Enhanced Phosphorylation and Altered Localization Lead to Impairment of p27kip Activity in CML Progenitor Cells Despite Enhanced Protein Translation and Expression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 999-999 ◽  
Author(s):  
Su Chu ◽  
Tinisha McDonald ◽  
Ravi Bhatia
Keyword(s):  
Protein Expression ◽  
Progenitor Cells ◽  
Nuclear Localization ◽  
Protein Translation ◽  
Pool Size ◽  
Mrna Levels ◽  
Cytoplasmic Localization ◽  
Hematopoietic Progenitor ◽  
Protein Levels ◽  
Cd34 Cells

Abstract The cyclin-dependent kinase inhibitor p27kip is a key regulator of hematopoietic progenitor proliferation and pool size. The activity of p27 can be regulated by modulation of its expression and localization in different cellular compartments. The levels of p27 protein expression are reported to be reduced in BCR-ABL expressing cell lines. In contrast p27 levels have been reported to be elevated in BCR/ABL expressing CML progenitor cells. However, CML progenitors paradoxically demonstrate enhanced proliferation despite having elevated levels of p27. Therefore the regulation of p27 protein expression and activity in primary CML progenitor cells is not well understood and requires further investigation. We have therefore performed a careful assessment of p27 expression, phosphorylation and localization in CD34+ cells from CML patients as well as in cord blood CD34+ cells transduced with retroviral vectors to ectopically express the BCR-ABL gene. We observed that p27 protein levels were markedly increased in both primary CML CD34+ cells and BCR/ABL transduced cells. CML CD34+ cells and BCR/ABL transduced CD34+ cells also demonstrated increased phosphorylation of p27 on T157 (an Akt-dependent phosphorylation site) and T187 (a cdk2-dependent site) on western blotting as well as increased tyrosine phosphorylation of p27 in immunoprecipitation studies. Immunofluorescence microscopy analysis of cells labeled with anti-p27 antibodies demonstrated primarily nuclear localization of p27 in normal CD34+ cells, but markedly reduced nuclear and increased cytoplasmic localization of p27 in CML CD34+ cells and BCR/ABL transduced CD34+ cells. Cdk2 activity was maintained, cdk4 activity was increased and phosphorylated RB levels were increased in BCR-ABL expressing hematopoietic cells, indicating CDKI activity was reduced in these cells despite increased total p27 levels. Consistent with this BCR-ABL expressing CD34+ cells demonstrated enhanced cell cycling compared to controls. Mutation of the Y177 residue in BCR-ABL (BCR-ABL-Y177F), which abrogates Grb2 binding and reduces Ras and Akt activation by BCR-ABL, reversed BCR-ABL induced abnormalities in p27 expression, phosphorylation and localization. Treatment of BCR-ABL transduced CD34+ cells with the PI-3K inhibitor LY294002 resulted in reduced phosphorylation of p27 on T157 but not T187, and restored nuclear localization of p27, further indicating an important role for AKT signaling in abnormal cytoplasmic localization of p27. Quantitative reverse transcription PCR analysis indicated that p27 mRNA levels were similar in BCR/ABL expressing and control CD34+ cells, suggesting that p27 expression is regulated mainly at the posttranscriptional level. Metabolic labeling of cells with S35-methionine showed that p27 translation was increased in BCR/ABL expressing cells, explaining the observed increase in total p27 protein levels. We conclude that p27 CDKI activity is reduced in BCR-ABL expressing progenitors as a result of altered p27 phosphorylation at important regulatory sites and a shift in cellular localization of p27 from the nucleus to the cytoplasm. The enhanced level of total p27 in primary CML progenitors reflects enhanced protein translation, which may be a compensatory response to reduced p27 CDKI activity. These results provide important insights into p27 deregulation in CML progenitors and its potential role in the expansion of the hematopoietic progenitor pool size in CML.

scholarly journals PBRM1 Cooperates with YTHDF2 to Control HIF-1α Protein Translation

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1425
Author(s):  
Alena Shmakova ◽  
Mark Frost ◽  
Michael Batie ◽  
Niall S. Kenneth ◽  
Sonia Rocha
Keyword(s):  
Protein Expression ◽  
Cellular Response ◽  
Transcriptional Activity ◽  
Mrna Translation ◽  
Protein Translation ◽  
Signalling Pathways ◽  
Independent Function ◽  
Protein Levels ◽  
Associated Proteins ◽  
Core Components

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here, we investigated how PBRM1 controls HIF-1α activity. We found that PBRM1 is required for HIF-1α transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1α mRNA translation, as absence of PBRM1 results in reduced actively translating HIF-1α mRNA. Interestingly, we found that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1α mRNA is m6A-modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1α mRNA and reduction of YTHDF2 results in reduced HIF-1α protein expression in cells. Our results identify a SWI/SNF-independent function for PBRM1, interacting with HIF-1α mRNA and the epitranscriptome machinery. Furthermore, our results suggest that the epitranscriptome-associated proteins play a role in the control of hypoxia signalling pathways.

scholarly journals Determination of CYP450 Expression Levels in the Human Small Intestine by Mass Spectrometry-Based Targeted Proteomics

2021 ◽  
Vol 22 (23) ◽  
pp. 12791
Author(s):  
Alexia Grangeon ◽  
Valérie Clermont ◽  
Azemi Barama ◽  
Fleur Gaudette ◽  
Jacques Turgeon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Intestine ◽  
Protein Expression ◽  
Mrna Levels ◽  
Targeted Proteomics ◽  
Tissue Samples ◽  
Human Organ ◽  
Expression Levels ◽  
Protein Levels ◽  
Human Small Intestine

The human small intestine can be involved in the first-pass metabolism of drugs. Under this condition, members of the CYP450 superfamily are expected to contribute to drug presystemic biotransformation. The aim of this study was to quantify protein expression levels of 16 major CYP450 isoforms in tissue obtained from nine human organ donors in seven subsections of the small intestine, i.e., duodenum (one section, N = 7 tissue samples), jejunum (three subsections (proximal, mid and distal), N = 9 tissue samples) and ileum (three subsections, (proximal, mid and distal), N = 9 tissue samples), using liquid chromatography tandem mass spectrometry (LC-MS/MS) based targeted proteomics. CYP450 absolute protein expression levels were compared to mRNA levels and enzyme activities by using established probe drugs. Proteins corresponding to seven of sixteen potential CYP450 isoforms were detected and quantified in various sections of the small intestine: CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, CYP3A5 and CYP4F2. Wide inter-subject variability was observed, especially for CYP2D6. CYP2C9 (p = 0.004) and CYP2C19 (p = 0.005) expression levels decreased along the small intestine. From the duodenum to the ileum, CYP2J2 (p = 0.001) increased, and a trend was observed for CYP3A5 (p = 0.13). CYP3A4 expression was higher in the jejunum than in the ileum (p = 0.03), while CYP4F2 expression was lower in the duodenum compared to the jejunum and the ileum (p = 0.005). CYP450 protein levels were better correlated with specific isoform activities than with mRNA levels. This study provides new data on absolute CYP450 quantification in human small intestine that could improve physiologically based pharmacokinetic models. These data could better inform drug absorption profiles while considering the regional expression of CYP450 isoforms.

scholarly journals Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Amy C. Ladd ◽  
Robert Pyatt ◽  
Andre Gothot ◽  
Susan Rice ◽  
Jon McMahel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Phase I ◽  
Progenitor Cells ◽  
Phase Ii ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cytokine Stimulation ◽  
Phase Iv

Abstract Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

scholarly journals Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy- 1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2842-2848 ◽  
Author(s):  
M Korbling ◽  
YO Huh ◽  
A Durett ◽  
N Mirza ◽  
P Miller ◽  
...  
Keyword(s):  
Body Weight ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Allogeneic Transplantation ◽  
Hematopoietic Progenitor Cells ◽  
Donor Blood ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
The Mean ◽  
Single Bone

Abstract Apheresis-derived hematopoietic progenitor cells have recently been used for allogeneic transplantation. Forty-one normal donors were studied to assess the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (12 micrograms/kg/d) on the peripheralization of hematopoietic progenitor cells and lymphoid subsets. The white blood cell, polymorphonuclear cell (PMNC), and lymphocyte concentrations at the peak of rhG-CSF effect in the donor's peripheral blood (PB) exceeded baseline by 6.4-, 8.0-, and 2.2-fold, respectively. Corresponding concentrations of PB CD34+ cells and primitive subsets such as CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells increased by 16.3-fold, 24.2-fold, and 23.2-fold, respectively in eight normal donors. The percentage of CD34+ Thy-1dim and CD34+ Thy- 1dim CD38- cells among CD34+ cells increased as well, suggesting an additional peripheralization effect of rhG-CSF on primitive CD34+ subsets. The preapheresis PB CD34+ and CD34+ Thy-1dim cell concentrations were predictive of their corresponding apheresis yield per liter of donor blood processed PB lymphoid subsets were not significantly affected by rhG-CSF treatment. The mean apheresis-derived yield of CD34+, CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 x 10(4) (n = 41), 27.2 x 10(4) (n = 10), and 1.9 x 10(4) (n = 10), respectively. As compared with 43 single bone marrow (BM) harvest, the CD34+ cell yield of peripheral blood progenitor cell allografts of 41 normal donors exceeded that of BM allografts by 3.7- fold and that of lymphoid subsets by 16.1-fold (CD3+), 13.3-fold (CD4+), 27.4-fold (CD8+), 11.0-fold (CD19+), and 19.4-fold (CD56+CD3-). All PBPC allografts were cryopreserved before transplantation. The mean recovery of CD34+ cells after freezing, thawing, and washing out dimethylsulfoxide was 86.6% (n = 31) and the recovery of lymphoid subsets was 115.5% (CD3+), 121.4% (CD4+), 105.6% (CD8+), 118.1% (CD19+), and 102.4% (CD56+CD3-). All donors were related to patients: 39 sibling-to-sibling, 1 parent-to-child, and 1 child-to-parent transplant. Thirty-eight transplants were HLA fully identical, two transplants differed in one and two antigens. Engraftment occurred in 38 recipients; two patients died too early to be evaluated, and one patient did not engraft. The lowest CD34+ cell dose transplanted and resulting in complete and sustained engraftment was 2.5 x 10(6)/kg of recipient body weight.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95)

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2871-2877 ◽  
Author(s):  
K Takenaka ◽  
K Nagafuji ◽  
M Harada ◽  
S Mizuno ◽  
T Miyamoto ◽  
...  
Keyword(s):  
Growth Factors ◽  
Progenitor Cells ◽  
Calf Serum ◽  
Functional Expression ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Progenitor ◽  
Fas Antigen ◽  
Cd34 Cells

Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.

scholarly journals Intracellular Immunization of Rhesus CD34+ Hematopoietic Progenitor Cells With a Hairpin Ribozyme Protects T Cells and Macrophages From Simian Immunodeficiency Virus Infection

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4822-4831 ◽  
Author(s):  
Michael Rosenzweig ◽  
Douglas F. Marks ◽  
Donna Hempel ◽  
Marina Heusch ◽  
Günter Kraus ◽  
...  
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hairpin Ribozyme ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Gene ◽  
Stem Cell Gene Therapy

Abstract Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

scholarly journals Aloe-Emodin Induces Breast Tumor Cell Apoptosis through Upregulation of miR-15a/miR-16-1 That Suppresses BCL2

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Xuefeng Jiang ◽  
Yusheng Liu ◽  
Guijuan Zhang ◽  
Shujun Lin ◽  
Jieyan Wu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Apoptosis ◽  
Breast Tumor ◽  
Protein Translation ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
Anticancer Activities ◽  
Bax Protein ◽  
Aloe Emodin ◽  
Mcf 7

Purpose. Aloe-emodin (AE) is a natural compound derived from aloe vera and palmatum rhubarb and shows anticancer activities in various cancers. Bcl-2 family is the main regulator of cell death or cell survival. This study describes the effects of AE on proliferation of breast tumor (BT) cells. Methods. MCF-10A, MCF-10AT, MCF-7, and MDA-MB-231 cell lines were exposed to AE. Cell proliferation and apoptosis were assessed by CCK-8 and flow cytometry. Protein levels were measured by Western blotting. The levels of mRNA and miRNA were examined by RT-PCR. Bioinformatics was applied to screen miRNAs that bind to 3′-UTR of mRNA. Results. The results showed that AE selective activity inhibited the proliferation and induced apoptosis of MCF-10AT and MCF-7 cells but exhibited no significant inhibition in MCF10A and MDA-MB-231 cells. Mechanistically, AE dose-dependently decreased the protein expression of Bcl-2 and Bcl-xl, while it increased Bax protein expression in MCF-10AT and MCF-7 cells. The levels of Bcl-xl and Bax mRNA were altered by AE treatment, which was consistent with the protein expression results. However, Bcl-2 mRNA levels were not affected in either cell line, suggesting that AE may modulate the protein translation of Bcl-2 through miRNAs. In all candidate miRNAs that bind to 3′-UTR of Bcl-2, miR-15a and miR-16-1 were dose-dependently downregulated by AE. Moreover, inhibition of miR-15a/16-1 could eliminate the inhibition of MCF-10AT and MCF-7 cells growth by AE and could reverse the downregulation of AE-induced Bcl-2 protein level. Conclusion. Our research provides an important basis that AE induces BT cell apoptosis through upregulation of miR-15a/miR-16-1 that suppresses BCL2.

scholarly journals Myeloperoxidase expression in CD34+ normal human hematopoietic cells

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2069-2078 ◽  
Author(s):  
H Strobl ◽  
M Takimoto ◽  
O Majdic ◽  
G Fritsch ◽  
C Scheinecker ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Transferrin Receptor ◽  
Staining Pattern ◽  
Hematopoietic Progenitor ◽  
Colony Forming Unit ◽  
Cd34 Cells ◽  
Normal Human ◽  
Colony Forming Capacity ◽  
Adult Peripheral Blood

Abstract Bone marrow (BM), adult peripheral blood (aPB), and umbilical cord blood (CB) samples contain small proportions of CD34+ cells that include virtually all hematopoietic progenitor cells. Myeloperoxidase (MPO) is considered to be selectively expressed in cells committed to granulomonocytic differentiation. Using flow cytometry and an antibody against MPO, we studied at which stage of normal hematopoietic differentiation CD34+ cells being to express MPO. We consistently observed a characteristic MPO/CD34 staining pattern and found that 35% +/- 9% of CD34+ BM cells express MPO. The MPO+ CD34+ subset and the CD33+ CD34+ subset were of similar size and overlapped considerably. MPO+ CD34+ cells expressed high levels of HLA-D molecules, were weakly CD71/transferrin receptor positive to negative, were CD45RA+ and lacked the CD45RO isoform of the leukocyte common antigen. Additionally, MPO+ CD34+ cells were on average larger in size than MPO- CD34+ cells. Virtually identical phenotypic features have previously been described for in vitro colony-forming granulomonocytic progenitor cells. In vitro clonogenic assays performed with MPO-enriched and MPO-depleted fractions of CD34+ BM cells performed by us also suggest, but do not formally prove, that at least a portion of MPO+ CD34+ cells have in vitro cluster (10 to 50 cells/colony) or colony-forming unit granulocyte-macrophage (> or = 50 cells/colony) forming capacity. CD34+ cells from CB and aPB resembled CD34+ BM cells in that considerable proportions of them coexpressed CD33. However, in contrast to BM, CD34+ cells from CB and aPB samples lacked significant MPO expression and, in line with this, the majority of them (CB, 59% +/- 7%; aPB, 66% +/- 5%) coexpressed CD45RO.

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