© 1991 S. Karger AG. Basel 0009-3157/91/0376-0441 $ 2.75/0
Chemotherapy 1991;37:441-448
Effects of Recombinant Human Cytokines on Cytarabine Activity in K562 Human Myeloid Leukaemia Cells HT. Hassan, H. R. Maurer Leukämie-Forschung. Institut für Pharmazie, Freie Universität Berlin, FRG
Key Words. Cytarabine • Cytokine • Interferon ■Interleukin • Myelocytic leukaemia
Introduction
The human myeloid leukaemia cell line K562, which was established from the pleural effusion of a chronic myeloid leu kaemia patient in blastic crisis [I], pro vides a unique population of primitive myeloid leukaemic cells [2]. K562 cells
were induced to differentiate along the erythroid, granulocytic, macrophage and megakaryocytic lineages in response to several agents [2], Cytarabine is not only the most widely used drug in the treat ment of myeloid leukaemia [3] but also one of the most potent inducers of crythroid differentiation of K562 cells [4] which
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 9:55:01 AM
Abstract. The K562 cell line provides a unique population of primitive human mye loid leukaemia cells which can be induced to differentiate along the erythroid, granulo cytic, macrophage and megakaryocytic lineages in response to several agents. Cytara bine is not only the most widely used drug in the treatment of myeloid leukaemia but also the most effective agent in K562 cells. The effects of five recombinant human cyto kines - interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GMCSF), interferon-a, interferon-|I and interferon-y on cytarabine-induced growth inhibi tion and differentiation of K562 cells was studied in liquid suspension cultures. GM-CSF and to a lesser extent IL-3 enhanced the antiproliferative effect of cytarabine in K562 cells, whereas the three interferons reduced it. The efficacy of cytarabine in inhibiting the growth of K562 cells was doubled by its combination with GM-CSF or IL-3 but was halved by its combination with interferons. The five cytokines did not significantly affect cytarabine-induced erythroid differentiation of K562 cells. The present results appear to favour the use of GM-CSF and IL-3 but not of interferons in future treatment strategies based on a combined cytokine and chemotherapy approach for myeloid leukaemia.
Hassan/Maurer
442
Materials and Methods Materials Benzidine, cytarabine, glacial acetic acid, hydro gen peroxide, RPMI 1640 culture medium and try pan blue were purchased from Sigma Chemicals. Fetal calf serum was purchased from Bochringer. Recombinant human GM-CSF (specific activity of 5 x 107 U/mg protein), IL-3 (specific activity of 5 x 107 U/mg protein), Interferon-p, (specific activity of 1 x 107 U/mg protein) and interferon-y (specific activity of 2.5 x 107 U/mg protein) were purchased from Genzyme Corporation. Recombinant human interferon-«,,, (specific activity of 4.2 x 107 U/mg protein) was purchased from Hoffmann-La Roche. Cytarabine was diluted with RPMI 1640 medium to 1 mM stock solution and kept at -20 °C. Prior to in cubation, cytarabine was freshly diluted with RPMI 1640 medium to working concentrations. Cytokines were diluted with RPMI 1640 medium containing 10% fetal calf serum 0.5 million U/ml stock solution and kept at -70°C. Prior to incubation cytokines were freshly diluted with RPMI 1640 medium con taining 10% fetal calf serum to working concentra tions. Cells K562 cells were grown in suspension culture in RPMI 1649 medium containing 10% fetal calf serum in 10 ml in 25-cm2 tissue culture flasks at 37 °C in a humidified incubator under 7.5% CO,. The cultures were diluted to 5 x 104 cells/ml in fresh medium twice weekly. Cell concentration and viability were assessed by the trypan blue exclusion test. Cell Culture K562 cells were cultured at a concentration of 5 x 104 cells/ml in 24 well tissue culture plates con taining RPMI 1640 medium supplemented with /glutamine and 10% fetal calf serum in a final vol ume of 1 ml. Each of the five recombinant human cytokines: GM-CSF, IL-3, interferon-a, -p and -y were added at graded concentrations (100-1,000 U/ ml) either alone or with cytarabine at graded con centrations (0.05, 0.5, 5 and 50 \xM). Also, cultures containing only cytarabine at the same graded con centrations and control cultures containing neither cytarabine nor GM-CSF nor IL-3 nor interferons were set up in conditions otherwise identical. Qua druplicate plates of each of the culture conditions
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 9:55:01 AM
was terminal, irreversible [5] and S phase dependent [6]. Several chemical and biological agents enhanced cytarabine activity in human myeloid leukaemia cells [7], Recombi nant human granulocyte-macrophage col ony stimulating factor (GM-CSF) not only enhanced vitamin D3-induced monocytic differentiation of U937 human monoblastic leukacmic cells [8] and rctinoic-acid-induced granulocytic differen tiation of human congenital agranulocy tosis bone marrow progenitor cells [9] but also the cytostatic activity of cytara bine and hydroxyurea in HL-60 [101 and AML [11, 12] cells. Moreover, GM-CSF co-operated with cytarabine in inducing differentiation of the resistant human myeloid luekaemic cell line KG1 [13] and with recombinant human erythropoietin in inducing erythroid differentiation in more than 80% of K562 clonogenic cells [14], Recombinant human interlcukin-3 (IL-3) enhanced the cytoxicity of cytara bine in AML cells [12, 15]. Recombinant human interferons enhanced retinoicacid-, vitamin-Dr and tumour-necrosisfactor-a-induced growth inhibition and differentiation of human myeloid leukae mia cells [7]. The interactions between cytarabine and recombinant human cytokines could provide the basis for improved therapeutic regimens based on a combined cytokine and chemotherapy approach for myeloid leukaemic patients. Therefore, we investi gated the effects of five recombinant hu man cytokines: GM-CSF, IL-3, interferon-a, interferon-ß and interferon-y on cytarabine-induced growth inhibition and differentiation of K562 human myeloid leukaemia cells.
443
Effects of Cytokines on Cytarabine Activity
Table 1. Effect of recombinant human cytokines in concentrations from 100 to 1.000 U/ml on K562 human myeloid leukaemia cell proliferation in liquid suspension culture Recombinant human cytokine
IL-3 GM-CSF Interferon-a,b Interferon-ß, Interferon-y
K562 cell count on day 4, % of control culture 1(H) U/ml
500 U/ml
1,000 U/ml
131.6 ± 5.1 II 8.4 ±5.2 86.8 ±7.0 138.5 ±6.1 94.0 ±4.7
134.2 ±9.0 125.0 ±7.1 76.6 ±5.6 146.1 ±8.0 83.6 ±5.8
139.5 ±5.0 128.9 ±8.2 69.2 ±3.8 148.7 ±7.9 73.9 ±7.8
Means ±SD of 12 determinations from three separate experiments.
Assessment o f K562 Cell Proliferation and Differentiation After 4 days of incubation, the total and viable cell numbers in each well of the quadruplicate plates were counted using a Coulter counter ZS and the trypan blue exlusion test. Also, the erythroid dif ferentiation of K562 cells was assessed by the hae moglobin production using the benzidine staining method as described before (4) in each well of the quadruplicate plates on day 4. and at least 500 cells were scored as benzidine positive (blue) or negative (yellow). Calculation o f 1C50 of Cytarabine IC50 are the concentrations of cytarabine induc ing 50% growth inhibition of K562 cells on day 4 compared with control cultures containing neither cytarabine nor GM-CSF nor 1L-3 nor interferons. Dose-response curves of the percentage of growth inhibition in relation to control cultures on day 4
were plotted versus the logarithm of concentra tions of cytarabine when used alone or with GMCSF, IL-3 or interferons, and IG*, values were cal culated from the logarithmic regression analysis of each dose-response curve using the cricket graph version 1.3.2 program on an Apple Macintosh computer. Statistical Analysis The results were represented as means ±standard deviation of 12 determinations of quadrupli cate cultures from three separate experiments, and the statistical significance was determined using the two-tailed Student t distribution test.
Results
The addition of IL-3 or GM-CSF to cul tures of K562 cells increased the cell num bers by 25.6-44.5 or 13.2-37.1%, respective ly, on day 4 (table 1). Also, the addition of intcrferon-()2, also known as interleukin-6, increased K562 cell counts by 32.4-56.6% on day 4 (table 1). On the other hand, both interferon-a and interferon-y re-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 9:55:01 AM
in three separate experiments were incubated for 4 days at 37 °C in a humidified incubator under 7.5% C 02. The time point was selected to allow ade quate time for growth inhibition and differentia tion but to avoid density arrest in more rapidly growing wells and to avoid problems with the limit ed in vitro stability of the recombinant human cy tokines.
444
Hassan/Maurcr
Table 2. Effect of recombinant human cytokines in concentrations from 0 to 1,000 U/ml on the anti proliferative activity of cytarabinc in K562 human myeloid leukaemia cells Recombinant human cytokine
Cytarabine IC5(, values. nM
IL-3 GM-CSF Interfcron-a,b lnterferon-|i, Intcrfcron-y
0 (alone)
+ 100 U/ml
+500 U/ml
+1,000 U/ml
32 32 32 32 32
30 28 57 51 47
29 24 62* 60* 63*
14* 17* 83* 62* 89*
IC^i values are cytarabinc concentrations which induce 50% inhibition of K562 human myeloid leukae mia cell proliferation. Means of 12 determinations from three separate experiments with standard deviations not exceeding 8% of the mean. * p
Chemotherapy 1991;37:441-448
Effects of Recombinant Human Cytokines on Cytarabine Activity in K562 Human Myeloid Leukaemia Cells HT. Hassan, H. R. Maurer Leukämie-Forschung. Institut für Pharmazie, Freie Universität Berlin, FRG
Key Words. Cytarabine • Cytokine • Interferon ■Interleukin • Myelocytic leukaemia
Introduction
The human myeloid leukaemia cell line K562, which was established from the pleural effusion of a chronic myeloid leu kaemia patient in blastic crisis [I], pro vides a unique population of primitive myeloid leukaemic cells [2]. K562 cells
were induced to differentiate along the erythroid, granulocytic, macrophage and megakaryocytic lineages in response to several agents [2], Cytarabine is not only the most widely used drug in the treat ment of myeloid leukaemia [3] but also one of the most potent inducers of crythroid differentiation of K562 cells [4] which
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 9:55:01 AM
Abstract. The K562 cell line provides a unique population of primitive human mye loid leukaemia cells which can be induced to differentiate along the erythroid, granulo cytic, macrophage and megakaryocytic lineages in response to several agents. Cytara bine is not only the most widely used drug in the treatment of myeloid leukaemia but also the most effective agent in K562 cells. The effects of five recombinant human cyto kines - interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GMCSF), interferon-a, interferon-|I and interferon-y on cytarabine-induced growth inhibi tion and differentiation of K562 cells was studied in liquid suspension cultures. GM-CSF and to a lesser extent IL-3 enhanced the antiproliferative effect of cytarabine in K562 cells, whereas the three interferons reduced it. The efficacy of cytarabine in inhibiting the growth of K562 cells was doubled by its combination with GM-CSF or IL-3 but was halved by its combination with interferons. The five cytokines did not significantly affect cytarabine-induced erythroid differentiation of K562 cells. The present results appear to favour the use of GM-CSF and IL-3 but not of interferons in future treatment strategies based on a combined cytokine and chemotherapy approach for myeloid leukaemia.
Hassan/Maurer
442
Materials and Methods Materials Benzidine, cytarabine, glacial acetic acid, hydro gen peroxide, RPMI 1640 culture medium and try pan blue were purchased from Sigma Chemicals. Fetal calf serum was purchased from Bochringer. Recombinant human GM-CSF (specific activity of 5 x 107 U/mg protein), IL-3 (specific activity of 5 x 107 U/mg protein), Interferon-p, (specific activity of 1 x 107 U/mg protein) and interferon-y (specific activity of 2.5 x 107 U/mg protein) were purchased from Genzyme Corporation. Recombinant human interferon-«,,, (specific activity of 4.2 x 107 U/mg protein) was purchased from Hoffmann-La Roche. Cytarabine was diluted with RPMI 1640 medium to 1 mM stock solution and kept at -20 °C. Prior to in cubation, cytarabine was freshly diluted with RPMI 1640 medium to working concentrations. Cytokines were diluted with RPMI 1640 medium containing 10% fetal calf serum 0.5 million U/ml stock solution and kept at -70°C. Prior to incubation cytokines were freshly diluted with RPMI 1640 medium con taining 10% fetal calf serum to working concentra tions. Cells K562 cells were grown in suspension culture in RPMI 1649 medium containing 10% fetal calf serum in 10 ml in 25-cm2 tissue culture flasks at 37 °C in a humidified incubator under 7.5% CO,. The cultures were diluted to 5 x 104 cells/ml in fresh medium twice weekly. Cell concentration and viability were assessed by the trypan blue exclusion test. Cell Culture K562 cells were cultured at a concentration of 5 x 104 cells/ml in 24 well tissue culture plates con taining RPMI 1640 medium supplemented with /glutamine and 10% fetal calf serum in a final vol ume of 1 ml. Each of the five recombinant human cytokines: GM-CSF, IL-3, interferon-a, -p and -y were added at graded concentrations (100-1,000 U/ ml) either alone or with cytarabine at graded con centrations (0.05, 0.5, 5 and 50 \xM). Also, cultures containing only cytarabine at the same graded con centrations and control cultures containing neither cytarabine nor GM-CSF nor IL-3 nor interferons were set up in conditions otherwise identical. Qua druplicate plates of each of the culture conditions
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 9:55:01 AM
was terminal, irreversible [5] and S phase dependent [6]. Several chemical and biological agents enhanced cytarabine activity in human myeloid leukaemia cells [7], Recombi nant human granulocyte-macrophage col ony stimulating factor (GM-CSF) not only enhanced vitamin D3-induced monocytic differentiation of U937 human monoblastic leukacmic cells [8] and rctinoic-acid-induced granulocytic differen tiation of human congenital agranulocy tosis bone marrow progenitor cells [9] but also the cytostatic activity of cytara bine and hydroxyurea in HL-60 [101 and AML [11, 12] cells. Moreover, GM-CSF co-operated with cytarabine in inducing differentiation of the resistant human myeloid luekaemic cell line KG1 [13] and with recombinant human erythropoietin in inducing erythroid differentiation in more than 80% of K562 clonogenic cells [14], Recombinant human interlcukin-3 (IL-3) enhanced the cytoxicity of cytara bine in AML cells [12, 15]. Recombinant human interferons enhanced retinoicacid-, vitamin-Dr and tumour-necrosisfactor-a-induced growth inhibition and differentiation of human myeloid leukae mia cells [7]. The interactions between cytarabine and recombinant human cytokines could provide the basis for improved therapeutic regimens based on a combined cytokine and chemotherapy approach for myeloid leukaemic patients. Therefore, we investi gated the effects of five recombinant hu man cytokines: GM-CSF, IL-3, interferon-a, interferon-ß and interferon-y on cytarabine-induced growth inhibition and differentiation of K562 human myeloid leukaemia cells.
443
Effects of Cytokines on Cytarabine Activity
Table 1. Effect of recombinant human cytokines in concentrations from 100 to 1.000 U/ml on K562 human myeloid leukaemia cell proliferation in liquid suspension culture Recombinant human cytokine
IL-3 GM-CSF Interferon-a,b Interferon-ß, Interferon-y
K562 cell count on day 4, % of control culture 1(H) U/ml
500 U/ml
1,000 U/ml
131.6 ± 5.1 II 8.4 ±5.2 86.8 ±7.0 138.5 ±6.1 94.0 ±4.7
134.2 ±9.0 125.0 ±7.1 76.6 ±5.6 146.1 ±8.0 83.6 ±5.8
139.5 ±5.0 128.9 ±8.2 69.2 ±3.8 148.7 ±7.9 73.9 ±7.8
Means ±SD of 12 determinations from three separate experiments.
Assessment o f K562 Cell Proliferation and Differentiation After 4 days of incubation, the total and viable cell numbers in each well of the quadruplicate plates were counted using a Coulter counter ZS and the trypan blue exlusion test. Also, the erythroid dif ferentiation of K562 cells was assessed by the hae moglobin production using the benzidine staining method as described before (4) in each well of the quadruplicate plates on day 4. and at least 500 cells were scored as benzidine positive (blue) or negative (yellow). Calculation o f 1C50 of Cytarabine IC50 are the concentrations of cytarabine induc ing 50% growth inhibition of K562 cells on day 4 compared with control cultures containing neither cytarabine nor GM-CSF nor 1L-3 nor interferons. Dose-response curves of the percentage of growth inhibition in relation to control cultures on day 4
were plotted versus the logarithm of concentra tions of cytarabine when used alone or with GMCSF, IL-3 or interferons, and IG*, values were cal culated from the logarithmic regression analysis of each dose-response curve using the cricket graph version 1.3.2 program on an Apple Macintosh computer. Statistical Analysis The results were represented as means ±standard deviation of 12 determinations of quadrupli cate cultures from three separate experiments, and the statistical significance was determined using the two-tailed Student t distribution test.
Results
The addition of IL-3 or GM-CSF to cul tures of K562 cells increased the cell num bers by 25.6-44.5 or 13.2-37.1%, respective ly, on day 4 (table 1). Also, the addition of intcrferon-()2, also known as interleukin-6, increased K562 cell counts by 32.4-56.6% on day 4 (table 1). On the other hand, both interferon-a and interferon-y re-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 9:55:01 AM
in three separate experiments were incubated for 4 days at 37 °C in a humidified incubator under 7.5% C 02. The time point was selected to allow ade quate time for growth inhibition and differentia tion but to avoid density arrest in more rapidly growing wells and to avoid problems with the limit ed in vitro stability of the recombinant human cy tokines.
444
Hassan/Maurcr
Table 2. Effect of recombinant human cytokines in concentrations from 0 to 1,000 U/ml on the anti proliferative activity of cytarabinc in K562 human myeloid leukaemia cells Recombinant human cytokine
Cytarabine IC5(, values. nM
IL-3 GM-CSF Interfcron-a,b lnterferon-|i, Intcrfcron-y
0 (alone)
+ 100 U/ml
+500 U/ml
+1,000 U/ml
32 32 32 32 32
30 28 57 51 47
29 24 62* 60* 63*
14* 17* 83* 62* 89*
IC^i values are cytarabinc concentrations which induce 50% inhibition of K562 human myeloid leukae mia cell proliferation. Means of 12 determinations from three separate experiments with standard deviations not exceeding 8% of the mean. * p
