electron microscopic localization of acridine orange ... · io"3 m acridine orange after...

[CANCER RESEARCH 31, 1128-1133 August 1971]

Electron Microscopic Localization of Acridine Orange Bindingto 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

An electron microscopic technique has been developed tovisualize the binding sites of acridine orange to DNA withinfixed human leukemic bone marrow cells. This electronmicroscopic technique provides both higher resolution andincreased chemical specificity for discriminating the nuclearbinding sites of acridine orange than does the previousfluorescent light microscopy. Acridine orange binds to DNAexclusively within the active extended euchromatin portion ofthe cell nucleus. This locale of binding is predicted by themolecular model of gene derepression within interphasemammalian chromatin and correlates with the effects of thisligand on RNA synthesis and on the conversion ofeuchromatin to heterochromatin by this and other nuclearligands.

INTRODUCTION

Acridine orange is a useful fluorescence microscopy probefor studying the changes in conformation of nuclear chromatinduring lymphocyte activation by phytohemagglutinin (19, 20),nucleated erythrocyte activation after cell hybridization (6),atypical activation of lymphocytes in infectiousmononucleosis (5), cell inactivation during spermatogenesis(17, 25), and cell inactivation during culture at high celldensities (4, 30).

When used in such microspectrofluorimetric analyses ofsingle fixed cells, acridine orange probes can physicallydistinguish single-stranded nucleic acid binding sites fromdouble-stranded sites (23) but cannot chemically distinguishDNA binding sites from RNA sites (23). With the increasingevidence for the intracellular presence of double-strandedRNA duplexes (13, 18) and single-stranded DNA loops (13),this low chemical specificity has become critical in the furtheruse of the probe. In addition, the low resolution of separatebinding sites possible with fluorescent light microscopysuggested the need for the development of a high resolutionelectron microscopic technique for detecting acridine orangebinding sites chemically specific for DNA. The development ofsuch an ultrastructural probe method has permitted highresolution studies of intranuclear binding sites within humanleukemic bone marrow cells (11).

' This investigation was supported in part by Research GrantCA-10174 from the National Cancer Institute and by a ResearchScholar Award from the Leukemia Society.

Received December 4, 1970; accepted April 13, 1971.

MATERIALS AND METHODS

Bone marrow samples were aspirated from untreatedleukemic patients undergoing diagnostic marrow examination.Informed consent was obtained from the patient in all cases. Atotal of 2.0 ml was aspirated in each case, of which a 1.0-mlaliquot was used for the acridine orange study. Particulatemarrow spicules were separated from aspirated blood byadhesion to an inclined slide and were allowed to react at 4Â°and pH 7.2 for 2 hr with 10~3 M acridine orange (K and K

Laboratories, Plainview, N. Y.; twice recrystallized) in Medium199 (Grand Island Biological Co., Grand Island, N. Y.) afterfixation at 4Â°with 5% glutaraldehyde in Medium 199 at pH

6.5 for 2 hr. The stained spicules were then washed 3 times inMedium 199 and incubated at 37Â°for 30 min in low-calciumspinner-type Eagle's minimal essential medium (Grand IslandBiological Co.) at pH 7.4 and 0.8 mM Mg" containing either

DNase I (Worthington Biochemical Corp., Freehold, N. J.;electrophoretically separated from any contaminating RNaseactivity), RNase (Worthington, electrophoretically pure),trypsin (Worthington, crystallized 3 times) at a concentrationof 1.0 mg/ml, or no enzyme in control aliquots. The incubatedspicules were then prepared for electron microscopy (16) bybeing postfixed in 1% Os04, dehydrated in ethanol, embeddedin Epon, sectioned 0.1 ÃŸthick, stained with 5% uranyl acetate,and examined at 80 kV under high resolution in a Siemens 1Aelectron microscope. Parallel microspectrofluorimetricexaminations (24) were performed on alternate 1.0 Â¿(-thicksections with a Zeiss MPM microspectrofluorimeter. Replicatecontrol samples from a single aspiration either omitted theacridine orange or substituted 10~3 M carbodiimide (1,26)

(Aldrich Chemical Co., Inc., Milwaukee, Wis.; twicerecrystallized) for the acridine orange.

RESULTS

Human bone marrow cells that are caused to react withIO"3 M acridine orange after glutaraldehyde fixation and are

then digested with DNase display a characteristicelectron-dense reaction product, approximately 0.1 n indiameter, which is clearly visible by high-resolution electronmicroscopy (Figs. 1 to 4). The reaction product is visible ineach of the cells of a particular marrow spicule (Fig. 1) and isconfined to the nucleus of each cell, never being found withinthe cytoplasm of such cells (Figs. 2 to 4). Within each cellnucleus, the reaction product is confined to the extendedeuchromatin portion of the cell nucleus (Figs. 2 to 4), never

1128 CANCER RESEARCH VOL. 31

Association for Cancer Research. by guest on August 29, 2020. Copyright 1971 Americanhttps://bloodcancerdiscov.aacrjournals.orgDownloaded from

https://bloodcancerdiscov.aacrjournals.org

Electron Microscopy of Acridine Orange Binding to DNA

being found within the condensed heterochromatin portion orwithin the nucleolar portion of the cell nucleus. The reactionproduct is found within all types of cells of the marrowspicule, 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), issubstituted for acridine orange, the characteristic reactionproduct is not observed (Table 1). Similarly, if DNase isomitted from the preparation sequence or if RNase or trypsinare substituted for DNase, the characteristic rea ion productis not observed (Table 1). These control data indicate thatboth reaction with acridine orange and digestion with DNaseare necessary for visualization of the reaction product (Table1) and strongly suggest that the reaction product is formed asa result of the interaction of acridine orange with DNAbinding sites within the euchromatin portion of the cellnucleus. In view of previous studies, which indicate resistanceto DNase digestion after glutaraldehyde fixation (3),additional studies are currently in progress to define themolecular composition of the reaction product with acridineorange binding and DNase digestion in isolated DNA andisolated euchromatin (16) systems.

DISCUSSION

Acridine orange binds to isolated DNA via each of 2physical binding modes: (a) a stacking interaction resultingfrom the intercalation of acridine orange molecules between

Table 1Reaction product formation after nuclear ligand binding

and enzymatic digestion

The concentration of the nuclear ligand in the reaction solution is1.0 mM. The concentration of the enzyme in the digestion solution is1.0 mp/ml.

Enzyme

Nuclear ligand None DNase RNase Trypsin

NoneAcridineorangeCarbodiimide0a000+0000000

" O, no reaction product visible; +, prominent reaction product

visible.

adjacent base pairs within the interior of the, DNA helix at lowratios of ligand to nucleic acid (21) and (b) an electrostaticinteraction between the basic groups of the acridine orangemolecule and the acidic phosphate groups on the exterior ofthe DNA helix at high ratios of ligand to nucleic acid (22). Theprior presence of polycationic proteins, such as histones, onthe DNA helix effectively decreases the reactivity of suchDNA to acridine orange, both by stabilizing the DNA helixagainst the strand separation (10) necessary to allowintercalation of acridine orange (21) and by neutralizing thephosphate groups on the exterior of the DNA helix capable ofreacting with acridine orange (22). As a consequence of suchinhibition of acridine orange binding to DNA by histones,acridine orange microscopic fluorescent probes have been usedto distinguish chroma tin states in which histones are tightlybound to underlying DNA helices from those in whichhistones are loosely bound to DNA (24).

The current molecular model of gene derepression withinmammalian chroma tin (10) indicates that histones withinactive extended euchromatin are less tightly bound tounderlying DNA than are histones within repressed condensedheterochromatin (9). On this basis, it might be expected that amolecular probe such as acridine orange, which requires accessto DNA in order to bind to DNA (24), would preferentiallybind to DNA within euchromatin rather than to DNA withinheterochromatin (13), although the largest part of nuclearDNA is contained within heterochromatin (13, 16). Thisexpectation is strikingly confirmed in the present study, inwhich the vast majority of the reaction product of acridineorange binding to DNA is found in the euchromatin portion ofthe cell nucleus, with little or none found in theheterochromatin portion (Figs. 2 to 4).

This distribution of acridine orange binding to DNA issimilar to the finding that actinomycin D, another ligand witha high affinity for DNA, similarly binds preferentially to theeuchromatin rather than to the heterochromatin portion ofthe cell nucleus (2). In fact (Table 2), both of these ligands toDNA, while localized preferentially to the euchromatinportion of the nucleus, are also similar in that both areinhibitors of RNA synthesis (13, 14) and both induce theconversion of euchromatin to heterochromatin following theirbinding to the DNA of euchromatin (8, 28). By contrast, bothphytohemagglutinin (7) and mercuric chloride (23) are nuclearligands that increase RNA synthesis (Table 2) and induce theconversion of heterochromatin to euchromatin (23, 29).Previous electron microscopic studies have shown that bothphytohemagglutinin (12, 27) and mercuric chloride (12, 14)

Table 2Correlation of nuclear binding site with ligand effect on RNA synthesis

Localization of nuclear ligand determined by electron microscopy and appropriate enzymaticdigestion (see text).

NuclearligandAcridine

orangeActinomycin DPhytohemagglutininMercuric chlorideNuclear

bindingsiteDNA

within euchromatin (this study)DNA within euchromatin (2)Histones within heterochromatin (27)Histones within heterochromatin ( 14)Ligand

effect onRNAsynthesisDecreases

(13)Decreases (13)Increases (7)Increases (23)

AUGUST 1971 1129



John H. Frenster

are localized within the heterochromatin portion of the cellnucleus of cells responding to these ligands. The binding ofeach of these 2 ligands is sensitive to trypsin digestion (14,27), suggesting that the binding site of bothphytohemagglutinin and mercuric chloride may be thehistones of condensed heterochromatin (14, 27). Suchhistones within condensed heterochromatin are more exposedto potential ligands than are the histones within extendedeuchromatin, since the latter are displaced from underlyingDNA helices by nuclear polyanions such as derepressor RNA(10), phosphoproteins, and acidic and hydrophobicnonhistone proteins (9).

These data indicate (Table 2) that DNA ligands (acridineorange, actinomycin D) localize preferentially to activeextended euchromatin, where they effect an inhibition ofRNA synthesis and a conversion of euchromatin toheterochromatin, while histone ligands (phytohemagglutinin,mercuric chloride) localize preferentially to repressedcondensed heterochromatin, where they effect an activation ofRNA synthesis and a conversion of heterochromatin toeuchromatin (12,14).

There thus appear to be a variety of nuclear ligands withspecific binding sites that can be visualized by high-resolutionelectron microscopy and with actions effecting cell activationor inactivation that are profound following such binding (14).Such ultrastructural probes of chromatin conformation statesare currently being utilized for analyses of neoplastic and ofdifferentiating cells (15).

ACKNOWLEDGMENTS

I thank the study patients for their kindness and generosity, thereferring physicians for their cooperation, and Marie A. Shatos andCheryl C. Hayden for their technical assistance in embedding,sectioning, and staining the marrow samples.

REFERENCES

1. Augusti-Tocco, G., and Brown, G. L. Reaction of jV-Cyclohexyl,jV-B (4-Methylmorpholinium) Ethyl Carbodiimide Iodide withNucleic Acids and Polynucleotides. Nature, 206: 683-685, 1965.

2. Berlowitz, L., Pallotta, D., and Sibley, C. H. Chromatin andHistones: Binding of Tritiated Actinomycin D to Heterochromatinin Mealy Bugs. Science, 164: 1527-1529,1969.

3. Bernard, W., and Granboulan, N. The Fine Structure of the CancerCell Nucleus. Exptl. Cell Res. Suppl., 9: 19-53, 1963.

4. Bolund, L., Darzynkiewicz, Z., and Ringertz, N. R. CellConcentration and the Staining Properties of NuclearDeoxyribonucleoprotein. Exptl. Cell Res., 62: 76-89, 1970.

5. Bolund, L., Gahrton, G., Killander, D., Rigler, R., and Wahren, B.Structural Changes in the Deoxyribonucleoprotein Complex ofLeukocytes from Patients with Infectious Mononucleosis. Blood,35: 322-332, 1970.

6. Bolund, L., Ringertz, N. R., and Harris, H. Changes in theCytochemical Properties of Erythrocyte Nuclei Reactivated by CellFusion. J. Cell Sci., 4: 71-87, 1969.

7. Cooper, H. L. Ribonucleic Acid Metabolism in LymphocytesStimulated by Phytohemagglutinin. J. Biol. Chem., 243: 34-43,

1968.8. DeMan, J. C. H., and Noorduyn, N. J. A. Light and Electron

Microscopic Radioautography of Hepatic Cell Nucleoli in MiceTreated with Actinomycin D. J. Cell Biol., 33: 489-496, 1967.

9. Frenster, J. H. Nuclear Polyanions as De-repressors of Synthesis ofRibonucleic Acid. Nature, 206: 680-683, 1965.

10. Frenster, J. H. A Model of Specific De-repression within InterphaseChromatin. Nature, 206: 1269-1270, 1965.

11. Frenster, J. H. Electron Microscopic Localization of AcridineOrange Binding within Nuclei of Human Leukemic Bone MarrowCells. J. Cell Biol., 43: 39a, 1969.

12. Frenster, J. H. Electron Microscopic Localization of NuclearBinding within Human Leukocytes. Blood, 34: 847, 1969.

13. Frenster, J. H. Biochemistry and Molecular Biophysics ofHeterochromatin and Euchromatin. In: A. Lima-de-Faria (ed.),Handbook of Molecular Cytology, pp. 251-276. Amsterdam:

North Holland Publishing Co., 1969.14. Frenster, J. H. Ultrastructural Binding Sites Correlate with Effects

of Nuclear Ligands on RNA Synthesis within Human Leukocytes.In: I. B. Zbarsky (ed.), Structure and Functions of the CellNucleus, pp. 129-147. Moscow: Institute of DevelopmentalBiology of the Academy of Sciences, 1970.

15. Frenster, J. H. Gene De-repression within Human Neoplasms andwithin Immunotherapeutic Human Lymphocytes. J. Cell Biol., 47:65a-66a, 1970.

16. Frenster, J. H., Allfrey, V. G., and Mirsky, A. E. Repressed andActive Chromatin Isolated from Interphase Lymphocytes. Proc.Nati. Acad. Sei. U. S., 50: 1026-1032, 1963.

17. Gledhill, B. L., Gledhill, M. P., Rigler, R., and Ringertz, N. R.Changes in Deoxyribonucleoprotein during Spermiogenesis in theBull. Exptl. Cell Res., 41: 652-665, 1966.

18. Harel, L, and Montagnier, L. Homology of Double Stranded RNAfrom Rat Liver Cells with the Cellular Genome. Nature, 229:106-108, 1971.

19. Keshgegian, A. A., Meisner, L. F., and Frenster, J. H. ThymidineReversal of Ribothymidine Inhibition of Lymphocyte Mitosis, in:O. R. Mclntyre (ed.), Proceedings of the Fourth Annual LeukocyteCulture Conference, pp. 361-366. New York: Appleton-Century-

Crofts, Inc., 1971.20. Killander, D., and Rigler, R. Activation of Deoxyribonucleoprotein

in Human Leukocytes Stimulated by Phytohemagglutinin. Exptl.Cell Res., 54: 163-170,1969.

21. Lerman, L. S. The Structure of the DNA-Acridine Complex. Proc.Nati. Acad. Sci. U.S., 49: 94-102, 1963.

22. Mason, S. F., and McCaffery, A. J. Optical Rotatory Power ofDNA and of Its Complex with Acridine Orange under StreamingConditions. Nature, 204: 468-470, 1964.

23. Pauly, J. L., CarÃ³n,G. A., and Suskind, R. R. Blast Transformationof Lymphocytes from Guinea Pigs, Rats, and Rabbits Induced byMercuric Chloride in Vitro. ÃŒ.Cell Biol., 40: 847-850, 1969.

24. Rigler, R. Acridine Orange in Nucleic Acid Analysis. Ann. N. Y.Acad. Sci., 157: 211-224, 1969.

25. Ringertz, N. R., Gledhill, B. L., and Darzynkiewicz, Z. Changes inDeoxyribonucleoprotein during Spermiogenesis in the Bull. Exptl.Cell Res., 62: 204-218, 1970.

26. Salganik, R. I., Dashkevich, V. S., and Dymshits, G. M. Studies ofReplicating DNA of Regenerating Rat Liver Using ChemicalModifications with Water-soluble Carbodiimide. Biochim. Biophys.Acta, 149: 603-606, 1967.

27. Stanley, D A., Frenster, J. H., and Rigas, D. A. Localization ofH3-Phytohemagglutinin within Human Lymphocytes and

Monocytes. In: O. R. Mclntyre (ed.), Proceedings of the Fourth




Annual Leukocyte Culture Conference, pp. 1-11. New York:Appleton-Century-Crofts, Inc. 1971.

28. Stevens, B. J. The Effects of Actinomycin D on Nucleolar andNuclear Fine Structure in Salivary Gland Cell of Chironomusthummi. ÃŒ.Ultrastruct Res., ;/: 329-353, 1964.

29. Tokuyasu, K., Madden, S. C., and Zeldis, L. J. Fine Structural

Electron Microscopy of Acridine Orange Binding to DNA

Alterations of Interphase Nuclei of Lymphocytes Stimulated toGrowth Activity in Vitro. J. Cell Biol., 39: 630-660, 1968.

30. Zetterberg, A., and Auer, G. Proliferative Activity andCytochemical Properties of Nuclear Chromatin Related to LocalDensity of Epithelial Cells. Exptl. Cell Res., 62: 262-270, 1970.

AUGUST 1971 1131



,'â€¢-

.â€¢. r

Fig. 1. Electron micrograph of a marrow spicule aspirated from an untreated patient with chronic myelogenous leukemia and caused to reactwith acridine orange followed by DNase digestion (see text). Electron-dense reaction products, 0.1 Min diameter, are confined to the euchromatinportion 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-densereaction products, 0.1 Min diameter, are confined to the euchromatin portion of the cell nucleus. X 10,000.




-â€¢â€¢ Â«-FI - ,

â€¢.*+. *â€¢'

Â«MAÂ»M- *

Fig. 3. Electron micrograph of a myelocyte from another untreated patient with chronic myelogenous leukemia prepared in the same manner asthose shown in Figs. 1 and 2. Electron-dense reaction products, 0.1 n in diameter, are confined to the euchromatin portion of the cellnucleus. X 7,500.

Fig. 4. Electron micrograph of a nucleated erythrocyte from a 3rd untreated patient with chronic myelogenous leukemia prepared in the samemanner 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 cellnucleus. X 10,000.

AUGUST 1971 1133



electron microscopic localization of acridine orange ... · io"3 m acridine orange after...

Documents