Tumor-specific cholinergic CD4+ T lymphocytes guide immunosurveillance of hepatocellular carcinoma.

Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca2+-NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.

Severe bilateral Legg-Calvé-Perthes resolved with pamidronate in combination with casts, physiotherapy and adductor tenotomy: a pictorial essay over 11 years.

We describe an 11-year prospective clinical and radiologic course of a 6-year-old boy with bilateral Legg-Calvé-Perthes disease, who was treated with intravenous pamidronate (IV-PAM). His baseline radiographs showed grade IV avascular necrosis/Catterall stage IV, and at worst he progressed to lateral pillar/Herring stage C bilaterally. His disease initially was extremely functionally limiting with expected poor outcome with eventual joint replacement. Because IV-PAM stops bone breakdown and allows for ongoing bone formation while revascularisation of bone occurs, we hypothesised that IV-PAM could act as an adjunct to traditional treatment to help heal the femoral heads. Our patient received nine once monthly doses of IV-PAM (1 mg/kg/dose) over 13 months, along with Petrie/broomstick casts and physiotherapy. Remarkably, over time, his femoral heads healed. Now, at 11-year follow-up, he has excellent functional and radiologic outcome with congruence between femoral head and acetabulum, no residual osteonecrosis and minimal loss of femoral head sphericity.

Choline acetyltransferase-expressing T cells are required to control chronic viral infection.

Although widely studied as a neurotransmitter, T cell-derived acetylcholine (ACh) has recently been reported to play an important role in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of Chat within the T cell compartment in mice ablated vasodilation in response to infection, impaired the migration of antiviral T cells into infected tissues, and ultimately compromised the control of chronic LCMV clone 13 infection. Our results reveal a genetic proof of function for ChAT in T cells during viral infection and identify a pathway of T cell migration that sustains antiviral immunity.

E3 ubiquitin ligase Mule targets ß-catenin under conditions of hyperactive Wnt signaling.

Wnt signaling, named after the secreted proteins that bind to cell surface receptors to activate the pathway, plays critical roles both in embryonic development and the maintenance of homeostasis in many adult tissues. Two particularly important cellular programs orchestrated by Wnt signaling are proliferation and stem cell self-renewal. Constitutive activation of the Wnt pathway resulting from mutation or improper modulation of pathway components contributes to cancer development in various tissues. Colon cancers frequently bear inactivating mutations of the adenomatous polyposis coli (APC) gene, whose product is an important component of the destruction complex that regulates ß-catenin levels. Stabilization and nuclear localization of ß-catenin result in the expression of a panel of Wnt target genes. We previously showed that Mule/Huwe1/Arf-BP1 (Mule) controls murine intestinal stem and progenitor cell proliferation by modulating the Wnt pathway via c-Myc. Here we extend our investigation of Mule's influence on oncogenesis by showing that Mule interacts directly with ß-catenin and targets it for degradation under conditions of hyperactive Wnt signaling. Our findings suggest that Mule uses various mechanisms to fine-tune the Wnt pathway and provides multiple safeguards against tumorigenesis.

Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies.

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-ß (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.

Mule Regulates the Intestinal Stem Cell Niche via the Wnt Pathway and Targets EphB3 for Proteasomal and Lysosomal Degradation.

The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.

Ablating the aryl hydrocarbon receptor (AhR) in CD11c+ cells perturbs intestinal epithelium development and intestinal immunity.

Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.

Idh1 mutations contribute to the development of T-cell malignancies in genetically engineered mice.

Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1) are key drivers of hematopoietic malignancies. Although these mutations are most commonly associated with myeloid diseases, they also occur in malignancies of the T-cell lineage. To investigate their role in these diseases and provide tractable disease models for further investigation, we analyzed the T-cell compartment in a conditional knock-in (KI) mouse model of mutant Idh1. We observed the development of a spontaneous T-cell acute lymphoblastic leukemia (T-ALL) in these animals. The disease was transplantable and maintained expression of mutant IDH1. Whole-exome sequencing revealed the presence of a spontaneous activating mutation in Notch1, one of the most common mutations in human T-ALL, suggesting Idh1 mutations may have the capacity to cooperate with Notch1 to drive T-ALL. To further investigate the Idh1 mutation as an oncogenic driver in the T-cell lineage, we crossed Idh1-KI mice with conditional Trp53 null mice, a well-characterized model of T-cell malignancy, and found that T-cell lymphomagenesis was accelerated in mice bearing both mutations. Because both IDH1 and p53 are known to affect cellular metabolism, we compared the requirements for glucose and glutamine in cells derived from these tumors and found that cells bearing the Idh1 mutation have an increased dependence on both glucose and glutamine. These data suggest that mutant IDH1 contributes to malignancy in the T-cell lineage and may alter the metabolic profile of malignant T cells.

Targeting Mitosis in Cancer: Emerging Strategies.

The cell cycle is an evolutionarily conserved process necessary for mammalian cell growth and development. Because cell-cycle aberrations are a hallmark of cancer, this process has been the target of anti-cancer therapeutics for decades. However, despite numerous clinical trials, cell-cycle-targeting agents have generally failed in the clinic. This review briefly examines past cell-cycle-targeted therapeutics and outlines how experience with these agents has provided valuable insight to refine and improve anti-mitotic strategies. An overview of emerging anti-mitotic approaches with promising pre-clinical results is provided, and the concept of exploiting the genomic instability of tumor cells through therapeutic inhibition of mitotic checkpoints is discussed. We believe this strategy has a high likelihood of success given its potential to enhance therapeutic index by targeting tumor-specific vulnerabilities. This reasoning stimulated our development of novel inhibitors targeting the critical regulators of genomic stability and the mitotic checkpoint: AURKA, PLK4, and Mps1/TTK.

Comparison of cast materials for the treatment of congenital idiopathic clubfoot using the Ponseti method: a prospective randomized controlled trial.

BACKGROUND: The Ponseti method of congenital idiopathic clubfoot correction has traditionally specified plaster of Paris (POP) as the cast material of choice; however, there are negative aspects to using POP. We sought to determine the influence of cast material (POP v. semirigid fibreglass [SRF]) on clubfoot correction using the Ponseti method. METHODS: Patients were randomized to POP or SRF before undergoing the Ponseti method. The primary outcome measure was the number of casts required for clubfoot correction. Secondary outcome measures included the number of casts by severity, ease of cast removal, need for Achilles tenotomy, brace compliance, deformity relapse, need for repeat casting and need for ancillary surgical procedures. RESULTS: We enrolled 30 patients: 12 randomized to POP and 18 to SRF. There was no difference in the number of casts required for clubfoot correction between the groups (p = 0.13). According to parents, removal of POP was more difficult (p < 0.001), more time consuming (p < 0.001) and required more than 1 method (p < 0.001). At a final follow-up of 30.8 months, the mean times to deformity relapse requiring repeat casting, surgery or both were 18.7 and 16.4 months for the SRF and POP groups, respectively. CONCLUSION: There was no significant difference in the number of casts required for correction of clubfoot between the 2 materials, but SRF resulted in a more favourable parental experience, which cannot be ignored as it may have a positive impact on psychological well-being despite the increased cost associated.

CONTEXTE: La méthode de Ponseti pour la correction du pied bot congénital idiopathique a de tout temps spécifié l'utilisation du plâtre de Paris comme matériau de choix; il y a toutefois certains inconvénients associés au plâtre de Paris. Nous avons voulu déterminer l'influence du matériau utilisé (plâtre de Paris c. fibre de verre semi-rigide) sur la correction du pied bot selon la méthode de Ponseti. MÉTHODES: Les patients ont été assignés aléatoirement soit au plâtre de Paris soit à la fibre de verre semi-rigide en vue de l'intervention de Ponseti. Le principal paramètre mesuré était le nombre de plâtres requis pour corriger le pied bot. Les paramètres secondaires incluaient le nombre de plâtres en fonction de la gravité, la facilité de retrait du plâtre, la nécessité de sectionner le tendon d'Achille, le port assidu de l'attelle, le retour de la difformité, la nécessité d'autres plâtres et interventions chirurgicales auxiliaires. RÉSULTATS: Nous avons inscrit 30 patients : 12 ont été assignés au plâtre de Paris et 18 à la fibre de verre. On n'a noté aucune différence entre les groupes quant au nombre de plâtres requis pour la correction du pied bot (p = 0,13). Selon les parents, le retrait du plâtre de Paris était plus difficile p < 0,001), prenait plus de temps (p < 0,001) et nécessitait le recours à plus d'une méthode (p < 0,001). Au moment du dernier suivi à 30,8 mois, les intervalles moyens avant un retour de la difformité nécessitant la pose d'un autre plâtre et/ou une chirurgie ont été de 18,7 et 16,4 mois dans les groupes traités au moyen de la fibre de verre semi-rigide et du plâtre de Paris, respectivement.. CONCLUSION: On n'a noté aucune différence significative entre les 2 matériaux quant au nombre de plâtres requis pour corriger le pied bot, mais la fibre de verre a donné lieu à une expérience plus agréable pour les parents, ce qui ne peut être ignoré en raison de l'impact potentiellement positif sur le bien-être psychologique, et ce, malgré un coût plus élevé.

Mule/Huwe1/Arf-BP1 suppresses Ras-driven tumorigenesis by preventing c-Myc/Miz1-mediated down-regulation of p21 and p15.

Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.

Quality indicators in pediatric orthopaedic surgery: a systematic review.

BACKGROUND: The ability to measure health system quality has become a priority for governments, the private sector, and the public. Quality indicators (QIs) refer to clear, measurable items related to outcomes. The use of QIs can initiate local quality improvement and track changes in quality over time as interventions are implemented. QUESTIONS/PURPOSES: We identified existing evidence-based indicators of quality pediatric orthopaedic care and evaluated published QIs that may be applicable to pediatric orthopaedic care. SEARCH STRATEGY: Using five standard search engines we searched the literature using terms such as "quality indicators," "orthopaedic surgery," and "pediatric." Study selection was performed in a stepwise manner, first by title, then abstract, and then full-text review. Of the 604 citations identified, 13 articles were selected for inclusion. Eight papers included only pediatric patients. RESULTS: The most commonly reported indicator was mortality followed by postoperative complications. Reoperation and readmission rates were also reported along with patient-centered QIs, although with less frequency. CONCLUSION: Although mortality and postoperative complications were the most frequently reported QIs, concern for their applicability was raised because of their relative infrequency in pediatrics. Patient-centered QIs appear to be the most useful tools reported, although their use is somewhat limited in the published literature. Although there are benefits and drawbacks to all reported QIs, patient-centered and surgeon-defined outcomes along with cost-effectiveness have important roles in evaluating the quality of pediatric orthopaedic care.

The alternative splice variant of protein tyrosine kinase 6 negatively regulates growth and enhances PTK6-mediated inhibition of ß-catenin.

Protein tyrosine kinase 6 (PTK6), also called breast tumor kinase (BRK), is expressed in epithelial cells of various tissues including the prostate. Previously it was shown that PTK6 is localized to epithelial cell nuclei in normal prostate, but becomes cytoplasmic in human prostate tumors. PTK6 is also primarily cytoplasmic in the PC3 prostate adenocarcinoma cell line. Sequencing revealed expression of wild type full-length PTK6 transcripts in addition to an alternative transcript lacking exon 2 in PC3 cells. The alternative transcript encodes a 134 amino acid protein, referred to here as ALT-PTK6, which shares the first 77 amino acid residues including the SH3 domain with full length PTK6. RT-PCR was used to show that ALT-PTK6 is coexpressed with full length PTK6 in established human prostate and colon cell lines, as well as in primary cell lines derived from human prostate tissue and tumors. Although interaction between full-length PTK6 and ALT-PTK6 was not detected, ALT-PTK6 associates with the known PTK6 substrates Sam68 and ß-catenin in GST pull-down assays. Coexpression of PTK6 and ALT-PTK6 led to suppression of PTK6 activity and reduced association of PTK6 with tyrosine phosphorylated proteins. While ALT-PTK6 alone did not influence ß-catenin/TCF transcriptional activity in a luciferase reporter assay, it enhanced PTK6-mediated inhibition of ß-catenin/TCF transcription by promoting PTK6 nuclear functions. Ectopic expression of ALT-PTK6 led to reduced expression of the ß-catenin/TCF targets Cyclin D1 and c-Myc in PC3 cells. Expression of tetracycline-inducible ALT-PTK6 blocked the proliferation and colony formation of PC3 cells. Our findings suggest that ALT-PTK6 is able to negatively regulate growth and modulate PTK6 activity, protein-protein associations and/or subcellular localization. Fully understanding functions of ALT-PTK6 and its impact on PTK6 signaling will be critical for development of therapeutic strategies that target PTK6 in cancer.

DDB2, an essential mediator of premature senescence.

Reactive oxygen species (ROS) is critical for premature senescence, a process significant in tumor suppression and cancer therapy. Here, we reveal a novel function of the nucleotide excision repair protein DDB2 in the accumulation of ROS in a manner that is essential for premature senescence. DDB2-deficient cells fail to undergo premature senescence induced by culture shock, exogenous oxidative stress, oncogenic stress, or DNA damage. These cells do not accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the antioxidant genes in vitro and in vivo. DDB2 represses antioxidant genes by recruiting Cul4A and Suv39h and by increasing histone-H3K9 trimethylation. Moreover, expression of DDB2 also is induced by ROS. Together, our results show that, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the antioxidant genes to cause persistent accumulation of ROS and induce premature senescence.

Single-dose versus multiple-dose antibiotic prophylaxis for the surgical treatment of closed fractures.

BACKGROUND AND PURPOSE: Recent meta-analyses have suggested similar wound infection rates when using single- or multiple-dose antibiotic prophylaxis in the operative management of closed long bone fractures. In order to assist clinicians in choosing the optimal prophylaxis strategy, we performed a cost-effectiveness analysis comparing single- and multiple-dose prophylaxis. METHODS: A cost-effectiveness analysis comparing the two prophylactic strategies was performed using time horizons of 60 days and 1 year. Infection probabilities, costs, and quality-adjusted life days (QALD) for each strategy were estimated from the literature. All costs were reported in 2007 US dollars. A base case analysis was performed for the surgical treatment of a closed ankle fracture. Sensitivity analysis was performed for all variables, including probabilistic sensitivity analysis using Monte Carlo simulation. RESULTS: Single-dose prophylaxis results in lower cost and a similar amount of quality-adjusted life days gained. The single-dose strategy had an average cost of $2,576 for an average gain of 272 QALD. Multiple doses had an average cost of $2,596 for 272 QALD gained. These results are sensitive to the incidence of surgical site infection and deep wound infection for the single-dose treatment arm. Probabilistic sensitivity analysis using all model variables also demonstrated preference for the single-dose strategy. INTERPRETATION: Assuming similar infection rates between the prophylactic groups, our results suggest that single-dose prophylaxis is slightly more cost-effective than multiple-dose regimens for the treatment of closed fractures. Extensive sensitivity analysis demonstrates these results to be stable using published meta-analysis infection rates.

Tumor suppression by ARF: gatekeeper and caretaker.

ARF is a vital tumor suppressor and its loss contributes significantly to cancer. The frequency in which ARF is mutated, deleted or silenced is second to the loss of p53. The most documented and widely accepted activity of ARF is mediated through its activation of the p53 transcriptional program by inhibiting MDM2 function. However, several lines of evidence have surfaced demonstrating that ARF possesses p53-independent functions. One of these p53-independent functions is ARF's regulation of the E2F family. The E2F/DP transcription factor is critical for cell cycle progression. The balance between activator and repressor E2Fs regulates the expression of E2F target genes and thus cell proliferation as well as other cellular functions such as checkpoint, chromosome assembly and repair. Through its ability to bind directly to DP1, ARF can cause dissociation of both activator and repressor E2Fs. While the regulation of the activator E2Fs is related to cell cycle arrest, there is evidence that the regulation of the repressors, E2F4 and E2F5, is significant in maintaining genomic stability.

Incidence and mortality of hip fractures in the United States.

CONTEXT: Understanding the incidence and subsequent mortality following hip fracture is essential to measuring population health and the value of improvements in health care. OBJECTIVE: To examine trends in hip fracture incidence and resulting mortality over 20 years in the US Medicare population. DESIGN, SETTING, AND PATIENTS: Observational study using data from a 20% sample of Medicare claims from 1985-2005. In patients 65 years or older, we identified 786,717 hip fractures for analysis. Medication data were obtained from 109,805 respondents to the Medicare Current Beneficiary Survey between 1992 and 2005. MAIN OUTCOME MEASURES: Age- and sex-specific incidence of hip fracture and age- and risk-adjusted mortality rates. RESULTS: Between 1986 and 2005, the annual mean number of hip fractures was 957.3 per 100,000 (95% confidence interval [CI], 921.7-992.9) for women and 414.4 per 100,000 (95% CI, 401.6-427.3) for men. The age-adjusted incidence of hip fracture increased from 1986 to 1995 and then steadily declined from 1995 to 2005. In women, incidence increased 9.0%, from 964.2 per 100,000 (95% CI, 958.3-970.1) in 1986 to 1050.9 (95% CI, 1045.2-1056.7) in 1995, with a subsequent decline of 24.5% to 793.5 (95% CI, 788.7-798.3) in 2005. In men, the increase in incidence from 1986 to 1995 was 16.4%, from 392.4 (95% CI, 387.8-397.0) to 456.6 (95% CI, 452.0-461.3), and the subsequent decrease to 2005 was 19.2%, to 369.0 (95% CI, 365.1-372.8). Age- and risk-adjusted mortality in women declined by 11.9%, 14.9%, and 8.8% for 30-, 180-, and 360-day mortality, respectively. For men, age- and risk-adjusted mortality decreased by 21.8%, 25.4%, and 20.0% for 30-, 180-, and 360-day mortality, respectively. Over time, patients with hip fracture have had an increase in all comorbidities recorded except paralysis. The incidence decrease is coincident with increased use of bisphosphonates. CONCLUSION: In the United States, hip fracture rates and subsequent mortality among persons 65 years and older are declining, and comorbidities among patients with hip fractures have increased.

A conserved phosphorylation site within the forkhead domain of FoxM1B is required for its activation by cyclin-CDK1.

The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G(1)/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G(2)/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G(2)/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.