scholarly journals Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl−/− mice

Blood ◽  
2008 ◽  
Vol 112 (8) ◽  
pp. 3148-3153 ◽  
Author(s):  
Maria Kauppi ◽  
James M. Murphy ◽  
Carolyn A. de Graaf ◽  
Craig D. Hyland ◽  
Kylie T. Greig ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein C ◽  
Lymphoid Cells ◽  
Wild Type ◽  
Gene Encoding ◽  
Mutagenesis Screen ◽  
Transcriptional Regulatory ◽  
Regulatory Complex ◽  
Transcriptional Coregulator ◽  
B Lymphoid

Abstract In an N-nitroso-N-ethylurea (ENU) mutagenesis screen using Mpl−/− mice, we isolated a semidominant suppressor of thrombocytopenia, termed Plt6. The gene mutated in Plt6 mice encodes the transcriptional coregulator p300, and the mutation, a tyrosine to asparagine substitution at amino acid 630 (Y630N), disrupts the interaction between p300 and c-Myb. Mpl−/−p300Plt6/+ mice displayed elevated platelet counts relative to Mpl−/−p300+/+ controls, whereas mice homozygous for the Plt6 mutation produced supraphysiological levels of circulating platelets. On a wild-type genetic background, mice homozygous for the p300Plt6 mutation, or recipients of Mpl+/+p300Plt6/Plt6 bone marrow, also exhibited thrombocytosis as well as deficiencies in B-lymphoid cells. Increased platelet numbers in Plt6 mutant mice were accompanied by significant increases in megakaryocyte progenitor cells within the bone marrow and spleen with concomitantly elevated numbers of megakaryocytes. The expansion of megakaryocytopoiesis and suppression of Mpl−/− thrombocytopenia in Plt6 mutants is highly reminiscent of that observed in mice with mutations affecting the p300 partner protein c-Myb, suggesting an indispensable repressive role for the c-Myb/p300 transcriptional regulatory complex in megakaryocyte develop-ment, the inhibition of which allows substantial thrombopoietin (TPO)–independent platelet production.

scholarly journals Adenovirus E1A specifically blocks SWI/SNF-dependent transcriptional activation.

1996 ◽  
Vol 16 (10) ◽  
pp. 5737-5743 ◽  
Author(s):  
M E Miller ◽  
B R Cairns ◽  
R S Levinson ◽  
K R Yamamoto ◽  
D A Engel ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Cellular Transformation ◽  
Transcriptional Coactivator ◽  
Gene Encoding ◽  
Adenovirus E1a ◽  
Transcriptional Regulatory ◽  
Genes Encoding ◽  
Regulatory Complex ◽  
Activation Pathways ◽  
Coactivator P300

Expression of the adenovirus E1A243 oncoprotein in Saccharomyces cerevisiae produces a slow-growth phenotype with accumulation of cells in the G1 phase of the cell cycle. This effect is due to the N-terminal and CR1 domains of E1A243, which in rodent cells are involved in triggering cellular transformation and also in binding to the cellular transcriptional coactivator p300. A genetic screen was undertaken to identify genes required for the function of E1A243 in S. cerevisiae. This screen identified SNF12, a gene encoding the 73-kDa subunit of the SWI/SNF transcriptional regulatory complex. Mutation of genes encoding known members of the SWI/SNF complex also led to loss of E1A function, suggesting that the SWI/SNF complex is a target of E1A243. Moreover, expression of E1A in wild-type cells specifically blocked transcriptional activation of the INO1 and SUC2 genes, whose activation pathways are distinct but have a common requirement for the SWI/SNF complex. These data demonstrate a specific functional interaction between E1A and the SWI/SNF complex and suggest that a similar interaction takes place in rodent and human cells.

scholarly journals Myofibroblastic stromal cells isolated from human bone marrow induce the proliferation of both early myeloid and B-lymphoid cells

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2396-2405 ◽  
Author(s):  
I Moreau ◽  
V Duvert ◽  
C Caux ◽  
MC Galmiche ◽  
P Charbord ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Cells ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Present System ◽  
Normal Human ◽  
B Lymphoid

Abstract Normal human bone marrow stromal cells (BMSC) were isolated from Dexter- type long-term cultures according to their capacity to adhere to plastic and to their lack of hematopoietic antigens. The BMSC displayed a homogeneous appearance and a myofibroblastic phenotype in culture. The stromal cells (SC) were shown to support the proliferation of purified CD34+ hematopoietic progenitors and permitted us to maintain myeloid cells for several weeks in culture. In addition, the BMSC induced the proliferation of purified CD10+ s mu- fetal BM B-cell precursors (BCP). The capacity of the BMSC to induce the proliferation of early myeloid cells was shared by several other human fibroblastic- like cell types. In contrast, the BMSC were far superior to other adherent cells for induction of BCP proliferation. This capacity was largely mediated by endogenously produced interleukin-7 (IL-7), because it could be inhibited by anti-IL-7 antibody. In line with this finding, addition of IL-7 considerably enhanced BCP proliferation in cocultures with skin fibroblasts or synoviocytes. Thus, production of IL-7 appears to be a critical parameter that determines the ability of fibroblastic- like cells to induce BCP proliferation. Taken together, our data show that normal human myofibroblastic BMSC induce the proliferation of both early myeloid and B-lymphoid cells in the absence of accessory hematopoietic cells. The present system should constitute a model to study interactions between native human BM myofibroblastic stroma and various hematopoietic cell subsets.

scholarly journals Expression of the c-myc oncogene under control of an immunoglobulin enhancer in E mu-myc transgenic mice.

1987 ◽  
Vol 7 (4) ◽  
pp. 1436-1444 ◽  
Author(s):  
W S Alexander ◽  
J W Schrader ◽  
J M Adams
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
B Lymphocyte ◽  
Lymphoid Cells ◽  
Lymphoid Tissues ◽  
Myc Oncogene ◽  
Feedback Model ◽  
B Lymphocyte Development ◽  
B Lymphoid ◽  
Spleen And Lymph Nodes

Transgenic mice bearing a cellular myc oncogene coupled to the immunoglobulin heavy-chain enhancer (E mu) exhibit perturbed B-lymphocyte development and succumb to B lymphoid tumors. To investigate how the enhancer has affected myc expression, we analyzed the structure and abundance of myc transcripts in tissues of prelymphomatous mice and in the lymphomas. Expression of the E mu-myc transgene appeared to be confined largely to B lymphoid cells, being dominant in bone marrow, spleen, and lymph nodes, with no detectable expression in T cells or other hematopoietic lineages examined. The myc transcripts initiated very predominantly at the normal myc promoters, although use of the more upstream myc promoter was accentuated and an enhancer-associated promoter may be used infrequently. The level of E mu-myc transcripts in the preneoplastic lymphoid tissues and in the E mu-myc tumors was not markedly higher than myc RNA levels in proliferating normal lymphocytes. Thus, enforced expression of structurally normal myc transcripts at only a modestly elevated level has profound biological consequences. The absence of detectable endogenous c-myc RNA in any tumor, or in preneoplastic bone marrow, supports a negative feedback model for normal c-myc regulation.

Stat3β Promotes Basal Granulopoiesis and Inhibits Emergency Granulopoiesis, While Stat3α Inhibits Basal Granulopoiesis and Promotes Emergency Granulopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3871-3871
Author(s):  
Michele Redell ◽  
S. Wen-Wen Chen ◽  
Marcos J. Ruiz ◽  
David J. Tweardy
Keyword(s):  
Bone Marrow ◽  
B Lymphocyte ◽  
Inflammatory Responses ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
Transactivation Domain ◽  
Wild Type ◽  
Alternative Mrna Splicing ◽  
Deficient Mice

Abstract Signal transducer and activator of transcription 3 (Stat3) is a key signaling intermediate that is activated by several cytokines that regulate hematopoiesis, including granulocyte-colony stimulating factor (G-CSF), interleukin 6, and stem cell factor (SCF). Studies using mice with Stat3 deletion targeted to hematopoietic cells have shown that Stat3 negatively regulates basal granulopoiesis but positively regulates emergency granulopoiesis. Stat3 also has been reported to promote B lymphocyte differentiation. Defining the hematopoietic functions of Stat3 is further complicated by the existence of two isoforms: full-length Stat3α (p92), and truncated Stat3β (p83). Stat3β is derived from alternative mRNA splicing resulting in replacement of the C-terminal transactivation domain with 7 unique amino acids (CT7), which have been demonstrated to confer markedly prolonged nuclear retention. Homozygous Stat3α-deficient mice are not viable, whereas Stat3β-deficient mice survive to adulthood and are fertile, but have increased inflammatory responses compared to wild-type mice. We compared basal granulopoiesis and lymphopoiesis, as well as emergency granulopoiesis, in homozygous Stat3β-deficient mice (βΔ/βΔ), which express only Stat3α, vs. their wild-type (+/+) littermates. We found that βΔ/βΔ mice were significantly leukopenic (2880 ± 1260/ml v. 4600 ± 1670/ml; p<0.05), with lower absolute neutrophil counts (ANC, 360 ± 180/ml v. 800 ± 380/ml, p<0.05) and B lymphocyte counts (780 ± 470/ml v. 1830 ± 1260/ml, p<0.05), compared to +/+ mice. Within the circulating B-lymphocyte population, the mature B220hi/IgM− cells were most dramatically reduced (170 ± 70/ml v. 480 ± 350/ml, p<0.05). Percentages of myeloid and lymphoid cells in the spleen and bone marrow were not significantly different between βΔ/βΔ and +/+ mice. Bone marrow from βΔ/βΔ mice generated significantly fewer myeloid colonies (CFU-GM) compared to wild-type marrow (28 ± 9 v. 42 ± 8 colonies per 20,000 cells, p<0.05). Additionally, βΔ/βΔ lineage-depleted bone marrow cells cultured in G-CSF and SCF produced significantly fewer CD11b+/Gr1+ myeloid cells compared to +/+ cells (52.8 ± 6.5% v. 68.3 ± 2.6%, p<0.05). In contrast, bone marrow from βΔ/βΔ and +/+ mice produced equal numbers of pro-B colonies in CFU assays containing the lymphopoietic cytokine IL-7. Finally, as a test of emergency granulopoiesis, we administered a single dose of G-CSF (250 μg/kg subcutaneously) or an equal volume of PBS, and 24 hr later measured the ANC, percentage of CD11b+/Gr1+ myeloid cells in the bone marrow, and CFU-GM generation. Mice of both genotypes responded to G-CSF stimulation with increases in ANC, percent of myeloid cells within the marrow, and CFU-GM. Bone marrow from βΔ/βΔ mice showed a larger G-CSF-induced increase in CFU-GM (PBS: 22 ± 5 v. G-CSF: 39 ± 1, p<0.05) compared to +/+ marrow (PBS: 24 ± 14 v. G-CSF: 31 ± 14, NS). Thus, Stat3β positively regulates basal granulopoiesis in the bone marrow, and may negatively regulate emergency granulopoiesis. This pattern is the opposite of that seen with deletion of both Stat3 isoforms, indicating that Stat3α’s function is to negatively regulate basal granulopoiesis and positively regulate emergency granulopoiesis. Stat3β also positively regulates circulating B lymphocyte numbers, via a mechanism other than B lymphocyte production in the bone marrow.

scholarly journals Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl.

1989 ◽  
Vol 9 (1) ◽  
pp. 67-73 ◽  
Author(s):  
W S Alexander ◽  
J M Adams ◽  
S Cory
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Bone Marrow Cells ◽  
Lymphocyte Transformation ◽  
Lymphoid Cells ◽  
B Lineage ◽  
B Lymphoid ◽  
Marrow Cells ◽  
B Lineage Cells

Although transgenic mice bearing a c-myc gene controlled by the immunoglobulin heavy-chain enhancer (E mu) eventually develop B-lymphoid tumors, B-lineage cells from preneoplastic bone marrow express the transgene but do not grow autonomously or produce tumors in mice. To determine whether other oncogenes can cooperate with myc to transform B-lineage cells, we compared the in vitro growth and tumorigenicity of normal and E mu-myc bone marrow cells infected with retroviruses bearing the v-H-ras, v-raf, or v-abl oncogene. The v-H-ras and v-raf viruses both generated a rapid polyclonal expansion of E mu-myc pre-B bone marrow cells in liquid culture and 10- to 100-fold more pre-B lymphoid colonies than normal in soft agar. The infected transgenic cells were autonomous, cloned efficiently in agar, and grew as tumors in nude mice. While many pre-B cells from normal marrow could also be induced to proliferate by the v-raf virus, these cells required a stromal feeder layer, did not clone in agar, and were not malignant. Most normal cells stimulated to grow by v-H-ras also cloned poorly in agar, and only rare cells were tumorigenic. With the v-abl virus, no more cells were transformed from E mu-myc than normal marrow and the proportion of tumorigenic pre-B clones was not elevated. These results suggest that both v-H-ras and v-raf, but apparently not v-abl, collaborate with constitutive myc expression to promote autonomous proliferation and tumorigenicity of pre-B lymphoid cells.

scholarly journals Clonal growth of murine pre-B colony-forming cells and their targeted infection by a retroviral vector: dependence on interleukin-7

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1132-1138
Author(s):  
DE Williams ◽  
AE Namen ◽  
DY Mochizuki ◽  
RW Overell
Keyword(s):  
Bone Marrow ◽  
Clonal Growth ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Mouse Bone Marrow ◽  
Retroviral Infection ◽  
Murine Bone Marrow ◽  
Interleukin 7 ◽  
B Lymphoid

The cDNA for interleukin-7 (IL-7) was recently isolated from a stromal cell line derived from a long-term B-lymphoid culture. We report that purified recombinant murine IL-7 can promote the clonal growth in semi- solid culture of a subpopulation of cells expressing the B220 surface antigen from normal murine bone marrow. These colony-forming cells (CFC- Pre-B) give rise to colonies of 20 to 1,000 cells after 7 days in culture. Morphologic examination of cells within the colonies showed a characteristic lymphoid morphology, and histochemical examination demonstrated an absence of markers associated with granulocyte, macrophage, eosinophil, or megakaryocyte differentiation, as well as an absence of hemoglobinization (indicative or erythroid differentiation). IL-7 was found to specifically enhance the infection of CFC-Pre-B but not CFU-GM when the cytokine was present during a 48-hour co- cultivation period between irradiated, retrovirus-producing psi 2 clones and normal mouse bone marrow cells. In contrast, IL-3 enhanced the infection of CFU-GM but not CFC-Pre-B. Thymidine suiciding studies suggest that this targeted infection is due to specific induction of cycling of CFC-Pre-B by IL-7 and CFU-GM by IL-3. These data demonstrate that IL-7 can target retroviral infection into a specific subpopulation of early B-lymphoid cells (CFC-Pre-B), and that IL-7 cannot directly promote the in vitro clonal growth of myeloid committed progenitor cells (ie, CFU-GM).

Oncogene cooperation in lymphocyte transformation: malignant conversion of E mu-myc transgenic pre-B cells in vitro is enhanced by v-H-ras or v-raf but not v-abl

1989 ◽  
Vol 9 (1) ◽  
pp. 67-73
Author(s):  
W S Alexander ◽  
J M Adams ◽  
S Cory
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Bone Marrow Cells ◽  
Lymphocyte Transformation ◽  
Lymphoid Cells ◽  
B Lineage ◽  
B Lymphoid ◽  
Marrow Cells ◽  
B Lineage Cells

Although transgenic mice bearing a c-myc gene controlled by the immunoglobulin heavy-chain enhancer (E mu) eventually develop B-lymphoid tumors, B-lineage cells from preneoplastic bone marrow express the transgene but do not grow autonomously or produce tumors in mice. To determine whether other oncogenes can cooperate with myc to transform B-lineage cells, we compared the in vitro growth and tumorigenicity of normal and E mu-myc bone marrow cells infected with retroviruses bearing the v-H-ras, v-raf, or v-abl oncogene. The v-H-ras and v-raf viruses both generated a rapid polyclonal expansion of E mu-myc pre-B bone marrow cells in liquid culture and 10- to 100-fold more pre-B lymphoid colonies than normal in soft agar. The infected transgenic cells were autonomous, cloned efficiently in agar, and grew as tumors in nude mice. While many pre-B cells from normal marrow could also be induced to proliferate by the v-raf virus, these cells required a stromal feeder layer, did not clone in agar, and were not malignant. Most normal cells stimulated to grow by v-H-ras also cloned poorly in agar, and only rare cells were tumorigenic. With the v-abl virus, no more cells were transformed from E mu-myc than normal marrow and the proportion of tumorigenic pre-B clones was not elevated. These results suggest that both v-H-ras and v-raf, but apparently not v-abl, collaborate with constitutive myc expression to promote autonomous proliferation and tumorigenicity of pre-B lymphoid cells.

Cytotoxic Injury to Thymic Stroma and Impaired Reconstitution.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1232-1232
Author(s):  
Susan E. Prockop ◽  
Richard J. O’Reilly ◽  
Howard Petrie
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
De Novo ◽  
Immunosuppressive Treatment ◽  
Lymphoid Cells ◽  
Cytotoxic Agents ◽  
Cell Transplant ◽  
Wild Type ◽  
Long Term Outcome ◽  
The Impact

Abstract A key component of long-term outcome after stem cell transplant (SCT) is successful reconstitution of the immune system. Effective reconstitution of antigen-specific T-cell immunity requires de novo T cell generation. Bone marrow derived progenitors seed the thymus and undergo a complex process involving lineage commitment, proliferation and selection. Coordinated interaction of marrow-derived lymphoid progenitors with thymic stromal cells is required for successful T lymphopoiesis in the post-natal thymus. Disruption of the microenvironment can result in disrupted T cell lymphopoiesis. One cause of prolonged defects in generating functional T lymphocytes after BMT is damage to the thymic microenvironment induced by radiation or cytotoxic therapy. However, the impact of individual agents, administered at myeloablative or non-myeloablative doses, on the thymic microenvironment has not been fully evaluated. In addition, mechanisms by which stromal injury modifies T cell production and maturation have only begun to be understood. We have developed a model system using immunodeficient mice as a platform on which to assess thymic reconstitution. The thymus of mice deficient for the alpha chain of the IL-7 receptor (IL7R−/−) is relatively depleted of lymphoid cells and can be reconstituted following transplant of wild type marrow administered without myeloablative or immunosuppressive treatment. Injection of low doses of wild type bone marrow into these mice results in low levels of marrow chimerism and a normally cellular thymus repopulated with donor-derived lymphocytes. The ability to achieve this reconstitution appears to depend on absolute numbers of early intra-thymic precursors, rather than on total thymic cellularity. We have exploited this model to differentially assess the effects of cytotoxic agents including radiation and immunosuppressive drugs, on the capacity of the thymic microenvironment to support the maturation of normal lympoid progenitors (Figure 1). We demonstrate that some agents do not affect the ability of the thymic microenvironment to support reconstitution (eg fludarabine), others nearly ablate it (cyclophosphamide). We are also able to show dose, schedule, and synergistic effects on the ability of the thymic microenvironment to support de novo T cell lymphopoeisis. Distinct morphologic and phenotypic effects can be demonstrated by different agents (eg busulfan versus thiotepa) with preliminary data suggesting that the effects are mediated by injury to different stromal subsets. It is anticipated that this information will lead to strategies to both minimize delayed immune reconstitution and to augment T cell lymphopoiesis post-transplant. In addition, further evaluation of impaired thymic reconstitution will augment the understanding of lymphostromal interactions crucial to normal T cell lymphopoiesis.

scholarly journals Clonal growth of murine pre-B colony-forming cells and their targeted infection by a retroviral vector: dependence on interleukin-7

Blood ◽  
1990 ◽  
Vol 75 (5) ◽  
pp. 1132-1138 ◽  
Author(s):  
DE Williams ◽  
AE Namen ◽  
DY Mochizuki ◽  
RW Overell
Keyword(s):  
Bone Marrow ◽  
Clonal Growth ◽  
Bone Marrow Cells ◽  
Lymphoid Cells ◽  
Mouse Bone Marrow ◽  
Retroviral Infection ◽  
Murine Bone Marrow ◽  
Interleukin 7 ◽  
B Lymphoid

Abstract The cDNA for interleukin-7 (IL-7) was recently isolated from a stromal cell line derived from a long-term B-lymphoid culture. We report that purified recombinant murine IL-7 can promote the clonal growth in semi- solid culture of a subpopulation of cells expressing the B220 surface antigen from normal murine bone marrow. These colony-forming cells (CFC- Pre-B) give rise to colonies of 20 to 1,000 cells after 7 days in culture. Morphologic examination of cells within the colonies showed a characteristic lymphoid morphology, and histochemical examination demonstrated an absence of markers associated with granulocyte, macrophage, eosinophil, or megakaryocyte differentiation, as well as an absence of hemoglobinization (indicative or erythroid differentiation). IL-7 was found to specifically enhance the infection of CFC-Pre-B but not CFU-GM when the cytokine was present during a 48-hour co- cultivation period between irradiated, retrovirus-producing psi 2 clones and normal mouse bone marrow cells. In contrast, IL-3 enhanced the infection of CFU-GM but not CFC-Pre-B. Thymidine suiciding studies suggest that this targeted infection is due to specific induction of cycling of CFC-Pre-B by IL-7 and CFU-GM by IL-3. These data demonstrate that IL-7 can target retroviral infection into a specific subpopulation of early B-lymphoid cells (CFC-Pre-B), and that IL-7 cannot directly promote the in vitro clonal growth of myeloid committed progenitor cells (ie, CFU-GM).

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