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.

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

Abstract 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.

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 The effects of tumor necrosis factor-alpha on early human hematopoietic progenitor cells treated with 4-hydroperoxycyclophosphamide

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 681-689
Author(s):  
J Moreb ◽  
JR Zucali ◽  
S Rueth
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Leukemic Cell ◽  
Tnf Alpha ◽  
Protective Effects ◽  
Necrosis Factor Alpha ◽  
Leukemic Cell Lines ◽  
Blast Cells ◽  
Factor Alpha

We have previously reported that 20 hours' preincubation of human bone marrow cells with interleukin-1 beta (IL-1) can protect early progenitor cells from 4-hydroperoxycyclophosphamide (4-HC) cytotoxicity. Since tumor necrosis factor-alpha (TNF alpha) shares many of the biologic properties of IL-1, we have compared the protective effects of TNF alpha with IL-1 against 4-HC. Incubation of human bone marrow mononuclear cells or an enriched progenitor population for 20 hours with either TNF alpha or IL-1 resulted in the survival of an increased number of single- and mixed-lineage colonies, including replatable blast cell colonies, while only rare colonies were seen in the control group. Antibodies to TNF alpha completely abolished the protection observed with IL-1, while antibodies to IL-1 alpha and IL-1 beta decreased but did not abolish the protection seen with TNF alpha. Combinations of low doses of TNF alpha and IL-1 showed synergy in their protective effects. Furthermore, no protection was observed by IL-1, IL- 1 bone-marrow-conditioned medium (IL-1-BMCM), or TNF alpha for HL-60, K562, KG1, KG1a, and DU.528 leukemic-cell lines or primary acute myelogenous leukemic (AML) blast cells from the lethal effects of 4-HC. In the case of HL-60 and KG1a cell lines, TNF alpha preincubation resulted in increased cytotoxicity. Furthermore, preincubation of a mixture of AML cells and normal bone-marrow cells with IL-1 + TNF alpha before 4-HC resulted in the protection of normal but not leukemic progenitors. These results suggest that TNF alpha is necessary for the protection of normal, early, human hematopoietic progenitors from 4-HC, while IL-1 is not mandatory but will synergize with TNF alpha to offer increased protection. In addition, no protection from 4-HC is observed by TNF alpha, IL-1, or IL-1-BMCM for primary leukemic blast cells or leukemic cell lines.

scholarly journals The effects of tumor necrosis factor-alpha on early human hematopoietic progenitor cells treated with 4-hydroperoxycyclophosphamide

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 681-689 ◽  
Author(s):  
J Moreb ◽  
JR Zucali ◽  
S Rueth
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Leukemic Cell ◽  
Tnf Alpha ◽  
Protective Effects ◽  
Necrosis Factor Alpha ◽  
Leukemic Cell Lines ◽  
Blast Cells ◽  
Factor Alpha

Abstract We have previously reported that 20 hours' preincubation of human bone marrow cells with interleukin-1 beta (IL-1) can protect early progenitor cells from 4-hydroperoxycyclophosphamide (4-HC) cytotoxicity. Since tumor necrosis factor-alpha (TNF alpha) shares many of the biologic properties of IL-1, we have compared the protective effects of TNF alpha with IL-1 against 4-HC. Incubation of human bone marrow mononuclear cells or an enriched progenitor population for 20 hours with either TNF alpha or IL-1 resulted in the survival of an increased number of single- and mixed-lineage colonies, including replatable blast cell colonies, while only rare colonies were seen in the control group. Antibodies to TNF alpha completely abolished the protection observed with IL-1, while antibodies to IL-1 alpha and IL-1 beta decreased but did not abolish the protection seen with TNF alpha. Combinations of low doses of TNF alpha and IL-1 showed synergy in their protective effects. Furthermore, no protection was observed by IL-1, IL- 1 bone-marrow-conditioned medium (IL-1-BMCM), or TNF alpha for HL-60, K562, KG1, KG1a, and DU.528 leukemic-cell lines or primary acute myelogenous leukemic (AML) blast cells from the lethal effects of 4-HC. In the case of HL-60 and KG1a cell lines, TNF alpha preincubation resulted in increased cytotoxicity. Furthermore, preincubation of a mixture of AML cells and normal bone-marrow cells with IL-1 + TNF alpha before 4-HC resulted in the protection of normal but not leukemic progenitors. These results suggest that TNF alpha is necessary for the protection of normal, early, human hematopoietic progenitors from 4-HC, while IL-1 is not mandatory but will synergize with TNF alpha to offer increased protection. In addition, no protection from 4-HC is observed by TNF alpha, IL-1, or IL-1-BMCM for primary leukemic blast cells or leukemic cell lines.

scholarly journals The effects of alkyl-lysophospholipids on leukemic cell lines. I. Differential action on two human leukemic cell lines, HL60 and K562

Blood ◽  
1981 ◽  
Vol 57 (4) ◽  
pp. 794-797 ◽  
Author(s):  
T Tidwell ◽  
G Guzman ◽  
WR Vogler
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Short Term ◽  
Cell Numbers ◽  
Human Leukemic Cell ◽  
Leukemic Cell Lines ◽  
Low Concentrations ◽  
Bone Marrow Cultures ◽  
Differential Action

Abstract The action of an alkyl-lysophospholipid (ALP), ET180CH3, on clonogenicity, 3H-TdR uptake, and cell numbers was tested in two human leukemic cell lines, HL60 and K562, and short-term human leukemic bone marrow cultures. ALP eliminated clonogenicity in HL60 but not in K562 cultures; 3H-TdR uptake and cell numbers were depressed at low concentrations of ET180CH3 in HL60, but not K562 cultures. The action of the lysophospholipid analog on human leukemic bone marrow short-term cultures at low concentrations was similar to its action on HL60 cultures; clonogenicity and 3H-TdR uptake were depressed, but cell numbers were not significantly affected. The demonstration of differential action of ALP on two cell lines should significantly simplify the investigation of the mechanism of the reported differential action of ET180CH3 on normal and leukemic cell membranes.

scholarly journals A combined approach for purging multidrug-resistant leukemic cell lines in bone marrow using a monoclonal antibody and chemotherapy

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 2079-2084 ◽  
Author(s):  
M Aihara ◽  
Y Aihara ◽  
G Schmidt-Wolf ◽  
I Schmidt-Wolf ◽  
BI Sikic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Multidrug Resistant ◽  
Selective Removal ◽  
Malignant Cells ◽  
Log Reduction ◽  
Leukemic Cell Lines ◽  
Surface Product ◽  
Rabbit Complement

Abstract Selective removal of malignant cells (purging) from bone marrow (BM) is a concern in autologous BM transplantation (ABMT). Use of vincristine, etoposide, or doxorubicin for purging could be rendered ineffective by the presence of multidrug-resistant (MDR) tumor cells. To circumvent this particular problem, we investigated whether 17F9, a monoclonal IgG2b antibody directed against the cell surface product of the MDR gene, P-glycoprotein, is effective in selective removal of MDR cells from BM when used with rabbit complement (C′). Using two different cell lines we have demonstrated that 17F9 + C′ selectively lyses MDR- positive cells. Three rounds of antibody + C′ resulted in 96.4% +/- 3.6% kill of K562/DOX and 100% +/- 0% of CEM/VLB cells. Mixtures of malignant cells and normal BM resulted in 99.85% removal of K562/DOX and 99.91% removal of CEM/VLB clonogenic cells. This treatment did not affect normal committed precursors compared with C′ alone. The addition of the cytotoxic agent etoposide (VP-16) following antibody purging results in a 4.6 log reduction of malignant cells. Furthermore, this antibody was effective when used against patients' leukemic blasts. These results suggest the use of 17F9 + C′ is effective and selective for removal of MDR cells from BM before ABMT and the addition of VP-16 enhances the purging efficacy.

Differential expression of a novel proline-rich homeobox gene (Prh) in human hematolymphopoietic cells

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1237-1245 ◽  
Author(s):  
G Manfioletti ◽  
V Gattei ◽  
E Buratti ◽  
A Rustighi ◽  
A De Iuliis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Maturation Stage ◽  
Lymphoid Tissues ◽  
Specific Cell ◽  
B Cell Differentiation ◽  
Leukemic Cell Lines

Proline-rich homeobox (Prh) is a novel human homeobox-containing gene recently isolated from the CD34+ cell line KG-1A, and whose expression appears mainly restricted to hematopoietic tissues. To define the pattern of Prh expression within the human hematopoietic system, we have analyzed its constitutive expression in purified cells obtained from normal hematopoietic tissues, its levels of transcription in a number of leukemia/lymphoma cell lines representing different lineages and stages of hematolymphopoietic differentiation, and its regulation during in vitro maturation of human leukemic cell lines. Prh transcripts were not detected in leukemic cells of T-lymphoid lineage, irrespective of their maturation stage, and in resting or activated normal T cells from peripheral blood and lymphoid tissues. In contrast, high levels of Prh expression were shown in cells representing early stages of B lymphoid maturation, being maintained up to the level of circulating and tissue mature B cells. Terminal B-cell differentiation appeared to be conversely associated with the deactivation of the gene, since preplasmacytic and plasmocytoma cell lines were found not to express Prh mRNA. Prh transcripts were also shown in human cell lines of early myelomonocytic, erythromegakaryocytic, and preosteoclast phenotypes. Prh expression was lost upon in vitro differentiation of leukemic cell lines into mature monocyte-macrophages and megakaryocytes, whereas it was maintained or upregulated after induction of maturation to granulocytes and osteoclasts. Accordingly, circulating normal monocytes did not display Prh mRNA, which was conversely detected at high levels in purified normal granulocytes. Our data, which show that the acquisition of the differentiated phenotype is associated to Prh downregulation in certain hematopoietic cells but not in others, also suggest that a dysregulated expression of this gene might contribute to the process of leukemogenesis within specific cell lineages.

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