Selective growth inhibition in BRAF mutant thyroid cancer by the mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244

CONTEXT: Activating mutations in the BRAF gene, primarily at V600E, are associated with poorer outcomes in patients with papillary thyroid cancer. MAPK kinase (MEK), immediately downstream of BRAF, is a promising target for ras-raf-MEK-ERK pathway inhibition. OBJECTIVE: The objective of the investigation was to study the efficacy of a MEK1/2 inhibitor in thyroid cancer preclinical models with defined BRAF mutation status. EXPERIMENTAL DESIGN: After treatment with the potent MEK 1/2 inhibitor AZD6244, MEK inhibition and cell growth were examined in four BRAF mutant (V600E) and two BRAF wild-type thyroid cancer cell lines and in xenografts from a BRAF mutant cell line. RESULTS: AZD6244 potently inhibited MEK 1/2 activity in thyroid cancer cell lines regardless of BRAF mutation status, as evidenced by reduced ERK phosphorylation. Four BRAF mutant lines exhibited growth inhibition at low doses of the drug, with GI50 concentrations ranging from 14 to 50 nm, predominantly via a G0/G1 arrest, comparable with findings in a sensitive BRAF mutant melanoma cell line. In contrast, two BRAF wild-type lines were significantly less sensitive, with GI50 values greater than 200 nm. Nude mouse xenograft tumors derived from the BRAF mutant line ARO exhibited dose-dependent growth inhibition by AZD6244, with effective treatment at 10 mg/kg by oral gavage. This effect was primarily cytostatic and associated with marked inhibition of ERK phosphorylation. CONCLUSION: AZD6244 inhibits the MEK-ERK pathway across a spectrum of thyroid cancer cells. MEK inhibition is cytostatic in papillary thyroid cancer and anaplastic thyroid cancer cells bearing a BRAF mutation and may have less impact on thyroid cancer cells lacking this mutation.     

Differential induction of TNF-alpha and NOS2 by mitogen-activated protein kinase signaling pathways during Mycobacterium bovis infection

The role of mitogen-activated protein kinase (MAPK) signaling pathways in the regulation of TNF-alpha and NOS2 production by human monocytes infected with Mycobacterium bovis BCG was examined. Inhibition studies showed that ERK1/2 and p38 MAPK activation were necessary for the monocyte response to M. bovis infection. Analysis of MAPK activation showed rapid phosphorylation of ERK1/2 and p38 in response to M. bovis BCG. Phosphorylation was not due to an autocrine effect of TNF-alpha secretion, since an anti-TNF-alpha antibody had no significant effect on the levels of p38 phosphorylation. The inhibitor PD98059 significantly reduced M. bovis BCG-induced TNF-alpha production and almost completely abrogated phosphorylation of ERK1/2; in addition the potent MEK inhibitor U0126 also abrogated phosphorylation. In contrast, studies using inhibitors selective for ERK1/2 and p38 showed that p38 plays an essential role in the induction of NOS2, whereas the role of ERK1/2 was minor. These results suggest that ERK1/2 and p38 kinases differentially regulate the M. bovis BCG-mediated induction of TNF-alpha and NOS2 in human monocytes.     

Selective inhibition of human leukemia cell growth and induction of cell cycle arrest and apoptosis by pseudolaric acid B

Purpose  

The leukemias account for the largest number of cases of childhood cancer and remain the primary cause of cancer-related mortality among children in the United States. There is a need for novel antileukemia agents due to toxicity and resistant to existing chemotherapeutic agents. In this study, the effects of pseudolaric acid B (PAB) on three human leukemia cell lines, acute promyelocytic leukemia HL-60 cells, acute lymphoblastic leukemia CCRF-CEM cells, and human chronic myeloid leukemia blast-phase K562 cells were investigated in vitro, compared to normal human peripheral blood mononuclear cells (PBMC).     

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.     

Activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal regulated kinase (ERK) signaling pathway is involved in myeloid lineage commitment

Common lymphoid progenitors (CLPs) are lymphoid-lineage-committed progenitor cells. However, they maintain a latent myeloid differentiation potential that can be initiated by stimulation with interleukin-2 (IL-2) via ectopically expressed IL-2 receptors. Although CLPs express IL-7 receptors, which share the common gamma chain with IL-2 receptors, IL-7 cannot initiate lineage conversion in CLPs. In this study, we demonstrate that the critical signals for initiating lineage conversion in CLPs are delivered via IL-2 receptor beta (IL-2R beta) intracellular domains. Fusion of the A region of the IL-2R beta cytoplasmic tail to IL-7R alpha enables IL-7 to initiate myeloid differentiation in CLPs. We found that Shc, which associates with the A region, mediates lineage conversion signals through the mitogen activated protein kinase (MAPK) pathway. Because mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitors completely blocked IL-2-mediated lineage conversion, MAPK activation, specifically via the MEK/ERK pathway, is critically involved in the initiation of this event. Furthermore, formation of granulocyte/macrophage (GM) colonies by hematopoietic stem cells, but not by common myeloid progenitors (CMPs), was severely reduced in the presence of MEK/ERK inhibitors. These results demonstrate that activation of MEK/ERK plays an important role in GM lineage commitment.     

Transport stress induces apoptosis in rat myocardial tissue via activation of the mitogen-activated protein kinase signaling pathways

The present study aimed to elucidate the mechanism of myocardial damage induced by simulated transport stress. Sprague–Dawley rats were subjected to 35 °C and 60 rpm (0.1×g rcf) on a constant temperature shaker. The blood samples were prepared for detection of epinephrine (E), norepinephrine (NE), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and serum cardiac troponin T (cTNT); myocardium samples were prepared for morphological examination and signaling protein quantitative. The result showed that plasma norepinephrine (NE) and epinephrine (E) concentrations increased in all stressed groups (P < 0.01). Levels of serum cardiac troponin T (cTNT) were elevated in both the S2d (P < 0.05) and S3d groups (P < 0.01). The concentration of plasma BNP was increased significantly in S3d group (P < 0.05); the difference in ANP was not remarkable. Morphological observation demonstrated obvious microstructure and ultrastructure damage after simulated transport stress. There was also a significant increase in the number of TUNEL-positive cardiomyocytes in stressed hearts. Western blot analysis found that the mitogen-activated protein kinase (MAPK) pathways were activated by strengthening phosphorylation of ASK-1, JNK, P38 and ERK in rat myocardial tissue after simulated transport stress (P < 0.05, P < 0.01). In addition, the ratio of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins was increased in stressed rats (P < 0.01), and the amount of cleaved-caspase3 increased in all stressed rats (P < 0.01). The expression of cleaved-caspase9 protein was also elevated in S2d and S3d groups (P < 0.01). Consequently simulated transport stress induced obvious myocardial damage, which may be attributed to the activation of caspase 9-mediated mitochondrial apoptotic pathway and MAPK pathways.     

Dual effects of arsenic trioxide (As2O3) on non-acute promyelocytic leukaemia myeloid cell lines: induction of apoptosis and inhibition of proliferation

Clinical efficacy of As2O3 has been shown in patients with relapsed acute promyelocytic leukaemia (APL). There is evidence that the effects of As2O3 are not restricted to events specific for APL. As2O3 might target mechanisms involved in the pathogenesis of other malignancies. We assessed susceptibility to induction of apoptosis by As2O3 and cytostatics in 22 myeloid and non-myeloid malignant cell lines. As2O3 was used in concentrations of 0.01-10 micromol/l. Cell lines displayed different kinetics of response and different sensitivity to As2O3. The minimum concentration of As2O3 for induction of apoptosis was 0.1 micromol/l. High concentrations of As2O3 (5 micromol/l) induced apoptosis in a large proportion of cells in all cell lines tested. Low (1 micromol/l As2O3) concentrations induced apoptosis in NB-4, HL-60, U-937, CEM, HL-60, KG-1a, PBL-985, ML-2 and MV-4-11, but not in HEL, K-562, KG-1 and Jurkat up to 35 d of incubation. However, the non-apoptotic population of 1 micromol/l As2O3-treated HEL, K-562, K-562 (0.02), K-562(0.1) and Jurkat showed reduced proliferation. CEM as well as its' multidrug-resistant derivatives were sensitive to 1 micromol/l As2O3. In summary, these data demonstrate that As2O3-induced apoptosis is not restricted to cell lines with t(15;17). Apoptosis was induced in vitro by As2O3 concentrations that are achievable in vivo after infusion of well-tolerated As2O3 doses. Thus, As2O3 might be a suitable therapeutic agent for malignancies other than APL provided the adequate dose and duration of As2O3 treatment are used.     

Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal-induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal-induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.     

Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase.

Mast cell growth factor (MGF, the ligand for c-kit receptor) can stimulate proliferation of factor dependent myeloid cell line, M07e, and MGF synergizes with granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3 in this effect. The effect of MGF on protein tyrosine kinase activity in M07e cells was investigated by immunoblotting with anti-phosphotyrosine mAb and this was compared with effects of GM-CSF. MGF stimulation rapidly induced or enhanced at least 12 tyrosine phosphorylated bands. Major bands had molecular weights of 145, 120, 110, 98, 62, 55 and 42 kD. P145, the most prominent phosphorylated protein, was identified as c-kit product using anti-c-kit-mAb (YB5.B8), suggesting ligand-dependent receptor autophosphorylation. Five of six tyrosine phosphorylated bands induced or enhanced by GM-CSF stimulation comigrated with those tyrosine phosphorylated by MGF (138, 120, 76, 55 and 42 kD). P42 was identified, at least in part, as mitogen-activated protein (MAP) kinase. MGF induced tyrosine phosphorylation of a complex of GTPase-activating protein (GAP, 120 kD) and GAP associated proteins (p62/p190) as detected by anti-GAP Ab immunoprecipitation followed by immunoblotting with anti-phosphotyrosine mAb. GM-CSF also stimulated slightly but consistently tyrosine phosphorylation of GAP and p190 but not p62. Both MGF and GM-CSF enhanced Raf-1 phosphorylation and increased Raf-1 associated kinase activity in vitro. Phosphoamino acid analysis revealed Raf-1 phosphorylation by these two growth factors occurred almost exclusively on serine residues. No tyrosine phosphorylation of Raf-1 protein was detected. These data suggest shared and unshared components of signaling pathways of both factors, which may be involved in cell proliferation.     

Mechanisms of adrenocorticotropin-induced activation of extracellularly regulated kinase 1/2 mitogen-activated protein kinase in the human H295R adrenal cell line

The role of ACTH in stimulating or inhibiting growth of adrenal cells has been a subject of some controversy. Reports that ACTH may stimulate ERK/MAPK in Y1 cells have suggested a role for cAMP in this process. In attempting to extend this work, the ACTH responses in the human H295R cell line have been studied. This cell line makes only a very modest cAMP response to ACTH, yet the ERK1/2 response is highly reproducible and immediate but not prolonged. It is minimally reduced by the protein kinase A inhibitor, H89, but unaffected by protein kinase C and calcium inhibitors. Inhibition of epidermal growth factor receptor or other tyrosine kinase receptor transactivation was without effect, as was inhibition of c-Src activity or c-Src phosphorylation. The most effective inhibitor of this pathway was dansylcadaverine, an inhibitor of receptor internalization. These findings imply that ACTH-induced ERK1/2 activation in H295R cells is dependent on a mechanism distinct from that by which most G protein-coupled receptors activate ERK1/2 but that nevertheless seems to depend on receptor internalization.     

Phospholipase A2-mediated activation of mitogen-activated protein kinase by angiotensin II

In renal proximal tubule epithelial cells, a membrane-associated phospholipase A₂ (PLA₂) is a major signaling pathway linked to angiotensin II (Ang II) type 2 receptor (AT₂). The current studies were designed to test the hypothesis that membrane-associated PLA₂-induced release of arachidonic acid (AA) and/or its metabolites may serve as an upstream mediator of Ang II-induced mitogen-activated protein kinase (MAPK) activation. Ang II stimulated transient dose-dependent phosphorylation of MAPK with a maximum at 1 μM (10 min). Inhibition of PLA₂ by mepacrine diminished both AA release and MAPK phosphorylation, induced by Ang II. Furthermore, AA itself induced time- and dose-dependent phosphorylation of MAPK, supporting the importance of PLA₂ as a mediator of Ang II signaling. The effects of both Ang II and AA on MAPK phosphorylation were protein kinase C independent and abolished by the inhibitor of cytochrome P450 isoenzyme, ketoconazole. Moreover, 5,6-epoxyeicosatrienoic acid and 14,15-epoxyeicosatrienoic acid, the cytochrome P450-dependent metabolites of AA, significantly stimulated MAPK activity in renal proximal tubule epithelial cells. These observations document a mechanism of Ang II-induced MAPK phosphorylation, mediated by PLA₂-dependent release of AA and cytochrome P450-dependent production of epoxy derivatives of AA.     

Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase

BACKGROUND & AIMS: Clostridium difficile toxin A causes marked apoptosis of colonocytes in vivo and in vitro, which contributes to the formation of ulcers and pseudomembranes. We investigated the role of p53-dependent pathways and p38 mitogen-activated protein kinase (p38) in toxin A-induced colonocyte apoptosis. METHODS: The effects of the activation of p53 and p53-dependent pathways including p21(WAF1/CIP1) were assessed in nontransformed human colonic NCM460 epithelial cells exposed to toxin A. Phosphorylation of p53 protein by p38 was measured by in vitro kinase assay, whereas p21 induction by activated p53 was determined by gel shift assays and RNA silencing (small interfering RNA). The relationship between colonocyte apoptosis and p38/p53-dependent pathways was studied in intact mice. RESULTS: Toxin A stimulated p38 and p53 activation and induced cell cycle arrest (G(2)-M) with persistent expression of p21(WAF1/CIP1). Blockage of p38 by SB203580 inhibited p53 phosphorylation and induction of p21(WAF1/CIP1). In intact mice, p38 blockade suppressed toxin A-mediated destruction of intestinal villi, p21(WAF1/CIP1) expression, and enterocyte apoptosis. In addition, toxin A-mediated p21(WAF1/CIP1) and Bak induction, cytochrome c release, and caspase-3 activation were markedly attenuated in p53-silenced colonocytes, despite active p38. Overexpression of p21(WAF1/CIP1) triggered apoptosis and increased toxin A-associated colonocyte apoptosis. CONCLUSIONS: The signaling pathway for colonocyte apoptosis following toxin A exposure involves p38-dependent activation of p53 and subsequent induction of p21(WAF1/CIP1), resulting in cytochrome c release and caspase-3 activation through Bak induction.     

Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival, such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover, constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation, although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor, C188-9, inhibited G-CSF-induced Stat3 phosphorylation, induced apoptosis in AML cell lines and primary samples, and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore, Stat3 inhibition may be a valuable strategy for targeted therapies for AML.     

Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis

Activity of the serine-threonine protein kinase PINOID (PID) has been implicated in the asymmetrical localization of the membrane-associated PINFORMED (PIN) family of auxin transport facilitators. However, the means by which PID regulates PIN protein distribution is unknown. We have used recombinant PID protein to dissect the regulation of PID activity in vitro. We demonstrate that intramolecular PID autophosphorylation is required for the ability of PID to phosphorylate an exogenous substrate. PID-like mammalian AGC kinases act in a phosphorylation cascade initiated by the phospholipid-associated kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds to the C-terminal hydrophobic PDK1-interacting fragment (PIF) domain found in PDK1 substrates. We find that Arabidopsis PDK1 interacts with PID, and that transphosphorylation by PDK1 increases PID autophosphorylation. We show that a PID activation loop serine is required for PDK1-dependent PID phosphorylation. This activation is rapid and requires the PIF domain. Cell extracts from flowers and seedling shoots dramatically increase PID phosphorylation in a tissue-specific manner. A PID protein variant in which the PIF domain was mutated failed to be activated by the seedling shoot extracts. PID immunoprecipitated from Arabidopsis cells in which PDK1 expression was inhibited by RNAi showed a dramatic reduction in transphosphorylation of myelin basic protein substrate. These results indicate that AtPDK1 is a potent enhancer of PID activity and provide evidence that phospholipid signaling may play a role in the signaling processes controlling polar auxin transport.     

Platelet-derived growth factor BB-induced p38 mitogen-activated protein kinase activation causes cell growth, but not apoptosis, in vascular smooth muscle cells

The aim of this experiment was to examine the regulation of p38 mitogen-activated protein (MAP) kinase by platelet-derived growth factor (PDGF)-BB and its biological effects on rat cultured vascular smooth muscle cells (VSMCs). VSMCs were obtained from aortae of male Wistar rats by the media explant technique. After being stimulated by PDGF-BB with or without the p38 MAP kinase-specific inhibitor, SB-203580, the cells were solubilized, and the levels of phosphorylated p38 MAP kinase were examined by immunoblot analysis. The amounts of DNA synthesis and content were measured by using [3H]-thymidine and Hoechst-33258 dye, respectively. The detection of apoptotic cells was evaluated by the TUNEL method. PDGF-BB could phosphorylate p38 MAP kinase dose-dependently, and the phosphorylation was specifically inhibited by SB-203580 in a dose-dependent manner. However, PDGF-BB did not affect the protein level of p38 MAP kinase. Both [3H]-thymidine incorporation and total cellular DNA content were increased by PDGF-BB, and these elevations were prevented by SB-203580. In contrast, PDGF-BB-stimulated VSMCs did not show apoptotic change in spite of the presence or absence of SB-203580. These results established that PDGF-BB activated p38 MAP kinase and subsequently regulated cell growth in VSMCs, providing a molecular mechanism by which p38 MAP kinase can cause the development of cardiovascular diseases, including atherosclerosis.     

Involvement of p38 mitogen-activated protein kinase signaling in transformed growth of a cholangiocarcinoma cell line

Although mitogen-activated protein kinase (MAPK) pathways play a key role in cell growth, their role in mediating the altered growth phenotype of transformed cells remains unclear. The p44/p42 MAPK signaling cascades are activated by mitogenic stimulation of human cholangiocytes. In contrast, the p38 MAPK pathway is activated by mitogen stimulation of malignant, but not nonmalignant cholangiocytes. Thus, our aims were to determine the role of p38 MAPK signaling in mediating the growth phenotype of transformed cholangiocytes. KMCH-1 malignant human cholangiocytes required the presence of serum for proliferation, but were able to grow in reduced serum conditions. Inhibition of p38 MAPK decreased serum-dependent proliferation of KMCH-1 cells. Furthermore, inhibition of p38 MAPK, but not of p44/p42 MAPK, reduced anchorage-independent growth of KMCH-1 cells. Although both p38 and p44/p42 MAPK are activated in response to mitogens, they have divergent effects on anchorage-independent growth. Inhibition of p38 MAPK, but not of p44/p42 MAPK signaling, decreased cell cycle progression and increased expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIPl). However, expression of p27(KIP1) or p16(INK4A) was not altered by either pathway. Thus, mitogen activation of p38 MAPK decreases expression of p21(WAF1/CIP1) and mediates growth independent of anchorage signals, whereas mitogen activation of p44/p42 MAPK mediates an anchorage signal-dependent growth pathway. These data provide a link between aberrant stress-activated cell signaling and the altered growth phenotype of transformed cells that may be important for the development of therapies to limit transformed cell growth.     

The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts

SU5416 and SU6668 are potent antiangiogenic small-molecule inhibitors of receptor tyrosine kinases, including those of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor, c-kit, is structurally related to these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemic blasts derived from 60% to 80% of acute myeloid leukemia (AML) patients. The c-kit kinase inhibitory activity of SU5416 and SU6668 was evaluated in MO7E cells, a human myeloid leukemia cell line. Tyrosine autophosphorylation of the receptor, induced by SCF, was inhibited in these cells by SU5416 and SU6668 in a dose-dependent manner (inhibitory concentration of 50% [IC(50)] 0.1-1 microM). Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, a signaling event downstream of c-kit activation, was also inhibited in a dose-dependent manner. Both compounds also inhibited SCF-induced proliferation of MO7E cells (IC(50) 0.1 microM for SU5416; 0.29 microM for SU6668). Furthermore, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner as measured by the increase in activated caspase-3 and the enhanced cleavage of its substrate poly(ADP-ribose) polymerase. These findings with MO7E cells were extended to leukemic blasts from c-kit(+) patients. In patient blasts, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies indicate that SU5416 and SU6668 inhibit biologic functions of c-kit in addition to exhibiting antiangiogenic properties and suggest that the combination of these activities may provide a novel therapeutic approach for the treatment of AML.