scholarly journals Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 100-106 ◽  
Author(s):  
HN Steinberg ◽  
AS Tsiftsoglou ◽  
SH Robinson
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Leukemic Cell Lines

Abstract The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

scholarly journals Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 100-106
Author(s):  
HN Steinberg ◽  
AS Tsiftsoglou ◽  
SH Robinson
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Leukemic Cell Lines

The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy.

scholarly journals Clonal analysis of the response of human myeloid leukemic cell lines to colony-stimulating activity

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 285-292 ◽  
Author(s):  
FW Ruscetti ◽  
SJ Collins ◽  
AM Woods ◽  
RC Gallo
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemic Cell ◽  
Conditioned Media ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Leukemic Cell Lines ◽  
Growth Promoting ◽  
Myeloid Leukemic Cell

Abstract The recent development of two continuously proliferating human myeloid leukemic cell lines (HL-60 and KG-1) that response to CSA provides an opportunity for a detailed study of the interaction of CSA with leukemic myeloid cells. Here we report on the colony-forming ability of HL-60 and KG-1 over an extended culture life of the cells. Several different sources of human CSA of different stages of purity enhanced colony formation of these cells. CSA, obtained from conditioned media from an SV-40 transformed human trophoblast, was partially purified, and its activity for normal bone marrow copurified with the activity that stimulated HL-60 colony formation. Over 100 clones of HL-60 were developed and tested for their response to CSA. All responded to CSA by showing an increase in colony size and number. However, none of the colonies formed from any of the 100 clones differentiated in response to CSA despite the fact that many chemical can induce differentiation of HL-60. since HL-60 forms spontaneous colonies without the addition of any exogenous stimulating factors, HL-60 conditioned media and cell extracts were tested for the production by these cells of their own endogenous growth-promoting activity (such as a CSA-like molecule). No growth-promoting endogenous activity was found that stimulated normal bone marrow or HL-60 colony formation even after concentration and fractionation methods were employed. These experiments suggest that: (1) the effect of CSA markedly favors proliferation over differentiation in these cell lines; (2) CSA is unlikely to suppress growth of the age of the type of leukemic myeloid cells that HL-60 and KG-1 represent; and (3) if HL-60 cells produce their own growth- promoting factor it is not detectable in the media.

scholarly journals Clonal analysis of the response of human myeloid leukemic cell lines to colony-stimulating activity

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 285-292
Author(s):  
FW Ruscetti ◽  
SJ Collins ◽  
AM Woods ◽  
RC Gallo
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemic Cell ◽  
Conditioned Media ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Leukemic Cell Lines ◽  
Growth Promoting ◽  
Myeloid Leukemic Cell

The recent development of two continuously proliferating human myeloid leukemic cell lines (HL-60 and KG-1) that response to CSA provides an opportunity for a detailed study of the interaction of CSA with leukemic myeloid cells. Here we report on the colony-forming ability of HL-60 and KG-1 over an extended culture life of the cells. Several different sources of human CSA of different stages of purity enhanced colony formation of these cells. CSA, obtained from conditioned media from an SV-40 transformed human trophoblast, was partially purified, and its activity for normal bone marrow copurified with the activity that stimulated HL-60 colony formation. Over 100 clones of HL-60 were developed and tested for their response to CSA. All responded to CSA by showing an increase in colony size and number. However, none of the colonies formed from any of the 100 clones differentiated in response to CSA despite the fact that many chemical can induce differentiation of HL-60. since HL-60 forms spontaneous colonies without the addition of any exogenous stimulating factors, HL-60 conditioned media and cell extracts were tested for the production by these cells of their own endogenous growth-promoting activity (such as a CSA-like molecule). No growth-promoting endogenous activity was found that stimulated normal bone marrow or HL-60 colony formation even after concentration and fractionation methods were employed. These experiments suggest that: (1) the effect of CSA markedly favors proliferation over differentiation in these cell lines; (2) CSA is unlikely to suppress growth of the age of the type of leukemic myeloid cells that HL-60 and KG-1 represent; and (3) if HL-60 cells produce their own growth- promoting factor it is not detectable in the media.

Identification of Pathogenetically Important Genes within the Common Deleted Region (CDR) of the 5q- Syndrome.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3439-3439
Author(s):  
Soren Lehmann ◽  
Sophie Raynaud ◽  
Julian C. Desmond ◽  
Phillip H. Koeffler
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Refractory Anemia ◽  
Normal Bone Marrow ◽  
Expression Data ◽  
Hematopoietic Stem ◽  
Normal Bone ◽  
Leukemic Cell Lines ◽  
Biological Studies

Abstract The 5q- syndrome is characterized by refractory anemia, normal or high platelet count, hypolobulated megakaryocytes, a good prognosis and a low risk of leukemic transformation. Although the CDR has been defined to a 1.5 Mb interval on the long arm on chromosome 5 (5q33.1), the molecular pathogenesis of the disease is still unknown. The CDR contains 39 known-genes of which 33 have been shown to be expressed in hematopoietic stem cells. In order to elucidate the molecular mechanisms behind the 5q- syndrome, we performed real-time quantitative PCR on these 33 genes. Samples from the bone marrow of 12 patients with a sole deletion of 5q and 14 patients with MDS with normal karyotype were initially analyzed. The genes that showed the most pronounced decrease in expression in the 5q- samples were: SLC36A1 (89% down-regulated compared to non 5q-), G3BP (79%), ATOX1 (76%), CSF1R (76%), RPS14 (74%), PDGFRB (73%), TNIP1 (72%), SPARC (71%), ANAX6 (69%), NSDT (66%) and TIGD (60%). SPARC expression was found to be higher in both types of MDS samples compared to normal bone marrow (n=18) as well as compared to seven leukemic cell lines (HL-60, NB4, HEL, KG1, K562, U937 and TP-1). ATOX1 expression was highly over-expressed (20- to 80-fold) in the leukemic cell lines and modestly but significantly higher in normal bone marrow compared to both types of MDS. For G3BP, the expression was similar in normal bone marrow compared to the non-5q- samples but 1- to 10-fold higher in the cell lines. RPS14 was down-regulated in both types of MDS compared to normal bone marrow and leukemic cell lines. Thus, we have identified the most significantly down-regulated genes within the CDR of the 5q- syndrome. Based on our expression data, their known biological functions and on publicly available tissue expression data, genes such as G3BP, ATOX1, TNIP1, RPS14 and CSF1R are interesting targets for further studies. Biological studies are currently being performed on these genes with respect to their role during hematopoiesis with special focus on erythropoiesis.

Pioglitazone Inhibits the Growth of Human Leukemic Cell Lines and Primary Leukemic Cells in Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4493-4493 ◽  
Author(s):  
Yoshihiro Hatta ◽  
Minoru Saiki ◽  
Yuko Enomoto ◽  
Shin Aizawa ◽  
Umihiko Sawada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Hematopoietic Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Peroxisome Proliferator ◽  
Leukemic Cell Lines ◽  
Ppar Γ

Abstract Troglitazone and pioglitazone are one of thiazolidinediones that are high affinity ligand for the nuclear receptor called peroxisome proliferator-activated receptor gamma (PPAR-γ). Troglitazone is a potent inhibitor of clonogenic growth of acute myeloid leukemia cells when combined with a retinoid. However, the effect of pioglitazone to neoplastic cells and normal hematopoietic cells has not been studied yet. Adult T-cell leukemia (ATL), prevalent in western Japan, is a highly aggressive malignancy of mature T lymphocyte. Therefore, we studied antitumor effect of pioglitazone against leukemic cells including ATL as well as normal hematopoietic cells. With 300 μM of pioglitazone, colony formation of ATL cell lines (MT1, MT2, F6T, OKM3T, and Su9T01) was completely inhibited. Colony formation of HUT102, another ATL cell line, was 12 % compared to untreated control. Clonogenic cells of other leukemic cell lines (K562, HL60, U937, HEL, CEM, and NALM1) was also inhibited to 0–30% of control. Colony formation of primary leukemic cells from 5 AML patients was decreased to 15 %. However, normal hematopoietic cells were weakly inhibited with 300 μM pioglitazone; 77 % of CFU-GM, 70 % of CFU-E, and 33 % of BFU-E survived. Cell cycle analysis showed that pioglitazone decreased the ratio of G2/M phase in HL60 cells, suggesting the inhibition of cell division. By Western blotting, PPAR-γ protein level was similar in all leukemic cells and normal bone marrow mononuclear cells. Taken together, pioglitazone effectively eliminate leukemic cells and could be used as an antitumor agent in vivo.

Targeting Aberrant Self-Renewal of Leukemic Cells with a Novel CBP/p300 Bromodomain Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3750-3750
Author(s):  
Angeliki Thanasopoulou ◽  
Katharina Dumrese ◽  
Sarah Picaud ◽  
Oleg Fedorov ◽  
Stefan Knapp ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Colony Formation ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines

Abstract The CBP/p300 histone acetyltransferases are key transcriptional regulators of hematopoiesis that have been found to be involved in AML-associated recurrent chromosomal translocations and shown to function as co-activators of leukemogenic fusion oncogenes, suggesting that specific targeting of CBP/p300 may be beneficial for therapy. We characterized the anti-leukemic potential of I-CBP112, a novel chemical inhibitory probe targeting the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. I-CBP112 represents a new, potent and selective class of BRD inhibitors (oxazepines) binding to recombinant CBP/p300 BRDs with a KD of 151nM and 157nM respectively. Initial characterization by FRAP and BRET assays revealed that I-CBP112 displaced the isolated BRD construct from chromatin but not the full length CBP. I-CBP112 also impaired the interaction of CBP/p300 with p53, resulting in reduced p53-K382 acetylation, reduced p21 expression, and high sensitivity to Doxorubicin-induced DNA damage. We started to explore the effects of the compound on leukemic cells by exposing a series of murine cell lines immortalized by the MLL-CBP fusion and other potent leukemia-associated oncogenes including the MLL-AF9, MLL-ENL, or the NUP98-HOXA9 fusion to increasing doses of I-CBP112. Interestingly, no significant cytotoxicity was observed up to concentrations of 5μM. However, in all cell lines we observed a significant reduced number of colonies formed in methylcellulose, associated with morphological differentiation as observed in Giemsa stained cytospots. Similar to the murine leukemic cell lines we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Reduced colony formation upon I-CBP112 treatment was also observed of human primary AML blasts but not of CD34+ hematopoietic stem cells from two healthy donors. I-CBP112 effects were studied in more detail in three human leukemia cell lines: SEM (MLL-AF4+), MOLM13 (MLL-AF9+) and Kasumi-1 (AML1-ETO+). Long-term exposure of these cells to I-CBP112 in liquid medium, resulted in a dose-dependent G1 cell cycle arrest, with Kasumi-1 being the most sensitive to the inhibitor, demonstrating further morphological signs of differentiation and apoptotic cell death. Importantly, combination of I-CBP112 with the BET-BRD inhibitor JQ1 or Doxorubicin revealed a clear synergistic effect on cell survival of the AML cell lines except for the combination of I-CBP112 with Doxorubicin on MOLM13. Surprisingly only modest effects of I-CBP112 exposure on the transcriptional programs of SEM, MOLM13 and Kasumi-1 cells were found by microarray expression profiling. Genes found affected were mainly immune response regulators or NFkappaB targets suggesting that attenuation of NFkappaB downstream signals might impair the leukemia initiation capacity reflected by reduced colony formation. Extreme limited dilution assays (ELDA) in methylcellulose, as well as bone marrow transplantations in limiting dilutions using MLL-AF9-transformed murine leukemic blasts revealed that I-CBP112 significantly impaired self-renewal of the leukemic stem cell compartment in vitro and reduced the leukemia-initiating potential in vivo. Taken together, these data demonstrate that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML and other human cancers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Utilization of a colony assay to assess the variables influencing elimination of leukemic cells from human bone marrow with monoclonal antibodies and complement

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 945-950
Author(s):  
TW LeBien ◽  
DE Stepan ◽  
RM Bartholomew ◽  
RC Stong ◽  
JM Anderson
Keyword(s):  
Bone Marrow ◽  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Colony Assay ◽  
Leukemic Cell Lines ◽  
Human Leukemic Cells ◽  
Cell Colony

We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB- NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.

Expression of MIF and CD74 in leukemic cell lines: correlation to DR expression destiny

2016 ◽  
Vol 397 (6) ◽  
pp. 519-528
Author(s):  
Mirella Georgouli ◽  
Lina Papadimitriou ◽  
Maria Glymenaki ◽  
Valia Patsaki ◽  
Irene Athanassakis
Keyword(s):  
Cell Lines ◽  
Immune Surveillance ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Leukemic Cell Lines ◽  
Hla Dr ◽  
High Affinity Receptor ◽  
Escape Process ◽  
Affinity Receptor

Abstract Invariant chain (Ii) or CD74 is a non-polymorphic glycoprotein, which apart from its role as a chaperone dedicated to MHCII molecules, is known to be a high-affinity receptor for macrophage migration inhibitory factor (MIF). The present study aimed to define the roles of CD74 and MIF in the immune surveillance escape process. Towards this direction, the cell lines HL-60, Raji, K562 and primary pre-B leukemic cells were examined for expression and secretion of MIF. Flow cytometry analysis detected high levels of MIF and intracellular/membrane CD74 expression in all leukemic cells tested, while MIF secretion was shown to be inversely proportional to intracellular HLA-DR (DR) expression. In the MHCII-negative cells, IFN-γ increased MIF expression and induced its secretion in HL-60 and K562 cells, respectively. In K562 cells, CD74 (Iip33Iip35) was shown to co-precipitate with HLA-DOβ (DOβ), inhibiting thus MIF or DR binding. Induced expression of DOα in K562 (DOα-DOβ+) cells in different transfection combinations decreased MIF expression and secretion, while increasing surface DR expression. Thus, MIF could indeed be part of the antigen presentation process.

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