The Effects of Drugs that Cause Neutropenia upon Colony Formation by Bone Marrow Cells in Semi-Solid Agar

1974 ◽  
Vol 46 (5) ◽  
pp. 619-628
Author(s):  
A. Howell ◽  
Susan Chinn ◽  
T. M. Andrews ◽  
R. W. E. Watts
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Mouse Bone Marrow ◽  
Sodium Aurothiomalate ◽  
Response Curves ◽  
Agar Culture ◽  
Drug Concentrations ◽  
Solid Agar ◽  
Semi Solid

1. The effect of drugs that cause neutropenia has been tested upon normal bone marrow colony formation in semi-solid agar. 2. Thiouracil, methimazole and phenylbutazone do not cause inhibition whereas sodium aurothiomalate, 6-mercaptopurine and chloramphenicol cause dose-related inhibition of colony formation at or below drug concentrations found in serum during therapy. Chloramphenicol also inhibits mouse bone marrow, L cells and Hela cells in agar culture. 3. Dose—response curves have been analysed by the probit method and found to be reproducible.

scholarly journals Effects of neuraminidase on the regulation of erythropoiesis

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 784-788 ◽  
Author(s):  
VF LaRussa ◽  
F Sieber ◽  
LL Sensenbrenner ◽  
SJ Sharkis
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Colony Growth ◽  
Mouse Bone Marrow ◽  
Continuous Exposure ◽  
Low Concentrations ◽  
High Concentrations ◽  
Marrow Cells

Abstract In this article, we present evidence that sialic acid-containing surface components play a role in the regulation of erythropoiesis. A 1- hr exposure of mouse bone marrow cells to high concentrations of neuraminidase reduced erythroid colony formation. Coculture of 10(6) untreated thymocytes with neuraminidase-treated bone marrow cells restored erythroid colony growth. Neuraminidase-treated thymocytes retained their ability to suppress erythroid colony formation by untreated marrow cells, but lost their ability to enhance erythroid colony formation. Continuous exposure to low concentrations of neuraminidase enhanced erythroid bone marrow cell colony growth in response to a suboptimal dose of erythropoietin.

scholarly journals Cell interactions influencing murine marrow megakaryocytes: nature of the potentiator cell in bone marrow

Blood ◽  
1981 ◽  
Vol 57 (1) ◽  
pp. 157-163 ◽  
Author(s):  
N Williams ◽  
H Jackson ◽  
P Ralph ◽  
I Nakoinz
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Buoyant Density ◽  
Colony Growth ◽  
Mouse Bone Marrow ◽  
Bone Marrow Cultures ◽  
Cell Conditioned Medium ◽  
Marrow Cells

Abstract Auxiliary bone marrow cells are required for optimal murine megakaryocyte colony formation in addition to progenitor cells and a colony stimulating activity (CSA) present in WEHI-3 cell conditioned medium. These auxiliary cells are adherent, with a sedimentation rate of 5.8 mm hr-1 and buoyant density of 1.065–1.078 gcm-3. The activity from bone marrow cells is loss at irradiation doses above 900 rad. Bone marrow cells with these characteristics, and supernatants from lung, bone shafts, and peritoneal exudate cells were all active in enhancing megakaryocyte colony incidences in mouse bone marrow cultures above those stimulated by an obligatory activity in WEHI-3 cell conditioned medium. Certain macrophage cell lines (J774, P388D1) could elaborate the activity. This study confirms that a potentiation activity enhances CSA stimulation of megakaryocyte colony formation. The potentiator is elaborated by bone marrow cells in limiting amounts requiring either high cell concentrations or an exogenous source of the activity for optimal colony growth.

scholarly journals Effects of neuraminidase on the regulation of erythropoiesis

Blood ◽  
1984 ◽  
Vol 63 (4) ◽  
pp. 784-788
Author(s):  
VF LaRussa ◽  
F Sieber ◽  
LL Sensenbrenner ◽  
SJ Sharkis
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Colony Growth ◽  
Mouse Bone Marrow ◽  
Continuous Exposure ◽  
Low Concentrations ◽  
High Concentrations ◽  
Marrow Cells

In this article, we present evidence that sialic acid-containing surface components play a role in the regulation of erythropoiesis. A 1- hr exposure of mouse bone marrow cells to high concentrations of neuraminidase reduced erythroid colony formation. Coculture of 10(6) untreated thymocytes with neuraminidase-treated bone marrow cells restored erythroid colony growth. Neuraminidase-treated thymocytes retained their ability to suppress erythroid colony formation by untreated marrow cells, but lost their ability to enhance erythroid colony formation. Continuous exposure to low concentrations of neuraminidase enhanced erythroid bone marrow cell colony growth in response to a suboptimal dose of erythropoietin.

scholarly journals Fundamental Studies on Granulocyte-Macrophage Colony Formation by Mouse Bone Marrow Cells

1986 ◽  
Vol 8 (2) ◽  
pp. 193-199
Author(s):  
Yuji YAMAGUCHl ◽  
Masahiro MISAGO ◽  
Makoto KIKUCHI ◽  
Shyozo CHIBA
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Mouse Bone Marrow ◽  
Macrophage Colony ◽  
Mouse Bone Marrow Cells ◽  
Marrow Cells

scholarly journals Enrichment for CFU-C from murine and human bone marrow using soybean agglutinin

Blood ◽  
1982 ◽  
Vol 59 (2) ◽  
pp. 360-363
Author(s):  
Y Reisner ◽  
N Kapoor ◽  
MZ Hodes ◽  
RJ O'Reilly ◽  
RA Good
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Graft Versus Host Reaction ◽  
Mouse Bone Marrow ◽  
Soybean Agglutinin ◽  
Mixed Cell ◽  
Agar Culture ◽  
Marrow Cells

Mouse bone marrow and spleen cells agglutinated by soybean agglutinin (SBA) or peanut agglutinin (PNA) were previously shown to be enriched for spleen colony-forming cells (CFU-S) and sufficiently depleted of graft-versus-host reaction producing cells to allow hematologic reconstitution of lethally irradiated allogeneic recipient mice. A similar enrichment for cells capable of forming colonies in soft agar culture (CFU-C) has now been found in the SBA-agglutinated fraction of mouse bone marrow cells, in contrast to the finding that in human bone marrow the majority of the CFU-C are in the fraction not agglutinated by SBA. Cytofluorometric studies with fluorescein-labeled SBA (FITC- SBA) revealed that the majority of both mouse and human bone marrow cells bind the lectin. Experiments mixing the human marrow fractions separated by SBA reveal that true enrichment for CFU-C is achieved in the unagglutinated fraction, as opposed to a possible depletion of a suppressor cell population. Granulocytic, monocytic, and mixed cell colonies were all enriched in the SBA-unagglutinated cell fraction from human bone marrow.

PTPN11 Mutations in JMML Confer Dasatinib Sensitivity in a TNK2-Dependent Manner

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3154-3154
Author(s):  
Chelsea Jenkins ◽  
Samuel B Luty ◽  
Julia E. Maxson ◽  
Christopher A. Eide ◽  
Bill H. Chang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Cell Transformation ◽  
Ex Vivo ◽  
Therapeutic Strategies ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Mapk Activation ◽  
Mouse Bone Marrow Cells

Abstract Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare disease characterized by an overproduction of granulocytes and monocytes, with cells displaying hypersensitivity to stimulation by the cytokine GM-CSF in culture. The only curative approach for JMML is allogeneic hematopoietic stem cell transplant, and disease recurrence is the leading cause of death in these patients, occurring within two years in 50% of patients. New therapeutic strategies are needed to improve treatment and survival. Methods: In order to better identify therapeutic strategies, our lab has developed ex vivo functional assays with small-molecule inhibitors and RNAi for the profiling of actionable targets in primary patient samples. In these assays, mononuclear cells from patient bone marrow or blood are cultured in the presence of small molecule inhibitor or RNAi libraries and cell viability is measured by colorimetric assay. To investigate targets identified in these assays, gene constructs are transfected into HEK293 T17 cells for immunoblots or transduced into mouse bone marrow cells for methylcellulose-based colony formation assays. Results: Mononuclear white blood cells from a patient with recurrent JMML were found to be sensitive to the multi-family kinase inhibitor dasatinib as well as siRNA targeting the dasatinib target tyrosine kinase non-receptor 2 (TNK2, also known as ACK1), suggesting an over-reliance on TNK2 for cell viability. TNK2 was recently demonstrated by our lab and others to regulate signaling downstream of signaling pathways including EGFR, ER, Src, and CSF3R. Based on the ex vivo functional data for this JMML specimen, the patient was placed on daily dasatinib and achieved marked improvement, which bridged the patient to an additional bone marrow transplant, resulting in disease-free survival for another year. This patient had a mutation in PTPN11 (also known as SHP2), which encodes a protein tyrosine phosphatase that is mutated in approximately 35% of JMML patients. PTPN11 is an auto-inhibited phosphatase that dephosphorylates targets in many signaling pathways including RAS-MAPK, with an overall positive effect on pathway activity and cellular proliferation. In JMML, mutations in residues of the N-terminal SH2 domain of PTPN11 result in a constitutively active form of the protein, which increases proliferation and cell survival and is linked to dysregulation of RAS/MAPK. Results of in vitro experiments demonstrate, for the first time, an interaction of TNK2 with mutant PTPN11. Co-expression of mutant-PTPN11 and TNK2 results in increased phospho-PTPN11 at two sites associated with activation of PTPN11 (Y542 and Y580), and co-immunoprecipitation experiments indicate direct interaction of TNK2 with PTPN11. These phosphorylation events coincide with significantly increased MEK/ERK signaling in the context of TNK2/mutant PTPN11 co-expression relative to expression of either gene alone. Expression of a TNK2 mutant harboring a loss-of-function mutation at a key activating residue (TNK2 Y284F) reduces PTPN11/MAPK activation, whereas expression of a gain-of-function, drug-resistant TNK2 gatekeeper mutant increases PTPN11/MAPK activation when compared to wild-type TNK2. Inhibition of TNK2 with either dasatinib or the TNK2-specific inhibitor AIM100 also reduces PTPN11/MAPK activation. Interestingly, phospho-TNK2 levels are significantly reduced in the presence of mutant-PTPN11, suggesting a feedback mechanism that has yet to be fully elucidated. Functional experiments suggest that mutant-PTPN11 and TNK2 work in a synergistic fashion to increase cell transformation and cytokine hypersensitivity. Mouse bone marrow cells transduced with both mutant PTPN11 and TNK2 have significantly more colonies in methylcellulose colony formation assays than with mutant- PTPN11 alone or with wild-type PTPN11 and TNK2. Treatment of cells in colony formation assays with either dasatinib or AIM100 inhibits colony formation, suggesting a dependence on TNK2 kinase activity for cell transformation. Conclusions: Taken together, the preliminary data suggest that PTPN11 mutations may be a functional marker for dasatinib sensitivity, and that the underlying mechanism for this sensitivity may be through the inhibition of TNK2 activity by dasatinib. Our aim is further validate the role of PTPN11 mutations as a marker for efficacy of dasatinib or other TNK2 inhibitors. Disclosures No relevant conflicts of interest to declare.

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