John H. Frenster, Shirley L. Nakatsu, and Marilyn A. Masek
Division of Medical Oncology, Department of Medicine,
Stanford University School of Medicine, Stanford, California 94305
II. Acridine Orange Binding to DNA Templates
A. Within
Isolated DNA:
B. Within Isolated Chromatin:
C. Within Isolated Cells:
III. DNase I-Sensitive Ultrastructural Probes of DNA Templates
A. Within Bone Marrow Cells:
(Electron Micrographs):
B. Within Lymph Node Lymphocytes:
(Electron Micrographs):
C. Within Dividing Cells:
D. Within Neoplastic Cells:
V. Summary:
In differentiated cells, only a fraction of the DNA within each cell is active as a template for RNA synthesis (Frenster et al, 1963), and this fraction may be characteristic for each different type of tissue (Paul and Gilmour, 1968).
Control of DNA template activity is evident in many biological systems. During the course of spermatogenesis, a progressive fall in DNA template activity and RNA synthesis results in the formation of mature sperm synthesizing almost no RNA (Ringertz et al, 1970). When such a mature sperm hybridizes with a mature ovum during fertilization, the sperm DNA templates are derepressed, allowing an equal maternal and paternal contribution to gene expression in all resulting cells of the embryo and the adult (Davidson, 1968). Similarly, during the course of embryogenesis in the developing liver, a progressive decrease in the diversity of RNA species being synthesized is observed, but when the adult liver is induced to regenerate following partial hepatectomy, derepression of previously repressed DNA templates again allows the reappearance of those RNA species characteristic of the embryonic state (Church and McCarthy, 1967).
Increasing derepression of previously repressed DNA templates is also noted if neoplastic cells are assayed for the diversity of RNA species being synthesized as the neoplasm progresses first to a benign nodule, then to a spontaneous neoplasm, and finally to a transplantable highly malignant neoplasm (Turkington, 1971). Such a derepression of normally repressed DNA templates also characterizes human leukemic lymphocytes (Neiman and Henry, 1969; Sawada et al, 1973), and may account for the reappearance within adult human neoplasms of cell surface antigens characteristic of normal fetal life (Gold, 1971). A quite similar reappearance of normal fetal antigens has been observed in both chemically-induced and virus-induced neoplasms in experimental antigens (Herstein and Frenster, 1972).
These findings suggest that variable DNA template activity and its control are central to molecular and cellular events occurring during embryogenesis, organ regeneration, and neoplasia, and have prompted the development of high-resolution techniques for assessing DNA template activity within single intact cells (Frenster, 1971).
II. Acridine Orange Binding to DNA Templates.
Acridine orange is a planar polycyclic molecule which binds with
high affinity to isolated DNA molecules (Rigler, 1969).The binding of acridine
orange to DNA involves at least two physical binding modes:
(a): a stacking interaction which involves the intercalation of
acridine orange molecules between adjacent base pairs within the interior
of the DNA helix and which occurs at low ratios of ligand to nucleic acid
(Lerman, 1963); 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, which becomes prominent at high ratios
of ligand to nucleic acid (Mason and McCaffery, 1964).
The prior presence of chromatin proteins, such as polycationic histones,
on the DNA helix effectively decreases the reactivity of such DNA to acridine
orange (Rigler, 1969). The mechanisms of such restriction of binding of
acridine orange are:
(a): the prevention by polycationic histones of local strand separations
within the DNA helix (Frenster, 1965b):
and (b): the neutralization by polycationic histones of phosphate
groups on the exterior of the DNA helix, which otherwise would be available
for reaction with the basic groups of the acridine orange molecule (Frenster,
1969). As a consequence of such inhibition of acridine orange binding by
histones, acridine orange microfluorescent probes (Rigler, 1969) have been
used to distinguish chromatin states in which histones are tightly bound
to underlying DNA helices from those in which histones are loosely bound
to DNA (Frenster, 1965a); this technique
distinguishes DNA templates that are inactive or active, respectively,
in RNA synthesis (Frenster et al, 1963; Frenster,
1965a).
Fluorescent acridine orange probes have made it possible to detect changes in DNA template activity in a wide variety of cell systems by means of microspectrofluorimetry. These cell systems include the increases in DNA template activity that occur in lymphocytes after phytohemagglutinin stimulation (Killander and Rigler, 1969), in nucleated erythrocytes after cell hybridization (Bolund et al, 1969), and in lymphocytes obtained from patients with infectious mononucleosis (Bolund et al, 1970). In addition, the same method has been used to detect decreases in DNA template activity occurring in differentiating spermatozoa during spermatogenesis (Ringertz et al, 1970) and in cells cultured at high cell densities (Zetterberg and Auer, 1970).
When used in such microspectrofluorimetric analyses of single fixed cells, acridine orange probes can distinguish single-stranded nucleic acid binding sites from double-stranded sites by physical means (Rigler, 1969), but cannot distinguish DNA binding sites from RNA binding sites by chemical means. Because of the increasing evidence for the natural occurrence of both double-stranded RNA-RNA duplexes (Jelinek and Darnell, 1972) and of single-stranded DNA loops (Saucier and Wang, 1972), this low chemical specificity becomes a limiting factor in such analyses.
Another limiting factor, the low resolution of separate binding sites possible with fluorescent light microscopy, suggested the need for development of a high-resolution electron microscopic technique for detecting acridine orange binding sites specific for DNA. It was also desirable that such techniques might be employed on section tissue so that in vivo topological relations between cells could be preserved for the DNA template analysis.
III. Ultrastructural Probes of DNA Templates
A technique has been developed for the aspiration of living bone marrow cells from healthy subjects or untreated patients (Frenster, 1971). The cells are obtained in bone marrow spicules which preserve the in vivo topological relationships between the cells. When such living bone marrow spicules are allowed to react with acridine orange and DNase (Frenster, 1972) and are then examined by high-resolution electron microscopic techniques, an electron-dense reaction product is localized over the sites of active DNA templates within the euchromatin portion of the cell nucleus (Frenster, 1971). The intensity of individual probe site accumulation can be analyzed further by measuring the size of the individual reaction products observed within single cells. Such individual probe site accumulations range between 0.025 and 0.1 um in diameter (B probes), 0.1-0.35 um in diameter (A probes), and >0.35 um in diameter (AA probes) and represent the reaction product formed between acridine orange and osmic acid (Nakatsu et al, 1974).
When the number of probe sites was counted within each of 123 individual
differentiating granulocytes (Fig. 1) from normal living human bone marrow,
it was found that the incidence of cells positive for either A or B probes
declined as cell differentiation progressed (Table I).
| Percent Cells
containing: |
Mean Site Count
per positive cell: |
|||
| Cells: | A Probes | B Probes | A Probes | B Probes |
| Promyelocytes | 91.5 | 100. | 9.55 | 35.5 |
| Myelocytes | 85.9 | 96.4 | 8.75 | 35.1 |
| Metamyelocytes | 47.6 | 85.7 | 3.9 | 36.3 |
| Band granulocytes | 0 | 20.0 | 0 | 8.0 |
| Segmented granulocytes | 0 | 12.2 | 0 | 10.1 |
In addition, the number of A probes per positive cell declined as cell differentiation progressed (Table I). These probe site data correlate well with the decline in RNA synthesis previously noted within such differentiating cells (Feinendegan et al, 1964), and they indicate that a progressive restriction of the number and activity of DNA templates occurs as a feature of normal granulocyte differentiation (Nakatsu et al, 1974).
Similarly, when the number of probe sites was determined within each
of 189 individual differentiating erythrocytes (Fig. 2) from normal living
human bone marrow, it was found that the incidence of cells positive for
either A or B probes declined as cell differentiation progressed (Table
II).
| Percent Cells
containing: |
Mean Site Count
per positive cell: |
|||
| Cells: | A Probes | B Probes | A Probes | B Probes |
| Proerythroblasts | 100. | 100. | 16.0 | 8.4 |
| Early erythroblasts | 84.7 | 91.6 | 8.5 | 16.3 |
| Late erythroblasts | 14.8 | 17.2 | 3.4 | 11.8 |
| Nucleated erythrocytes | 0. | 8.3 | 0. | 12.0 |
In addition, the number of A probes per positive cell declined as cell differentiation progressed (Table II). These probe site data correlate well with the decline in RNA synthesis previously noted within such differentiating cells (Feinendegan er al, 1964), and indicate that a progressive restriction of the number and activity of DNA templates occurs as a feature of normal erythrocytic differentiation.
When the number of probe sites within each of 97 individual mononuclear
cells (Figs. 3-6) from normal living human bone marrow was determined (Table
III):
| Percent Cells
containing: |
Mean Site Count
per positive cell: |
|||
| Cells: | A Probes | B Probes | A Probes | B Probes |
| Monocytes | 77.3 | 100. | 7.3 | 43.4 |
| Macrophages | 100. | 100. | 11.1 | 17.4 |
| Endothelial cells | 90.0 | 100. | 8.8 | 16.8 |
| Reticulum cells | 52.5 | 81.2 | 12.2 | 64.3 |
| Plasma cells | 46.2 | 84.6 | 5.5 | 26.5 |
| Lymphocytes | 11.1 | 44.4 | 4.0 | 20.5 |
it was found that cells active in phagocytosis (monocytes, macrophages, endothelial cells, reticulum cells) had a consistently higher incidence of cells positive for either A or B probes than did nonphagocytic cells (lymphocytes, plasma cells). In addition, the number of A probes per positive cell was higher for phagocytic cells than for nonphagocytic cells (Table III). These probe site data suggest that phagocytic cells possess more numerous and more active DNA templates than do nonphagocytic cells in normal human bone marroe. By contrast, small numbers of undifferentiated cells (? stem cells: Fig. 4B) had very high incidences of either A or B probes, and a high number of A probes per positive cell, suggesting that such undifferentiated marrow cells have a very numerous and very active DNA templates in normal human bone marrow.
B: Within Lymph Node Lymphocytes:
A technique has been developed for the surgical removal and analysis of living lymph node cells from untreated patients with Hodgkin's disease (Archibald and Frenster, 1973). The cells are obtained in cubes of lymph node aliquots which preserve the in vivo topological relationships between the cells. When such lymph node aliquots are fixed in glutaraldehyde (Archibald and Frenster, 1973), then reacted with acridine orange and DNase, and finally examined by high-resolution electron microscopic techniques, an electron-dense reaction product is localized over the sites of active DNA templates within the euchromatin portion of the cell nucleus (Frenster, 1972).
Lymph node lymphocytes in Hodgkin's disease are believed to represent
host immunoactive cells directed against neoplastic Reed-Sternberg cells
(Archibald and Frenster, 1973). When the number of
probe sites was determined within each of 194 individual immunoactive lymphocytes
(Fig. 7) from intact lymph nodes of untreated patients with Hodgkin's disease,
it was found that the incidence of cells positive for either A or AA probes
increased with the incidence of cells activated from monoribosomal lymphocytes
to polyribosomal lymphocytes (Table IV).
| Percent Cells
containing: |
Mean Site Count
per positive cell: |
|||
| Lymphocytes: | AA Probes | A Probes | AA Probes | A Probes |
| Monoribosomal | 53.2 | 94.8 | 2.7 | 18.1 |
| Mixed | 67.8 | 100. | 3.2 | 18.7 |
| Polyribosomal | 79.5 | 100. | 3.5 | 17.1 |
In addition, the number of AA probes per positive cell increased as lymphocyte activation increased (Table IV). These probe site data correlate well with the increase in polyribosomes (Tokuyasy et al, 1968) and in acridine orange binding (Killander and Rigler, 1969) previously noted within such lymphocytes during activation, and indicate that a progressive increase in the number and activity of DNA templates occurs as a feature of lymphocyte immune activation (Stanley et al, 1971; Frenster and Rogoway, 1970).
When probe sites were analyzed within a number of large nonlymphocytic polyribosomal cells in the lymph nodes of untreated patients with Hodgkin's disease (Fig. 8), it was found that the number of A or AA probe sites per positive cell decreases as these cells enter the various mitotic phases of cell division. The reaction products within mitotic cells (Fig. 8B) are confined to noncondensed areas of the reconstituting nucleus and correlate with the previous finding that only a small amount of RNA is synthesized by dividing cells (Jakob, 1972). This suggests that a significant restriction in the number and activity of DNA templates occurs as a feature of metaphase and anaphase during mitotic division (Keshgegian et al, 1971; Nakatsu et al, 1974).
When the probe sites within a number of mononuclear or polynuclear Reed-Sternberg cells (Bernhard and Leplus, 1964: Mori and Lennart, 1969) in the lymph nodes of untreated patients with Hodgkin's disease were examined (Fig. 9), it was found that the incidence of mononuclear Reed-Sternberg cells positive for either A or AA probes was quite high (Fig. 9A), while a significant number of polynuclear Reed-Sternberg cells were found to be completely negative for A or AA probes (Fig. 9B). These probe site data correlate well with the decreased incidence of DNA synthesis observed in polynuclear Reed-Sternberg cells as compared to mononuclear Reed-Sternberg cells (Peckham and Cooper, 1969), and suggest that during the course of neoplastic cell survival a significant fraction of advanced neoplastic cells leave the proliferative cycle by virtue of decreasing their DNA template activity (Saunders and Mauer, 1969: Peckham and Cooper, 1969; Ahearn and Trujillo, 1972).
A number of direct ligands to DNA consist of small molecules that are potentially capable of being utililized as probes of DNA primary, secondary, or tertiary structure (Frenster, 1965b). They can be divided into ligands binding preferentially to single-stranded or to double-stranded DNA, and by means of such preference effecting either increases or decreases when added to sensitive cells (Frenster, 1965b). None so far has achieved the utility, high-resolution, and specificity of acridine orange, but studies in their possible use are continuing (Frenster and Herstein, 1973).
Gene expression via RNA synthesis requires RNA precursor substrates, RNA polymerases, and active DNA templates for effective transcription of genetic information. In many biological systems, restriction of DNA template activity, as detected by fluorescent or ultrastructural probes of active DNA template sites, presents a contolling factor in gene expression and RNA synthesis.
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Additional References:
0. Electron Microscopy of Human Lymphocytes before and after Activation by PHA (Busch H, 1974).
1. DNase I-Sensitive Ultrastructural Probes of Active DNA Templates (Electron Micrographs):