Essential role for the p110delta isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110alpha, p110beta, p110gamma, and p110delta) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110delta isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110delta-selective inhibitor, suppressed both constitutive and Flt-3-stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110delta as a potential therapeutic target in AML.     

PI3K/AKT抑制剂对肺腺癌细胞株生长的影响

目的检测磷脂酰肌醇3-激酶（PI3K/AKT）特异性抑制剂Ly294002对肺腺癌细胞系XWLC-05增殖和凋亡的作用。方法 MTT法检测Ly294002对肺腺癌细胞XWLC-05体外细胞培养株诱导凋亡作用,流式细胞术测定不同条件下细胞的周期分布比例和细胞凋亡情况。结果 Ly294002对XWLC-05细胞株生长有抑制作用,但Ly294002需达到50μmol.L-1才能产生明显差异。Ly294002处理XWLC-05细胞24 h、48 h后G2/M期细胞相对增加,有统计学意义（P〈0.05）,凋亡细胞增加,但无统计学意义（P〉0.05）。结论 LY294002对肺腺癌细胞有抑制生长的作用,且抑制细胞增殖与导致细胞凋亡并无直接关系。     

Insulin-like growth factor signaling pathways in rat hepatic stellate cells: importance for deoxyribonucleic acid synthesis and hepatocyte growth factor production

It has been shown recently that insulin-like growth factor 1 (IGF-1) increases both DNA synthesis and hepatocyte growth factor (HGF) production in cultured hepatic stellate cells. In this study, we used selective blockers to investigate crucial signaling pathways for these effects of IGF-1 in cultured rat hepatic stellate cells. Both LY 294002 [a phosphatidylinositol 3-kinase (PI3-K) inhibitor], and rapamycin [a blocker of activation of the serine/threonine p70 S6 kinase (p70S6K), a molecule downstream from PI3-K] completely reversed the IGF-1-induced stimulation of DNA synthesis. Mitogen-activated protein kinase (MAPK) inhibition by PD 98059 had a less pronounced suppressory effect, although the used PD 98059 dose was fully effective in inhibiting MAPK phosphorylation. Both LY 294002 and PD 98059 lowered the IGF-1-induced increase of HGF in the medium by about 40%, but LY 294002 was 10 times more potent than PD 98059. Inhibition of p70S6K activation by rapamycin blocked IGF-1-induced DNA synthesis but not the increase in HGF. In conclusion, PI3-K (and, to some extent, MAPK) signaling pathways seem to be important for IGF-1-stimulated DNA synthesis and HGF production. DNA synthesis also seems to be dependent on rapamycin-sensitive activation of the PI3-K effector p70S6K.     

Phosphatidylinositol 3-kinases are involved in the all-trans retinoic acid-induced upregulation of CD38 antigen on human haematopoietic cells

All-trans retinoic acid (ATRA) is a specific inducer of CD38 antigen on marrow CD34+ cells as well as on blast cells in acute promyelocytic and myeloblastic leukaemia. The CD38 antigen contributes to the control of blast cell proliferation, and the upregulation of CD38 might constitute an element in the pathogenesis of retinoic acid syndrome. The aim of this study was to determine whether phosphoinositide 3-kinase (PI3-K) is involved in the modification of CD38 antigen expression on myeloid cells, as PI3-K plays a major role in the ATRA-induced granulocytic differentiation of HL-60 cells. We evaluated the effects of PI3-K inhibitors (wortmannin and LY294002) on the levels of CD38 antigen and mRNA in HL-60 and normal marrow CD34+ cells exposed to ATRA (1 micromol/l). The inhibitors prevented increase in CD38 mRNA expression and the overexpression of membrane CD38 antigen, without modification of the cytoplasmic level of this antigen. Interestingly, PI3-K activity was also necessary for CD38 expression on normal marrow CD34+ cells and for the ATRA-induced upregulation of CD157, a CD38-related antigen. In conclusion, PI3-K activity plays an essential role in the regulation of CD38 expression on human haematopoietic cells, and might constitute an interesting therapeutic target in haematological disorders involving CD38 overexpression.     

Biological characteristics of CD7 positive acute myelogenous leukaemia

We studied the biological characteristics of CD7+ acute myelogenous leukaemia (AML). We diagnosed nine out of 88 consecutive AML cases as CD7+ AML based on myeloperoxidase positivity and surface antigen expression. In eight of these nine cases more than 20% of leukaemic blasts were found to coexpress both CD7 and a myeloid-associated antigen, CD33, by a two-colour flow-cytometric assay, while in the remaining case more than 90% of blasts were positive for CD7 and myeloperoxidase. CD7+ AML was most frequently observed in M1 among AML subtypes according to the FAB classification. An early stage-specific antigen, CD34 was also expressed on leukaemic blasts from eight of these nine cases. Neither the T-cell receptor (TcR)-beta nor the TcR-gamma gene was clonally rearranged in any of the cases. We then studied the proliferative responses to stimulation by various growth factors. Among interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF), IL-3 showed the strongest stimulatory effect on DNA synthesis and leukaemic blast colony formation in 8/9 and 6/8 CD7+ AML cases examined, respectively. On the other hand, the strongest stimulatory effect exerted by IL-3 on blast colony formation was observed in only six out of the 33 CD7- AML cases examined. Furthermore, CD7+ AML blasts could proliferate in response to stem cell factor (SCF); SCF alone showed stimulatory effects on blast colony formation (7/8 cases), and in 5/7 SCF-responding cases, stimulatory effects of SCF were more potent than those of IL-3. In addition, SCF enhanced blast colony formation synergistically with IL-3 in four of these seven cases. These data suggest that progenitor cells of CD7+ AML may possess the biological properties characteristic of immature haematopoietic stem cells.     

Autoimmunity, intestinal lymphoid hyperplasia, and defects in mucosal B-cell homeostasis in patients with PTEN hamartoma tumor syndrome

The Phosphatase And Tensin Homolog Deleted On Chromosome 10 (PTEN) regulates the phosphoinositol-3-kinase (PI3K)-AKT signaling pathway. In a series of 34 patients with PTEN mutations, we described gastrointestinal lymphoid hyperplasia, extensive hyperplastic tonsils, thymus hyperplasia, autoimmune lymphocytic thyroiditis, autoimmune hemolytic anemia, and colitis. Functional analysis of the gastrointestinal mucosa-associated lymphoid tissue revealed increased signaling via the PI3K-AKT pathway, including phosphorylation of S6 and increased cell proliferation, but also reduced apoptosis of CD20(+)CD10(+) B cells. Reduced activity of PTEN therefore affects homeostasis of human germinal center B cells by increasing PI3K-AKT signaling via mammalian target of rapamycin as well as antiapoptotic signals.     

Effects of interleukin-4 and interleukin-6 on the proliferation of CD34+ and CD34- blasts from acute myelogenous leukemia.

We studied the effects of interleukin-4 (IL-4) and IL-6 on the growth of leukemic blasts from 40 patients with acute myelogenous leukemia (AML). Patients were selected on the basis of negativity for a series of B-cell antigens including CD10 and CD19. Twenty-one cases were CD34-positive (CD34+) (greater than 15% of blasts) and the remaining 19 were CD34-negative (CD34-) (less than 3% of blasts). IL-4 alone (100 U/ml) could stimulate either DNA synthesis (with greater than 2.0 stimulation index) or leukemic blast colony formation in 24 of 40 AML patients. In the presence of other growth factors, IL-4 showed divergent effects on IL-3-, granulocyte-macrophage colony-stimulating factor-, granulocyte colony-stimulating factor-, or erythropoietin-dependent colony formation. These effects of IL-4 were observed in both CD34+ and CD34- AML cases. IL-6 (100 U/mL) alone could not stimulate DNA synthesis and blast colony formation except for one CD34+ case. On the other hand, IL-6 showed synergistic effects on IL-3- and IL-4-dependent blast colony formation in 10 of 12 and 7 of 9 CD34+ AML cases, respectively. Among CD34- AML cases, such synergism was seen only in 1 of 12 cases for IL-3-dependent colony formation and in 3 of 7 cases for IL-4-dependent colony formation. The divergent effect of IL-4 and the synergistic effect of IL-6 were also observed in purified CD34+ leukemic blast populations, indicating that these phenomena are not mediated by accessory cells. The present study suggests that IL-4, alone or in combination with other growth factors, has divergent effects on the growth of AML progenitors irrespective of the CD34 expression, and that IL-6 acts synergistically with IL-3 or IL-4 on the growth of leukemic progenitors preferentially in CD34+ AML.     

PI-103在体外抗急性髓细胞白血病效应的实验研究

目的研究急性髓细胞白血病（AML）细胞P13K-Akt—mTOR信号转导通路各基因的表达及PI-103在体外对AML细胞增殖、凋亡及细胞周期的影响。方法RT-PCR法检测AML细胞P13K、Akt、mTORmRNA的表达;四甲基偶氮唑蓝法检测PI-103对AML细胞的增殖作用;流式细胞仪检测PI-103对AML细胞的凋亡率和细胞周期的影响。结果①81．25％的AML患者表达P13K基因,87．5％的患者表达mTOR基因,50％的患者同时表达此两种基因。②PI-103能明显提高PI3K—Akt-mTOR信号通路持续活化的AML细胞的增殖抑制率和凋亡率、阻滞细胞于G0／G1期（P均〈0．05）,且与剂量显著相关（P均〈0．05）。结论大部分AML患者存在PI3K-Akt-mTOR信号转导通路的异常活化;PI-103可通过PI3K、mTOR信号通路抑制AML细胞增殖、促进其凋亡。     

Role for interleukin-6 and insulin-like growth factor-I via PI3-K/Akt pathway in the proliferation of CD56- and CD56+ multiple myeloma cells

OBJECTIVES: Several studies including ours have suggested that lack of CD56 in multiple myeloma (MM) defines a unique patient subset with poorer prognosis. However, the mechanism underlying this aggressive behavior of CD56(-) MM has not been well elucidated. Interleukin-6 (IL-6) or insulin-like growth factor I (IGF-I) induce proliferation of MM cells. In this study, we report about the relationship between CD56 expression and responsiveness to these cytokines. METHODS: We sorted out both CD56(-) and CD56(+) fractions from MM cell lines such as KMS-21-BM and U-266, and investigated their different responsiveness to IL-6 or IGF-I. Furthermore, we compared the effects of these cytokines on the regulation of cell-cycle distribution between CD56(-) and CD56(+) cells. RESULTS: Although CD56(-) cells in both KMS-21-BM and U-266 cells responded significantly to IL-6, CD56(+) cells did not. Ki-67(+) cells in the CD56(-) cells were significantly increased by IL-6. Western blotting showed that IL-6 phosphorylated Akt, and upregulated and downregulated the level of cyclin D1 and p27 protein in the CD56(-) KMS-21-BM cells, respectively. LY-294002 completely blocked these effects of IL-6. On the other hand, Ki-67(+) cells in the CD56(+) cells did not respond to IL-6. Anti-IGF-I mAb significantly reduced Ki-67(+) cells only in the CD56(+) cells. IGF-I phosphorylated Akt and upregulated cyclin D1 in the CD56(+) KMS-21-BM cells, which was completely blocked by LY294002. CONCLUSIONS: These results suggest that CD56(-) and CD56(+) MM cells could be stimulated by IL-6 and IGF-I, respectively, via PI3-K/Akt pathway, and provide useful information for anticytokine therapies.     

PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival

Growth factors are known to favor both proliferation and survival of hepatocytes. In this work, we investigated the role of 2 main signaling pathways, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK), in these processes. First, evidence was provided that the PI3K cascade as well as the MEK/ERK cascade is a key transduction pathway controlling hepatocyte proliferation, as ascertained by arrest of DNA synthesis in the presence of LY294002, a specific PI3K inhibitor. Inhibition of FRAP/mTOR by rapamycin also abrogated DNA replication and protein synthesis induced by growth factor. We showed that expression of cyclin D1 at messenger RNA (mRNA) and protein levels was regulated by this pathway. We highlighted that 4E-BP1 phosphorylation was not activated by epidermal growth factor (EGF) but was under an insulin-regulation mechanism through a PI3K-FRAP/mTOR activation that could account for the permissive role of insulin on hepatocyte proliferation. No interference between the MEK/ERK pathway and 4E-BP1 phosphorylation was detected, whereas p70S6K phosphorylation induced by EGF was under a U0126-sensitive regulation. Last, we established that the antiapoptotic function of EGF was dependent on MEK, whereas LY294002 and rapamycin had no direct effect on cell survival. Taken together, these data highlight the regulation and the role of 2 pathways that mediate growth-related response by acting onto distinct steps. In conclusion, hepatocyte progression in late G1 phase induced by EGF generates survival signals depending on MEK activation, whereas PI3K and MEK/ERK cascades are both necessary for hepatocyte replication.     

Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin

Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolytic/gluconeogenic pathway that can be dephosphorylated and activated in response to insulin. However, the signaling cascades involved in this insulin effect have not been established. In this work we have investigated the potential involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mitogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nM, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nM wortmannin. Likewise, wortmannin at 100 and 500 nM, elicited partial and total inhibitions of insulin-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could be involved in PK-L activation by insulin. Three facts appear to reinforce this hypothesis: 1) the selective and complete inhibition of the PI 3-K/protein kinase B pathway by LY294002 (50 microM) was accompanied by a partial blockade of insulin-induced PK-L activation; 2) when signaling through the MAPK cascade was selectively suppressed by the presence of PD98059 (50 microM), a 50% reduction of insulin-induced activation of PK-L was observed; and 3) the effect of PD98059 (50 microM) on PK-L activation was reinforced by the additional presence of 100 nM wortmannin. We also observed that the blockade of p70 S6-kinase by rapamycin did not affect the activation of PK-L by insulin. From these findings it can be concluded that both PI 3-K and MAPK pathways, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin in rat hepatocytes.     

HIV-Nef enhances interleukin-2 production and phosphatidylinositol 3-kinase activity in a human T cell line

OBJECTIVE: The Nef protein has a major influence on disease pathogenesis in HIV-infected individuals. The objective of the present study was to examine the effects of Nef on T lymphocyte activation and associated signalling events. DESIGN: A recombinant vaccinia expression system was used to express Nef in a human T cell line. Stimulation of these cells with anti-CD28 antibody, and either phorbol 12-myristate 13-acetate (PMA) or anti-CD3, activates signal transduction pathways and results in IL-2 production and IL-2 receptor alpha-chain (CD25) expression. Cellular responses were examined in cells expressing either Nef or an irrelevant control protein. METHODS: Activation of signalling was assessed by immunoblot analysis, or by in-vitro phosphatidylinositol 3-kinase (PI3K) assays. IL-2 production was measured by enzyme-linked immunosorbent assay, and CD25 cell surface expression was examined using flow cytometry. RESULTS: Infection of cells with recombinant vaccinia expressing HIV-nef resulted in a marked increase in the production of IL-2 when cells were activated. The enhanced IL-2 response was accompanied by an increase in the level of PI3K activity. IL-2 production remained sensitive to inhibition with the PI3K competitive inhibitor Ly294002, and to the fungal macrolide, rapamycin. In contrast, CD25 expression was not affected, and there were no measurable changes to nuclear factor kappaB (NFkappaB) activation pathways. CONCLUSION: Enhanced IL-2 production in stimulated T cells expressing HIV-Nef is associated with increased activation of PI3K-dependent signalling pathways. The results support a model in which Nef affects HIV disease progression by distorting T cell responses.     

Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL- and mutant FLT3-expressing cells

Mediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhi-bitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL–, and induce apoptosis of BCR-ABL–expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL– and mutant FLT3-expressing cells both in vitro and in vivo.     

PI3K在CD_3mAb活化T细胞信号转导中的作用

目的探讨磷脂酰肌酶-3激酶（PI3K）在CD3mAb活化T细胞信号转导途径中的作用。方法分离获取健康人外周血单个核细胞（PBMC）,用不同浓度的PI3K特异性抑制剂LY294002处理后再用CD3mAb活化T细胞。0、6、12、24、48、72h后检测总T细胞CD69分子的表达及IL-2表达情况,培养10d后计数总T细胞的增殖情况。结果LY294002呈浓度依赖性地抑制总T细胞CD69的表达、IL-2的产生和T细胞增殖。结论PI3K参与CD3mAb诱导T淋巴细胞活化的信号转导途径,对T细胞的充分活化必不可少。     

The soluble form of interleukin-6 receptor modulates cell proliferation by acute myeloblastic leukemia blast cells

 As interleukin-6 (IL-6) has been shown to have diverse effects on blast cell growth in acute myeloblastic leukemia (AML), and as a soluble (s) form of IL-6 receptor (IL-6R) agonizes IL-6 effects in many cell types, we investigated whether sIL-6R was able to modulate clonogenic blast cell growth in AML. The proliferation responses of eight autonomously growing AML cell lines and eight primary AML blast cell samples were compared with their IL-6 and sIL-6R expression. Only three of the 16 AML samples were influenced by IL-6, two of them being stimulated and one inhibited by it. The sIL-6R-induced responses were more frequent, however, and, in contrast to those by IL-6, always stimulatory: clonogenic cell growth in six of the 16 AML samples was stimulated by sIL-6R treatment. All the cell lines and four of the seven primary blast cell samples analyzed expressed IL-6, and the expression was associated with unresponsiveness to exogenous IL-6. sIL-6R was also frequently expressed by AML cells: only one of the samples was negative for it. However, there was no correlation between sIL-6R expression and the responsiveness of cells to exogenous sIL-6R. The work presented here shows that sIL-6R is able to stimulate blast cell growth in AML. As AML blast cells are provided by exogenous IL-6 and sIL-6R in a bone marrow environment, and as many of them also express IL-6 and sIL-6R themselves in vitro, it is possible that signaling through the IL-6/sIL-6R system plays a role in maintaining their growth also in vivo. Received: February 20, 1998 / Accepted: November 26, 1998     

mTOR信号通路抑制剂和DNA甲基转移酶抑制剂对人胃癌细胞株Syk表达的影响

背景：哺乳动物雷帕霉素靶蛋白（roTOR）信号途径和DNA甲基化均参与肿瘤的发生、发展。脾酪氨酸激酶（Syk）是一种候选抑癌基因,与肿瘤发生有关。目的：研究mTOR信号通路抑制剂和DNA甲基转移酶抑制剂对人胃癌细胞株Syk表达的调控作用。方法：分别单独或联合应用mTOR抑制剂雷帕霉素（RAPA）、DNA甲基转移酶抑制剂5-氮杂-2’-脱氧胞苷（5-Aza—dC）、磷脂酰肌醇-3-激酶／丝苏氨酸蛋白激酶（P13K／Akt）抑制剂LY294002干预人胃癌细胞株MKN45和SGC7901,以细胞计数法检测细胞增殖能力,实时定量PCR法检测Syk表达情况,亚硫酸氢盐修饰后测序法检测DNA甲基化改变。结果：单独应用5-Aza—dC对细胞增殖抑制不明显,与RAPA、LY294002联合则显著抑制细胞增殖。单独应用5-Aza-dC可通过抑制DNA甲基化上调胃癌细胞的Syk表达,联合应用5-Aza—dc、RAPA和LY294002则显著上调Syk表达,但未进一步抑制DNA甲基化。结论：抑制mTOR信号通路可间接增强DNA甲基转移酶抑制剂上调Syk表达,从而抑制胃癌细胞生长。     

Interleukin-1 alpha also induces granulocyte-macrophage colony-stimulating factor in immature normal bone marrow cells.

The cytokine interleukin-1 (IL-1) plays a role in the regulation of normal as well as leukemic hematopoiesis. In acute myeloid leukemia (AML), IL-1 induces autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF) production, and these factors may then synergistically induce proliferation in AML blast cells. In this report, we show that IL-1 stimulates DNA synthesis of highly enriched normal bone marrow blast cells (CD34 positive, adherent cell depleted, CD3/CD14/CD15 negative). The stimulative effect of IL-1 can be blocked with neutralizing anti-TNF alpha and anti-GM-CSF antibodies and, most efficiently, by the combination of anti-TNF alpha and anti-GM-CSF, but not with anti-G-CSF antibody, suggesting that IL-1-induced proliferation was initiated through TNF and GM-CSF release. Concentrations of TNF and GM-CSF increased in the culture medium of normal bone marrow blast cells after IL-1 induction. Of the IL-1-induced cells, 12% were positive for GM-CSF mRNA by in situ hybridization, as opposed to 6% of non-induced cells. Thus, in addition to its effect on leukemic blast cells, IL-1 also acts on normal marrow blast cells. We propose a scheme where IL-1 stimulation of normal bone marrow blast cells leads to the induction of TNF alpha and GM-CSF, which in association stimulate DNA synthesis efficiently according to a paracrine or autocrine mechanism within the marrow blast cell compartment.     

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce cell cycle progression through the synthesis of c-Myc protein by internal ribosome entry site-mediated translation via phosphatidylinositol 3-kinase pathway in human factor-dependent leukemic cells

To investigate the roles of c-myc during hematopoietic proliferation induced by growth factors, we used factor-dependent human leukemic cell lines (MO7e and F36P) in which proliferation, cell cycle progression, and c-Myc expression were strictly regulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). In these cell lines, both c-myc mRNA and c-Myc protein stability were not affected by GM-CSF and IL-3, suggesting a regulation of c-Myc protein at the translational level. However, rapamycin, an inhibitor of cap-dependent translation, did not block c-myc induction by GM-CSF and IL-3. Thus, we studied the cap-independent translation, the internal ribosome entry site (IRES), during c-Myc protein synthesis using dicistronic reporter gene plasmids and found that GM-CSF and IL-3 activated c-myc IRES to initiate translation. c-myc IRES activation, c-Myc protein expression, and cell cycle progression were all blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002. In another factor-dependent cell line, UT7, we observed the cell cycle progression and up-regulation of c-Myc protein, c-myc mRNA, and c-myc IRES simultaneously, which were all inhibited by LY294002. Results indicate that hematopoietic growth factors induce cell cycle progression via IRES-mediated translation of c-myc though the PI3K pathway in human factor-dependent leukemic cells.