End Of G1 Research Articles

Role of the Holoenzyme PP1-SPN in the Dephosphorylation of the RB Family of Tumor Suppressors During Cell Cycle.

Simple SummaryCell cycle progression is highly regulated by modulating the phosphorylation status of retinoblastoma (RB) family proteins. This process is controlled by a balance in the action of kinases, such as the complexes formed by cyclin-dependent kinases (CDKs) and cyclins, and phosphatases, mainly the protein phosphatase 1 (PP1). However, while the phosphorylation of the RB family has been largely studied, its dephosphorylation is less known. Recently, the PP1-Spinophilin (SPN) holoenzyme has been described as the main phosphatase responsible for the dephosphorylation of RB proteins during the G0/G1 transition and at the end of G1. Here, we describe the regulation of the phosphorylation status of RB family proteins, giving importance not only to their inactivation by phosphorylation but also to their dephosphorylation to restore the cell cycle.Cell cycle progression is highly regulated by modulating the phosphorylation status of the retinoblastoma protein (pRB) and the other two members of the RB family, p107 and p130. This process is controlled by a balance in the action of kinases, such as the complexes formed by cyclin-dependent kinases (CDKs) and cyclins, and phosphatases, mainly the protein phosphatase 1 (PP1). However, while the phosphorylation of the RB family has been largely studied, its dephosphorylation is less known. Phosphatases are holoenzymes formed by a catalytic subunit and a regulatory protein with substrate specificity. Recently, the PP1-Spinophilin (SPN) holoenzyme has been described as the main phosphatase responsible for the dephosphorylation of RB proteins during the G0/G1 transition and at the end of G1. Moreover, SPN has been described as a tumor suppressor dependent on PP1 in lung and breast tumors, where it promotes tumorigenesis by increasing the cancer stem cell pool. Therefore, a connection between the cell cycle and stem cell biology has also been proposed via SPN/PP1/RB proteins.

Down-regulation of Cdk1 activity in G1 coordinates the G1/S gene expression programme with genome replication.

Cell division is regulated by cyclin-dependent kinases (Cdks) and requires the periodic activation and inactivation of transcription factors that generate waves of gene expression in different cell-cycle phases. In fission yeast, the MCB-binding transcription factor (MBF) is activated at the end of G1 and regulates the expression of a set of genes that encode for proteins involved in the G1/S transition and DNA replication. Here, we review the importance of controlling MBF by Cdk activity at the onset of S phase. Furthermore, we emphasize that MBF regulation by Cdk is particularly critical under conditions in which G1 is extended, such as in nitrogen-poor environments, where down-regulation of Cdk activity in G1 is crucial to generate a proper wave of MBF-dependent transcription at the end of G1, which is critical to promote a successful S phase.

Nutritional cell cycle reprogramming reveals that inhibition of Cdk1 is required for proper MBF-dependent transcription.

In nature, cells and in particular unicellular microorganisms are exposed to a variety of nutritional environments. Fission yeast cells cultured in nitrogen-rich media grow fast, divide with a large size and show a short G1 and a long G2. However, when cultured in nitrogen-poor media, they exhibit reduced growth rate and cell size and a long G1 and a short G2. In this study, we compared the phenotypes of cells lacking the highly conserved cyclin-dependent kinase (Cdk) inhibitor Rum1 and the anaphase-promoting complex/cyclosome (APC/C) activator Ste9 in nitrogen-rich and nitrogen-poor media. Rum1 and Ste9 are dispensable for cell division in nitrogen-rich medium. However, in nitrogen-poor medium they are essential for generating a proper wave of MluI cell-cycle box binding factor (MBF)-dependent transcription at the end of G1, which is crucial for promoting a successful S phase. Mutants lacking Rum1 and Ste9 showed premature entry into S phase and a reduced wave of MBF-dependent transcription, leading to replication stress, DNA damage and G2 cell cycle arrest. This work demonstrates how reprogramming the cell cycle by changing the nutritional environment may reveal new roles for cell cycle regulators.

Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.

Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner—both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.

Genes adopt non‐optimal codon usage to generate cell cycle‐dependent oscillations in protein levels

The cell cycle is a temporal program that regulates DNA synthesis and cell division. When we compared the codon usage of cell cycle-regulated genes with that of other genes, we discovered that there is a significant preference for non-optimal codons. Moreover, genes encoding proteins that cycle at the protein level exhibit non-optimal codon preferences. Remarkably, cell cycle-regulated genes expressed in different phases display different codon preferences. Here, we show empirically that transfer RNA (tRNA) expression is indeed highest in the G2 phase of the cell cycle, consistent with the non-optimal codon usage of genes expressed at this time, and lowest toward the end of G1, reflecting the optimal codon usage of G1 genes. Accordingly, protein levels of human glycyl-, threonyl-, and glutamyl-prolyl tRNA synthetases were found to oscillate, peaking in G2/M phase. In light of our findings, we propose that non-optimal (wobbly) matching codons influence protein synthesis during the cell cycle. We describe a new mathematical model that shows how codon usage can give rise to cell-cycle regulation. In summary, our data indicate that cells exploit wobbling to generate cell cycle-dependent dynamics of proteins.

Transcription Factor Epiprofin Is Essential for Tooth Morphogenesis by Regulating Epithelial Cell Fate and Tooth Number

In tooth morphogenesis, the dental epithelium and mesenchyme interact reciprocally for growth and differentiation to form the proper number and shapes of teeth. We previously identified epiprofin (Epfn), a gene preferentially expressed in dental epithelia, differentiated ameloblasts, and certain ectodermal organs. To identify the role of Epfn in tooth development, we created Epfn-deficient mice (Epfn-/-). Epfn-/- mice developed an excess number of teeth, enamel deficiency, defects in cusp and root formation, and abnormal dentin structure. Mutant tooth germs formed multiple dental epithelial buds into the mesenchyme. In Epfn-/- molars, rapid proliferation and differentiation of the inner dental epithelium were inhibited, and the dental epithelium retained the progenitor phenotype. Formation of the enamel knot, a signaling center for cusps, whose cells differentiate from the dental epithelium, was also inhibited. However, multiple premature nonproliferating enamel knot-like structures were formed ectopically. These dental epithelial abnormalities were accompanied by dysregulation of Lef-1, which is required for the normal transition from the bud to cap stage. Transfection of an Epfn vector promoted dental epithelial cell differentiation into ameloblasts and activated promoter activity of the enamel matrix ameloblastin gene. Our results suggest that in Epfn-deficient teeth, ectopic nonproliferating regions likely bud off from the self-renewable dental epithelium, form multiple branches, and eventually develop into supernumerary teeth. Thus, Epfn has multiple functions for cell fate determination of the dental epithelium by regulating both proliferation and differentiation, preventing continuous tooth budding and generation.

The DNA Damage Response Mediator MDC1 Directly Interacts with the Anaphase-promoting Complex/Cyclosome

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (gamma-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of gamma-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.

Multi-Kinase Phosphorylation of the APC/C Activator Cdh1 Revealed by Mass Spectrometry

by activating a large ubiquitin ligase called the anaphase-promoting complex, or cyclosome (APC/C). At the end of G1, APC/CCdh1 is inhibited by cyclin-dependent kinase (CDK) phosphorylation of Cdh1. The specific Cdh1 phosphorylation sites used to regulate APC/CCdh1 activity have not been directly identified. Here, we used a mass spectrometric approach to identify the in vivo phosphorylation sites on yeast Cdh1. Surprisingly, in addition to several expected CDK phosphorylation sites, we discovered numerous non-CDK phosphorylation sites. In total, at least 19 serine and threonine residues on Cdh1 are phosphorylated in vivo. Seventeen of these sites are located in the N-terminal half of Cdh1, outside the highly conserved WD40 repeats. The pattern of phosphorylation was the same when Cdh1 was purified from yeast cultures arrested in S, early M and late M. Mutation of CDK consensus sequences eliminated detectable phosphorylation at many of the non-CDK sites. In contrast, mutation of non-CDK sites had no significant effect on CDK phosphorylation. We conclude that phosphorylation of CDK sites promotes the subsequent recognition of Cdh1 by at least one additional kinase. The function of non-CDK phosphorylation may differ from CDK phosphorylation because mutation of non-CDK sites did not result in constitutive activation of APC and consequent cell cycle arrest. These results suggest that phosphoregulation of APC/CCdh1 activity is much more complex than previously thought.

Phosphoinositide 3-kinase and Forkhead, a switch for cell division.

Cell cycle progression is a tightly controlled process. To initiate cell division, mitogens trigger a number of early signals that promote the G(0)-G(1) transition by inducing cell growth and the activation of G(1) cyclins. Activation of cyclin E/cdk2 (cyclin-dependent kinase 2) at the end of G(1) is then required to trigger DNA synthesis (S phase entry). Among the early signals induced by mitogens, activation of PI3K (phosphoinositide 3-kinase) appears essential to induce cell cycle entry, as it regulates cell growth signalling pathways, which in turn determine the rate of cell cycle progression. Another mechanisms by which PI3K and its downstream effector protein kinase B regulate cell cycle entry is by inactivation of the FOXO (Forkhead Box, subgroup O) transcription factors, which induce expression of quiescence genes such as those encoding p27(kip), p130 and cyclin G2. PI3K/FOXO then work as a complementary switch: when PI3K is active, FOXO transcription factors are inactive. The switch is turned on and off at different phases of the cell cycle, thus regulating cell cycle progression.

Functional and molecular identification of intermediate-conductance Ca(2+)-activated K(+) channels in breast cancer cells: association with cell cycle progression.

We have previously reported that the hEAG K(+) channels are responsible for the potential membrane hyperpolarization that induces human breast cancer cell progression into the G1 phase of the cell cycle. In the present study, we evaluate the role and functional expression of the intermediate-conductance Ca(2+)-activated K(+) channel, hIK1-like, in controlling cell cycle progression. Our results demonstrate that hIK1 current density increased in cells synchronized at the end of the G1 or S phase compared with those in the early G1 phase. This increased current density paralleled the enhancement in hIK1 mRNA levels and the highly negative membrane potential. Furthermore, in cells synchronized at the end of G1 or S phases, basal cytosolic Ca(2+) concentration ([Ca(2+)](i)) was also higher than in cells arrested in early G1. Blocking hIK1 channels with a specific blocker, clotrimazole, induced both membrane potential depolarization and a decrease in the [Ca(2+)](i) in cells arrested at the end of G1 and S phases but not in cells arrested early in the G1 phase. Blocking hIK1 with clotrimazole also induced cell proliferation inhibition but to a lesser degree than blocking hEAG with astemizole. The two drugs were essentially additive, inhibiting MCF-7 cell proliferation by 82% and arresting >90% of cells in the G1 phase. Thus, although the progression of MCF-7 cells through the early G1 phase is dependent on the activation of hEAG K(+) channels, when it comes to G1 and checkpoint G1/S transition, the membrane potential appears to be primarily dependent on the hIK1-activity level.

Cell-cycle-dependent expression of the large Ca 2+-activated K + channels in breast cancer cells

In a previous work, we have reported that the ionic nature of the outward current recorded in MCF-7 cells was that of a K + current. In this study, we have identified a Ca 2+-activated K + channel not yet described in MCF-7 human breast cancer cells. In cells arrested in the early G1 (depolarized cells), increasing [Ca 2+] i induced both a shift in the I– V curve toward more negative potentials and an increase in current amplitude at negative and more at positive potential. Currents were inhibited by r-iberiotoxin (r-IbTX, 50 nM) and charybdotoxin (ChTX, 50 nM). These data indicate that human breast cancer cells express large-conductance Ca 2+-activated K + (BK) channels. BK current-density increased in cells synchronized at the end of G1, as compared with those in the early G1 phase. This increased current-density paralleled the enhancement in BK mRNA levels. Blocking BK channels with r-IbTX, ChTX or both induced a slight depolarization in cells arrested in the early G1, late G1, and S phases and accumulated cells in the S phase, but failed to induce cell proliferation. Thus, the expression of the BK channels was cell-cycle-dependent and seems to contribute more to the S phase than to the G1 phase. However, these K + channels did not regulate the cell proliferation because of their minor role in the membrane potential.

Phosphorylation of the Septin Cdc3 in G1 by the Cdc28 Kinase Is Essential for Efficient Septin Ring Disassembly

The septins constitute a family of filament-forming proteins ubiquitous in eukaryotic species. We demonstrate here that the Saccharomyces cerevisiae septin, Cdc3, is a substrate of the cell cycle regulatory cyclin-dependent kinase (Cdk), Cdc28. Two serines near the C-terminus of Cdc3 are phosphorylated in a Cdc28-dependent manner. Analysis of a mutant allele that cannot be phosphorylated at these sites revealed an effect of Cdc28 phosphorylation of Cdc3 at the time of budding. Immunofluorescence analysis of wild-type and mutant Cdc3 indicated that prevention of phosphorylation at Cdc28-dependent sites impairs the disassembly of the old septin ring, which is inherited at mitosis but which usually disappears immediately prior to assembly of a new ring. Furthermore, immunofluorescence analysis of septin ring dynamics in a G1 cyclin (Cln) mutant suggests that G1 cyclin function is required for efficient ring disassembly. Thus, phosphorylation of Cdc3 by the Cdc28 kinase at the end of G1 may facilitate initiation of a new cell cycle by promoting disassembly of the obsolete septin ring from the previous cell cycle.Key WordsG1, Cdc3, Septin Ring, Drosophila, Cytokinesis

Induction of anergy in Th1 cells associated with increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1.

Th1 cells exposed to Ag and the G(1) blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21(Cip1) and p27(Kip1). Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21(Cip1) and p27(Kip1). However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21(Cip1) and p27(Kip1) when examined 24 h after Ag stimulation. The increase in p21(Cip1) observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27(Kip1) observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21(Cip1) and perhaps the downstream failure to maintain low levels of p27(Kip1). Increased levels of both p21(Cip1) and p27(Kip1) at the end of G(1) could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.

Stepwise assembly of initiation proteins at budding yeast replication origins in vitro.

The initiation of DNA replication in the budding yeast Saccharomyces cerevisiae occurs in two sequential and mutually exclusive steps. Prereplicative complexes (pre-RCs) containing origin recognition complex (ORC), Cdc6p, and the MCM2-7 proteins assemble only under conditions of low cyclin-dependent kinase (Cdk) activity during G(1), whereas origin activation is driven by the increase in Cdk activity at the end of G(1). As a first step toward the reconstitution of this two-step process in vitro, we describe a system in which extracts prepared from G(1)-arrested cells promote sequential assembly of ORC, Cdc6p, and MCM2-7 proteins onto exogenously added origin-containing DNA. This reaction requires an intact ARS consensus sequence and requires ATP for two distinct steps. Extracts from cells arrested in mitosis also can support the binding of ORC but are unable to load either Cdc6p or MCM2-7 proteins. This system should be useful for studying the mechanism and regulation of pre-RC assembly.

Cyclin E–Cdk2 Phosphorylation Promotes Late G1-Phase Degradation of MyoD in Muscle Cells

Proliferating myoblasts already express MyoD before the induction of differentiation. Overexpression of MyoD in normal and transformed cell lines was shown to block cells from entering S phase, suggesting that the MyoD growth suppressive effect must be tightly controlled in growing myoblasts. Here we show that during G1 phase, but not in G2, MyoD abundance is down-regulated by the ubiquitin–proteasome pathway through phosphorylation of serine 200. Roscovitine, a specific inhibitor of cyclin–Cdk2 complexes, prevents both phosphorylation and degradation of MyoD in G1. Inhibition of the ubiquitin-dependent proteasome pathway by MG132 results in stabilization of MyoD-wt, with little effect on a MyoD mutant where serine 200 is replaced by an alanine. Our results show that MyoD Ser200 is the substrate for phosphorylation by cyclin E–Cdk2 stimulating its degradation by the ubiquitin–proteasome system which controls MyoD levels in G1. Phosphorylation/degradation of MyoD at the end of G1 thus represents the regulatory checkpoint in growing myoblasts allowing progression into S phase in a manner similar to the recently examplified cdk2-phosphorylation/degradation of p27Kip1.

The puc1 cyclin regulates the G1 phase of the fission yeast cell cycle in response to cell size.

Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1(+) gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.

Cdk Phosphorylation Triggers Sequential Intramolecular Interactions that Progressively Block Rb Functions as Cells Move through G1

We present evidence that phosphorylation of the C-terminal region of Rb by Cdk4/6 initiates successive intramolecular interactions between the C-terminal region and the central pocket. The initial interaction displaces histone deacetylase from the pocket, blocking active transcriptional repression by Rb. This facilitates a second interaction that leads to phosphorylation of the pocket by Cdk2 and disruption of pocket structure. These intramolecular interactions provide a molecular basis for sequential phosphorylation of Rb by Cdk4/6 and Cdk2. Cdk4/6 is activated early in G1, blocking active repression by Rb. However, it is not until near the end of G1, when cyclin E is expressed and Cdk2 is activated, that Rb is prevented from binding and inactivating E2F.

Skp2 induction and phosphorylation is associated with the late G1 phase of proliferating rat hepatocytes

The changes in phosphoproteins purified with the affinity peptide p9CKShs1 were analyzed from extracts of regenerating rat livers in order to define some G1 and G1/S regulations characteristic of mature hepatocytes stimulated to proliferate. We observed a 47 kDa phosphoprotein that occurred first at the end of G1 before peaking in the S phase. P47 was also found to be phosphorylated in late G1 in primary hepatocyte cultures stimulated with mitogens. P47 was still phosphorylated in extracts depleted of Cdc2, but to a lesser extent after Cdk2 depletion. This phosphoprotein was identified as Skp2. (i) P47 shared the same electrophoretic mobility than Skp2, a cell cycle protein essential for S phase entry in human fibroblasts; (ii) Skp2, like P47, started to be expressed and was highly phosphorylated during the G1/S transition of hepatocytes stimulated to proliferate in vivo and in vitro; (iii) P47 was specifically immunoprecipitated by an antibody directed against Skp2. In addition, cyclin A/Cdk2 complexes from regenerating liver clearly interacted with Skp2. This is the first demonstration that Skp2 is induced and phosphorylated in the late G1 and S phase of hepatocytes in vivo in regenerating liver as well as in vitro in mitogen-stimulated hepatocytes.

Toxin variability during the cell cycle of the dinoflagellate Alexandrium fundyense

Cultures of the toxic dinoflagellate Alexandrium fundyense Balech were subjected to conditions that induced two synchronized divisions over a period of 48 h. Before, during, and after this interval, toxin content, toxin composition, and several other physiological parameters were monitored every 2 h for 94 h. Toxin production was discontinuous, induced by light, and always occurred during a defined time frame within the G1 phase of the cell cycle. Specific toxin production rates were positive for a period of ∼8–10 h in early G1 and dropped to zero for the remainder of the interphase and mitosis. Analysis of toxin composition showed that cellular concentrations of all the saxitoxin derivatives followed a similar pattern of increase, stabilization, and decrease throughout one generation time. A putative sequence of interconversions between the derivatives could be established, with C2 as the first compound to appear. Division of a subset of the population during the first 24 h of the experiment and the ensuing total synchrony of the culture suggest the existence of two transition points in the cell cycle of this dinoflagellate. The first transition point, at the beginning of G1, is light‐dependent and holds the cells in a G0‐like period. The second block point at the end of G1 is size‐dependent and arrests the cells in G1. We propose a model of the cell cycle of A. fundyense in which progression through the cell cycle can be arrested at two different transition points located in G1 and toxin production is induced by light during G1. The restriction off toxin production to a relatively short segment of the cell cycle provides a tool for comparing cells that are and are not synthesizing toxin.

Proliferation markers

Types of growth include embryonic, fetal, neonatal, juvenile and mature. Until full differentiation is achieved, cells grow through proliferation from progenitor cells. At maturity, the cellular genome is fixed with committed patterns of cell cycle duration and adaptation, ranging from static to renewing type 3. The static cell type cannot proliferate and adapts through hypertrophy. The renewing type continuously proliferates even without stimulus. In all cell types the processes of differentiation and proliferation are mutually exclusive. Cellular kinetics involve (a) the duration of the cell cycle, (b) the birth rate of cells, and (c) the growth rate fractions. The duration of the cell cycle is 2-4 days. All growth factors (GF) exert their influence during G1 phase. Release a GF by one cell type can influence the proliferation of another (= paracrine stimulation). At the end of G1 is the point of highest sensitivity to toxicity. Tumor suppressor genes act here through tyrosine phosphorylation. During S, the cell replicates its chromosomes. During G2 the immune surveillance and DNA damage repair mechanisms operate. Injured cells stay here longer enabling repair of their damaged DNA. Cell division involves both nuclear (mitosis) and cytoplasmic (cytokinesis) phases giving rise to 2 new cells. The cell cycle has 2 checkpoints. The first involves the G1-S transition and the second the G2-M transition. The types of cell cycle inhibition include (a) cycle- and phase-specific inhibition; (b) cycle-and nonphase-specific inhibition; (c) noncycle-and nonphase-specific inhibition, and finally (d) noncycle, nonphase-, and nonorgan-specific inhibition. Proliferation is a circadian process and it is stimulated by a variety of stimuli which include (1) interference with hormonal feedback pathways; (2) inhibition of the tissue trophic activity; (3) sustained presence of antigenic substances; (4) tissue ischemia; (5) changes of conditions luminally or on surfaces of tissues; (6) sustained cytotoxicity; (7) cell death; and (8) surgical resection. Proliferation can be arrested through senescence, apoptosis, injury or even during the development of immune cells. In the past, tissue/cell kinetics have been studied by tritiated thymidine histoautoradiography. Recently, monoclonal antibodies to proliferation-associated antigens, have been successfully employed. These antigens are cycle-associated proteins and include (1) PCNA; (2) p53; (3) Ki67; (4) AGNOR; (5) Statin; and (6) BrdU. Practical examples are given comparing PCNA and BrdU markers from 3 tissues, i.e. liver, glandular stomach, and uterus, across 2 or 3 strains of rats. Mean values of labeling indices are cited. Within the PCNA marker, 2 different clones are compared from the glandular stomach of SD rats of 2 different ages. Gender and across species comparisons are also made. All these comparisons denote that in every study where markers are used (a) there is a need for a concurrent study control group of the same age; (b) there is a need for in-house control data for this particular organ by species, strain, gender and age; (c) there is ancillary assessment of the trophic status of the target tissue; (d) there is a need for at least 2 different time points during assessment; (e) there is a need for such proliferation data prior to commencing the 2 year rodent bioassay; and (f) that PCNA is the most reliable and versatile of all markers used, capable of rendering good results even from archival specimens.