases.""* PCNA was originally identified as the target of autoantibodies from human patient with systemic lupus erythematosus. The half-life of the PCNA protein has been estimated to be 20 hours in vivo, making expression in the G, and Gy phases of daughter cells possible.* PCNA has been a useful marker for immunohisto- chemical studies of a wide variety of proliferative le- sions. ‘H-thymidine or BrdU incorporation yields r sults similar to those of PCNA.!"™""! Several commer- cial monoclonal antibodies raised against PCNA are effective in multiple species and useful in studies using formalin-fixed, paraftin-embedded archival spe mens.'®"”' Although PCNA has been used primarily for determining proliferating fractions m immunohis- tochemical studies, the identification of cells in their respective cell cycle stages is also possible:*"" To- pographic staining pattems of PCNA allow it to be used to further define proliferative fractions in tissue sections, Because DNA polymerase 6 functions in DNA syn- thesis and repair, caution must be used in the interp tation of PCNA immunoreactivity.” Resting cells ex- posed to a DNA-damaging agent can synthesize and express PCNA withut initiation of the Gell cycle. For determination of proliferative index in tumors as a prognostic factor, this probably is of litle significance unless the patient had received radio- or chemotherapy prior to tumor excision or biopsy. However, this index The Cell Cycle a must be taken into account in toxicologic studies, where an acute proliferative response should be distin- guished from a DNA repair response." Ki 67 is a nuclear antigen of unknown function originally identified by investigators screening for a marker for Reed-Sternberg cells." It is expressed in G, through M phase but is not expressed in Gy or terminally differentiated cells.” Antibodies against the Ki-67 antigen recognize doublets of apparent molec ular masses of 320 and 350 kd.” Ki-67 is a highly labile antigen with a half-life of 1-2 hours or less." It is synthesized continuously in cycling cells, with less than 5 minutes of *°S-methionine incorporation heing sufficient for labeling Ki-67 appears to be in dispensable for cell division, as antisense oligonucleo- tides produce rapid decreases in cellular ‘H-thymidine incorporation.“ Expression of Ki-67 antigen provides information similar to that provided by PCNA immunoreactivity and BrdU incorporation.*** Ki-67. because of its short half-life and rapid synthesis, provides more accurate ‘measures of proliferative indices than does PCNA. The 20-hour half-life of PCNA may allow carryover of PCNA into the subsequent generation, especially cells, in log-phase growth. However, Ki-67 does present some difficulties. Because it has unknown functions, interpretation of results in acute proliferative responses and other situations must include some uncertainties. In addition, the short half-life makes detection diffi- cult. The original antibodies raised against the Ki-67 antigen were useful only in nonfixed cryostat sections. More recently developed antibodies can be used on formalin-fixed, paraffin-embedded sections, provided the appropriate antigen retrieval techniques are em- ployed.*'""" Although the literature contains a pau- city of applications in nonhuman species, the Ki-67 antigen is conserved across mammalian species, and Ki-67 antibodies have been successfully used in vet- cerinary applications.°*****® Because of the difficulty with preservation of Ki-67 immunoreactivity, its ap- phication may be more suitable for prospective studies, where tissue fixation and processing can be more pre- cisely controlled than in retrospective studies, Recently, immunohistochemical detection of cyclins and caks has emerged as a third alternative for me: surement of proliferative indices. Antibodies reactive to cdki and cdk2 and cyclins A, B1, Di, D3, and E have been developed that are useful in immunohisto- chemical studies and are reactive against epitopes in formalin-fixed, paraffin-embedded tissues." Alihough largely restticted (© investigations of human tissues, the conservation of cyclins and edks across species suggests that the utility of antibodies raised against them could include veterinary applications. A few investigators have demonstrated the successful usean of some of these antibodies in mouse studies." These antibodies probably will become useful in other spe cies as well ‘Cyelins and cdks are valuable markers of cellular proliferation, They are expressed exclusively in cy cling cells and, in the instances of the cyclins, are ex- pressed for only a particular portion of the cell cycle. ‘This differential expression may allow further char- acterization, although crude, of the cell cycle in tissue However, cyclin data must be used selectively. Cy- clin have predominately heen tested as prognostic fac tors in human neoplasias. In many tumors and tumor cell lines, unscheduled expression of cyclins has been detected.” Unscheduled expression refers to synthesis of a particular cyclin protein during an inappropriate phase of the cell cycle, such as the expression of cyclin D late in the cell cycle. This unscheduled expression may provide a falsely high index of proliferation in some cells as compared with cells that have normally scheduled expression of the cyclin. The use of cyclins as prognostic factors in neoplasia may be appropriate as long as the possibility of unscheduled expression is, Kept in mind during interpretation of results. ‘Although the cyclins have been used largely in stud ies of neoplasia, they have potential utility for unde standing acute proliferative reactions to a variety of toxicants. Because PCNA can be induced by DNA- damaging agents and the function of Ki-67 is un- Known, the determination of the cellular content of ci- ther cyclins or edks may be a viable alternative be- cause of the uncertainties in results that may be raised by the use of PCNA or Ki-67. If such studies use cell cultures, they must first demonstrate normally sched- uled cyclin synthesis. Unscheduled cyclin expression should not be a concern in whole animal studies. Acknowledgements ‘This review was prepared with the support of the Ofice of Health and Environmental Research, US Department of En- ergy, under eontsact ED-ACOS-76EVO1O13, While prepasin, this manuscript, I was a graduate student in the Lovelace Respiratory Research Institute/Purdue University Experi- ‘mental Pathology Program located in Albuquerque, NM. thank P. Bradley and W. Piper for technical assistance and Drs, FE Hahn, E. B, Janovitz, N. F Johnson, and L. Tierney for helpful discussions. 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