Journal of Leukocyte Biology 37:407-422 (1985)

Human Leukemic Models of

Myelomonocytic Development: A Review
of the HL-60 and U937 Cell Lines

Paul Harris and Peter Ralph

Cornell University Medical College, Sloan-Kettering Division, Department of
Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center,
New York

The human leukemic myeloblast HL-6O and monoblast U937 cell lines have
made important contributions to the disciplines of cancer, hematology, and
immunology. As sources of leukemic cells, they have been used for the study
of neoplasia and therapeutics. As sources of hemic cells, they have been used
for biochemical and biological analysis of regulation and differentiation in
myelopoiesis. When stimulated with immunomodulatory factors, the cell lines
develop properties of host-defense effector cells. They are also a source of
cytokines that affect other cell types.

Key words: HL-60, U937, differentiation, cytokines

Research into the biology of human leukemia has led not only to important
advances in the understanding of these diseases but has provided some tremendously
useful models for the study of cellular development. The human cell lines HL-60 and
U937 are two such examples. HL-60 was isolated from the peripheral blood of a
patient with acute promyelocytic leukemia [28]. U937 originated from the pleural
fluid of a patient with diffuse histiocytic lymphoma [76]. At present these lines are
homogenous, readily maintainable in culture, and, most important, exist in an arrested
yet pliant state of maturation. Phenotypically, HL-60 and U937 resemble blast cells
of their lineage and are believed to be the neoplastic derivatives of committed
progenitors of granulocytes and monocytes, respectively. Initially, these cells were
the focus of study designed to delineate the nature of their neoplastic transformation

Received iune 18, 1984; accepted August 1, 1984.

Peter Ralph is currently with the Department of Cell Biology, Cetus Corporation 1400 53rd Street,
Emeryville, CA.
Reprint requests: Peter Ralph, Department of Cell Biology, Cetus Corporation, 1400 53rd Street, Emery-
yule, CA 94608.

© 1985 Alan R. Liss, Inc.

408 Harris and Ralph

and define the conditions under which their malignancy could be reversed [72] . An
additional goal was to achieve this reversal without the use of nonspecific cytotoxic
drug regimes used in current leukemotherapy. Such conditions have been met in vitro
[39,41] and in animal models [35] and have become the basis of several clinical trials.
A wide range of substances, both exogenous and endogenous (lymphokines and other
biological response modifiers), have been identified that can achieve a myelomono-
cytic-specific inhibition of cellular division (see Fig. 1). As was discovered, the loss
of proliferative capacity was often accompanied by a series of phenotypic transfor-
mations that appeared to reflect normal ontogeny. The systems herein described are
of potential value to any investigator interested in the regulatory biology of hemato-
poietic development or lymphokines and afford both the convenience (and shortcom-
ings) of cell lines.

INTRODUCTION

The purpose of this review is to briefly survey the literature of phenotypic

modulation in HL-60 and U937 and, wherever possible, provide a factual and
theoretical nexus between what is known about their biology and the behavior of
normal cells of the myelomonocytic lineage. Emphasis will be placed on those
inducible changes that parallel normal maturation and on those inducers that may
have physiological significance. The references cited are representative rather than
exhaustive.

PHENOTYPE OF UNINDUCED CELLS

The phenotypic character of HL-60 and U937 reflects their leukemic origins.
The histochemistry and morphology of these cells typify them as immature cells of
myelomonocytic lineage. Tables 1 and 2 summarize many of the characteristics and
inducers that have been studied in these cell lines. A brief discussion of the biochem-
ical profile will follow.
The HL-60 cell line has a population doubling time of 20-24 hr. In culture, HL-
60 cells stain as myeloblasts and promyelocytes, though approximately 10% of these
cells appear to have passed beyond this stage forming more mature elements [28].
Culture in exhausted media may increase this number. HL-60 is a constitutive
producer of a growth factor on which it is dependent for colony growth [9]. This
example of autocrine growth regulation is felt to be an important factor in the
maintenance of neoplasia 175]. The cells are characterized by a lack of lymphoid
markers, growth as compact colonies in semisolid culture, and nonadherence to
substrata. The cells possess few Fc-IgG1 and C3b receptors, and weakly phagocytize
latex or yeast particles. HL-60 cells also possess receptors for the chemotactic
tripeptide formyl-methionyl-leucyl-phenylalanine [53] and insulin [2].
Histochemically, HL-60 has a profile positive for myeloperoxidase, ASD chlo-
roacetate esterase, and Sudan Black B. Normal promyelocytes stain well for alkaline
phosphatase whereas HL-60 cells do not [28,86]. Acid phosphatase, an enzyme
abundant in macrophages, is found in minute quantities. The enzymatic activities of
beta-glucuronidase, lysozyme, and G6PD found in the granules of normal granulo-
cytes are low in HL-60, as is the capacity for their degranulation. There is evidence
that these enzymes are already synthesized in HL-60, stored within the granules as
 Models of Myelomonocytic Development 409

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zymogens, and are only converted to active enzymes by stimulation [87]. Lastly, HL-
60 cells have a marginal capacity to produce H20, and 0-, having a low level of
HMPS activity [52]. The ability to kill staphylococcus and other microorganisms is
also muted in these cells 128].
The U937 cell line is characterized by a population doubling time originally
reported of 95 hr. Recent passages of the line grow more quickly, 20-48 hr per
population doubling. Morphologically the cells are monoblastic and the histochemical
profile, ANAE, NASDAE, and j3-glucuronidase reveal monocytic lineage [76,86].
Acid and alkaline phosphatase are detected weakly. U937 releases lysozyme into
culture medium, a monocyte-specific characteristic [67]. The neutral protease elastase
is present within the cells. The surface of these cells bear few Fc,C3, and chemotactic
peptide receptors when compared to normal monocytes [76]. Histamine and insulin
receptors are also expressed [1]. Only a small percentage of cells are phagocytic and
U937 only weakly produces H2O2 and 02-. The capacity for microorganism and
tumor cell killing is absent [43,68].
With this brief outline of the biological profile of uninduced HL-60 and U937
cells as an introduction, the wide range of inducible properties will be discussed.

MORPHOLOGICAL CHANGES

The most readily observable maturational changes in U937 and HL-60 cell lines
are morphological. Significant increases in substratum adherence are regularly ob-
served when the cells are maturationally induced. The HL-60 cell line changes
appearance from promyelocytic to later stages of granulocyte development when
treated with 1 % dimethyl sulfoxide (DMSO) [18]. HL-60 cells, when exposed to
phorbol ester, transform in a monocytoid character as evidenced by the disappearance
of azurophilic granules, appearance of pseudopodia, and a cerebriform nucleus. There
exists some evidence that these cells can transform along the eosinophil line in
response to colony stimulating activity (CSA) from human placental-conditioned
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412 Harris and Ralph

media [49] . One anomaly of maturation is that HL-60 cells fail to develop the
secondary granules of polymorphonuclear cells (PMNs), perhaps due to incomplete
maturation [52] . The U937 cell line, under maturational influence, increases in size,
acquires a lobated nucleus, and its cytoplasmic granules are replaced by vacuoles,
mimicking the monoblast-to-monocyte transformation. Tetramyristic phorbol acetate
(TPA) and DMSO induce strong adherence in these cells. Note, however, that any
agent that alters membrane hydrophobicity is likely to increase the number of adherent
cells [38], as is seen with B-cell lines, and, alone, adherence should not serve as a
criterion of differentiation.

HISTOCHEMISTRY AND BIOCHEMICAL CHANGES DURING HL-60

DIFFERENTIATION

As a less subjective approach to studying maturational change, a variety of

enzymatic activities and their by-products have been measured. The more frequently
studied activities are often the constituents of the lysosomal compartment. These are
granulocyte lysozyme, acid phosphatase, and 13-glucuronidase. When HL-60 is in-
duced with l08 M TPA the activity of these enzymes is increased up to 20-fold in the
surrounding media [69] These activities
. are usually increased after induction with
any of the manifold agents used in maturational studies, such as TPA, DMSO,
Lymphokine (LK), vitamin D3 (VD3), with retinoic acid (RA) being the least potent.
The presence of acid phosphatase, a distinctive monocyte lysosomal enzyme, can be
detected after exposure to TPA for several days. The elaboration of this enzyme into
surrounding media is also a monocyte-associated characteristic and accompanies the
TPA-induced maturation of these cells. Myeloperoxidase, an enzyme specific to the
myelomonocytic lineage, is constitutively expressed in HL-60. Myeloperoxidase
activity is lost after induction with PMA, LK, CSA, or RA, but is unaffected when
DMSO or cAMP inducing agents are used [41,70]. Myeloperoxidase is also lost
during the development of normal macrophages [15]. The staining for chloroacetic
esterase is variable after induction. Agents such as TPA and LK decrease its presence,
suggesting monocytic development, whereas DMSO, RA, and CSA preparations
increase the frequency of granulocytelike positive cells. The ability to form granulo-
cytic or monocytic elements, as determined by the maturational agent used, is the
experimental basis for the belief that HL-60 cells retain some bipotentiality in their
inducible character [22]. A similar murine cell line explanted into embryos can later
give rise to chimeric populations of granulocytes or macrophages [83].
The isozymes of lactate dehydrogenase are present in a specific pattern in
uninduced HL-60 cells, yet change after induction with DMSO. The resultant pattern,
however, is not characteristic of mature granulocytes or macrophages but intermedi-
ate, suggesting incomplete maturation [62]. Simultaneous expression of other granu-
locytic and monocytic specific markers may also be the result of incomplete
maturation. Important in this respect is the fact that cell division is not required for
the induction of many of these characteristics [8] and may account for uncoordinate
expression. Intracellular NADase, a catabolic enzyme, has been shown to increase in
response to TPA and RA but not DMSO induction [34].
Nucleic acid metabolism is altered during maturation of HL-60 and such data
suggest that differentiation, rather than modulation, of phenotype is occurring. Mes-
senger RNA species have been isolated, translated in vitro, and analyzed by two-
 Models of Myelomonocytic Development 413

dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

after TPA or DMSO induction. The TPA-induced protein pattern differed significantly
from the DMSO pattern and was characterized as monocytoid and myeloid, respec-
tively, [17] . Poly ADP-ribose metabolism has been linked to the maturation of
granulocyte-macrophage progenitors. ADP-ribosyl transferase is a chromatin-bound
enzyme catalyzing the transfer ofADP-ribose to chromatin proteins. The activity of this
enzyme increases during the CSF-stimulated differentiation of marrow precursors to
monocytes [25]. The level of poly ADP-ribose was measured during the DM50-in-
duced differentiation of HL-60 cells. It was reported that the levels increased 800%
concomitantly with morphological differentiation to mature granulocyte elements [37].
One of the most important microbicidal products of granulocytes and macro-
phages is hydrogen peroxide and superoxide anion. Experimentally, one of the most
frequent and convenient qualitative measures of reactive oxygen intermediates is the
reduction of nitroblue tetrazolium (NBT) to a black formizan precipitate. A variety of
factors can induce the capacity in HL-60 to produce H202 upon PMA or zymosan slim-
ulation. Notably, TPA, DMSO, LK, RA, and cAMP all increase the proportion of NBT-
positive cells. Most recently a DIF factor has been characterized. Obtained from a T-
cell line, this factor can induce the maturation of HL-60 cells alone or in synergy with
RA, as assessed by an increase in the percentage of NBT-positive staining cells [591.
The microbicidal capacity ofgranulocytes and HL-60 cells is metabolically linked to the
hexose monophosphate shunt (HMPS) activity. This pathway produces reducing equiv-
alents used in the production of superoxide anion. HL-60 HMPS activity is increased
maximally 6 days post-DMSO treatment [28]. The capacity of LK size fractions to in-
duce H2O2 production in HL-60 cells has led to the preliminary identification of an ac-
tive LK species which does not appear to be y-interferon (IFN) [29]. The release of
arachidonate acid-derived metabolites has been followed after induction of maturation
with several agents. Preliminary results suggest that there are changes in the released
pattern of arachidonate derivatives [31].

HISTOCHEMICAL AND BIOCHEMICAL CHANGES DURING U937

DIFFERENTIATION

The U937 cells have a clear monocytic lineage derivation but have not received
the same intense biochemical characterization, perhaps due to the rarity of monocyte-
related leukemia/lymphoma from which it originated. Maturation of these cells is
characterized usually by quantitative rather than qualitative changes, as the cells are
constitutive producers of a variety of enzymes. Because U937 cells are of monocytic
derivation, agents that effect a maturational change may have potent immunoadjuvant
uses, as well as a role in leukemotherapy. Peripheral blood monocytes and U937 cells
are characterized by their content of fluoride-inhibitable esterase, alkaline and acid
phosphatase, and f3-glucuronidase. Agents able to increase the expression of these
activities include TPA, VD3, CSA, y-IFN, and LK [32].
The expression in U937 of nonspecific esterases (NASDAE and ANAE) is
increased in response to TPA or LK incubation [54]. Other enzymatic activities that
have been measured include elastase and urokinase. U937 elastase is not released
constitutively and is not readily modulatable [73]. The role of phospholipid methyla-
tion has been examined in the induction of maturation. Of the agents studied, a-IFN,
f.3-IFN, DMSO, and PMA, only PMA was able to induce a decrease of methyl-group
414 Harris and Ralph

incorporation [38] . Phospholipid methylation in normal monocyte ontogeny decreases

with maturation [7].
The production of H202 and superoxide has been studied extensively in U937.
The production of reactive oxygen species is an important effector function of the
line and is a characteristic of maturation. The percentage of NBT-positive cells is
increased by a variety of agents such as TPA, DMSO, LK, a- and f3-IFN, RA, VD3,
prostaglandin E2 (PGE2) and other cAMP-inducing agents, Ara-C, and differentiation
inducing factor (DIF) [32,58,59]. One source of DIF, Hut 102 T-cell line, was
reported to increase the percentage of NBT-positive cells only after RA priming [59].
Other DIF activities appear independent of RA priming [32].
On a more quantitative basis, the activity of HMP shunt has been examined as
well. Notably, dibutryl cyclic adenosine monophosphate (db cAMP) and PGE are
able to increase activity of the shunt but cannot induce other characteristics of maturity
in U937 [56]. The ability of U937 cells to respond to TPA, opsonized zymosan, or
chemotactic peptide receptor (fMLP) with a respiratory burst has been measured.
After induction with LK fractions or conditioned media of a T-cell line, U937 cells
could be stimulated tenfold in H202 production [29,32]. Other inducers possessing
this activity are conditioned media from human cell lines (as sources of differentia-
tion-inducing factor), such as SK-Hep, 5637, or CSA from GCT [26,32]. Purified ‘y-
IFN (100 U/ml) has little activity in inducing the production of H2O2, though at this
concentration it increases immunoglobulin receptor (FcR) dramatically. Higher con-
centration of ‘y-IFN (1000 U/ml) can induce peroxide production [32]. In contrast,
mature peripheral blood monocytes show greatly increased peroxide production after
several days of incubation with ‘y-IFN. Production of arachidonate acid (AA) metab-
olites spontaneously with stimulation and after induction has been studied. Early
studies demonstrated that U937 cells could not produce POE2 in response to lipopo-
lysaccharide (LPS) or Con A stimulation [421. The uninduced U937 cells differ from
peripheral blood monocytes and macrophages in this respect. If supplied with exoge-
nous AA, the cells can release POE7 [16]. The failure to release esterified AA to the
cyclooxygenase pathway was believed to be a property shared by U937 cells and
certain macrophage subsets. Recently it has been shown that U937 cells can be
induced to produce various arachidonate metabolites, both without stimulation and in
response to Con A stimulation. Induction protocols included sources of DIF and ‘y-
IFN, both of which had strong ability to effect thromboxane release into culture
media [31]. Release of PGE2 and other derivatives appears to be another maturation-
ally acquired phenotype.

ELABORATION OF CYTOKINES

The HL-60 cell line has received scant attention in regard to the production of
biologically active macromolecules. The best characterized species is a leukemia-
associated inhibitor of normal myelopoiesis [55]. HL-60 cells constitutively produce
this factor, which was later characterized as an acidic isoferritin. This molecule is
active on marrow myelopoietic progenitors called the GM-CFU-c. Acidic isoferritins
(AIF) can reduce the proportion of these cells found in S phase. The molecule is
believed to be an important down regulator in the homeostasis of normal myelopoiesis
and is responsible for the profound granulocytopenia seen in myeloid leukemia [10].
HL-60 has also been reported to produce a CSA active on normal marrow cells, but
 Models of Myelomonocytic Development 415

only after TPA induction [77] . Elaborated molecules may be subject to abnormal
glycosylations; a-l-antitrypsin and haptoglobin, as produced by HL-60, have subtle
structural differences as compared to their normal granulocyte counterparts [4]. The
phospholipid mediator platelet activating factor has been demonstrated in HL-60
conditioned media following macrophagelike differentiation in TPA [12].
U937 is a source of a variety of biologically active macromolecules. The
monokine interleukin- 1 (IL-l) (a.k.a. endogenous pyrogen, or lymphopine activating
factor, LAF) is constitutively released in small amounts. The release of IL-I can be
augmented by the addition of TPA for a few days [60]. U937, like HL-60, secretes a
CSA that acts as its own growth factor. Moreover, LF, TF, and AIF down regulate
its release, as is seen in normal myelopoietic in vitro regulation [10] . This CSA is not
active on normal GM-CFC [42] . U937 is a producer of the second component of
complement. The release of C2 could be augmented sevenfold by inducing maturation
of U937 with PMA or LK. Actual synthetic activity rather than increased release was
shown using cycloheximide [60] . U937 cells can also be induced to produce a factor
(MCF, distinct from IL-l?) capable of increasing the production of collagenase and
PGE2 in cultured synovial cells [3]. U937 production of an inhibitor of lymphocyte
proliferation was isolated and characterized [84] as Con A inducible, 65 KD M.W.,
heat resistant, and distinct from lymphotoxin or IFN. Lastly, erythropoietin activity
is released into growth medium by U937 cells [5].

MODULATION AT THE CELL SURFACE OF HL-60

Changes in the plasma membrane constituents of HL-60, resulting from matur-

ation, have been reported. The expression of “fast-eluting” glycopeptides was studied
after induction with DM50 or TPA [81]. When the elution profiles of fucosyl-labeled
material were compared to normal granulocytes and macrophages, it was noted that
a) HL-60 glycopeptides were larger and more complex than polymorphonuclear cells
or less mature band elements; b) DMSO exposure did not induce normalization of
elution profile to a mature shape, but instead augmented the differences; c) TPA
induction yielded a small reduction in size and complexity of the eluate but remained
larger than monocyte controls. It has been noted that immature myeloid elements
express these fast-eluting glycopeptides prior to marrow egress, and for a limited
period of time in the periphery, after which they are lost [80]. DMSO-induced changes
in the cell surface glycoproteins have been reported using SDS-PAGE. Following
neuraminidase treatment, HL-60 cells were labeled by galatose oxidase/sodium bo-
rohydride. The most reproducible result was the appearance of a OP 130 along with
the disappearance of a OP 160 [27]. The time course of appearance of OP 130 was
concomitant with chemotactic ability. The authors note that patients with impaired
granulocyte chemotaxis have greatly reduced amounts of OP 130 in their granulocytes
[71]. Cytoskeletal proteins of HL-60 have been studied [6]. During induced matura-
tion, there was increased synthesis of vimentin and other structural proteins. Profiles
of cytoskeletal proteins induced by DMSO resembled granulocytes, and TPA induc-
tion resembled macrophages.
The modulation of several antigens has been characterized with the use of
monoclonal antibodies. In one study [63] two monoclonals, B9.8. 1 (specific for
monocytes and metamyelocytes) and Bl3 .4.1 (monocytes and myelocytes), were used
to characterize maturational changes. Treatment with RA, DMSO, or TPA induced a
416 Harris and Ralph

neoexpression of the antigens. In a similar study, reactivity with the monoclonals Mo

1 and Mo 2, recognizing determinants specific to the myelomonocytic lineage, could
be induced with TPA as well as with LK [78] . The expression of other monocyte-
specific antigens has been reported after induction with TPA or LK [20]. Other
monoclonals recognizing antigens lost or newly expressed have been generated, and
to discuss them all is beyond the scope of this review. The reader is referred to the
literature [21,51].
HL-60 expresses antigens from the MHC. HLA-A and B are constituent, while
induction of HLA-DR determinants has been reported [20] . Normal myelomonocytic
precursors express the DR antigens twice: transiently during early development
(CFU-c-OM) [23] and later as monocytes. HLA-DR determinants have been detected
following 5- to 6-day treatment with LK [20] . These determinants cannot be induced,
however, with RA or DMSO, concordant with the belief that these agents induce
granulocytic development.
7-Interferon is a component of LK. Though the role of ‘y-IFN in the maturation
induction of HL-60 has not been completely assessed, it has been demonstrated that
‘y-IFN can induce dramatic increases in the expression of the immunoglobulin recep-
tor (FcR) [30]. Peripheral blood monocytes display a similar response to ‘y-IFN . a-
and fi-IFN are less effective in HL-60 modulation. Leukocyte a-IFN may not be
targeted for monocyte activation, as it fails to induce the aforementioned differentia-
tion activities [44] or, alternatively, may block their induction.
Concomitant with the increased expression of DR, the HL-60 cell surface
demonstrated greater amounts of Fc and C receptor. Increased expression of comple-
ment receptor (C3) can also be demonstrated in response to treatment with postendo-
toxin serum [14]. Postendotoxin serum, like LK, though generated in vivo, is thought
to contain DIF activity. These markers are not monocytespecific and, as expected,
can be induced with agents other than TPA or LK. Dimethyl sulfoxide, RA, VD3,
and db cAMP are also effective. Glucocorticoids have been demonstrated as antago-
nists to FcR induction, decreasing the tonically expressed level in HL-60 [19]. The
vitamin 1, 25-dihydrocholecalciferol (VD3) is reported to induce a monocytelike path
of maturation. The expression of antigens reactive with OKM1 and four other mono-
cyte-specific antibodies were increased after treatment with 10-8 M VD3. Staining for
FI-ANAE (monocyte specific) increases from 0 to 73% positive after VD3 treat-
ment [47].
The surface receptors for insulin have been studied. The HL-60 cell line can be
induced to decrease its expression of these receptors after treatment with TPA [61].
The receptor for fMLP is expressed in control cultures of HL-60, but only in the
subpopulation having more mature morphology. The receptor could be induced by
treatment with DMF [53], DIF activities, LK and y-IFN [32]. In contrast, y-IFN
could not induce the receptor in U937 cells [32]. Other markers examined have been
0KM 1 and Leu M2, which are up regulated with 5637 CM, LK, VD3, TPA, and
DMSO [32].

MODULATION AT THE CELL SURFACE OF U937

The major cell surface glycoproteins of U937 have been characterized before
and after induction. The use of SDS-PAGE demonstrated the effects of TPA or LK
treatment. Uninduced cells revealed material with apparent M.W.s of 160-145 Kd
 Models of Myelomonocytic Development 417

(strongest), 210, 200 and 190, and 90 Kd. Following TPA induction, the 200 Kd band
disappeared, while the 90 and 160-145 bands increased in intensity. New bands at
180, 140, and 85 Kd were detected. Lymphokine treatment showed similar results.
The postinduction banding pattern resembled that of peripheral blood monocytes [54].
The expression of Fc and C3 in U937 receptors has been studied. The receptor
of IgG1 can be induced by treatment with TPA, LK, and recombinant ‘y-IFN (30),
CM from SK hepatoma and 5637 cell lines (sources of DIF activity) [32], VD3, and
DMSO [41] . The receptor for IgE is also present but its modulation has not been
studied [48]. The C3b receptor (as recognized by EAC or Mac-l monoclonal anti-
body) is inducible in a similar manner [66]. The Mac-3 cell surface antigen has also
been studied. Mac-i and macrophage-restricted Mac-3 are TPA- and LK-inducible
antigens. If Mac-i antigen is complexed with its complementary monoclonal, the
induction of Mac-3 is blocked [66].
The fMLP receptor is weakly expressed in U937 (7,505 sites/cell). Seventy-two
hours post-LK induction, 38,000 sites per cell were detected [65]. Other inducers
studied include lymphokine titrated with anti-y antibody (0 units y-IFN activity),
OCT-CM, SK-Hep CM, and 5637 CM [32]. Dexamethasone, TPA, and SK-Hep CM
are intermediate in activity. Crude y-IFN and recombinant y-IFN have no activity in
fMLP induction [32]. The expression of HLA-DR determinants in response to
treatment with y-IFN seems variable but has precedent [64] . 132-Microglobulin
expression has also been reported to increase in response to LK [54]. Insulin may
play an important role in the growth regulation of U937 cells. The number of insulin
receptors is reduced after incubation with TPA or RA [52] and may have a causal
role in the observed growth inhibition.

COLONY GROWTH, MOBILITY, AND EFFECTOR FUNCTIONS OF HL-60

As HL-60 cells are cloned in soft agar, they form small compact colonies.
These colonies, under the influence of maturational agents, undergo a morphological
shift to a more diffuse/larger organization. Differentiation induction factor, for ex-
ample, has such activity, and in the mouse it may [11] or may not [50] be closely
associated with a type of G-CSF that induces granulocyte colonies from normal
marrow precursors. There is evidence that CSF-like activities transiently increase the
proliferation of HL-60, have maturational activity, and result in an extinction of
proliferative capacity. By measuring the cloning efficiency and the ratio of diffuse to
compact colonies, the potency of an inducing agent may be ascertained. Other agents
active in this assay include TPA, CSAs, RA, and VD3. This assay is the basis of
biochemical purification of DIF factors found in 5637 and SK-Hep CMs 142].
Increased migration of cells to form diffuse colonies may be the result of increased
mobility and chemotactic responsiveness. In a modified chemotaxis assay, HL-60
cells increased their migration in response to fMLP after a 5-day induction with db
cAMP [13] or DMF [24], but it is not known if other maturational agents act similarly.
Phagocytosis of latex beads or opsonized yeast and the capacity to kill microor-
ganisms are inducible functions in myelomonocytic cell lines like HL-60. Phagocytic
ability is readily induced by TPA, DMSO, RA, VD3, db cAMP, LK, and DIFs.
Phagocytosis may be one of the earliest acquired effector functions of both granulo-
cytes and monocytes. Tetramyristic phorbol acetate- or DMSO-induced HL-60 cells
effectively kill Staphylococcus bacteria [18,40]. Recently it has been demonstrated
418 Harris and Ralph

that HL-60 cells can mediate monocyte antibody dependent cellular cytotoxicity
(ADCC)-like reaction against antibody-coated chicken erythrocytes. Following treat-
ment with LK, HL-60 demonstrated ADCC concomitantly with neoexpression of
HLA-Dr determinants. An unresponsive HL-60 subpopulation did not mature and
continued to proliferate [20]. Similarly a “blast” subclone of HL-60 has been isolated
and characterized as induction-resistant by DMSO but responds to TPA. This variant
has been studied as a model of leukemic progression [46,79]. Continued in vitro
passage may select for “unresponsive” lines, as early freeze-backs show inductions
more dramatically.

EFFECTOR FUNCTIONS OF U937

The U937 cell line is a popularly accepted model of monocyte ADCC effector
functions. First reports of ADCC capacity used LK preparations to activate or induce
the cells [43] . Lymphokine (devoid of CSA) and 5637 CM have been assessed of
their capacity to induce direct and ADCC. Using a mouse leukemia target, LK and
5637 CM induced ADCC, TPA induced both CMC and ADCC, and LPS was
ineffective in either assay [68] . When LK size fractions were tested, it was discovered
that individual cuts could not induce ADCC in spite of the presence of y-IFN [48].
The roles of a-, j3-, and y-IFN as inducers of cytotoxicity have been evaluated. a-
and 13-IFN (400-1,000 units/ml) can induce ADCC against antibody-coated chicken
erythrocyte targets [33]. As inducers of Fc receptors, a- and /3-IFN are only active at
greater than 300 units/mI. Recombinant y-IFN induces erythrocyte ADCC capacity
in U937 cells. With as littleas 10 units/ml a sevenfold increase in FcR expression
correlated with a sevenfold increase in cytotoxicity against antibody-coated chick
erythrocytes [74]. In this same study, dexamethasone did not block the increase in
FcR or ADCC. It is possible that the cellular cytotoxic mechanisms required to lyse
erythrocytes are different from those that will destroy a tumor cell. Lastly, U937 cells
have been studied in the context of their capacity to support the intracellular multipli-
cation of T. Gondii. A small portion of U937 cells can phagocytize these protozoans.
After 24 hr those cells have either lysed or contain numerous trophozoites within in
their vacuoles. A 72-hr incubation with LK could significantly decrease the number
of organisms found within the vacuoles in spite of a generalized increase in phagocy-
tosis [85].

CONCLUSIONS

HL-60 and U937 cells can be used in the screening of novel chemotherapeutic
agents, but it is recognized that these studies are not readily extrapolated to human
leukemia. A powerful application of these cell lines lies in their use for the study of
cytokines and biological response modifiers. It has been observed that growth inhibi-
tion often heralds the acquisition of differentiation markers. If a factor-induced,
leukemia-specific growth inhibition can be achieved in a patient, then an important
goal will be realized. Alternatively, can these factors be used to enhance host defense?
It has been shown that various cytokines can augment or induce effector functions,
such as direct tumor cytotoxicity and ADCC, in these cell lines. Studies have begun
to define the role of these factors in normal hematopoiesis and immunity. The rigorous
characterization of those biochemical events that define cellular development and
 Models of Myelomonocytic Development 419

maturation may point to areas responsive to pharmacologic manipulation and broaden

our basic understanding of hematopoiesis and differentiation.

ACKNOWLEDGMENTS

We thank M.A.S. Moore for support and advice. This work was supported by
grant CH-3O from the American Cancer Society and by the Oar Reichman Foundation.

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