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Differentiation of human promyelocytic leukemia cells in vitro by 6-thioguanine
33
Cancer Letters, 10 (1980) 33-38 o Elsevier/North-Holland Scientific Publisher Ltd.
DIFFERENTIATION OF HUMAN PROMYELOCYTIC LEUKEMIA CELLS IN VITRO BY 6-THIOGUANINE*
ROSE, J. PAPAC
Medical Service, Department of Medicine, West Haven, CT 06516 (U.S.A.)
Veterans Administration
Hospital,
ALFRED E. BROWN, EDWARD L. SCHWARTZ and ALAN C. SARTORELLI
Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Received 17 March 1980) (Accepted 18 March 1980)
SUMMARY
antineoplastic agent, 64hiogaunine, was found to be a highly potent inducer of differentiation of HL60 human leukemia cells in vitro, producing cells with predominantly myeloid characteristics, as judged by morphological, functional, and cytochemical criteria. Maturation of HL60 was attainable without significant cytotoxicity as measured by cell number; moreover, maximal differentiation occurred at a level of the purine antimetabolite below that producing 50% growth inhibition. The 6-thioguanine-induced differentiated cells had distinctive granulocytic morphology, and were capable of generating superoxide anion during the respiratory burst typical of mature phagocytic cells.
INTRODUCTION
The human cell line HL60, developed from peripheral blood leukocytes of a patient with acute promyelocytic leukemia, proliferates in suspension culture while maintaining the-morphological and cytochemical properties of acute leukemia [1,2]. Dimethylsulfoxide (DMSO), butyric acid, and a number of other polar compounds induce the differentiation of HL60 to *Supported in part by U.S. Public Health Service Grants CA-62817 and CA-16369 from the National Cancer Institute and a grant from the Bristol-Myers Company. E.L.S. is a fellow in Cander Research supported by Grant DRG-408F of the Damon Runyon-Waiter Winchell Cancer Fund.
34
mature granulocytes [ 3,4]. These agents also cause maturation of other neoplasms in culture, including murine erythroleukemia [ 51, neuroblastoma [6] and rodent myeloid leukemia cell lines [ 71. In addition, tumor promoters such as the phorbol diesters and mezerein induce changes in HL60 to forms that are characteristic of either myelocytes [ 81 or macrophages [ 91. The antileukemic agent 6-thioguanine (6-TG) at a concentration of 0.24 mM has been shown to induce greater than 80% erythroid differentiation in a clone of the Friend murine leukemia deficient in hypoxanthine-guanine phosphoribosyltransferase [lo]. Because of the clinical utility of 6-TG in the treatment of acute leukemias, we have compared the effectiveness of this purine analog with DMSO as an inducer of the differentiation of HL60. MATERIALS
AND METHODS
HL60 cells, subcultured from passage 27 of the original cell line, were seeded in T30 Falcon plastic flasks at 2.5 X 10’ cells/flask in 10 ml of RPMI-1640 medium (GIBCO) supplemented with 20% heat-inactivated fetal calf serum (Flow Laboratories) and 40 pg/ml gentamycin. The flasks were incubated at 37°C in a humidified 5% CO, atmosphere. Cell number and size were determined with a model ZBI Coulter particle counter equipped with a Channelyzer; trypan blue dye exclusion was employed as a measure of cell viability (i.e., membrane intactness). Morphological evaluation was assessed by differential counts (200 cells) of l-ml aliquots of cell suspension stained with Wright’s stain at various times after continuous exposure to either 6-TG or DMSO. Differentiated cells include myelocytes, metamyelocytes, band forms and segmented neutrophils. Phagocytosis-associated oxidative metabolism was assessed by incubating l-2 X lo6 cells with 0.1% nitro blue tetrazolium (NBT) and 1 /Ig 12-Otetradecanoylphorbol-13-acetate (TPA) in a final vol. of 1 ml. After 20 min (37”C), the cells were centrifuged, treated with Wrights stain, and evaluated for the percentage of cells containing reduced formazan granules. RESULTS
Untreated cells increased in number essentially logarithmically until the 6th day when a saturation density of 2.5-3.2 X lo6 cells/ml developed. A slight decrease in cell growth was observed in the presence of 0.5 PM 6-TG while a more pronounced decrease in cellular replication occurred with concentrations of 1 FM purinethione (Fig. 1). The ability of cells to exclude trypan blue at these concentrations of 6-TG was 90% and 65%, respectively. DMSO (140 mM) did not cause a detectable decrease in either cell replication or trypan blue uptake. A higher concentraton of DMSO (245 mM) caused substantial growth inhibition and cell death. Morphologically, untreated control cultures consisted predominantly (85-95s) of cells that appeared to be promyelocytic, with a small number
35 +20
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si $
r
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-4O_
.g _
L
._ti
5-60-g
iTi iii 0
6 #
-8O-
-100
s
-
0
2
6
4
6
Day
Fig. 1. Cell growth and differentiation of HL60 cells. Growth is expressed as the percentage of cells excluding trypan blue relative to untreated controls. Morphologic evaluation of differentiation is based upon 200 cell counts of Wright’s stained smears; differentiated forms include myelocytes, metamyelocytes, band forms and segmented neutrophils. x , control; A and A, 0.1 PM 6-TG; c and n ,0.5 PM 6-TG; c and l, 140 mM DMSO.
of blasts. These cells stained positive for naphthol ADS chloroacetate esterase, Sudan black and myeloperoxidase. In untreated cultures, approx. 5-10% of cells underwent spontaneous maturation, usually to myelocytes and metamyelocytes, although occasional band forms and monocytes were observed. The time course of differentiation of cells exposed to 6-TG paralleled that of DMSO-treated cells, (Fig. 1). The maximum number of differentiated cells occurred in cultures treated with these agents for 7-8 days. At concentrations of 0.1 PM 6-TG, approx. 25% of the cells differentiated beyond the myelocytes stage on day 8; a few monocytoid cells were also noted. At a level of 0.5 PM 6-TG, from 50 to 75% of the population consisted of differentiated cells. The extent of differentiation at 1 PM 6-TG was difficult to quantitate accurately due to the observed cytotoxicity, with cellular debris, extruded nuclei, and some giant cells present; a few macrophage-like cells were also noted. Both 6-TG and DMSO treatment for 5 days increased the number of cells containing formazan granules after TPA stimulation. The percentages of cells reducing NBT were 4% for untreated control, 16% for 0.5 PM 6-TG and 31% for 140 PM DMSO treated cells, values that were well correlated with the percentage of differentiated cells in these populations as detected morphologically. Differences occurred in the size of the cellular population treated with DMSO as compared to that of untreated control and 6-TG treated cultures (Fig. 2). Thus, cell size diminished with exposure to DMSO, and a homogenously sized population not seen in control or 6-TG treated cells developed after 4-5 days. In contrast, the decreased size observed during 6-TG induced
36
600
-
E 400 z!
-
**
2 G v 200
-
0’
I
0
1
2
I
1
4
1
I
I
6
Day Fig. 2. Cell volume of HL60 cells in suspension culture. HL60 cells were seeded in T30 plastic flasks at 2.5 x 10’ cells /flask in 10 ml of WMI-1640 medium supplemented with 20% heat-inactivated fetal calf serumand 40 rg/ml gentamycin. The flasks were incubated at 37°C in a humidified 5% CO, atmosphere. The cell number and volume were determined with a model ZBI Coulter particle counter equipped with a Channelyzer. x , control ; r: , 0.1 PM 6-TG; A, 0.5 PM 6-TG; 9, 1 NM 6-TG; l ,140 mM DMSO; m, 245 mM DMSO.
differentiation was not significantly different from the progressive decrease in size that occurred in the control population. At the highest concentration of 6-TG tested (i.e., 1 PM 6-TG), an increase in ceil volume was produced. DISCUSSION
Collins et al. [ 31 using DMSO and other polar solvents and Huberman and Callaham [8] using a number of phorbol esters have reported that HL60 cells which underwent differentiation had myeloid characteristics, as judged by morphological, histochemical, and functional criteria. In contrast, Rovera et al. [9] have described differentiation of HL60 in the presence of phorbol diesters and mezerein as being monocyte-macrophage-like. The purine analog 6-TG has been found in the present studies to yield predominantly myeloid differentiation. However, with DMSO, we observed, as did Collins et al. [ 31, a small number of monocytic forms. The mechanism(s) by which different agents act and the target(s) involved are not yet clear. A common cellular target for the various inducers may exist or various agents may bring about maturation through alternate pathways. Alternatively, the untreated cell population may be multipotent, so that selective differentiation develops
37
along established pathways in the presence of the appropriate inducing agents. In the work described in the present report, significant differentiation of HL60 cells occurred at a concentration of 6-TG (0.1 PM) which produced minimal cytotoxicity, as measured by cell number; the level of 6-TG at which differentiation was maximal was less than that causing more than 50% growth inhibition. That the concentration of 6-TG that induces differentiation of HL60 cells is less than that producing significant cytotoxicity differs from findings with the Friend erythroleukemia, in which the development of resistance to the cytotoxic actions of the 6thiopurine was required to permit the observance of a significant degree of erythroid development [lo]. There is evidence supportive of the concept of a maturation block in myeloid leukemias of animals [ 111 and humans [12], with a variety of maturation patterns which suggest there may be different blocks in the process of differentiation. Inducing agents, including colony stimulating factor, may mediate all or some stages in the maturation process in HL60 [ 131 as well as other human myeloid leukemias [ 14,153. Present day therapy of acute myeloid leukemia is based on cytodestruction, generally achieved with maximal tolerated doses of antineoplastic drug combinations. However, it is conceivable that some human leukemias, under appropriate conditions, will undergo differentiation. The observation that relatively low levels of 6-TG are capable of inducing significant maturation of human leukemic cells in vitro with minimal cellular destruction is intriguing, in that relatively noncytotoxic doses of 6-TG [ 41 or other chemotherapeutic agents [4,13] might be employed to induce differentiation of human leukemia cells in patients. A suitable condition in which a test of this hypothesis may be readily obtained is the preleukemic syndrome, since in vitro cellular maturation has been documented in some instances [ 161. A possible therapeutic approach to these conditions could well entail the use of minimal doses of cytotoxic agents that have the potential of inducing differentiation in vivo. REFERENCES Collins, S.J., Gallo, R.C. and Gallagher, R.E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature, 270,347-349. Gallagher, R., Collins, S., Trujillo, J., McCredie, K., Ahearn, M., Tsai, S., Metzgar, R., Aulakh, G., Ting, R., Ruscetti, F. and Gallo, R. (1979) Characterization of the continuous differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood, 54,713-733. Collins, S.J., Ruscetti, F.W., Gallagher, R.E. and Gallo, R.C. (1978) Terminal differentiation of human promyelocytic leukemia cells induced by dimethylsulfoxide and other polar compounds. Rot. Natl. Acad. Sci., 75, 2458-2462. Gallo, R.C. and Ruscetti, F.W. (1980) New human hematopoietic cell systems for the study of growth, differentiation, and involved factors. In: Molecular Actions and Targets for Cancer Chemotherapeutic Agents. Editors: A.C. Sartorelli, J.S. Laze, and J.R. Bertino. Academic Press, N.Y. (in press). Friend, C., Scher, W., Holland, J.G. and Soto, T. (1971) Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Roe. Natl. Acad. Sci., 68,378-382.
6 Kimhi, Y., Palfrey, C., Spector, I., Barrah, Y. and Littauer, U.Z. (1976) Maturation of neuorblastoma cells in the presence of dimethylsulfoxide. Proc. Natl. Acad. Sci., 73, 462466. 7 Krytosek, A. and Sachs, L. (1976) Control of lysozyme induction in the differentiation of myeloid leukemic cells. Cell, 9,675-684. 8 Huberman, E. and Callaham, M.F. (1979) Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Proc. Natl. Acad. Sci., 76.1293-1297. 9 Rovera, G., Santoli, D. and Damsky, C. (1979) Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with phorbol diester. Proc. Natl. Acad. Sci., 76,2779-2783. 10 Gusella, J.F. and Housman, D. (1976) Induction of erythroid differentiation in u&o by purines and purine analogues. Cell, 0,263-269. 11 Lotem, J. and Sachs, L. (1977) Genetic dissection of the control of normal differentiation in myeloid leukemic cells. Proc. Natl. Acad. Sci., 74, 5554-5558. 12 Palu, G., Powles, R., Selby, P., Summersgill, B.M. and Alexander, P. (1979) Patterns of maturation in short-term culture of human acute myeloid leukemic cells. Br. J. Cancer, 40, 719-730. 13 Lotem, J. and Sachs, L. (1979) Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion. Proc. Natl. Acad. Sci., 76, 5158-5162. 14 Koeffler, H.P. and Golde, D.W. (1978) Acute myelogenous leukemia: a human cell line responsive to colony stimulating activity. Science, 200, 1153-1154. 15 Brennan, J.K., DiPersio, J.F., Abboud, C.N. and Lichtman, M.A. (1979) The exceptional responsiveness of certain human myeloid leukemia cells to colony-stimulating activity. Blood, 54, 1230-1239. 16 Koeffler, H.P. and Golde, D.W. (1978) Cellular maturation in human leukemia. Blood, 52, 355-361.
Cancer Letters, 10 (1980) 33-38 o Elsevier/North-Holland Scientific Publisher Ltd.
DIFFERENTIATION OF HUMAN PROMYELOCYTIC LEUKEMIA CELLS IN VITRO BY 6-THIOGUANINE*
ROSE, J. PAPAC
Medical Service, Department of Medicine, West Haven, CT 06516 (U.S.A.)
Veterans Administration
Hospital,
ALFRED E. BROWN, EDWARD L. SCHWARTZ and ALAN C. SARTORELLI
Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT 06510 (U.S.A.) (Received 17 March 1980) (Accepted 18 March 1980)
SUMMARY
antineoplastic agent, 64hiogaunine, was found to be a highly potent inducer of differentiation of HL60 human leukemia cells in vitro, producing cells with predominantly myeloid characteristics, as judged by morphological, functional, and cytochemical criteria. Maturation of HL60 was attainable without significant cytotoxicity as measured by cell number; moreover, maximal differentiation occurred at a level of the purine antimetabolite below that producing 50% growth inhibition. The 6-thioguanine-induced differentiated cells had distinctive granulocytic morphology, and were capable of generating superoxide anion during the respiratory burst typical of mature phagocytic cells.
INTRODUCTION
The human cell line HL60, developed from peripheral blood leukocytes of a patient with acute promyelocytic leukemia, proliferates in suspension culture while maintaining the-morphological and cytochemical properties of acute leukemia [1,2]. Dimethylsulfoxide (DMSO), butyric acid, and a number of other polar compounds induce the differentiation of HL60 to *Supported in part by U.S. Public Health Service Grants CA-62817 and CA-16369 from the National Cancer Institute and a grant from the Bristol-Myers Company. E.L.S. is a fellow in Cander Research supported by Grant DRG-408F of the Damon Runyon-Waiter Winchell Cancer Fund.
34
mature granulocytes [ 3,4]. These agents also cause maturation of other neoplasms in culture, including murine erythroleukemia [ 51, neuroblastoma [6] and rodent myeloid leukemia cell lines [ 71. In addition, tumor promoters such as the phorbol diesters and mezerein induce changes in HL60 to forms that are characteristic of either myelocytes [ 81 or macrophages [ 91. The antileukemic agent 6-thioguanine (6-TG) at a concentration of 0.24 mM has been shown to induce greater than 80% erythroid differentiation in a clone of the Friend murine leukemia deficient in hypoxanthine-guanine phosphoribosyltransferase [lo]. Because of the clinical utility of 6-TG in the treatment of acute leukemias, we have compared the effectiveness of this purine analog with DMSO as an inducer of the differentiation of HL60. MATERIALS
AND METHODS
HL60 cells, subcultured from passage 27 of the original cell line, were seeded in T30 Falcon plastic flasks at 2.5 X 10’ cells/flask in 10 ml of RPMI-1640 medium (GIBCO) supplemented with 20% heat-inactivated fetal calf serum (Flow Laboratories) and 40 pg/ml gentamycin. The flasks were incubated at 37°C in a humidified 5% CO, atmosphere. Cell number and size were determined with a model ZBI Coulter particle counter equipped with a Channelyzer; trypan blue dye exclusion was employed as a measure of cell viability (i.e., membrane intactness). Morphological evaluation was assessed by differential counts (200 cells) of l-ml aliquots of cell suspension stained with Wright’s stain at various times after continuous exposure to either 6-TG or DMSO. Differentiated cells include myelocytes, metamyelocytes, band forms and segmented neutrophils. Phagocytosis-associated oxidative metabolism was assessed by incubating l-2 X lo6 cells with 0.1% nitro blue tetrazolium (NBT) and 1 /Ig 12-Otetradecanoylphorbol-13-acetate (TPA) in a final vol. of 1 ml. After 20 min (37”C), the cells were centrifuged, treated with Wrights stain, and evaluated for the percentage of cells containing reduced formazan granules. RESULTS
Untreated cells increased in number essentially logarithmically until the 6th day when a saturation density of 2.5-3.2 X lo6 cells/ml developed. A slight decrease in cell growth was observed in the presence of 0.5 PM 6-TG while a more pronounced decrease in cellular replication occurred with concentrations of 1 FM purinethione (Fig. 1). The ability of cells to exclude trypan blue at these concentrations of 6-TG was 90% and 65%, respectively. DMSO (140 mM) did not cause a detectable decrease in either cell replication or trypan blue uptake. A higher concentraton of DMSO (245 mM) caused substantial growth inhibition and cell death. Morphologically, untreated control cultures consisted predominantly (85-95s) of cells that appeared to be promyelocytic, with a small number
35 +20
7 J
-7 -2o-
si $
r
O-
I
f
I-
-4O_
.g _
L
._ti
5-60-g
iTi iii 0
6 #
-8O-
-100
s
-
0
2
6
4
6
Day
Fig. 1. Cell growth and differentiation of HL60 cells. Growth is expressed as the percentage of cells excluding trypan blue relative to untreated controls. Morphologic evaluation of differentiation is based upon 200 cell counts of Wright’s stained smears; differentiated forms include myelocytes, metamyelocytes, band forms and segmented neutrophils. x , control; A and A, 0.1 PM 6-TG; c and n ,0.5 PM 6-TG; c and l, 140 mM DMSO.
of blasts. These cells stained positive for naphthol ADS chloroacetate esterase, Sudan black and myeloperoxidase. In untreated cultures, approx. 5-10% of cells underwent spontaneous maturation, usually to myelocytes and metamyelocytes, although occasional band forms and monocytes were observed. The time course of differentiation of cells exposed to 6-TG paralleled that of DMSO-treated cells, (Fig. 1). The maximum number of differentiated cells occurred in cultures treated with these agents for 7-8 days. At concentrations of 0.1 PM 6-TG, approx. 25% of the cells differentiated beyond the myelocytes stage on day 8; a few monocytoid cells were also noted. At a level of 0.5 PM 6-TG, from 50 to 75% of the population consisted of differentiated cells. The extent of differentiation at 1 PM 6-TG was difficult to quantitate accurately due to the observed cytotoxicity, with cellular debris, extruded nuclei, and some giant cells present; a few macrophage-like cells were also noted. Both 6-TG and DMSO treatment for 5 days increased the number of cells containing formazan granules after TPA stimulation. The percentages of cells reducing NBT were 4% for untreated control, 16% for 0.5 PM 6-TG and 31% for 140 PM DMSO treated cells, values that were well correlated with the percentage of differentiated cells in these populations as detected morphologically. Differences occurred in the size of the cellular population treated with DMSO as compared to that of untreated control and 6-TG treated cultures (Fig. 2). Thus, cell size diminished with exposure to DMSO, and a homogenously sized population not seen in control or 6-TG treated cells developed after 4-5 days. In contrast, the decreased size observed during 6-TG induced
36
600
-
E 400 z!
-
**
2 G v 200
-
0’
I
0
1
2
I
1
4
1
I
I
6
Day Fig. 2. Cell volume of HL60 cells in suspension culture. HL60 cells were seeded in T30 plastic flasks at 2.5 x 10’ cells /flask in 10 ml of WMI-1640 medium supplemented with 20% heat-inactivated fetal calf serumand 40 rg/ml gentamycin. The flasks were incubated at 37°C in a humidified 5% CO, atmosphere. The cell number and volume were determined with a model ZBI Coulter particle counter equipped with a Channelyzer. x , control ; r: , 0.1 PM 6-TG; A, 0.5 PM 6-TG; 9, 1 NM 6-TG; l ,140 mM DMSO; m, 245 mM DMSO.
differentiation was not significantly different from the progressive decrease in size that occurred in the control population. At the highest concentration of 6-TG tested (i.e., 1 PM 6-TG), an increase in ceil volume was produced. DISCUSSION
Collins et al. [ 31 using DMSO and other polar solvents and Huberman and Callaham [8] using a number of phorbol esters have reported that HL60 cells which underwent differentiation had myeloid characteristics, as judged by morphological, histochemical, and functional criteria. In contrast, Rovera et al. [9] have described differentiation of HL60 in the presence of phorbol diesters and mezerein as being monocyte-macrophage-like. The purine analog 6-TG has been found in the present studies to yield predominantly myeloid differentiation. However, with DMSO, we observed, as did Collins et al. [ 31, a small number of monocytic forms. The mechanism(s) by which different agents act and the target(s) involved are not yet clear. A common cellular target for the various inducers may exist or various agents may bring about maturation through alternate pathways. Alternatively, the untreated cell population may be multipotent, so that selective differentiation develops
37
along established pathways in the presence of the appropriate inducing agents. In the work described in the present report, significant differentiation of HL60 cells occurred at a concentration of 6-TG (0.1 PM) which produced minimal cytotoxicity, as measured by cell number; the level of 6-TG at which differentiation was maximal was less than that causing more than 50% growth inhibition. That the concentration of 6-TG that induces differentiation of HL60 cells is less than that producing significant cytotoxicity differs from findings with the Friend erythroleukemia, in which the development of resistance to the cytotoxic actions of the 6thiopurine was required to permit the observance of a significant degree of erythroid development [lo]. There is evidence supportive of the concept of a maturation block in myeloid leukemias of animals [ 111 and humans [12], with a variety of maturation patterns which suggest there may be different blocks in the process of differentiation. Inducing agents, including colony stimulating factor, may mediate all or some stages in the maturation process in HL60 [ 131 as well as other human myeloid leukemias [ 14,153. Present day therapy of acute myeloid leukemia is based on cytodestruction, generally achieved with maximal tolerated doses of antineoplastic drug combinations. However, it is conceivable that some human leukemias, under appropriate conditions, will undergo differentiation. The observation that relatively low levels of 6-TG are capable of inducing significant maturation of human leukemic cells in vitro with minimal cellular destruction is intriguing, in that relatively noncytotoxic doses of 6-TG [ 41 or other chemotherapeutic agents [4,13] might be employed to induce differentiation of human leukemia cells in patients. A suitable condition in which a test of this hypothesis may be readily obtained is the preleukemic syndrome, since in vitro cellular maturation has been documented in some instances [ 161. A possible therapeutic approach to these conditions could well entail the use of minimal doses of cytotoxic agents that have the potential of inducing differentiation in vivo. REFERENCES Collins, S.J., Gallo, R.C. and Gallagher, R.E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature, 270,347-349. Gallagher, R., Collins, S., Trujillo, J., McCredie, K., Ahearn, M., Tsai, S., Metzgar, R., Aulakh, G., Ting, R., Ruscetti, F. and Gallo, R. (1979) Characterization of the continuous differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood, 54,713-733. Collins, S.J., Ruscetti, F.W., Gallagher, R.E. and Gallo, R.C. (1978) Terminal differentiation of human promyelocytic leukemia cells induced by dimethylsulfoxide and other polar compounds. Rot. Natl. Acad. Sci., 75, 2458-2462. Gallo, R.C. and Ruscetti, F.W. (1980) New human hematopoietic cell systems for the study of growth, differentiation, and involved factors. In: Molecular Actions and Targets for Cancer Chemotherapeutic Agents. Editors: A.C. Sartorelli, J.S. Laze, and J.R. Bertino. Academic Press, N.Y. (in press). Friend, C., Scher, W., Holland, J.G. and Soto, T. (1971) Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Roe. Natl. Acad. Sci., 68,378-382.
6 Kimhi, Y., Palfrey, C., Spector, I., Barrah, Y. and Littauer, U.Z. (1976) Maturation of neuorblastoma cells in the presence of dimethylsulfoxide. Proc. Natl. Acad. Sci., 73, 462466. 7 Krytosek, A. and Sachs, L. (1976) Control of lysozyme induction in the differentiation of myeloid leukemic cells. Cell, 9,675-684. 8 Huberman, E. and Callaham, M.F. (1979) Induction of terminal differentiation in human promyelocytic leukemia cells by tumor-promoting agents. Proc. Natl. Acad. Sci., 76.1293-1297. 9 Rovera, G., Santoli, D. and Damsky, C. (1979) Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with phorbol diester. Proc. Natl. Acad. Sci., 76,2779-2783. 10 Gusella, J.F. and Housman, D. (1976) Induction of erythroid differentiation in u&o by purines and purine analogues. Cell, 0,263-269. 11 Lotem, J. and Sachs, L. (1977) Genetic dissection of the control of normal differentiation in myeloid leukemic cells. Proc. Natl. Acad. Sci., 74, 5554-5558. 12 Palu, G., Powles, R., Selby, P., Summersgill, B.M. and Alexander, P. (1979) Patterns of maturation in short-term culture of human acute myeloid leukemic cells. Br. J. Cancer, 40, 719-730. 13 Lotem, J. and Sachs, L. (1979) Regulation of normal differentiation in mouse and human myeloid leukemic cells by phorbol esters and the mechanism of tumor promotion. Proc. Natl. Acad. Sci., 76, 5158-5162. 14 Koeffler, H.P. and Golde, D.W. (1978) Acute myelogenous leukemia: a human cell line responsive to colony stimulating activity. Science, 200, 1153-1154. 15 Brennan, J.K., DiPersio, J.F., Abboud, C.N. and Lichtman, M.A. (1979) The exceptional responsiveness of certain human myeloid leukemia cells to colony-stimulating activity. Blood, 54, 1230-1239. 16 Koeffler, H.P. and Golde, D.W. (1978) Cellular maturation in human leukemia. Blood, 52, 355-361.