[CANCER RESEARCH 31, 1128-1133 August 1971]
Electron Microscopic Localization of Acridine Orange Binding
to DNA within Human Leukemic Bone Marrow Cells1
John H. Frenster
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305
SUMMARY
MATERIALS
An electron microscopic technique has been developed to
visualize the binding sites of acridine orange to DNA within
fixed human leukemic bone marrow cells. This electron
microscopic technique provides both higher resolution and
increased chemical specificity for discriminating the nuclear
binding sites of acridine orange than does the previous
fluorescent light microscopy. Acridine orange binds to DNA
exclusively within the active extended euchromatin portion of
the cell nucleus. This locale of binding is predicted by the
molecular model of gene derepression within interphase
mammalian chromatin and correlates with the effects of this
ligand on RNA synthesis and on the conversion of
euchromatin to heterochromatin
by this and other nuclear
ligands.
Bone marrow samples were aspirated from untreated
leukemic patients undergoing diagnostic marrow examination.
Informed consent was obtained from the patient in all cases. A
total of 2.0 ml was aspirated in each case, of which a 1.0-ml
aliquot was used for the acridine orange study. Particulate
marrow spicules were separated from aspirated blood by
adhesion to an inclined slide and were allowed to react at 4°
and pH 7.2 for 2 hr with 10~3 M acridine orange (K and K
INTRODUCTION
Acridine orange is a useful fluorescence microscopy probe
for studying the changes in conformation of nuclear chromatin
during lymphocyte activation by phytohemagglutinin (19, 20),
nucleated erythrocyte activation after cell hybridization (6),
atypical
activation
of
lymphocytes
in
infectious
mononucleosis (5), cell inactivation during spermatogenesis
(17, 25), and cell inactivation during culture at high cell
densities (4, 30).
When used in such microspectrofluorimetric
analyses of
single fixed cells, acridine orange probes can physically
distinguish single-stranded nucleic acid binding sites from
double-stranded sites (23) but cannot chemically distinguish
DNA binding sites from RNA sites (23). With the increasing
evidence for the intracellular presence of double-stranded
RNA duplexes (13, 18) and single-stranded DNA loops (13),
this low chemical specificity has become critical in the further
use of the probe. In addition, the low resolution of separate
binding sites possible with fluorescent light microscopy
suggested the need for the development of a high resolution
electron microscopic technique for detecting acridine orange
binding sites chemically specific for DNA. The development of
such an ultrastructural probe method has permitted high
resolution studies of intranuclear binding sites within human
leukemic bone marrow cells (11).
' This investigation was supported in part by Research Grant
CA-10174 from the National Cancer Institute and by a Research
Scholar Award from the Leukemia Society.
Received December 4, 1970; accepted April 13, 1971.
1128
AND METHODS
Laboratories, Plainview, N. Y.; twice recrystallized) in Medium
199 (Grand Island Biological Co., Grand Island, N. Y.) after
fixation at 4°with 5% glutaraldehyde in Medium 199 at pH
6.5 for 2 hr. The stained spicules were
Medium 199 and incubated at 37°for
spinner-type Eagle's minimal essential
Biological Co.) at pH 7.4 and 0.8 mM
then washed 3 times in
30 min in low-calcium
medium (Grand Island
Mg" containing either
DNase I (Worthington Biochemical Corp., Freehold, N. J.;
electrophoretically separated from any contaminating RNase
activity), RNase (Worthington,
electrophoretically
pure),
trypsin (Worthington, crystallized 3 times) at a concentration
of 1.0 mg/ml, or no enzyme in control aliquots. The incubated
spicules were then prepared for electron microscopy (16) by
being postfixed in 1% Os04, dehydrated in ethanol, embedded
in Epon, sectioned 0.1 ß
thick, stained with 5% uranyl acetate,
and examined at 80 kV under high resolution in a Siemens 1A
electron
microscope.
Parallel
microspectrofluorimetric
examinations (24) were performed on alternate 1.0 ¿(-thick
sections with a Zeiss MPM microspectrofluorimeter.
Replicate
control samples from a single aspiration either omitted the
acridine orange or substituted 10~3 M carbodiimide (1,26)
(Aldrich Chemical Co., Inc., Milwaukee,
recrystallized) for the acridine orange.
Wis.;
twice
RESULTS
Human bone marrow cells that are caused to react with
IO"3 M acridine orange after glutaraldehyde fixation and are
then
digested
with
DNase display
a characteristic
electron-dense reaction product, approximately
0.1 n in
diameter, which is clearly visible by high-resolution electron
microscopy (Figs. 1 to 4). The reaction product is visible in
each of the cells of a particular marrow spicule (Fig. 1) and is
confined to the nucleus of each cell, never being found within
the cytoplasm of such cells (Figs. 2 to 4). Within each cell
nucleus, the reaction product is confined to the extended
euchromatin portion of the cell nucleus (Figs. 2 to 4), never
CANCER RESEARCH VOL. 31
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Electron Microscopy of Acridine Orange Binding to DNA
adjacent base pairs within the interior of the, DNA helix at low
ratios of ligand to nucleic acid (21) and (b) an electrostatic
interaction between the basic groups of the acridine orange
molecule and the acidic phosphate groups on the exterior of
the DNA helix at high ratios of ligand to nucleic acid (22). The
prior presence of polycationic proteins, such as histones, on
the DNA helix effectively decreases the reactivity of such
DNA to acridine orange, both by stabilizing the DNA helix
against the strand separation (10) necessary to allow
intercalation of acridine orange (21) and by neutralizing the
phosphate groups on the exterior of the DNA helix capable of
reacting with acridine orange (22). As a consequence of such
inhibition of acridine orange binding to DNA by histones,
acridine orange microscopic fluorescent probes have been used
to distinguish chroma tin states in which histones are tightly
bound to underlying DNA helices from those in which
histones are loosely bound to DNA (24).
The current molecular model of gene derepression within
mammalian chroma tin (10) indicates that histones within
active extended euchromatin
are less tightly bound to
underlying DNA than are histones within repressed condensed
heterochromatin (9). On this basis, it might be expected that a
molecular probe such as acridine orange, which requires access
to DNA in order to bind to DNA (24), would preferentially
bind to DNA within euchromatin rather than to DNA within
heterochromatin
(13), although the largest part of nuclear
DISCUSSION
DNA is contained within heterochromatin
(13, 16). This
expectation is strikingly confirmed in the present study, in
Acridine orange binds to isolated DNA via each of 2
which the vast majority of the reaction product of acridine
physical binding modes: (a) a stacking interaction resulting
orange binding to DNA is found in the euchromatin portion of
from the intercalation of acridine orange molecules between
the cell nucleus, with little or none found in the
heterochromatin portion (Figs. 2 to 4).
This distribution of acridine orange binding to DNA is
Table 1
similar to the finding that actinomycin D, another ligand with
Reaction product formation after nuclear ligand binding
a high affinity for DNA, similarly binds preferentially to the
and enzymatic digestion
euchromatin
rather than to the heterochromatin portion of
The concentration of the nuclear ligand in the reaction solution is
1.0 mM. The concentration of the enzyme in the digestion solution is the cell nucleus (2). In fact (Table 2), both of these ligands to
1.0 mp/ml.
DNA, while localized preferentially to the euchromatin
portion of the nucleus, are also similar in that both are
Enzyme
inhibitors of RNA synthesis (13, 14) and both induce the
conversion of euchromatin to heterochromatin following their
Nuclear ligand
None
DNase
RNase
Trypsin
binding to the DNA of euchromatin (8, 28). By contrast, both
NoneAcridine
phytohemagglutinin (7) and mercuric chloride (23) are nuclear
orangeCarbodiimide0a000+0000000
ligands that increase RNA synthesis (Table 2) and induce the
conversion of heterochromatin
to euchromatin (23, 29).
" O, no reaction product visible; +, prominent reaction product
Previous electron microscopic studies have shown that both
phytohemagglutinin (12, 27) and mercuric chloride (12, 14)
visible.
being found within the condensed heterochromatin portion or
within the nucleolar portion of the cell nucleus. The reaction
product is found within all types of cells of the marrow
spicule,
including
nucleated
erythrocytes,
myelocytes,
megakaryocytes, reticulum cells, histiocytes, and lymphocytes
(Figs. 1 to 4).
If acridine orange is omitted from the preparation sequence
(Table 1) or if carbodiimide, another ligand to DNA(1, 26), is
substituted for acridine orange, the characteristic reaction
product is not observed (Table 1). Similarly, if DNase is
omitted from the preparation sequence or if RNase or trypsin
are substituted for DNase, the characteristic rea ion product
is not observed (Table 1). These control data indicate that
both reaction with acridine orange and digestion with DNase
are necessary for visualization of the reaction product (Table
1) and strongly suggest that the reaction product is formed as
a result of the interaction of acridine orange with DNA
binding sites within the euchromatin portion of the cell
nucleus. In view of previous studies, which indicate resistance
to DNase digestion after glutaraldehyde
fixation (3),
additional studies are currently in progress to define the
molecular composition of the reaction product with acridine
orange binding and DNase digestion in isolated DNA and
isolated euchromatin (16) systems.
Table 2
Correlation of nuclear binding site with ligand effect on RNA synthesis
Localization of nuclear ligand determined by electron microscopy and appropriate enzymatic
digestion (see text).
AUGUST
effect on
synthesisDecreases
RNA
ligandAcridine
Nuclear
siteDNA
orange
Actinomycin D
Phytohemagglutinin
Mercuric chlorideNuclear
within euchromatin (this study)
(13)
DNA within euchromatin (2)
Decreases (13)
Histones within heterochromatin (27)
Increases (7)
Histones within heterochromatin ( 14)Ligand Increases (23)
binding
1971
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1971 American Association for Cancer Research.
1129
John H. Frenster
are localized within the heterochromatin portion of the cell
nucleus of cells responding to these ligands. The binding of
each of these 2 ligands is sensitive to trypsin digestion (14,
27),
suggesting
that
the
binding
site
of both
phytohemagglutinin
and mercuric chloride may be the
histones of condensed heterochromatin
(14, 27). Such
histones within condensed heterochromatin are more exposed
to potential ligands than are the histones within extended
euchromatin, since the latter are displaced from underlying
DNA helices by nuclear polyanions such as derepressor RNA
(10),
phosphoproteins,
and acidic
and hydrophobic
nonhistone proteins (9).
These data indicate (Table 2) that DNA ligands (acridine
orange, actinomycin
D) localize preferentially to active
extended euchromatin, where they effect an inhibition of
RNA synthesis and a conversion of euchromatin
to
heterochromatin,
while histone ligands (phytohemagglutinin,
mercuric chloride)
localize preferentially
to repressed
condensed heterochromatin, where they effect an activation of
RNA synthesis and a conversion of heterochromatin
to
euchromatin (12,14).
There thus appear to be a variety of nuclear ligands with
specific binding sites that can be visualized by high-resolution
electron microscopy and with actions effecting cell activation
or inactivation that are profound following such binding (14).
Such ultrastructural probes of chromatin conformation states
are currently being utilized for analyses of neoplastic and of
differentiating cells (15).
ACKNOWLEDGMENTS
I thank the study patients for their kindness and generosity, the
referring physicians for their cooperation, and Marie A. Shatos and
Cheryl C. Hayden for their technical assistance in embedding,
sectioning, and staining the marrow samples.
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1131
,
•
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.
r
Fig. 1. Electron micrograph of a marrow spicule aspirated from an untreated patient with chronic myelogenous leukemia and caused to react
with acridine orange followed by DNase digestion (see text). Electron-dense reaction products, 0.1 Min diameter, are confined to the euchromatin
portion of the cell nucleus of each cell in the spicule. X 2,500.
Fig. 2. Electron micrograph of a nucleated erythrocyte from the same marrow spicule as in Fig. 1 prepared in the same manner. Electron-dense
reaction products, 0.1 Min diameter, are confined to the euchromatin portion of the cell nucleus. X 10,000.
1132
CANCER RESEARCH VOL. 31
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•
.*+.
'
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«MA»
M- *
Fig. 3. Electron micrograph of a myelocyte from another untreated patient with chronic myelogenous leukemia prepared in the same manner as
those shown in Figs. 1 and 2. Electron-dense reaction products, 0.1 n in diameter, are confined to the euchromatin portion of the cell
nucleus. X 7,500.
Fig. 4. Electron micrograph of a nucleated erythrocyte from a 3rd untreated patient with chronic myelogenous leukemia prepared in the same
manner as those shown in Figs. 1 to 3. Electron-dense reaction products, 0.1 u in diameter, are confined to the euchromatin portion of the cell
nucleus. X 10,000.
AUGUST 1971
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1971 American Association for Cancer Research.
1133
Electron Microscopic Localization of Acridine Orange Binding
to DNA within Human Leukemic Bone Marrow Cells
John H. Frenster
Cancer Res 1971;31:1128-1133.
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