Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells

McCubrey, JA; Steelman, LS; Hoyle, Paul; Blalock, William L.; Weinstein‐Oppenheimer, Caroline; Franklin, Richard A.; Cherwinski, Holly; Bosch, Elizabeth; McMahon, Martin

doi:10.1038/sj.leu.2401215

Cited by 100 publications

(170 citation statements)

References 91 publications

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“…233,240,241 Furthermore, introduction of activated Ras, Raf, MEK genes into hematopoietic cells makes them sensitive to MEK inhibitors. 198,[242][243][244][245] BCR-ABL-transformed hematopoietic cells are usually highly sensitive to inhibitors such as imatinib, providing that the particular BCR-ABL gene present in the cells does not have a mutation that eliminates sensitivity to the inhibitor.…”

Section: Roles Of the Ras/raf/mek/erk Pathway In Leukemiamentioning

confidence: 99%

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

et al. 2008

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Mutations and chromosomal translocations occur in leukemic cells that result in elevated expression or constitutive activation of various growth factor receptors and downstream kinases. The Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways are often activated by mutations in upstream genes. The Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways are regulated by upstream Ras that is frequently mutated in human cancer. Recently, it has been observed that the FLT-3 and Jak kinases and the phosphatase and tensin homologue deleted on chromosome 10 ( PTEN) phosphatase are also frequently mutated or their expression is altered in certain hematopoietic neoplasms. Many of the events elicited by the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways have direct effects on survival pathways. Aberrant regulation of the survival pathways can contribute to uncontrolled cell growth and lead to leukemia. In this review, we describe the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT signaling cascades and summarize recent data regarding the regulation and mutation status of these pathways and their involvement in leukemia

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Section: Roles Of the Ras/raf/mek/erk Pathway In Leukemiamentioning

confidence: 99%

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

et al. 2008

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“…26 Primer pairs were as follows: N-terminus: 5 0 -CATGGAGGAACTTAATACATAC-3 0 (exon 3) and 5 0 -ATGCCAAACCTCTTGTCCACAG-3 0 (exons 10/11); Internal: 5 0 -GCCTTATCAATAGAATTGCCCAGA-3 0 (exon 5) and 5 0 -AGAATCATCACTGGTCTCAGGA-3 0 (exon 11); C-terminus: 5 0 -CAATTGGCCGCTAAACTTGCAT-3 0 (exon 6) and 5 0 -CATAGTCTTGCGAAGAAATCTG-3 0 (exons 14/15); N-terminus through internal: 5 0 -CATGGAGGAACTTAATACATAC-3 0 (exon 3) and 5 0 -CATAGTCTTGCGAAGAAATCTG-3 0 (exons 14/15); internal through the stop codon: 5 0 -CAATTGGCCGCTAAACTT GCAT-3 0 (exon 6) and 5 0 -AAGGGCTCTAACATGTGTGTCG-3 0 (exon 17); and GAPDH: 5 0 -CGATGCTGGCGCTGAGTAC-3 0 and 5 0 -CGTTCAGCTCAGGGATGAC-3 0 .…”

Section: Reverse Transcriptase-pcr Of Pkr Mrnamentioning

confidence: 99%

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress

et al. 2010

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A number of cancers possess constitutive activity of the dsRNA-dependent kinase, PKR. Inhibition of PKR in these cancers leads to tumor cell death. We recently reported the increased presence of PKR phosphorylated on Thr451 (p-T451 PKR) in clinical samples from myelodysplastic syndrome (MDS) patients and acute leukemia cell lines. Whereas p-T451 PKR in low-risk patient samples or PTEN-positive acute leukemia cell lines was mostly cytoplasmic, in high-risk patient samples and acute leukemia cell lines deficient in PTEN, p-T451 PKR was mainly nuclear. As nuclear activity of PKR has not been previously characterized, we examined the status of nuclear PKR in acute leukemia cell lines. Using antibodies to N-terminus, C-terminus and the kinase domain in conjunction with a proteomics approach, we found that PKR exists in diverse molecular weight forms in the nucleus. Analysis of PKR transcripts by reverse transcriptase-PCR, and PKR-derived peptides by MS/MS revealed that these forms were the result of post-translational modifications (PTMs). Biochemical analysis demonstrated that nuclear PKR is an active kinase that can respond to stress. Given the association of PKR with PTEN and the Fanconi complex, these results indicate that PKR likely has other previously unrecognized roles in nuclear signaling that may contribute to leukemic development.

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“…Immune complexes were collected, washed in lysis buffer, and the activities of ⌬Raf:ER proteins were assessed by incubation for 30 min at 30°C in a reaction mix containing 25 mM Hepes (pH 7.4), 10 mM MgCl 2 , 1 mM DTT, 50 M ATP and 10 M ␥-32 P-ATP (3000 C1/mmol; NEN) with 12.5 g of purified recombinant enzymatically unactive GST-MEK1 (Upstate Biotechnology (UBI), Lake Placid NY, USA) as a substrate as described previously. 14,[26][27][28][29][30] The reactions were analyzed by SDS-polyacrylamide gel electrophoresis and electrotransference to polyvinylidene difluoride membranes (PVDF, Immunobilon P; Millipore, Medford, MA, USA). Western blots were first exposed to X-ray film to quantitate GST-MEK1 phosphorylation and subsequently probed with an ␣-hbER Ab to quantitate the amount of the ⌬Raf:ER protein in each immunoprecipitate.…”

Section: Determination Of Raf Kinase Activitymentioning

confidence: 99%

“…Thirty-five cycles of PCR were performed to detect the cDNAs as described. 12,[14][15][16] The PCR products were electrophoresed on 1% agarose gels and visualized after ethidium bromide staining of the gel.…”

Section: Polymerase Chain Reaction Amplification Of Cytokine Mrna Tramentioning

confidence: 99%

“…11 This cell line has a low spontaneous frequency of conversion to factor independence and TF-1 cells do not normally form tumors after injection in immunocompromised mice. [11][12][13][14][15][16][17] Thus, these cell lines represent models for analysis of the effects of aberrant oncogene expression on human hematopoietic cell growth, differentiation and malignant transformation.…”

Section: Introductionmentioning

confidence: 99%

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Effects of deregulated Raf activation on integrin, cytokine-receptor expression and the induction of apoptosis in hematopoietic cells

Weinstein‐Oppenheimer

Steelman

Algate

et al. 2000

Leukemia

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The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by ␤-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (⌬Raf:ER). Continuous ⌬Raf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/⌬Raf:ER cells as they grew in large aggregated masses (Ͼ100 cells) whereas the parental cytokinedependent FDC-P1 cells grew in smaller grape-like clusters (Ͻ10 cells). FD/⌬Raf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and ␤-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of ⌬Raf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3 H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation. Leukemia

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Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells

Cited by 100 publications

References 91 publications

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

Multiple forms of PKR present in the nuclei of acute leukemia cells represent an active kinase that is responsive to stress

Effects of deregulated Raf activation on integrin, cytokine-receptor expression and the induction of apoptosis in hematopoietic cells

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