CD38 Regulates Homing and Engraftment of CLL Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3873-3873
Author(s):  
Tiziana Vaisitti ◽  
Sara Serra ◽  
Valentina Audrito ◽  
Chris Pepper ◽  
Davide Rossi ◽  
...  
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
In Vitro Assays ◽  
Wild Type ◽  
Proliferating Cells

Abstract Abstract 3873 Chronic lymphocytic leukemia (CLL) is considered the result of a dynamic balance between proliferating cells in lymphoid organs and circulating cells resisting apoptosis. Re-circulation of leukemic cells from blood to growth-permissive niches represents an essential step in the maintenance and progression of the disease. This equilibrium is finely tuned by a set of surface molecules expressed by CLL cells and modulated in response to environmental conditions. We previously reported that CD38, an enzyme and a receptor, functionally cooperates with the CXCL12/CXCR4 axis, enhancing the ability of CLL cells to home to bone marrow and lymph nodes. In addition, the use of anti-CD38 mAbs can enhance or impair the chemotactic behavior of the neoplastic cells. New evidence also indicates that CD38 synergizes with the CD49d integrin, increasing adhesion of CLL cells to VCAM-1 or the CS-1 fibronectin fragment, two known ligands of CD49d. To complete the picture, CD38 expression denotes a CLL subset with increased activity of the matrix metalloproteinases MMP-9. Ligation of CD38 with specific antibodies increases MMP-9 secretion and the invasive properties of CLL cells, using in vitro assays. The effects on chemotaxis, adhesion and invasion are obtained through modulation of a ERK1/2-dependent pathway. To further confirm the involvement of CD38 in CLL homing to specific niches, in vivo experiments have been set using NOD/SCID/γ chain−/− (NSG) mice. The CLL-like cell line Mec-1, constitutively CD38−/CD49d+, was adopted as a model and compared to transfectants stably expressing wild-type (wt) CD38, as well a mutant lacking enzyme activities. Results after i.v. injections of tumor cells indicate that de novo expression of CD38 by Mec-1 cells increases growth kinetics in vivo with a higher proliferation rate and metastatic potential, as compared to the Mec-1 mock-trasfected cells. Both these features are lost when the animals are injected with the enzyme-deficient variant of CD38, suggesting that the enzymatic activity is critical for in vivo growth and re-circulation of Mec-1 cells. Microarray data confirm that the genetic signature of the CD38-enzyme mutant overlaps with the wild-type cell line, clearly distinct from cells transfected with CD38. The latter cell line shows up-modulation of several genes involved in chemotaxis and adhesion. All together, these results support the notion that CD38 is part of a complex network of molecules and signals, that regulate homing of CLL cells to growth-permissive niches, suggesting a relationship between the expression of CD38, the ability to migrate and invade and the poor clinical outcome of the CD38+ subset of patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals De novo disease-associated mutations in KIF1A dominant negatively inhibit axonal transport of synaptic vesicle precursors

2021 ◽  
Author(s):  
Yuzu Anazawa ◽  
Tomoki Kita ◽  
Kumiko Hayashi ◽  
Shinsuke Niwa
Keyword(s):  
Synaptic Vesicle ◽  
Axonal Transport ◽  
De Novo ◽  
Molecular Motor ◽  
Model Systems ◽  
In Vitro Assays ◽  
In Vivo Model ◽  
Wild Type

KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations.

scholarly journals Identification of a Novel Indel Mutation in GATA-1 Gene In Vivo and Verified Its Defective Function in Vitro

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4801-4801
Author(s):  
Junbin Huang ◽  
Lifeng Huang ◽  
Yingsi Lu ◽  
Chengming Zhu ◽  
Chun Chen
Keyword(s):  
Cell Line ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Male Infant ◽  
Hela Cell Line ◽  
Wild Type ◽  
K562 Cell Line ◽  
Indel Mutation

GATA-1 is a zinc finger TF encoded by the GATA-1 gene located on the short-arm of the X-chromosome. Most of GATA-1 mutations were located in exon2 or 3. We describe a male infant presented with dyserythropoietic anemia, who is harbored a novel GG deletion of exon6 in GATA-1 gene and varified its disrupted function in vitro. We confirmed that: (1) by using an unique GATA-1 antibody, which corresponded to amino acids 394-413 of human GATA1, the mutation of the proband led to GATA-1 protein and mRNA defective expressions in both peripheral blood and bone marrow; (2) demonstrated the expression of the GATA-1 mutated form is restricted to erythroblasts and red blood cells, consisted with the proband's abnormal erythropoiesis; (3) temporary transfection of GATA-1-wt(wild type) and GATA-1-indel in Hela cell line, which has no expression of GATA-1 originally, resulted in normal and defective expression both in protein and mRNA leve, respectively; (4) in a manipulated K562 cell line, that its GATA-1 had been knocked down by shRNA, stable transfection GATA-1-indel can not rescue K562 from GATA-1 deficiency.. Our reseach provide new insights into the clinically relevant in vivo function of the C-terminal domain of GATA-1 in human hematopoiesis and the results were validated in two seprerated cell line. Disclosures No relevant conflicts of interest to declare.

Human ROR1 Activates AKT and Accelerates Leukemia Cell Proliferation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3872-3872
Author(s):  
Suping Zhang ◽  
Liguang Chen ◽  
Ling Zhang ◽  
Jian Yu ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Embryonic Cells ◽  
Control Vector

Abstract Abstract 3872 ROR1 is an orphan-receptor-tyrosine-kinase-like developmental surface-antigen that is expressed by embryonic cells, chronic lymphocytic leukemia (CLL) cells, and neoplastic cells of many other types of cancer, but not by virtually all normal adult tissues. ROR1 can serve as a receptor for Wnt5a and potentially complex with other surface receptors for growth/survival factors elaborated by the tumor microenvironment or expressed by the tumor itself. In prior studies, we and others found that silencing ROR1 could impair tumor-cell growth and/or survival in vitro and in vivo. Although the CLL cells of ≥96% of all examined patients (N= 800) expressed ROR1, we found that a B-cell line derived from CLL cells, namely MEC1 (Leuk Res 23:127, 1999), lacked expression of this protein. This provided us with an opportunity to assess how introduction of ROR1 could affect the biology of this CLL-cell line, which has relatively slow doubling-time of ≈40 hours. We transfected MEC1 cells with either an expression vector encoding human ROR1 or a control vector, and then selected for stable transfectants in selection medium. MEC1 cells transfected with the ROR1 vector expressed high-levels of ROR1, but not MEC1 cells transfected with the control vector. MEC1 cells made to express ROR1 had higher levels of phosphorylated and activated-AKT and activated cAMP response-element-binding (CREB) protein than non-transfected MEC1 cells or cells transfected with the control vector. Furthermore, MEC1 made to express ROR1 had significantly greater proportions of cells in S/G2/M phase than did control-transfected cells 16 hours after transfer from serum-free medium to complete growth medium, indicating that MEC1 cells made to express ROR1 had increased relative proportions of cells undergoing cell division. Consistent with this, we found that ROR1+ MEC1 cells had significantly shorter doubling times in culture than did comparably cultured parental MEC1 cells or MEC1 cells transfected with the control vector. This study indicates that expression of ROR1 in the MEC1 CLL cell line can activate AKT/CREB and accelerate leukemia cell growth, providing us with a model system with which to interrogate the function of ROR1 in vitro. Disclosures: No relevant conflicts of interest to declare.

Induction of resistance to the Abelson inhibitor STI571 in human leukemic cells through gene amplification

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1758-1766 ◽  
Author(s):  
Philipp le Coutre ◽  
Elena Tassi ◽  
Marileila Varella-Garcia ◽  
Rossella Barni ◽  
Luca Mologni ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Amplification ◽  
Marker Chromosome ◽  
Kinase Domain ◽  
Leukemic Cells ◽  
Tyrosine Kinase Domain ◽  
Fish Analysis ◽  
Human Leukemic Cells

The 2-phenylaminopyrimidine derivative STI571 has been shown to selectively inhibit the tyrosine kinase domain of the oncogenicbcr/abl fusion protein. The activity of this inhibitor has been demonstrated so far both in vitro with bcr/abl expressing cells derived from leukemic patients, and in vivo on nude mice inoculated with bcr/abl positive cells. Yet, no information is available on whether leukemic cells can develop resistance to bcr/ablinhibition. The human bcr/abl expressing cell line LAMA84 was cultured with increasing concentrations of STI571. After approximately 6 months of culture, a new cell line was obtained and named LAMA84R. This newly selected cell line showed an IC50 for the STI571 (1.0 μM) 10-fold higher than the IC50 (0.1 μM) of the parental sensitive cell line. Treatment with STI571 was shown to increase both the early and late apoptotic fraction in LAMA84 but not in LAMA84R. The induction of apoptosis in LAMA84 was associated with the activation of caspase 3–like activity, which did not develop in the resistant LAMA84R cell line. LAMA84R cells showed increased levels of bcr/abl protein and mRNA when compared to LAMA84 cells. FISH analysis with BCR- and ABL-specific probes in LAMA84R cells revealed the presence of a marker chromosome containing approximately 13 to 14 copies of the BCR/ABL gene. Thus, overexpression of the Bcr/Abl protein mediated through gene amplification is associated with and probably determines resistance of human leukemic cells to STI571 in vitro.

scholarly journals Immunologic and Cytogenetic Studies of Chronic Lymphocytic Leukemic Cells

Blood ◽  
1965 ◽  
Vol 26 (2) ◽  
pp. 121-132 ◽  
Author(s):  
JOOST J. OPPENHEIM ◽  
JACQUELINE WHANG ◽  
EMIL FREI
Keyword(s):  
Tritiated Thymidine ◽  
Lymphocyte Transformation ◽  
Normal Subjects ◽  
Peripheral Circulation ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Serum Factors ◽  
Cytogenetic Studies

Abstract The lymphocyte transformation response of 17 chronic lymphocytic leukemia patients when tested in the short-term tissue culture with PHA-M, and PPD was found to be significantly decreased when compared to normal subjects. Serum factors were not found to be responsible for this cellular hyporesponsiveness. The proportions of immunoresponsive lymphocytes found in the patients’ peripheral circulation decreased as their white blood cell count increased. The transformation response to PHA-M was generally better than to PPD. Neither the PPD negative patients nor the normal PPD negative subjects’ cells responded to PPD stimulation in vitro. Monocytes usually would phagocytize particles added to the cultures and could thus be distinguished from the nonphagocytic proliferating lymphocytes which were the only cells that incorporated thymidine H3. Radioautographs of tritiated thymidine also revealed the rate of PPD lymphocyte transformation to be slower than with PHA-M. There were no significant differences in the proportions or the degree of leukemic and normal transformed lymphocyte labeling with tritiated thymidine. Cytogenetic studies revealed that the patients’ mitotic indices both in vivo and in vitro were markedly depressed. The modal chromosome number was 46 in each patient, and no cytogenetic abnormalities other than those due to exposure to radiation were found.

scholarly journals Complement alternative pathway activation by chronic lymphocytic leukemia cells: its role in their hepatosplenic localization

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 463-467 ◽  
Author(s):  
F Praz ◽  
G Karsenty ◽  
JL Binet ◽  
P Lesavre
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Alternative Pathway ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Complement Alternative Pathway ◽  
Acetylneuraminic Acid ◽  
Pathway Activation ◽  
Do So

Abstract Using affinity-purified 125I-F(ab')2 anti-human C3, we have investigated the ability of various leukemic cells to activate complement. Lymphocytes from patients with chronic lymphocytic leukemia (CLL) activated the alternative pathway, but cells from patients with other forms of leukemia or normal lymphocytes did not do so. The amount of C3 deposited on the CLL cells was significantly higher in patients with organomegaly (i.e., splenomegaly and/or hepatomegaly). Activation of complement by CLL cells as assessed by C3 deposition on the membrane occurred both in vivo and in vitro and was not related to the N- acetylneuraminic acid content of the membrane.

scholarly journals Substitutions in the Periplasmic Domain of Low-Abundance Chemoreceptor Trg That Induce or Reduce Transmembrane Signaling: Kinase Activation and Context Effects

2001 ◽  
Vol 183 (2) ◽  
pp. 671-679 ◽  
Author(s):  
Bryan D. Beel ◽  
Gerald L. Hazelbauer
Keyword(s):  
Context Effects ◽  
High Abundance ◽  
In Vitro Assays ◽  
Wild Type ◽  
Kinase Activation ◽  
Interaction Sites ◽  
Natural Enzyme ◽  
Signaling Receptors

ABSTRACT We extended characterization of mutational substitutions in the ligand-binding region of Trg, a low-abundance chemoreceptor ofEscherichia coli. Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class of substitutions made the receptor insensitive, reducing ligand-induced signaling, and another mimicked ligand occupancy, inducing signaling in the absence of ligand. We tested these deductions with in vitro assays of kinase activation and found that insensitive receptors activated the kinase as effectively as wild-type receptors and that induced-signaling receptors exhibited the low level of kinase activation characteristic of occupied receptors. Differential activation by the two mutant classes was not dependent on high-abundance receptors. Cellular context can affect the function of low-abundance receptors. Assays of chemotactic response and adaptational modification in vivo showed that increasing cellular dosage of mutant forms of Trg to a high-abundance level did not significantly alter phenotypes, nor did the presence of high-abundance receptors significantly correct phenotypic defects of reduced-signaling receptors. In contrast, defects of induced-signaling receptors were suppressed by the presence of high-abundance receptors. Grafting the interaction site for the adaptational-modification enzymes to the carboxyl terminus of induced-signaling receptors resulted in a similar suppression of phenotypic defects of induced-signaling receptors, implying that high-abundance receptors could suppress defects in induced-signaling receptors by providing their natural enzyme interaction sites intrans in clusters of suppressing and suppressed receptors. As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of phenotypic defects in induced-signaling forms of Trg involved assistance in adaptation, not signaling.

Recombinant mammalian DNA methyltransferase activity on model transcriptional gene silencing short RNA–DNA heteroduplex substrates

2010 ◽  
Vol 432 (2) ◽  
pp. 323-332 ◽  
Author(s):  
Jason P. Ross ◽  
Isao Suetake ◽  
Shoji Tajima ◽  
Peter L. Molloy
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Double Helix ◽  
In Vitro Assays ◽  
Enzyme Analysis ◽  
Transcriptional Gene Silencing ◽  
Restriction Enzyme Analysis

The biochemical mechanism of short RNA-induced TGS (transcriptional gene silencing) in mammals is unknown. Two competing models exist; one suggesting that the short RNA interacts with a nascent transcribed RNA strand (RNA–RNA model) and the other implying that short RNA forms a heteroduplex with DNA from the unwound double helix, an R-loop structure (RNA–DNA model). Likewise, the requirement for DNA methylation to enact TGS is still controversial. In vitro assays using purified recombinant murine Dnmt (DNA methyltransferase) 1-dN (where dN indicates an N-terminal truncation), 3a and 3b enzymes and annealed oligonucleotides were designed to question whether Dnmts methylate DNA in a RNA–DNA heteroduplex context and whether a RNA–DNA heteroduplex R-loop is a good substrate for Dnmts. Specifically, model synthetic oligonucleotides were used to examine methylation of single-stranded oligonucleotides, annealed oligonucleotide duplexes, RNA–DNA heteroduplexes, DNA bubbles and R-loops. Dnmt methylation activity on the model substrates was quantified with initial velocity assays, novel ARORA (annealed RNA and DNA oligonucleotide-based methylation-sensitive restriction enzyme analysis), tBS (tagged-bisulfite sequencing) and the quantitative PCR-based method MethylQuant. We found that RNA–DNA heteroduplexes and R-loops are poor substrates for methylation by both the maintenance (Dnmt1) and de novo (Dnmt3a and Dnmt3b) Dnmts. These results suggest the proposed RNA/DNA model of TGS in mammals is unlikely. Analysis of tagged-bisulfite genomic sequencing led to the unexpected observation that Dnmt1-dN can methylate cytosines in a non-CpG context in DNA bubbles. This may have relevance in DNA replication and silencing of transcriptionally active loci in vivo.

Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hsiangyu Hu ◽  
Nirmalya Saha ◽  
Yuting Yang ◽  
Sierrah Marie Grigsby ◽  
Rolf Marschalek ◽  
...  
Keyword(s):  
Cell Growth ◽  
Acute Leukemia ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Fusion Partner ◽  
Wild Type

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

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