Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.
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PMID:Activated Raf-1 causes growth arrest in human small cell lung cancer cells. 942 77

A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expression of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphorylated the retinoblastoma protein inefficiently. Activation of the MEK1/ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27(Kip1)) nor led to activation of cyclin E- and A-dependent CDK2, and such cells did not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27(Kip1) was mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes were activated; and serum-starved cells entered S phase. Thus, although the activity of p27(Kip1) normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequestered p27(Kip1) and reduced the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27(Kip1)-mediated inhibition.
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PMID:Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1). 944 90

Members of the erbB family of receptor tyrosine kinases are commonly overexpressed in human breast cancer. However, the relative contribution of particular signalling pathways activated downstream of these receptors to the mitogenic response of transformed breast epithelial cells remains poorly characterized. Administration of heregulin-beta2 (HRG), a ligand for erbB3 and erbB4, to growth arrested T-47D human breast cancer cells leads to activation of both the PI3-kinase and MAP kinase signalling pathways and potent stimulation of cell cycle progression. Specific inhibitors were used to assess the role of these pathways in HRG-induced mitogenesis and to identify underlying mechanisms in terms of regulation of gene expression. Treatment with the MEK inhibitor PD98059 led to a complete block of HRG-induced entry into S-phase, whilst administration of the PI3-kinase inhibitor wortmannin resulted in only modest inhibition. In addition, administration of PD98059 8 h after HRG was equipotent with simultaneous administration in inhibiting entry into S-phase. However, delaying addition for 14-16 h after HRG, when the cells were entering S-phase, was without effect. HRG stimulation led to sequential induction of c-myc, cyclin D1, cyclin E and cyclin A gene expression and hyperphosphorylation of the retinoblastoma protein pRB. p21 (WAF1/CIP1/SDI1) gene expression was rapidly induced by HRG, but significant changes in p27 (KIP1) protein levels were not detected. Preincubation with PD98059 blocked the HRG-dependent induction of cyclin D1 mRNA, p21 and c-Myc protein and pRB phosphorylation. These findings demonstrate that MEK activation is critical to HRG-induced S-phase entry in these cells whilst PI3-kinase plays a minor role. Moreover, these data are compatible with HRG-induced activation of MEK being critical for a mid-G1 transition point and implicate c-myc and cyclin D1 as key targets of the MAP kinase pathway involved in this response.
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PMID:Inhibition of the MAP kinase cascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cells. 965 48

With increasing size, multicellular prostate tumor spheroids develop regions of quiescent, multidrug-resistant cells expressing the cyclin-dependent kinase inhibitor p27(kip1). Treatment of small (diameter 60 +/- 20 micrometer) spheroids with 200 microM hydrogen peroxide (H(2)O(2)) resulted in cell cycle arrest owing to up-regulation of p27(kip1) and down-regulation of the transcription factor c-Fos. Incubation with 100 nM-1 microM H(2)O(2) led to up-regulation of c-Fos and enhanced tumor growth. Growth stimulation was inhibited by bisindolylmaleimide I, indicating a role for protein kinase C in the signaling cascade that involved the mitogen-activated protein kinase members MEK1,2, ERK1, -2, and c-Jun N-terminal kinase. Changes in Ca(2+) influx underlined the differential effects of H(2)O(2). Incubation with 200 microM H(2)O(2) released [Ca(2+)](i) from intracellular stores followed by prolonged Ca(2+) influx. Inhibition of influx by Ca(2+)-free media or Ni(2+), La(3+), Mn(2+) and SKF-96365 prevented the induction of quiescence and stimulated spheroid growth. Consequently, treatment with 200 microM H(2)O(2) in Ca(2+)-free media down-regulated p27(kip1) and increased Fos protein. ATP exerted effects comparably to those observed with H(2)O(2). Encoding growth stimulation by [Ca(2+)](i) release and induction of cell quiescence by prolonged Ca(2+) influx may provide a general mechanism for the control of tumor growth.
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PMID:Growth stimulation versus induction of cell quiescence by hydrogen peroxide in prostate tumor spheroids is encoded by the duration of the Ca(2+) response. 1048 20

The present report delineates the critical pathway in the G(1) phase involved in downregulation of p27(Kip1), a cyclin-dependent kinase inhibitor, which plays a pivotal role in controlling entry into the S phase of the cell cycle. In resting CCL39 fibroblasts and IEC-6 intestinal epithelial cells, protein levels of p27(Kip1) were elevated but dramatically decreased on serum stimulation, along with hyperphosphorylation of pRb and increased CDK2 activity. In both cell types, expression of ras resulted in an increase of basal and serum-stimulated E2F-dependent transcriptional activity and a reduction in p27(Kip1) protein levels as well. The role of the mitogen-activated protein (MAP) kinase cascade in p27(Kip1) reduction and S phase reentry was reinforced by the blockades of serum-induced E2F-dependent transcriptional activity and p27(Kip1) downregulation with the MKK-1/2 inhibitor PD-98059. In both cell lines, downregulation of p27(Kip1) was associated with a repression of its synthesis, an event mediated by the p42/p44 MAP kinase pathway. Using an antisense approach, we demonstrated that p27(Kip1) may control cell cycle exit in both cell types. These data indicate that activation of the MAP kinase cascade is required for S phase entry and p27(Kip1) downregulation in fibroblasts and epithelial cells.
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PMID:MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells. 1051 95

Vascular endothelial cells are unique in that they exit from the cell cycle when they come into contact with each other. Although the phenomenon is called "contact inhibition," little is known about the cellular mechanisms involved. Here we show that the phosphatase inhibitor sodium orthovanadate (SOV) induced the reentry of contact-inhibited human umbilical vascular endothelial cells (HUVECs) into the cell cycle and that reentry was associated with activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-K)/Akt pathways. SOV stimulated [(3)H]thymidine uptake of contact-inhibited HUVECs in a time- and dose-dependent manner. SOV-induced increase in [(3)H]thymidine uptake was significantly inhibited by the mitogen-activated protein kinase kinase inhibitor PD98059 and by the PI 3-K inhibitor LY294002. SOV also stimulated the expression of cyclin D1, cyclin E, and cyclin A, and the activity of CDK2 kinase, whereas it decreased the expression of p27(kip1). In marked contrast, growth media alone did not induce these changes. Furthermore, these SOV-induced changes were abolished by pretreatment with PD98059 and LY294002. SOV stimulated phosphorylation of ERK and Akt in contact-inhibited HUVECs, while growth media alone did not. This phosphorylation was associated with inhibition of phosphatase activity in the cells. Finally, overexpression of high cell density-enhanced protein tyrosine phosphatase 1 inhibited c-fos and cyclin A promoter activity. Taken together, our results suggest that in contact-inhibited HUVECs, increased phosphatase activity suppressed the ERK and PI 3-K/Akt pathways, resulting in exit from the cell cycle by down-regulation of cyclin D1, cyclin E, and cyclin A and by up-regulation of p27(kip1).
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PMID:Reentry into the cell cycle of contact-inhibited vascular endothelial cells by a phosphatase inhibitor. Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. 1065 60

Tumor necrosis factor-alpha (TNF-alpha) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)-immortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifies the mechanism of growth stimulation. TNF-alpha promoted cell cycle progression by increasing expression of HPV-16 E6/E7 RNAs and enhancing activity of cyclin-dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p21(waf) and p27(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activated Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited growth. Thus, activation of ras promotes expression of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates growth stimulation of immortal keratinocytes by TNF-alpha. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. 27:97-109, 2000. Published by Wiley-Liss, Inc.
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PMID:Tumor necrosis factor-alpha promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by a ras-dependent pathway. 1065 2

The elements of the cell cycle regulatory machinery activated by the oncogenic form of Ras, [Lys61]N-Ras, have been analysed in NIH3T3 cells. We demonstrate that [Lys61]N-Ras expression is able to induce full cdk4 activation. As already reported, oncogenic Ras expression was sufficient to induce cyclin D1 and p21cip1 expression and their association with cdk4. Furthermore, serum-starved [Lys61]N-Ras NIH3T3 cells showed nuclear accumulation of cyclin D1 and cdk4 not observed in serum-starved NIH3T3 cells. This accumulation of cdk4 into the cell nucleus observed in serum-starved [Lys61]N-Ras NIH3T3 cells was inhibited by a microinjection of neutralizing anti-Ras antibodies. Thus, active [Lys61]N-Ras was a sufficient signal to induce nuclear accumulation of cyclin D1/cdk4, leading to its full activation. Transfection of [Lys61]N-Ras NIH3T3 cells with an inactive form of MEK or their treatment with PD 98059, showed that nuclear translocation of cdk4 was MEK dependent. Interestingly, cells constitutively expressing [Lys61]N-Ras did not inactivate pRb and did not proliferate in the absence of serum. This may be due to the fact that although association of cdk2 with cyclin E and the translocation of those complexes to the nucleus were achieved, [Lys61]N-Ras expression was not sufficient to induce cdk2 activation. The high levels of p27(kip1) that were found in cyclin E/cdk2 complexes may be responsible for the inability of oncogenic Ras to activate this kinase. In consequence, oncogenic alterations that lead to a decrease in p27kip1 bound to cyclin E may cooperate with Ras to induce full cdk2 activation, pRb inactivation and thus cell proliferation.
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PMID:[Lys61]N-Ras is able to induce full activation and nuclear accumulation of Cdk4 in NIH3T3 cells. 1069 14

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.
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PMID:Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity. 1070 62

Angiotensin II (AngII) induces G(1) phase arrest and hypertrophy of cultured renal proximal tubular cells. In previous studies, it was shown that these effects depend on oxygen radical-mediated induction of p27(Kip1), an inhibitor of cyclin-dependent kinases. The present study was undertaken to investigate whether mitogen-activated protein (MAP) kinases serve as signaling intermediates between AngII-induced oxidative stress and induction of p27(Kip1). AngII (10(-7) M) induces a biphasic phosphorylation pattern of p44/42 MAP kinase with an early phosphorylation after 2 min and a later, second phosphorylation peak after prolong incubation (12 h) in cultured proximal tubular cells from two different species (MCT and LLC-PK(1) cells). Total protein expression of MAP kinase was not changed by AngII. These phosphorylation patterns of p44/42 MAP kinase caused activation of the enzyme, as detected by phosphorylated MAP substrate Elk-1 after immuno-precipitation of MAP kinase. Exogenous H(2)O(2) also stimulates a biphasic phosphorylation of p44/42 MAP kinase. The flavoprotein inhibitor diphenylene iodinium, as well as the antioxidant N-acetylcysteine, prevented AngII-induced p44/42 MAP kinase phosphorylation, indicating involvement of reactive oxygen species generated by membrane-bound NAD(P)H oxidase. The MAP kinase kinase inhibitor PD98059 completely inhibits AngII-induced p27(Kip1) expression and (3)[H]leucine incorporation into proteins as a previously established marker of cell hypertrophy. PD98059 did not attenuate AngII-stimulated intracellular synthesis of oxygen radicals. Transient transfection with p44/42 MAP kinase antisense, but not sense, phosphorothioate-modified oligonucleotides also prevented AngII-induced MAP kinase phosphorylation, p27(Kip1) expression, and cell hypertrophy. Furthermore, induction of p27(Kip1) by H(2)O(2) was also abolished in the presence of PD98059. Although AngII induces phosphorylation of the stress-activated p38 MAP kinase, inhibition of this enzyme with SB203580 failed to attenuate induced p27(Kip1) expression and hypertrophy. These data provide evidence that AngII- mediated oxygen stress leads to the phosphorylation of p44/42 MAP kinase in proximal tubular cells. Activation of this enzyme is essential for p27(Kip1) expression, G(1) phase arrest, and hypertrophy of proximal tubular cells. These findings may lead to new concepts concerning interference of the development of proximal tubular hypertrophy, which may eventually turn into a maladaptive process in vivo leading ultimately to tubular atrophy and tubulointerstitial fibrosis.
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PMID:Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27(Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells. 1090 52


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