[CANCER RESEARCH 47, 140-144, January 1, 1987]
Correlation between the Molecular Weight and Potency of Polar Compounds
Which Induce the Differentiation of HL-60 Human Promyelocytic Leukemia
Cells1
Simon P. Langdon2 and John A. Hickman3
Cancer Research Campaign Experimental Chemotherapy Group, Pharmaceutical Sciences Institute, Aston University, Birmingham B4 7ET, United Kingdom
in the expression of certain oncogenes (4-7). These differen
tiating agents bring about, in an as yet undefined manner,
changes in gene expression and the consequent suppression of
the malignant phenotype. Such observations have generated an
increasing interest not only in the mechanism whereby such
agents change gene expression but also in the potential for the
treatment of human cancers by a strategy of noncytotoxic
therapy which would promote the terminal differentiation of
certain human tumors to a benign state (3, 8, 9). We, and
others, have been interested in the mechanisms of action and
the development of analogues of the polar solvents, in particular
of yVA-dimethylformamide and yV-methylformamide, with the
prospect of entering less toxic and more effective compounds
into clinical trial (9, 10). A study of the structure-activity
relationships in a series of congeners may provide information
about the molecular requirements necessary for the induction
of differentiation and may give clues as to their mechanism of
action, for example, as to whether or not some type of receptor
is involved. Collins et al. have shown previously that the in vitro
exposure of the HL-60 human promyelocytic leukemia cells to
dimethyl sulfoxide, and eight other polar compounds, promoted
the appearance of cells which resembled mature, nonproliferating granulocytes (11). We have extended this to a study of
the polar solvents related to yvyV-dimethylformamide and have
found that all of the compounds tested were capable of inducing
the differentiation of HL-60 cells. Additionally, the activity of
other polar solvents was examined, for example, methanol,
ethanol, and acetone.
ABSTRACT
Structure-activity studies of a series of polar organic compounds,
including V.V-<l¡im-(li\ltormam¡(li\
A'-methylformamide, and related
ureas and acetamides, were performed with regard to their ability to
promote the terminal differentiation of the human promyelocytic leuke
mia cell line HL-60 to granulocyte-like cells. Functional and morpholog
ical criteria were used to assess the percentage of differentiated cells
which arose from their continuous incubation with different concentra
tions of each agent over a period of 96 h. All of the alkylformamides,
alkylacetamides, and alkylureas tested were found to induce differentia
tion, regardless of structure. Inspection of the results showed that there
was a linear relationship (r = —0.937)between the molecular weight of
each compound and the logarithm of the concentration which was required
to bring about the differentiation of the greatest number of cells, while
viability was generally maintained at >85%. Once established, this
relationship was used to predict the potency of several polar solvents
which were structurally unrelated to the formamides. For example,
methanol, ethanol, and acetone were all inducers of differentiation with
a potency predictable from their molecular weight alone. The terminal
differentiation induced by all of the compounds was only accomplished
by cells which were capable of replication prior to differentiation. At
concentrations which prevented a single replication and brought about a
fall in cell viability over 96 h, no differentiation was observed. A corre
lation was observed between the molecular weight of each compound and
the logarithm of its concentration to bring about cytotoxicity without
differentiation (r = —0.935),and the line was almost parallel to that
defining the concentration required for optimal differentiation (slope
values of -0.02126 and -0.02288). A poorer (r = -0.6654) correlation
was found between the logarithm of the octanol-water partition coefficient
and the logarithm of the concentration required for optimal differentia
tion, when the data for 12 of the polar organic compounds were analyzed.
The results suggest that no special structural requirements are necessary
for the alkylformamides, -acetamides, -ureas, and related compounds to
induce the terminal differentiation of HL-60 cells to granulocyte-like
cells, but that the activity of each compound could be predicted from
their molecular weight. The concentrations required to induce differentia
tion were marginally lower than those which were cytostatic or cytotoxic,
which suggested that a toxic threat to the cells was sufficient to induce
differentiation. Retrospective analysis of a wide variety of compounds
which have been reported to induce the differentiation of HL-60 cells
revealed that, up to a molecular weight of approximately 400, a significant
(P = 0.0182) but poorer correlation (r = -0.665) existed for the relation
ship between molecular weight and the logarithm of the concentration
which induced optimal differentiation.
MATERIALS AND METHODS
Culture of HL-60 Cells. HL-60 human promyelocytic leukemia cells
were provided by Dr Geoffrey Brown, Birmingham University, United
Kingdom, and were maintained in UI'M I Medium 1640 supplemented
with 10% fetal calf serum (Gibco, Glasgow, Scotland), at 37°C,5%
COj, 100% humidity. Cells were routinely maintained in logarithmicphase growth between 2 x IO4 and 1 x 10' cells/ml by biweekly
subculture in 10-ml flasks and had a doubling time of approximately
28 h. Cells were counted by a Coulter Counter Model ZBI cell counter
or hemocytometer, and viability was assessed by the exclusion of a
0.1 % solution of trypan blue.
Assessment of Differentiation. A biochemical and a functional assay
were used to assess the percentage of cells which had undergone
differentiation to granulocyte-like cells. HL-60 cells which when differ
entiated were potentially capable of producing Superoxide aniónwere
incubated at 10* cells/ml for 0.5 h at 37°Cwith l mg/ml NBT4 and
12-O-tetradecanoylphorbol-13-acetate (5 Mg/ml) in PBS. Positive cells
contained intracellular, black formazan granules and were assessed by
light microscopy. Cells which were functionally capable of performing
phagocytosis of rabbit complement-coated yeast cells were assessed by
the method of Shaala et al. (12).
Induction of Differentiation. Five x IO4 HL-60 cells/ml were incu
INTRODUCTION
A number of antiproliferative drugs and chemical com
pounds, such as the polar solvents dimethyl sulfoxide and NJVdimethylformamide, are able to promote the terminal differ
entiation of certain malignant cell lines in vitro (1-3). In some
cases the induction of differentiation has coincided with a fall
Received 1/21/86; revised 6/30/86; accepted 9/30/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by Grant SP 1518 from the Cancer Research Campaign.
1 Present address: ICRF Medical Oncology Unit, Western General Hospital,
Edinburgh EH4 2XU, Scotland.
3To whom requests for reprints should be addressed.
bated continuously with each compound (see Fig. 1) for 4 days. The
cells were counted every 48 h, and after 96 h the percentage of viability
4 The abbreviations used are: NBT, nitrobluetetrazolium; PBS, phosphatebuffered saline (0.2 g KC1, 0.2 g KH2PO4, 8 g NaCl, and 2.2 g NaHPO4 7H2O
per liter).
140
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DIFFERENTIATION
OF HL-60 CELLS BY SOLVENTS
and differentiation were assessed. All experiments were repeated at
least 3 times.
Partition Coefficients. Octanol-water partition coefficients were de
termined by dissolution of each compound, approximately 20 ¿/I.
in 5
ml of PBS to which were added 5 ml of octanol. The mixture was
vortexed for 5 min and then centrifugea at 9000 x g for 2 min, a
process which ensured the separation of emulsions. Samples were taken
in duplicate from the aqueous and octanol phases and diluted 10-fold
with acetone containing dimethylacetamide (or ¿V-methylformamide
when the partition coefficient of dimethylacetamide was determined)
as internal standard. The concentration was determined by gas-liquid
chromatography, as described previously (16). Experimental results
were validated by repetition of the experiment under conditions where
the compound was first added to the octanol. Total recovery from the
two phases was complete in all experiments.
Compounds. Compounds were synthesized in our laboratories (13)
or were purchased from British Drug Houses (Poole, United Kingdom)
or Aldrich Chemical Company (Gillingham, United Kingdom).
Statistical Analyses. Simple regression analysis was performed by
the use of a Statsgraphics computer program purchased from STSC,
Inc., Rockville, MD.
RESULTS
The concentration
of each compound which gave "optimal"
differentiation was first determined. The optimum was that
concentration which allowed a maximum percentage of cells to
express both biochemical and functional characteristics of dif
ferentiation (which in the untreated cells was <5%) while gen
erally retaining a viability of >85% as assessed by the exclusion
of trypan blue. Fig. 1 illustrates this with regard to the effects
of ethanol: an optimum of 1.25% (v/v, 213 HIM)gave a maxi
mum of 75% NBT-positive cells, and 69% of cells were capable
of phagocytosis, while viability was 90%. The optimal concen
tration for differentiation was similarly defined for all of the
compounds (see legend to Fig. 2). The actual value for the
percentage of cells which underwent differentiation at the op
timum was variable according to each compound, an observa
tion common to all such studies (11, 14, 15) (Table 1). All of
the compounds, with the exception of formamide and urea,
were able, at the optimum, to induce between 50 and 90% of
the cells to differentiate. This variability is discussed further
below. Some compounds, for example, i-butylformamide, did
not allow the maintenance of viability >80%, and the optimum
was taken at the point of minimum toxicity. The relationship
between the logarithm of the optimum concentration for each
compound and its molecular weight is shown in Fig. 2A. Anal
ysis of the data for these 17 compounds by simple regression
analysis shows the regression line to have a slope of -0.02288,
% 100
cells
80
60
40
20
0.5
1.0
1.5
2.0
% ethanol v/v
Fig. 1. Biochemical and functional measurements of differentiation and via
bility of HL-60 cells after treatment with various concentrations of ethanol for
96 li. O, viability; D, production of Superoxide anión(NBT positive); •functional
phagocytosis. Points, mean; bars, SE.
intercept 3.573, and a correlation coefficient (r) of —0.937.An
original line was drawn, using the data from experiments with
the formamides, ureas, and acetamides, which allowed predic
tion of the potencies of the activity of the solvents methanol,
ethanol, and acetone. The commonly used inducer of differen
tiation, dimethyl sulfoxide (M, 78), is not shown on Fig. 2A
but, in common with others, we find an optimum differentiation
concentration of 180 mM (log = 2.26), which fits the regression
line (see "Discussion").
All of the compounds used (see legend to Fig. 2A) were
ultimately cytostatic and, at higher concentrations, were cytotoxic to HL-60 cells, when used at concentrations greater than
the optimum required to induce differentiation. Daily estima
tions of cell numbers showed that those cells which had begun
to differentiate by 96 h had undergone at least one doubling.
Fig. 3 illustrates, as an example, the dose-response curve for
the effects of ethanol on cell replication, viability, and differ
entiation. A relationship between the logarithm of the concen
tration of each compound which caused cytotoxicity and/or
cytostasis (i.e.. no doubling in cell numbers from the initial
density of 5 x 10" HL-60 cells/ml, with a loss of cell viability
at 48 h and no indication of differentiation) and molecular
weight produced, by simple regression analysis, a line which,
within experimental error, was almost parallel to that for dif
ferentiation (Fig. 2B). This line had a slope of —0.02126, an
intercept of 3.658, and a correlation coefficient (r) of —0.935.
Determination of the octanol-water partition coefficients of
12 of the compounds and simple regression analysis of the
relationship between the logarithm of the concentration to bring
about optimal differentiation and the logarithm of the partition
coefficient gave the relationship shown in Fig. 4. The regression
line has a slope of -0.0145, intercept 1.501, and a correlation
coefficient of —0.6654.When regression analysis of these same
12 compounds was run as in Fig. 2.1, that is, for the relationship
between logarithm of the concentration to bring about optimal
differentiation and their molecular weight, the line had a slope
of—0.0213, intercept 3.369, and correlation coefficient —0.851.
DISCUSSION
Continuous incubation of HL-60 cells with a variety of polar
compounds of the alkylformamide, -acetamide, and -urea class
brought about cellular differentiation of the HL-60 leukemia in
all cases (Fig. 2A). No structural requirements existed for this
activity. For example, there was no requirement as to the size
of the alkyl groupings, or their number, or whether formamides,
acetamides, or ureas were used. This contrasts strongly with
the structure-activity studies which we have performed with
these compounds using a murine tumor model in vivo for the
measurement of antitumor effect (13, 16), and it suggests that
the singular activity of A'-methylformamide against the murine
models may not be related to the effects on differentiation
observed here. This supposition is additionally supported by
arguments derived from pharmacokinetic data; in the mouse,
maximum plasma levels of about 7 mM are obtainable at a dose
of 400 mg/kg, which proved to be an optimal antitumor dose
(17), yet in vitro, in order to achieve any measure of differentia
tion, a concentration of greater than 100 mM was required for
a minimum of 24 h,5 and the compound was optimally active
at a concentration of 180 mM for 96 h (Table 1).
The observation that there appears to be no structural re
quirement for the activity of these agents resembles the findings
s S. P. Langdon, F. M. Deane, and J. A. Hickman, unpublished observation.
141
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DIFFERENTIATION
Fig. 2. I. relationship between molecular
weight and the logarithm of the concentration
of each agent required to promote optimal
terminal differentiation (see "Materials and
Methods"). /, methanol: 2, formamide: 3.
ethanol; 4, acetone; 5, acetamide; 6, /V-methylformamide; 7, urea; 8, AVV-dimethylformamide; 9. A'-ethylformamide; 10, /V-methylacetamide; //, iVjv-dimethylacetamide; 12, 1,1dimethylurea: 13, 1,3-dimethylurea; 14, r-butylformamide; 15, diethylformamide; 16, diethylacetamide; 17, tetramethylurea. Correla
tion coefficient r = -0.937. B, relationship
between molecular weight and the concentra
tion of compounds outlined in A above, which
prevent a single doubling of 5 x IO4 HL-60
cells/ml, without induction of differentiation.
Correlation coefficient r = -0.935.
OF HL-60 CELLS BY SOLVENTS
log.
optimal
3
concentration
for
differentiation
(mMI
102«
31
• concentration29
7m
3« bf
imMI4*
6*
212«10«
8*
21Mlog.
5¿3»
46» „9*
for
cytostasis
12«10*
.¡;'11«-30
13*
'3j
15«16*
117»1
i
l
5090molecular
70
i
90
iDr
110
30
50
weight
70
molecular weight•16«17«I110
Table 1 Percentage of differentiation of HL-60 cells induced by various compounds
The percentage of differentiated cells at 96 h was assessed by production of Superoxide anión and the capability to phagocytose yeast observed at the optimal
differentiation concentration (see "Materials and Methods").
of viability
of NBT positive
concentration
no.
phagocytosis
h°98±2C66
at 96
h*072
at 96
at96h*068
h)7.3(at 96
CompoundNoneMethanolFormamideEthanolAcetoneAcetamidejV-MethylformamideUrea/VyV-Dimethylformamide/V-EthylformamideA'-Methylacetamide/vyV-Dimethylacetamide
(mm)625400213138250180300100100252050254025105Cell
10s1.8
±1.7 x
I0!1.0
±0.1 x
±367
±640
±622
IO32.6
±0.3 x
±890
1275
±
±569
IO52.2
±0.3 x
172±
1074
±
±767
10*1.9
±0.4 x
±991
±482
±374
10*1.6
±0.5 x
±691
±385
±770
10'3.3
±0.5 x
±395
±511
±315±670
IO51.7
±0.4 x
±494
±485
10*1.6±0.5x
±0.3 x
±396
173±
±650
IO52.1
±289
1266
±
±955
10*1.2
±0.2 x
177±
±351
1840
±
10'1.8
±0.6 x
±985
1566
±
±2160
IO51.9
±0.3 x
1390
±
1767
±
1868
±
10*8.3
±0.5 x
±2465
±857
±847
IO42.5
±1.8 x
±887
±967
1840
±
10'1.8
±0.8 x
±685
1576
±
14ND*59
±
10*2.6
±0.9 x
±594
±468
±0.6 x 10*%
±3%
±IS%o(
±13
" Assessed by exclusion of trypan blue.
* See "Materials and Methods" for details.
' Mean ±SE(n = minimum of 3 experiments). Initial cell number, 5 ±0.3 x IO4at 0 h.
* ND, not done.
cells/ml
«v
3.0
•
1
•2
•5
log.
2-°
•
8
optimal
concentration
for
differentiation
(mM)
10s
10
•
•
'
•15
•16
1.0
•17
48
96 Hours
Fig. 3. Effect of various concentrations of ethanol on the growth, viability,
and differentiation of HL-60 cells. Numbers in parentheses, viability; percentage
of NBT-positive cells indicated at 96 h. •,342 mM; O, 256 mM; A, 213 HIM;A,
171 HIM:•.128 mM.
of a similar study of a series of amides which were screened for
their ability to promote the terminal differentiation of the
Friend murine erythroleukemia cell line in vitro (14, 15). In
spection of our data, however, suggested that a relationship
existed between the logarithm of the in vitro concentration
which was required to give optimal differentiation and its
-LO
o
+1.0
Log Pon nyo
Fig. 4. Relationship between the logarithm of the octanol-water partition
coefficient of compounds (key in legend to Fig. 2) and the logarithm of the
concentration of each agent required to promote optimal differentiation. Corre
lation coefficient r = -0.6654.
molecular weight (Fig. 2A), and once established we were able
to predict the optimum concentration of related compounds as
inducers of terminal differentiation. The activity of compounds
which were structurally unrelated to the amides and ureas, such
142
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DIFFERENTIATION
OF HL-60 CELLS BY SOLVENTS
as methanol and ethanol, was also predicted in this way, solely
by reference to their molecular weight.
Although excellent dose-response relationships existed for
each compound, so as to give an optimum concentration for
differentiation, the relationship was such as to generate steep
dose-response curves (e.g., Fig. 1) from which it can be seen
that, to a degree, there is an element of estimation regarding
the precise value of the optimum. Whether this is a satisfactory
explanation for the variation between each compound regarding
the percentage of cells observed to differentiate (Table 1) re
mains to be established. For example, formamide consistently
induced only a small percentage of cells to differentiate, despite
attempts to assay for increases in differentiation when small
incremental increases in formamide concentrations were made.
We are currently investigating the basis of this variability in
potency and believe it to be an inherent property of some of the
molecules rather than an artifact of the assay system. Schwartz
et al. have noted that a rough classification of the comparative
potency of inducers can be made so as to classify them as
"partial" or "strong" inducers (18). From the data in Table 1,
we consider all compounds except urea and formamide to be
strong inducers, the variability in potency of which largely
depends on the problem of estimating the optimum concentra
tion, as discussed above. Urea and formamide may be partial
inducers either because they alone lack alkyl groupings or,
possibly, because they are more easily metabolized by the cell,
in comparison to their alkyl derivatives. Rovera and Surrey (19)
classified inducers of the differentiation of Friend erythroleukemia cells into three groups according to their cross-resistance
patterns. They noted that the polar solvents, hypoxanthine and
6-thioguanine, belonged to one class, but, like our results with
HL-60 cells, they found that the actual percentage of cells
which underwent differentiation in response to these agents, as
measured by the induction of hemoglobin synthesis, varied from
compound to compound.
The finding of a relationship between the molecular weight
of polar compounds and the optimum concentration required
to induce HL-60 cell differentiation suggested that a simple
physicochemical property of these molecules may be important
for their pharmacological effect. Such a hypothesis parallels
that for the action of the general anesthetics, where activity
increased roughly in proportion to partition coefficient (re
viewed in Ref. 20). We investigated the octanol-water partition
coefficients for the compounds used in the present study but
found a poorer correlation (r = —0.6654) with the optimal
concentration required for differentiation (Fig. 4) when com
pared to the correlation coefficient for the data which relates
the logarithm of optimum concentration for differentiation to
molecular weight (r= —0.851).Although a correlation has been
reported, for some of the molecules used here, between their
molecular weight and their diffusion coefficients across biolog
ical membranes (21-23) it is unlikely that diffusion coefficient
plays a role in the induction of differentiation because the
compounds are present continuously for 96 h, a time in which
equilibrium of distribution would have been established for each
compound (24). In experiments with A'-methylformamide, un
der the conditions used here to obtain optimal differentiation,
the minimum time required to induce commitment of the cells
to differentiation was 26 h and, using I4C /V-methylformamide,
this was found to be a time sufficient for the compound
diffuse into the cell and reach equilibrium.5
to
Despite the fact that we cannot rationalize, in mechanistic
terms, the relationship between the logarithm of the concentra
tion required for a variety of polar compounds to induce opti
mum differentiation and their molecular weight, the relation
ship is revealing in that it allows prediction of an active concen
tration and also suggests to us that a specific receptor for each
molecule is an unlikely proposition. It additionally shows that,
for efficacy in this series, an increase in molecular weight is
advantageous. We have investigated, retrospectively, whether
this relationship holds for different types of compounds which
induce the differentiation of HL-60 cells (Fig. 5). Not surpris
ingly, since the data are taken from a number of studies which
have used conditions rather different from those described here
by us (reviewed in Ref. 24) and since many of these disparate,
higher molecular weight compounds have a very much more
complex chemistry, the relationship is not as good as with the
small molecular weight polar compounds (see Fig. 2). Never
theless, up to a molecular weight of about 400 (Compounds 1
to 12 in Fig. 5) there appears to be a trend relating activity to
molecular weight: regression analysis gives a slope of —0.0144
with a probability level of 0.0182; intercept 1.501; and corre
lation coefficient of—0.6654. The line drawn through the points
in Fig. 5 represents the line derived from the data of Fig. 2, i.e.,
for the polar compounds (y = - 0.02288* + 3.573). Clearly,
some compounds fall quite far from this line (particularly cyclic
AMP), and above a molecular weight of 400 the line reaches a
plateau. The significance of this sudden break point is unclear.
HL-60 cells have been reported to undergo terminal differ
entiation to granulocytic cells in response to a wide variety of
compounds in addition to the polar solvents, such as antimetabolites, anthracyclines, and retinoids (25-27). The question
arises as to how agents as disparate as methotrexate (26) and
tunicamycin (28) may bring about the common end-point of
HL-60 cell differentiation. One property showed by all of these
agents, including the polar compounds used here, is that they
are cytotoxic and/or cytostatic. Our data clearly show (Fig. 2)
•10
log.
optimal
concentration
for
-4
differentiation
(mM)
•6
«14
1213
15
16
•
17
8
-10
-12
•14
100
200
300
400
500 600
700
molecular weight
800
12001255
Fig. 5. Retrospective analysis of the relationship between the logarithm of the
concentration of compounds reported (25) to induce the differentiation of HL-60
cells and their molecular weight. /, sodium butyrate; 2, hypoxanthine; 3, 6mercaptopurine; 4, ethionine; 5, 6-thioguanine; 6, hexamethylenebisacetamide;
7, 5-deazauridine; S, retinoic acid; 9. bromodeoxyuridine; 10, cyclic 3',5'-adenosine monophosphate; //, prostaglandin E2; 12, 1,25-dihydroxyvitamin D; 13,
methotrexate; 14, dibutyryl-3',5'-adenosine monophosphate; 15, 12-O-tetradecanoylphorbol-13-acetate; 16, aclacinomycin A; 17, actinomycin D. Correlation
coefficient for Compounds 1 to 12 r = -0.665.
, that derived from the data
in Fig. 2A, i.e., y = -0.02288* + 3.573.
143
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DIFFERENTIATION
OF HL-60 CELLS BY SOLVENTS
that differentiation induced by the polar compounds occurs at
a concentration of agent which is marginally, and a nearly
constant fraction (0.9768, comparing intercepts), below that
which brings about cytotoxicity.
It has been argued that the events of differentiation and
cytotoxicity need not be related (1,3). However, while it is true
that the toxicity of drugs to HL-60 cells need not be associated
with differentiation, for example, Adriamycin and 6-thioguanine are toxic but have been reported not to induce differentiation
(27, 29), the converse does not appear to be true. When an
agent, under whatever conditions, is able to induce differentia
tion it does so at concentrations just below that required to
prevent cell replication and to induce cell death. Our results
suggest that granulocyte-like differentiation of HL-60 cells by
polar compounds may only depend upon the compound, irre
spective of its structure, bringing about events which are bor
derline to cytotoxicity. What these events are remains unclear.
It is our current premise that an adaptive response of some type
may be induced, perhaps by a multitude of mechanisms, in
response to different toxic threats, and that this response cul
minates in changes of gene expression leading to terminal
differentiation. Preliminary evidence5 suggests that the induc
tion of thermotolerance may be one such change, and we are
actively investigating the relationships between such "stress"
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
responses and differentiation.
18.
ACKNOWLEDGMENTS
19.
We thank Eamonn O'Reilly and Phil Kestell for assistance with
20.
measurements of partition coefficients, Andreas Gescher and Frances
Deane for helpful discussion, Pat Preedy for secretarial assistance, and
the Cancer Research Campaign for financial support.
21.
22.
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1987 American Association for Cancer Research.
Correlation between the Molecular Weight and Potency of Polar
Compounds Which Induce the Differentiation of HL-60 Human
Promyelocytic Leukemia Cells
Simon P. Langdon and John A. Hickman
Cancer Res 1987;47:140-144.
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