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Supplementary Figures S1-S2 from Epigenetic Down-Regulation of ARF Expression Is a Selection Step in Immortalization of Human Fibroblasts by c-Myc

Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates processive phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.

A significant hurdle to understanding the functions of deubiquitinases (DUBs) is the identification of their in vivo substrates. Substrate identification can be difficult for two reasons. First, many proteins that are degraded by the ubiquitin-proteasome system are expressed at relatively low levels in the cell, and second, redundancy between DUBs complicates loss of function screening approaches. Here, we describe a systematic overexpression approach that takes advantage of genome-wide resources available in S. cerevisiae to overcome these challenges and identify DUB substrates in cells.

Cells exposed to environmental stress arrest the cell cycle until they have adapted to their new environment. Cells adjust the length of the arrest for each unique stressor, but how they do this is not known. Here, we investigate the role of the stress-activated phosphatase calcineurin (CN) in controlling cell cycle arrest in Saccharomyces cerevisiae. We find that CN controls arrest duration through activation of the G1 cyclin–dependent kinase inhibitor Cip1. Our results demonstrate that multiple stressors trigger a G1/S arrest through Hog1-dependent down-regulation of G1 cyclin transcription. When a stressor also activates CN, this arrest is lengthened as CN prolongs Hog1-dependent phosphorylation of Cip1. Cip1 plays no role in response to stressors that activate Hog1 but not CN. These findings illustrate how stress response pathways cooperate to tailor the stress response and suggest that Cip1 functions to prolong cell cycle arrest when a cell requires additional time for adaptation.

Protein degradation during the cell cycle is controlled by the opposing activities of ubiquitin ligases and deubiquitinating enzymes (DUBs). Although the functions of ubiquitin ligases in the cell cycle have been studied extensively, the roles of DUBs in this process are less well understood. Here, we used an overexpression screen to examine the specificities of each of the 21 DUBs in budding yeast for 37 cell cycle–regulated proteins. We find that DUBs up-regulate specific subsets of proteins, with five DUBs regulating the greatest number of targets. Overexpression of Ubp10 had the largest effect, stabilizing 15 targets and delaying cells in mitosis. Importantly, UBP10 deletion decreased the stability of the cell cycle regulator Dbf4, delayed the G1/S transition, and slowed proliferation. Remarkably, deletion of UBP10 together with deletion of four additional DUBs restored proliferation to near–wild-type levels. Among this group, deletion of the proteasome-associated DUB Ubp6 alone reversed the G1/S delay and restored the stability of Ubp10 targets in ubp10Δ cells. Similarly, deletion of UBP14, another DUB that promotes proteasomal activity, rescued the proliferation defect in ubp10Δ cells. Our results suggest that DUBs function through a complex genetic network in which their activities are coordinated to facilitate accurate cell cycle progression.

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors., Author summary In order to survive exposure to environmental stress, cells transiently arrest the cell division cycle while they adapt to the stress. Several kinases and phosphatases are known to control stress adaptation programs, but the extent to which these signaling pathways work together to tune the stress response is not well understood. This study investigates the role of the phosphatase calcineurin in the stress response and shows that calcineurin inhibits the cell cycle in part by stimulating the activity of the Hog1/p-38 stress-activated MAPK in both yeast and human cells. Crosstalk between stress response pathways may help cells mount specific responses to diverse stressors and to survive changes in their environment.

Intrinsically disordered transcription factor transactivation domains (TADs) function through structural plasticity, adopting ordered conformations when bound to transcriptional co-regulators. Many transcription factors contain a negative regulatory domain (NRD) that suppresses recruitment of transcriptional machinery through autoregulation of the TAD. We report the solution structure of an autoinhibited NRD-TAD complex within FoxM1, a critical activator of mitotic gene expression. We observe that while both the FoxM1 NRD and TAD are primarily intrinsically disordered domains, they associate and adopt a structured conformation. We identify how Plk1 and Cdk kinases cooperate to phosphorylate FoxM1, which releases the TAD into a disordered conformation that then associates with the TAZ2 or KIX domains of the transcriptional co-activator CBP. Our results support a mechanism of FoxM1 regulation in which the TAD undergoes switching between disordered and different ordered structures.

SummaryUpon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between diverse stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the stress-activated phosphatase calcineurin (CN) in this process and show that CN utilizes multiple pathways to control the cell cycle. Upon activation, CN inhibits transcription factors (TFs) that regulate the G1/S transition through activation of the stress-activated MAPK Hog1. In contrast, CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner at multiple nodes of the cell cycle-regulatory network to rewire gene expression and arrest cells in response to stress. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.

Use of chemo-genetic interaction mediated synthetic lethality represents a strategy for mechanistic validation of targets and gene-based patient stratification for clinical development. Given that core pathways supporting cancer development and progression (cell cycle regulation, genome integrity, metabolism) are highly conserved, orthogonal model organisms represent powerful systems in which to identify synthetic lethal pairs for highly conserved human drug targets. BPM42522 is an enzyme in the ubiquitin proteasome system whose anti-cancer potential has been validated through genetic and pharmacologic modulation. The S. cerevisiae homolog of BPM42522 (yBPM42522) is a critical cell cycle-regulatory gene with 273 genetic interactions affecting fitness (Costanzo 2016). Negative genetic interactors regulate cellular processes validated for therapeutic intervention in oncology such as DNA replication and repair, protein turnover, and mitosis. Thus, mapping yBPM42522 negative genetic interactors to their human homologs and curating tumors in which they are altered may aid in identification of clinical contexts more susceptible to pharmacologic inhibition of BPM42522. To this end, conservation analysis of genetic interactors of yBPM42522 was performed, and coding mutations and deletion events were then characterized using The Cancer Genome Atlas (TCGA) tumor data. The most frequently altered genetic interactors across human tumors were reviewed for the direction and strength of the genetic interaction between their yeast homologs and yBPM42522, and mutations were curated by their likelihood to result in a loss of function. Those with strong negative genetic interactions with yBPM42522 and frequent mutation or deletion in its human homolog were prioritized. The resulting candidates included tumor suppressors (FBXW7, NF1), cell cycle regulators (CCNB1, CCNB3, BUB1B), and the NIMA kinase and GTPase-activating protein families (NEK3, NEK4, RASA1, RASAL2). TCGA database was used to determine if alterations in these candidate genes were prevalent in specific tumor types and whether they co-occurred with alterations in established cancer driver genes. Several candidate genetic interactors identified were frequently mutated or deleted in specific tumor types including uterine carcinosarcoma, uterine corpus endometrial carcinoma, ovarian serous cystadenocarcinoma, and mesothelioma. Moreover, these alterations were mutually exclusive with 96 reported cancer driver genes. Validation of the candidate genetic interactors for synthetic lethality with BPM42522 and their role in specific cancer types highlights the approach for rapid identification of synthetic lethality and its potential use to stratify patient populations most likely to benefit from therapeutic agents targeting highly conserved drug targets. Citation Format: Anne R. Diers, Kris Richardson, Leonardo O. Rodrigues, Rangaprasad Sarangarajan, Niven R. Narain, Jennifer A. Benanti, Stephane Gesta. Utility of S. cerevisiae genetic interactions in the mechanistic validation and therapeutic potential of highly conserved targets for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2982.

Progression through the cell cycle is controlled by a network of transcription factors that coordinate gene expression with cell-cycle events. One transcriptional activator in this network in budding yeast is the forkhead protein Hcm1, which controls the expression of genes that are transcribed during S-phase. Hcm1 activity is coordinated with the cell cycle via its regulation by cyclin-dependent kinase (Cdk1), which both activates Hcm1 and targets it for degradation, through phosphorylation of distinct sites. The mechanisms controlling the differential phosphorylation timing of the activating and destabilizing phosphosites are not clear. However, a recent study shows that the phosphatase calcineurin specifically removes activating phosphates from Hcm1 when cells are exposed to environmental stress, thus extinguishing its activity and slowing proliferation under unfavorable growth conditions. This regulatory mechanism, whereby a phosphatase actively alters the distribution of phosphosites on a cell cycle-regulatory transcription factor to elicit a change in cellular proliferation, adds an additional layer of complexity to the regulatory network controlling the cell cycle. Furthermore, this regulatory paradigm is likely to be a conserved mode of phosphoregulation that controls the cell cycle in diverse systems.

The transcription factor Hcm1 is a key regulator of chromosome segregation and genome stability. The phosphatase calcineurin directly inactivates Hcm1 in response to environmental stress, which inhibits proliferation. Hcm1 functions as a rheostat, whose phosphorylation state affects the rate of proliferation., Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation.

During mitosis chromosomes are condensed to facilitate their segregation, through a process mediated by the condensin complex. Although several factors that promote maximal condensin activity during mitosis have been identified, the mechanisms that downregulate condensin activity during interphase are largely unknown. Here, we demonstrate that Ycg1, the Cap-G subunit of budding yeast condensin, is cell cycle-regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. This cyclical expression pattern is established by a combination of cell cycle-regulated transcription and constitutive degradation. Interestingly, overexpression of YCG1 and mutations that stabilize Ycg1 each result in delayed cell-cycle entry and an overall proliferation defect. Overexpression of no other condensin subunit impacts the cell cycle, suggesting that Ycg1 is limiting for condensin complex formation. Consistent with this possibility, we find that levels of intact condensin complex are reduced in G1 phase compared to mitosis, and that increased Ycg1 expression leads to increases in both levels of condensin complex and binding to chromatin in G1. Together, these results demonstrate that Ycg1 levels limit condensin function in interphase cells, and suggest that the association of condensin with chromosomes must be reduced following mitosis to enable efficient progression through the cell cycle., Author Summary Chromosome conformation is cell cycle-regulated so that chromosomes are highly compacted to facilitate their segregation during mitosis, and decondensed during interphase to facilitate DNA-dependent processes such as replication and transcription. Understanding how chromosomes transition between these different states is important for understanding how cells maintain a stable genome. The condensin complex is an essential five-subunit complex that controls chromosome condensation in all eukaryotes. In this study, we show that expression of the Cap-G/Ycg1 subunit of condensin in budding yeast is cell cycle-regulated, and that its reduced expression during interphase limits condensin function. When this regulation is disrupted, and Ycg1 is constitutively expressed, progression through interphase is delayed. Emerging evidence indicates that individual condensin subunits are also expressed at limiting levels in metazoan cells, which suggests that cell-cycle regulation of an individual condensin subunit is a conserved mechanism that coordinates condensin function with the cell cycle.

Cik1, in association with the kinesin Kar3, controls both the mitotic spindle and nuclear fusion during mating. Here, we show that there are two Cik1 isoforms, and that the mitotic form includes an N-terminal domain required for ubiquitination by the Anaphase-Promoting Complex/Cyclosome (APC/C). During vegetative growth, Cik1 is expressed during mitosis and regulates the mitotic spindle, allowing for accurate chromosome segregation. After mitosis, APC/C(Cdh1) targets Cik1 for ubiquitin-mediated proteolysis. Upon exposure to the mating pheromone alpha factor, a smaller APC/C-resistant Cik1 isoform is expressed from an alternate transcriptional start site. This shorter Cik1 isoform is stable and cannot be ubiquitinated by APC/C(Cdh1). Moreover, the two Cik1 isoforms are functionally distinct. Cells that express only the long isoform have defects in nuclear fusion, whereas cells expressing only the short isoform have an increased rate of chromosome loss. These results demonstrate a coupling of transcriptional regulation and APC/C-mediated proteolysis.

Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated beta-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS.

Human fibroblasts undergo cellular senescence after a finite number of divisions, in response to the erosion of telomeres. In addition to being terminally arrested in the cell cycle, senescent fibroblasts express genes that are normally induced upon wounding, including genes that remodel the extracellular matrix. We have identified the novel zinc finger protein APA-1, whose expression increased in senescent human fibroblasts independent of telomere shortening. Extended passage, telomerase-immortalized fibroblasts had increased levels of APA-1 as well as the cyclin-dependent kinase inhibitor p16. In fibroblasts, APA-1 was modified by the ubiquitin-like protein SUMO-1, which increased APA-1 half-life, possibly by blocking ubiquitin-mediated degradation. Overexpression of APA-1 did not cause cell cycle arrest; but, it induced transcription of the extracellular matrix-remodeling genes MMP1 and PAI2, which are associated with fibroblast senescence. MMP1 and PAI2 transcript levels also increased in telomerase-immortalized fibroblasts that had high levels of APA-1, demonstrating that the matrix-remodeling phenotype of senescent fibroblasts was not induced by telomere attrition alone. APA-1 was able to transactivate and bind to the MMP1 promoter, suggesting that APA-1 is a transcription factor that regulates expression of matrix-remodeling genes during fibroblast senescence.

The relative contributions of CD11a/CD18 and CD11b/CD18 to the dynamics and strength of neutrophil adhesion to intercellular adhesion molecule (ICAM)-1–transfected cells were examined over the time course of chemotactic stimulation. Suspensions of neutrophils and transfectants were sheared in a cone-plate viscometer, and formation of heterotypic aggregates was measured by 2-color flow cytometry. The 2-body collision theory was used to compute adhesion efficiency, defined as the proportion of collisions between neutrophils and target cells that resulted in capture. ICAM-1 surface density and shear rate both regulated adhesion efficiency. Target cells expressing approximately 1000 ICAM-1 sites/μm2 (Ilow) were captured with an efficiency of 0.15 at 100 s−1, which decreased to zero at 300 s−1. At 8-fold higher ICAM-1 expression (Ihigh) corresponding to levels measured on interleukin-1–stimulated endothelium, efficiency was 0.3 at 100 s−1 and remained above background to 900 s−1. Shear alone was sufficient for CD11a/CD18-mediated adhesion to ICAM-1, and stimulation with formyl-methionyl-leucyl-phenylalanine boosted capture efficiency through CD11a/CD18 by 4-fold. In comparison, CD11b/CD18 supported one third of this efficiency, but was necessary for aggregate stability over several minutes of shear and at shear stresses exceeding 5 dyne/cm2. Hydrodynamics influenced capture efficiency predominantly through the collisional contact duration, predicted to be approximately 9 milliseconds for successful capture of Ilow and 4 milliseconds for Ihigh. The implication is that an increase in ICAM-1 from resting levels to those on inflamed endothelium effectively increases the permissible shear in which capture through β2-integrins may occur. Neutrophil adhesion to ICAM-1 appears to be a cooperative and sequential process of CD11a-dependent capture followed by CD11b-mediated stabilization.

The 33 genes in the Saccharomyces cerevisiae mitotic CLB2 transcription cluster have been known to be downregulated by the DNA damage checkpoint for many years. Here, we show that this is mediated by the checkpoint kinase Rad53 and the dedicated transcriptional activator of the cluster, Ndd1. Ndd1 is phosphorylated in response to DNA damage, which blocks recruitment to promoters and leads to the transcriptional downregulation of the CLB2 cluster. Finally, we show that downregulation of Ndd1 is an essential function of Rad53, as a hypomorphic ndd1 allele rescues RAD53 deletion.

Normal cells are thought to protect against transformation by undergoing a permanent cell cycle arrest, cellular senescence, in response to the expression of activated oncogenes such as RAS. We recently found that freshly established neonatal human fibroblasts are resistant to RAS-induced senescence. Moreover, extended passaging of normal fibroblasts leads to increased levels of the cyclin dependent kinase inhibitor p16 and sensitizes cells to senescence induced by RAS. These findings implicate exogenous stress as a necessary cofactor in RAS-induced senescence and demonstrate that RAS expression can promote some characteristics of transformation in the absence of other genetic changes.

The coupling of cellular growth and division is crucial for a cell to make an accurate copy of itself. Regulated protein degradation by the ubiquitin-proteasome system (UPS) plays an important role in the coordination of these two processes. Many ubiquitin ligases, in particular the Skp1-Cullin-F-box protein (SCF) family and Anaphase-Promoting Complex (APC), couple growth and division by targeting cell cycle and metabolic regulators for degradation. However, many regulatory proteins are targeted by multiple ubiquitin ligases. As a result, we are only just beginning to understand the complexities of the proteolytic regulatory network that connects cell growth and the cell cycle.

The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation., Author Summary Telomeres, the ends of linear chromosomes, are essential for chromosome stability. Telomerase is the enzyme that is responsible for lengthening telomeres in most organisms, including humans. One mechanism of survival for many human cancers is increased expression of telomerase. In baker's yeast, telomerase acts only late in the cell cycle, even though the catalytic subunit of telomerase is telomere bound throughout most of the cell cycle. Pif1p is a yeast helicase that limits telomerase by removing it from DNA ends. We demonstrate that Pif1p abundance is cell cycle regulated with its highest expression at the same time when telomerase acts. Consistent with this expression pattern, Pif1p is able to remove the active form of telomerase from DNA ends. Reducing the amount of telomere-bound telomerase either by Pif1p overexpression or by mutation in strains with defective telomere end protection causes death. Moreover, reducing Pif1p levels in the same end protection mutants improves their growth. These experiments suggest that compared to wild-type cells, strains with defective end protection require more telomere-bound telomerase for the proper replication or proper protection of their chromosome ends.

Entry into the cell cycle is regulated by nutrient availability such that cells do not divide when resources are limited. The Skp1-Cul1-F-box (SCF) ubiquitin ligase with the F-box protein Grr1 (SCF(Grr1)) controls the proteolytic turnover of regulators of cell-cycle entry and a glucose sensor, suggesting that it links the cell cycle with nutrient availability. Here, we show that SCF(Grr1) broadly regulates cellular metabolism. We have developed a proteomic screening method that uses high-throughput quantitative microscopy to comprehensively screen for ubiquitin-ligase substrates. Seven new metabolic targets of SCF(Grr1) were identified, including two regulators of glycolysis--the transcription factor Tye7 and Pfk27. The latter produces the second messenger fructose-2,6-bisphosphate that activates glycolysis and inhibits gluconeogenesis. We show that SCF(Grr1) targets Pfk27 and Tye7 in response to glucose removal. Moreover, Pfk27 is phosphorylated by the kinase Snf1, and unphosphorylatable Pfk27 is stable and inhibits growth in the absence of glucose. These results demonstrate a role for SCF(Grr1) in regulating the glycolytic-gluconeogenic switch.

While p53 activity is critical for a DNA damage-induced G(1) checkpoint, its role in the G(2) checkpoint has not been compelling because cells lacking p53 retain the ability to arrest in G(2) following DNA damage. Comparison between normal human foreskin fibroblasts (HFFs) and HFFs in which p53 was eliminated by transduction with human papillomavirus type 16 E6 showed that treatment with adriamycin initiated arrest in G(2) with active cyclin B/CDC2 kinase, regardless of p53 status. Both E6-transduced HFFs and control (LXSN)-transduced cells maintained a prolonged arrest in G(2); however cells with functional p53 extinguished cyclin B-associated kinase activity. Down regulation was mediated by p53-dependent transcriptional repression of the CDC2 and cyclin B promoters. In contrast, cells lacking p53 showed a prolonged G(2) arrest despite high levels of cyclin B/CDC2 kinase activity, at least some of which translocated into the nucleus. Furthermore, the G(2) checkpoint became attenuated as p53-deficient cells aged in culture. Thus, at late passage, E6-transduced HFFs entered mitosis following DNA damage, whereas the age-matched parental HFFs sustained a G(2) arrest. These results indicate that normal cells have p53-independent pathways to maintain DNA damage-induced G(2) arrest, which may be augmented by p53-dependent functions, and that cells lacking p53 are at greater risk of losing the pathway that protects against aneuploidy.

In this issue of Molecular Cell, Kimata et al. (2008) show that Cdc20 functions not only in the recruitment of substrates to the anaphase-promoting complex but also in its activation.