Protein kinase C-α isoform is involved in erythropoietin-induced erythroid differentiation of CD34+ progenitor cells from human bone marrow

Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in many cellular responses. Although the analysis of PKC activity in many systems has provided crucial insights to its biologic function, the precise role of different isoforms on the differentiation of normal hematopoietic progenitor cells into the various lineages remains to be investigated. The authors have assessed the state of activation and protein expression of PKC isoforms after cytokine stimulation of CD34+ progenitor cells from human bone marrow. Freshly isolated CD34+ cells were found to express PKC-, PKC-β2, and PKC-ɛ, whereas PKC-δ, PKC-γ, and PKC-ζ were not detected. Treatment with erythropoietin (EPO) or with EPO and stem cell factor (SCF) induced a predominantly erythroid differentiation of CD34+ cells that was accompanied by the up-regulation of PKC- and PKC-β2 protein levels (11.8- and 2.5-fold, respectively) compared with cells cultured in medium. Stimulation with EPO also resulted in the nuclear translocation of PKC- and PKC-β2 isoforms. Notably, none of the PKC isoforms tested were detectable in CD34+ cells induced to myeloid differentiation by G-CSF and SCF stimulation. The PKC inhibitors staurosporine and calphostin C prevented EPO-induced erythroid differentiation. Down-regulation of the PKC-, PKC-β2, and PKC-ɛ expression by TPA pretreatment, or the down-regulation of PKC- with a specific ribozyme, also inhibited the EPO-induced erythroid differentiation of CD34+ cells. No effect was seen with PKC-β2–specific ribozymes. Taken together, these findings point to a novel role for the PKC- isoform in mediating EPO-induced erythroid differentiation of the CD34+ progenitor cells from human bone marrow.

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Arsenic Primes Human Bone Marrow CD34+ Cells for Erythroid Differentiation

Bioinorganic Chemistry and Applications ◽

10.1155/2015/751013 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

Yuanyuan Zhang ◽

Shasha Wang ◽

Chunyan Chen ◽

Xiao Wu ◽

Qunye Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Arsenic Trioxide ◽

Erythroid Differentiation ◽

Human Bone ◽

Human Bone Marrow ◽

Hematopoietic Progenitor Cells ◽

Therapeutic Effects ◽

Hematopoietic Progenitor ◽

Cd34 Cells

Arsenic trioxide exhibits therapeutic effects on certain blood malignancies, at least partly by modulating cell differentiation. Previousin vitrostudies in human hematopoietic progenitor cells have suggested that arsenic may inhibit erythroid differentiation. However, these effects were all observed in the presence of arsenic compounds, while the concomitant cytostatic and cytotoxic actions of arsenic might mask a prodifferentiating activity. To eliminate the potential impacts of the cytostatic and cytotoxic actions of arsenic, we adopted a novel protocol by pretreating human bone marrow CD34+ cells with a low, noncytotoxic concentration of arsenic trioxide, followed by assaying the colony forming activities in the absence of the arsenic compound. Bone marrow specimens were obtained from chronic myeloid leukemia patients who achieved complete cytogenetic remission. CD34+ cells were isolated by magnetic-activated cell sorting. We discovered that arsenic trioxide enhanced the erythroid colony forming activity, which was accompanied by a decrease in the granulomonocytic differentiation function. Moreover, in erythroleukemic K562 cells, we showed that arsenic trioxide inhibited erythrocyte maturation, suggesting that arsenic might have biphasic effects on erythropoiesis. In conclusion, our data provided the first evidence showing that arsenic trioxide could prime human hematopoietic progenitor cells for enhanced erythroid differentiation.

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Distribution of Protein Kinase C Isoforms after Infection of Macrophages with Leishmania major

Infection and Immunity ◽

10.1128/iai.66.4.1795-1799.1998 ◽

1998 ◽

Vol 66 (4) ◽

pp. 1795-1799 ◽

Cited By ~ 13

Author(s):

Sabine Pingel ◽

Zhi-En Wang ◽

Richard M. Locksley

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Protein Kinase C ◽

Protein Kinase ◽

Leishmania Major ◽

Murine Bone Marrow ◽

Kinase C ◽

Pkc Isoforms ◽

Protein Kinase C Isoforms

ABSTRACT We characterized the effects of Leishmania infection on activation-induced translocation of protein kinase C (PKC) isoforms in murine bone marrow-derived macrophages. Although PKC-dependent gene expression was attenuated by infection, the distribution and translocation of PKC isoforms were unaffected. However, subsequent dissociation from membranes was substantially delayed for some isoforms, particularly PKCβ.

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Gene Expression Profiles for Normal Human Bone Marrow-Derived CD34+ Stem Cells and CD34−/CD33+ Myeloid-Committed Progenitor Cells in Response to Daily Granulocyte-Colony-Stimulating Factor (G-CSF) Treatment.

Blood ◽

10.1182/blood.v104.11.4162.4162 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4162-4162

Author(s):

Andrew A.G. Aprikyan ◽

David Pritchard ◽

Conrad W. Liles ◽

Steve Schwartz ◽

David C. Dale

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Transcription Factors ◽

Protein Kinase C ◽

Protein Kinase ◽

Progenitor Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Kinase C ◽

Chronic Neutropenia

Abstract G-CSF is widely used to accelerate marrow recovery after cancer chemotherapy, to facilitate collection of hematopoietic progenitor cells, and to treat severe chronic neutropenia. Although G-CSF was originally defined as a stimulus for myeloid cell proliferation, it has potent anti-apoptotic properties, affects synthesis of proteins stored in neutrophil granules, and has many other effects on cells of the myeloid lineage. To improve understanding of the molecular and cellular effects of G-CSF, particularly related to its use for the treatment of severe chronic neutropenia, we performed gene expression profile studies using Affymetrix oligonucleotide arrays and purified bone marrow cell sub-populations from normal volunteers treated with daily subcutaneous G-CSF (300 mcg/sc/qd) for five days. Under local anaesthesia, paired marrow aspirates were obtained from the posterior iliac crest before and after 5 daily doses of G-CSF. CD34+ and CD34−/CD33+ cells were purified using Miltenyi immunomagnetic beads. Two rounds of amplification of total RNA isolated from purified CD34+ or CD33+cells was used to obtain sufficient cRNA for hybridization. Expression data from scanned chips were first analyzed using the RMA algorithm. The limma package of the Bioconductor project was used to identify differentially expressed genes. Limma uses an empirical Bayes method to moderate the standard errors of the estimated log-fold changes. The statistical analysis of CD33+ cells revealed that 150 of more than 12,000 genes examined were up- or down-regulated >2-fold in response to G-CSF treatment. The top 10 genes with up- or down-regulated level of expression include clusterin, neutrophil elastase, two transcription factors, gelsolin, Grb2, phospholipase D3, protein kinase C, the major vault protein, and serine-threonine kinase. In the myeloid-committed CD34-/CD33+ progenitor cells, genes with altered expression level represent those with gene products involved in the cell cycle, regulation of apoptosis, the cytoskeleton, the inflammatory response, or serine proteases and transcription factors. Most of the genes up-regulated in CD33+ cells (e.g. neutrophil elastase, phospholipase D, protein kinase C) were down-regulated in CD34-positive cells in response to G-CSF. The results of the comparative analyses revealed the normal signature gene expression profiles for CD34+ and CD34−/CD33+ cells and identified genes that may mediate specific G-CSF effects.

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Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic Lineage Commitment of Human Bone Marrow Progenitor Cells

Blood ◽

10.1182/blood.v94.6.1971.418k34_1971_1978 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1971-1978 ◽

Cited By ~ 3

Author(s):

Gösta Bergh ◽

Mats Ehinger ◽

Inge Olsson ◽

Sten Eirik W. Jacobsen ◽

Urban Gullberg

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Differentiation ◽

Progenitor Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Lineage Commitment ◽

Flt3 Ligand ◽

Monocytic Differentiation ◽

Cd34 Cells

The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

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Involvement of the Retinoblastoma Protein in Monocytic and Neutrophilic Lineage Commitment of Human Bone Marrow Progenitor Cells

Blood ◽

10.1182/blood.v94.6.1971 ◽

1999 ◽

Vol 94 (6) ◽

pp. 1971-1978 ◽

Cited By ~ 24

Author(s):

Gösta Bergh ◽

Mats Ehinger ◽

Inge Olsson ◽

Sten Eirik W. Jacobsen ◽

Urban Gullberg

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Cell Differentiation ◽

Progenitor Cells ◽

Human Bone ◽

Human Bone Marrow ◽

Lineage Commitment ◽

Flt3 Ligand ◽

Monocytic Differentiation ◽

Cd34 Cells

Abstract The retinoblastoma gene product (pRb) is involved in both cell cycle regulation and cell differentiation. pRb may have dual functions during cell differentiation: partly by promoting a cell cycle brake at G1 and also by interacting with tissue-specific transcription factors. We recently showed that pRb mediates differentiation of leukemic cell lines involving mechanisms other than the induction of G1 arrest. In the present study, we investigated the role of pRb in differentiation of human bone marrow progenitor cells. Human bone marrow cells were cultured in a colony-forming unit–granulocyte-macrophage (CFU-GM) assay. The addition of antisense RB oligonucleotides (-RB), but not the addition of sense orientated oligonucleotides (SO) or scrambled oligonucleotides (SCR), reduced the number of colonies staining for nonspecific esterase without affecting the clonogenic growth. Monocytic differentiation of CD34+ cells supported by FLT3-ligand and interleukin-3 (IL-3) was correlated to high levels of hypophosphorylated pRb, whereas neutrophilic differentiation, supported by granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF), was correlated to low levels. The addition of -RB to liquid cultures of CD34+ cells, supported with FLT3-ligand and IL-3, inhibited monocytic differentiation. This was judged by morphology, the expression of CD14, and staining for esterase. Moreover, the inhibition of monocytic differentiation of CD34+ cells mediated by -RB, which is capable of reducing pRb expression, was counterbalanced by an enhanced neutrophilic differentiation response, as judged by morphology and the expression of lactoferrin. CD34+ cells incubated with oligo buffer, -RB, SO, or SCR showed similar growth rates. Taken together, these data suggest that pRb plays a critical role in the monocytic and neutrophilic lineage commitment of human bone marrow progenitors, probably by mechanisms that are not strictly related to control of cell cycle progression.

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Rapid and efficient homing of human CD34+CD38−/lowCXCR4+stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice

Blood ◽

10.1182/blood.v97.10.3283 ◽

2001 ◽

Vol 97 (10) ◽

pp. 3283-3291 ◽

Cited By ~ 228

Author(s):

Orit Kollet ◽

Asaf Spiegel ◽

Amnon Peled ◽

Isabelle Petit ◽

Tamara Byk ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Protein Kinase C ◽

Protein Kinase ◽

Immunodeficient Mice ◽

Human Cd34 ◽

Kinase C ◽

Cd34 Cells

Abstract Stem cell homing into the bone microenvironment is the first step in the initiation of marrow-derived blood cells. It is reported that human severe combined immunodeficient (SCID) repopulating cells home and accumulate rapidly, within a few hours, in the bone marrow and spleen of immunodeficient mice previously conditioned with total body irradiation. Primitive CD34+CD38−/lowCXCR4+ cells capable of engrafting primary and secondary recipient mice selectively homed to the bone marrow and spleen, whereas CD34−CD38−/lowLin− cells were not detected. Moreover, whereas freshly isolated CD34+CD38+/high cells did not home, in vivo stimulation with granulocyte colony-stimulating factor as part of the mobilization process, or in vitro stem cell factor stimulation for 2 to 4 days, potentiated the homing capabilities of cytokine-stimulated CD34+CD38+ cells. Homing of enriched human CD34+ cells was inhibited by pretreatment with anti-CXCR4 antibodies. Moreover, primitive CD34+CD38−/lowCXCR4+cells also homed in response to a gradient of human stromal cell-derived factor 1 (SDF-1), directly injected into the bone marrow or spleen of nonirradiated NOD/SCID mice. Homing was also inhibited by pretreatment of CD34+ cells with antibodies for the major integrins VLA-4, VLA-5, and LFA-1. Pertussis toxin, an inhibitor of signals mediated by Gαiproteins, inhibited SDF-1–mediated in vitro transwell migration but not adhesion or in vivo homing of CD34+ cells. Homing of human CD34+ cells was also blocked by chelerythrine chloride, a broad-range protein kinase C inhibitor. This study reveals rapid and efficient homing to the murine bone marrow by primitive human CD34+CD38−/lowCXCR4+cells that is integrin mediated and depends on activation of the protein kinase C signal transduction pathway by SDF-1.

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Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽

10.1182/blood.v91.10.3693 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3693-3701 ◽

Cited By ~ 13

Author(s):

Ponnazhagan Veena ◽

Christie M. Traycoff ◽

David A. Williams ◽

Jon McMahel ◽

Susan Rice ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Progenitor Cells ◽

Cultured Cells ◽

Retroviral Vector ◽

Human Bone ◽

Human Bone Marrow ◽

Transduction Efficiency ◽

Cd34 Cells

Abstract Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

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Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽

10.1182/blood.v91.10.3693.3693_3693_3701 ◽

1998 ◽

Vol 91 (10) ◽

pp. 3693-3701

Author(s):

Ponnazhagan Veena ◽

Christie M. Traycoff ◽

David A. Williams ◽

Jon McMahel ◽

Susan Rice ◽

...

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Progenitor Cells ◽

Cultured Cells ◽

Retroviral Vector ◽

Human Bone ◽

Human Bone Marrow ◽

Transduction Efficiency ◽

Cd34 Cells

Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

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