The miR-106a~363Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27Kip1 cell cycle inhibitor.

The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363 Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363 Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27 Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.

Prognostic significance of acquired copy-neutral loss of heterozygosity in acute myeloid leukemia.

BACKGROUND: Chromosomal abnormalities are important in the diagnosis and prognosis of patients with acute myeloid leukemia (AML). Genomic microarray techniques detect recurrent copy-neutral loss of heterozygosity (cnLOH) in addition to copy number aberrations. However, the clinical utility has not been fully established. Therefore, in the current study, the authors examined the prognostic impact of acquired cnLOH in patients with AML, including complete remission (CR) rate, duration of CR, and overall survival (OS). METHODS: A total of 112 consecutive patients with AML who were undergoing chromosome genomic array testing (CGAT) at the Seattle Cancer Care Alliance were included in the current study. DNA from the bone marrow or blood was analyzed with a microarray platform with both single-nucleotide polymorphism (SNP) probes and non-SNP probes to identify acquired cnLOH. Results were correlated with cytogenetic, molecular, immunophenotypic, and other clinicopathological findings. RESULTS: Patients with cnLOH demonstrated a shorter duration of CR (hazard ratio, 1.87; P =.04) and worse OS (HR, 1.82; P = .03). Multivariate analyses confirmed the independent predictive value of cnLOH for early disease recurrence (P =.02). These results largely reflected those in patients with intermediate and unfavorable cytogenetics. Most strikingly, 13q cnLOH was found to demonstrate a 6.64-fold higher rate of disease recurrence (P =.006) and 3.45-fold worse OS (P = .02) and was enriched with the FLT3-ITD (Fms-related tyrosine kinase 3-internal tandem duplication) mutation. CONCLUSIONS: CnLOH has important prognostic significance in patients with AML. CGAT can replace imbalance fluorescence in situ hybridization and the authors recommend the routine use of CGAT to detect cnLOH, particularly among patients with intermediate-risk cytogenetics.

Prognostic value and functional consequences of cell cycle inhibitor p27Kip1 loss in medulloblastoma.

BACKGROUND: The cyclin-dependent kinase inhibitor p27Kip1 functions during normal cerebellar development and has demonstrated tumor suppressor functions in mouse models of medulloblastoma. Because P27 loss is associated with increased proliferation, we assessed whether P27 absence in surgical medulloblastoma specimens correlated with response to therapy in pediatric patients enrolled in two large studies. Additionally, we examined the functional consequence of p27Kip1 loss in the SmoA1 medulloblastoma model to distinguish whether p27Kip1 reduces tumor initiation or slows tumor progression. FINDINGS: Analysis of 87 well-characterized patient samples identified a threshold of P27 staining at which significant P27 loss correlated with poor patient outcome. The same criteria, applied to a second test set of tissues from 141 patients showed no difference in survival between patients with minimal P27 staining and others, suggesting that P27 levels alone are not a sufficient prognostic indicator for identifying standard-risk patients that may fail standard therapy. These findings were in contrast to prior experiments completed using a mouse medulloblastoma model. Analysis of cerebellar tumor incidence in compound mutant mice carrying the activated Smoothened (SmoA1) allele that were heterozygous or nullizygous for p27Kip1 revealed that p27Kip1 loss did not alter the frequency of tumor initiation. Tumors haploinsufficient or nullizygous for p27Kip1 were, however, more invasive and displayed a higher proliferative index, suggesting p27Kip1 loss may contribute to SmoA1 medulloblastoma progression. CONCLUSIONS: These studies revealed P27 loss affects medulloblastoma progression rather than initiation and that this putative biomarker should not be used for stratifying children with medulloblastoma to risk-based therapeutic regimens.

T-cell intrinsic and extrinsic mechanisms of p27Kip1 in the regulation of CD8 T-cell memory.

CD8 T cells exhibit dynamic alterations in proliferation and apoptosis during various phases of the CD8 T-cell response, but the mechanisms that regulate cellular proliferation from the standpoint of CD8 T-cell memory are not well defined. The cyclin-dependent kinase inhibitor p27(Kip1) functions as a negative regulator of the cell cycle in T cells, and it has been implicated in regulating cellular processes, including differentiation, transcription and migration. Here, we investigated whether p27(Kip1) regulates CD8 T-cell memory by T-cell-intrinsic or T-cell-extrinsic mechanisms, by conditional ablation of p27(Kip1) in T cells or non-T cells. Studies of T-cell responses to an acute viral infection show that p27(Kip1) negatively regulates the proliferation of CD8 T cells by T-cell-intrinsic mechanisms. However, the enhanced proliferation of CD8 T cells induced by T-cell-specific p27(Kip1) deficiency minimally affects the primary expansion or the magnitude of CD8 T-cell memory. Unexpectedly, p27(Kip1) ablation in non-T cells markedly augmented the number of high-quality memory CD8 T cells by enhancing the accumulation of memory precursor effector cells without increasing their proliferation. Further studies show that p27(Kip1) deficiency in immunizing dendritic cells fail to enhance CD8 T-cell memory. Nevertheless, we have delineated the T-cell-intrinsic, anti-proliferative activities of p27(Kip1) in CD8 T cells from its role as a factor in non-T cells that restricts the development of CD8 T-cell memory. These findings have implications in vaccine development and understanding the mechanisms that maintain T-cell homeostasis.

p27(Kip1) is a microtubule-associated protein that promotes microtubule polymerization during neuron migration.

The migration of cortical interneurons is characterized by extensive morphological changes that result from successive cycles of nucleokinesis and neurite branching. Their molecular bases remain elusive, and the present work describes how p27(Kip1) controls cell-cycle-unrelated signaling pathways to regulate these morphological remodelings. Live imaging reveals that interneurons lacking p27(Kip1) show delayed tangential migration resulting from defects in both nucleokinesis and dynamic branching of the leading process. At the molecular level, p27(Kip1) is a microtubule-associated protein that promotes polymerization of microtubules in extending neurites, thereby contributing to tangential migration. Furthermore, we show that p27(Kip1) controls actomyosin contractions that drive both forward translocation of the nucleus and growth cone splitting. Thus, p27(Kip1) cell-autonomously controls nucleokinesis and neurite branching by regulating both actin and microtubule cytoskeletons.

Proliferative reactive gliosis is compatible with glial metabolic support and neuronal function.

BACKGROUND: The response of mammalian glial cells to chronic degeneration and trauma is hypothesized to be incompatible with support of neuronal function in the central nervous system (CNS) and retina. To test this hypothesis, we developed an inducible model of proliferative reactive gliosis in the absence of degenerative stimuli by genetically inactivating the cyclin-dependent kinase inhibitor p27Kip1 (p27 or Cdkn1b) in the adult mouse and determined the outcome on retinal structure and function. RESULTS: p27-deficient Müller glia reentered the cell cycle, underwent aberrant migration, and enhanced their expression of intermediate filament proteins, all of which are characteristics of Müller glia in a reactive state. Surprisingly, neuroglial interactions, retinal electrophysiology, and visual acuity were normal. CONCLUSION: The benign outcome of proliferative reactive Müller gliosis suggests that reactive glia display context-dependent, graded and dynamic phenotypes and that reactivity in itself is not necessarily detrimental to neuronal function.

p27(Kip1) is required to maintain proliferative quiescence in the adult cochlea and pituitary.

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27(Kip1) Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27(Kip1) in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27(Kip1) even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27(Kip1) expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27(Kip1) expression in animals with established pituitary tumors, in an inducible "knock-on" model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27(Kip1) did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27(Kip1) expression in the maintenance of cellular quiescence and terminal differentiation.

Effect of Xpcl1 activation and p27(Kip1) loss on gene expression in murine lymphoma.

Mice lacking the p27(Kip1) Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a~363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27(Kip1) knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a~363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27(Kip1) deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27(Kip1) null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27(Kip1) deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a~363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a~363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a~363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a~363.

Comparative analysis of risk factors for acute graft-versus-host disease and for chronic graft-versus-host disease according to National Institutes of Health consensus criteria.

Risk factors for grades 2-4 acute graft-versus-host disease (GVHD) and for chronic GVHD as defined by National Institutes of Health consensus criteria were evaluated and compared in 2941 recipients of first allogeneic hematopoietic cell transplantation at our center. In multivariate analyses, the profiles of risk factors for acute and chronic GVHD were similar, with some notable differences. Recipient human leukocyte antigen (HLA) mismatching and the use of unrelated donors had a greater effect on the risk of acute GVHD than on chronic GVHD, whereas the use of female donors for male recipients had a greater effect on the risk of chronic GVHD than on acute GVHD. Total body irradiation was strongly associated with acute GVHD, but had no statistically significant association with chronic GVHD, whereas grafting with mobilized blood cells was strongly associated with chronic GVHD but not with acute GVHD. Older patient age was associated with chronic GVHD, but had no effect on acute GVHD. For all risk factors associated with chronic GVHD, point estimates and confidence intervals were not significantly changed after adjustment for prior acute GVHD. These results suggest that the mechanisms involved in acute and chronic GVHD are not entirely congruent and that chronic GVHD is not simply the end stage of acute GVHD.

Gene regulation and epigenetic remodeling in murine embryonic stem cells by c-Myc.

BACKGROUND: The Myc oncoprotein, a transcriptional regulator involved in the etiology of many different tumor types, has been demonstrated to play an important role in the functions of embryonic stem (ES) cells. Nonetheless, it is still unclear as to whether Myc has unique target and functions in ES cells. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the role of c-Myc in murine ES cells, we mapped its genomic binding sites by chromatin-immunoprecipitation combined with DNA microarrays (ChIP-chip). In addition to previously identified targets we identified genes involved in pluripotency, early development, and chromatin modification/structure that are bound and regulated by c-Myc in murine ES cells. Myc also binds and regulates loci previously identified as Polycomb (PcG) targets, including genes that contain bivalent chromatin domains. To determine whether c-Myc influences the epigenetic state of Myc-bound genes, we assessed the patterns of trimethylation of histone H3-K4 and H3-K27 in mES cells containing normal, increased, and reduced levels of c-Myc. Our analysis reveals widespread and surprisingly diverse changes in repressive and activating histone methylation marks both proximal and distal to Myc binding sites. Furthermore, analysis of bulk chromatin from phenotypically normal c-myc null E7 embryos demonstrates a 70-80% decrease in H3-K4me3, with little change in H3-K27me3, compared to wild-type embryos indicating that Myc is required to maintain normal levels of histone methylation. CONCLUSIONS/SIGNIFICANCE: We show that Myc induces widespread and diverse changes in histone methylation in ES cells. We postulate that these changes are indirect effects of Myc mediated by its regulation of target genes involved in chromatin remodeling. We further show that a subset of PcG-bound genes with bivalent histone methylation patterns are bound and regulated in response to altered c-Myc levels. Our data indicate that in mES cells c-Myc binds, regulates, and influences the histone modification patterns of genes involved in chromatin remodeling, pluripotency, and differentiation.

Evaluation of NIH consensus criteria for classification of late acute and chronic GVHD.

Historically, graft-versus-host disease (GVHD) beyond 100 days after hematopoietic cell transplantation (HCT) was called chronic GVHD, even if the clinical manifestations were indistinguishable from acute GVHD. In 2005, the National Institutes of Health (NIH) sponsored a consensus conference that proposed new criteria for diagnosis and classification of chronic GVHD for clinical trials. According to the consensus criteria, clinical manifestations rather than time after transplantation should be used in clinical trials to distinguish chronic GVHD from late acute GVHD, which includes persistent, recurrent, or late-onset acute GVHD. We evaluated major outcomes according to the presence or absence of NIH criteria for chronic GVHD in a retrospective study of 740 patients diagnosed with historically defined chronic GVHD after allogeneic HCT between 1994 and 2000. The presence or absence of NIH criteria for chronic GVHD showed no statistically significant association with survival, risks of nonrelapse mortality or recurrent malignancy, or duration of systemic treatment. Antecedent late acute GVHD was associated with an increased risk of nonrelapse mortality and prolonged treatment among patients with NIH chronic GVHD. Our results support the consensus recommendation that, with appropriate stratification, clinical trials can include patients with late acute GVHD as well as those with NIH chronic GVHD.

Treatment change as a predictor of outcome among patients with classic chronic graft-versus-host disease.

We analyzed outcomes for 668 patients who had systemic treatment for chronic graft-versus-host disease (cGVHD) to assess the utility of early treatment change for exacerbation of cGVHD as a surrogate for survival endpoints in clinical trials. Fifty-six percent of patients had treatment change within 2 years after diagnosis of cGVHD. The median onset of treatment change was 4.4 months (range: 0.3-50 months). The cumulative incidence of nonrelapse mortality (NRM) at 2 years was 16%, and overall survival (OS) at 2 years was 74%. In time-dependent Cox models, treatment change was associated with an increase in risk of NRM (hazard ratio, 2.53; 95% confidence interval, 1.7-3.7; P < .0001). The hazard ratio was attenuated by 6% per month of delay in treatment change. Our results confirm that exacerbation of cGVHD is associated with an increased risk of NRM and with decreased OS, but the strength of this association is not large enough to allow the use of early exacerbation as a surrogate for survival endpoints in clinical trials. Other measures of clinical benefit, such as response, will need to be developed as endpoints in phase II trials for patients with cGVHD.

A mechanism misregulating p27 in tumors discovered in a functional genomic screen.

The cyclin-dependent kinase inhibitor p27(KIP1) is a tumor suppressor gene in mice, and loss of p27 protein is a negative prognostic indicator in human cancers. Unlike other tumor suppressors, the p27 gene is rarely mutated in tumors. Therefore misregulation of p27, rather than loss of the gene, is responsible for tumor-associated decreases in p27 protein levels. We performed a functional genomic screen in p27(+/-) mice to identify genes that regulate p27 during lymphomagenesis. This study demonstrated that decreased p27 expression in tumors resulted from altered transcription of the p27 gene, and the retroviral tagging strategy enabled us to pinpoint relevant transcription factors. inhibitor of DNA binding 3 (Id3) was isolated and validated as a transcriptional repressor of p27. We further demonstrated that p27 was a downstream target of Id3 in src-family kinase Lck-driven thymic lymphomagenesis and that p27 was an essential regulator of Lck-dependent thymic maturation during normal T-cell development. Thus, we have identified and characterized transcriptional repression of p27 by Id3 as a new mechanism decreasing p27 protein in tumors.

Differential gene expression of p27Kip1 and Rb knockout pituitary tumors associated with altered growth and angiogenesis.

Mice lacking the p27Kip1 Cdk inhibitor, like mice lacking Rb, develop pituitary tumors involving pars intermedia melanotrophs, yet p27(Kip1) tumors are genetically distinct from Rb derived tumors as they exhibit haploid insufficiency. We compared tumors from mice with p27( Kip1) constitutive and tissue specific null mutations to tumors arising in tissue specific Rb knockout mice with the aim of determining whether they are distinguished by quantitative or qualitative differences. The rate of p27Kip1 knockout tumor development was strongly influenced by strain background due to polygenic strain modifiers in the C57BL/6J versus 129S4 strains but, unlike a prior report of Rb mutants, this impacted tumor incidence but not the tumor spectrum. p27Kip1 tumors were oligoclonal or polyclonal based on studies of X-chromosomal inactivation of Dock11. In contrast, Rb null tissue developed monoclonal neoplasms even in the absence of a requirement for Rb mutant clonal selection. Rb null tumors exhibited a higher proliferation rate and developed ischemic necrosis associated with an aberrant vasculature. p27Kip1 null tumors maintained normal vascular density, through a tumor cell dependent mechanism, but were more often hemorrhagic. Gene expression profiles distinguished p27Kip1 from Rb null tumors including significant differences in expression of Rb and E2F signature genes. Rb null tumors expressed higher levels of VEGF which, in other systems, is associated with dilated vessels, ineffective perfusion and tissue hypoxia. Mouse models lacking p27Kip1 and Rb may help us better understand the pathophysiology of MEN syndromes, retinoblastoma and other cancers that disrupt these important cell cycle inhibitors.

Genetic mosaics reveal both cell-autonomous and cell-nonautonomous function of murine p27Kip1.

Loss of the cyclin-dependent kinase inhibitor p27(Kip1) leads to an overall increase in animal growth, pituitary tumors, and hyperplasia of hematopoietic organs, yet it is unknown whether all cells function autonomously in response to p27(Kip1) activity or whether certain cells take cues from their neighbors. In addition, there is currently no genetic evidence that tumor suppression by p27(Kip1) is cell-autonomous because biallelic gene inactivation is absent from tumors arising in p27(Kip1) hemizygous mice. We have addressed these questions with tissue-specific targeted mouse mutants and radiation chimeras. Our results indicate that the suppression of pars intermedia pituitary tumors by p27(Kip1) is cell-autonomous and does not contribute to overgrowth or infertility phenotypes. In contrast, suppression of spleen growth and hematopoietic progenitor expansion is a consequence of p27(Kip1) function external to the hematopoietic compartment. Likewise, p27(Kip1) suppresses thymocyte hyperplasia through a cell-nonautonomous mechanism. The interaction of p27(Kip1) loss with epithelial cell-specific cyclin-dependent kinase 4 overexpression identifies the thymic epithelium as a relevant site of p27(Kip1) activity for the regulation of thymus growth.

Negative cell-cycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are capable of shifting from a state of relative quiescence under homeostatic conditions to rapid proliferation under conditions of stress. The mechanisms that regulate the relative quiescence of stem cells and its association with self-renewal are unclear, as is the contribution of molecular regulators of the cell cycle to these decisions. Understanding the mechanisms that govern these transitions will provide important insights into cell-cycle regulation of HSCs and possible therapeutic approaches to expand HSCs. We have investigated the role of two negative regulators of the cell cycle, p27(Kip1) and MAD1, in controlling this transition. Here we show that Mad1(-/-)p27(Kip1-/-) bone marrow has a 5.7-fold increase in the frequency of stem cells, and surprisingly, an expanded pool of quiescent HSCs. However, Mad1(-/-)p27(Kip1-/-) stem cells exhibit an enhanced proliferative response under conditions of stress, such as cytokine stimulation in vitro and regeneration of the haematopoietic system after ablation in vivo. Together these data demonstrate that the MYC-antagonist MAD1 and cyclin-dependent kinase inhibitor p27(Kip1) cooperate to regulate the self-renewal and differentiation of HSCs in a context-dependent manner.

Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis.

The p27(Kip1) protein is a cyclin-dependent kinase inhibitor that blocks cell division in response to antimitogenic cues. p27 expression is reduced in many human cancers, and p27 functions as a tumor suppressor that exhibits haploinsufficiency in mice. Despite the well characterized role of p27 as a cyclin-dependent kinase inhibitor, its mechanism of tumor suppression is unknown. We used Moloney murine leukemia virus to induce lymphomas in p27+/+ and p27-/- mice and observed that lymphomagenesis was accelerated in the p27-/- animals. To identify candidate oncogenes that collaborate with p27 loss, we used a high-throughput strategy to sequence 277 viral insertion sites derived from two distinct sets of p27-/- lymphomas and determined their chromosomal location by comparison with the Celera and public (Ensembl) mouse genome databases. This analysis identified a remarkable number of putative protooncogenes in these lymphomas, which included loci that were novel as well as those that were overrepresented in p27-/- tumors. We found that Myc activations occurred more frequently in p27-/- lymphomas than in p27+/+ tumors. We also characterized insertions within two novel loci: (i) the Jun dimerization protein 2 gene (Jundp2), and (ii) an X-linked locus termed Xpcl1. Each of the loci that we found to be frequently involved in p27-/- lymphomas represents a candidate oncogene collaborating with p27 loss. This study illustrates the power of high-throughput insertion site analysis in cancer gene discovery.

MAD1 and p27(KIP1) cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity.

To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27(KIP1). Generation of Mad1/p27(KIP1) double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27(KIP1), including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27(KIP1) double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27(KIP1) single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27(KIP1) operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.